Principles of Multiple-Liquid Separation Systems: Interaction, Application and Advancement [1 ed.] 0323917283, 9780323917285

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Principles of Multiple-Liquid Separation Systems

Principles of Multiple-Liquid Separation Systems Interaction, Application and Advancement

Edited by

Kit Wayne Chew Xiamen University, Malaysia

Shir Reen Chia Universiti Tenaga Nasional, Malaysia

Pau Loke Show University of Nottingham, Malaysia

Elsevier Radarweg 29, PO Box 211, 1000 AE Amsterdam, Netherlands The Boulevard, Langford Lane, Kidlington, Oxford OX5 1GB, United Kingdom 50 Hampshire Street, 5th Floor, Cambridge, MA 02139, United States Copyright © 2023 Elsevier Inc. All rights reserved. No part of this publication may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopying, recording, or any information storage and retrieval system, without permission in writing from the publisher. Details on how to seek permission, further information about the Publisher’s permissions policies and our arrangements with organizations such as the Copyright Clearance Center and the Copyright Licensing Agency, can be found at our website: www.elsevier.com/permissions. This book and the individual contributions contained in it are protected under copyright by the Publisher (other than as may be noted herein). Notices Knowledge and best practice in this field are constantly changing. As new research and experience broaden our understanding, changes in research methods, professional practices, or medical treatment may become necessary. Practitioners and researchers must always rely on their own experience and knowledge in evaluating and using any information, methods, compounds, or experiments described herein. In using such information or methods they should be mindful of their own safety and the safety of others, including parties for whom they have a professional responsibility. To the fullest extent of the law, neither the Publisher nor the authors, contributors, or editors, assume any liability for any injury and/or damage to persons or property as a matter of products liability, negligence or otherwise, or from any use or operation of any methods, products, instructions, or ideas contained in the material herein. ISBN: 978-0-323-91728-5 For Information on all Elsevier publications visit our website at https://www.elsevier.com/books-and-journals Publisher: Susan Dennis Acquisitions Editor: Anita Koch Editorial Project Manager: Catherine Costello Production Project Manager: Sruthi Satheesh Cover Designer: Harris Greg Typeset by Aptara, New Delhi, India

Contents

Contributors

xi

1.

1

2.

3.

Polymer–polymer interaction Kah Rong Chew, Revathy Sankaran, Kit Wayne Chew and Pau Loke Show 1.1 Introduction 1.2 Phase diagram 1.3 Parameters influencing phase diagram 1.4 Application of aqueous two-phase system 1.5 Genetic materials 1.6 Future perspective 1.7 Conclusion References Polymer–salt interaction Jun Wei Roy Chong, Zatul Iffah Mohd Arshad, Kit Wayne Chew and Pau Loke Show 2.1 Introduction 2.2 Mechanism and working principles 2.3 Key process parameters 2.4 Applications 2.5 Limitation and future challenges 2.6 Conclusion References Alcohol–salt interaction Hui Yi Leong, Chih-Kai Chang, Krisya Nicole Garcia Aung, Dong-Qiang Lin and Pau Loke Show 3.1 Introduction 3.2 Background and basic principle of alcohol/salt-based liquid biphasic system 3.3 Influence of key parameters 3.4 Applications of alcohol/salt-based LBS 3.5 Limitations and advancements to the alcohol/salt-based liquid biphasic system 3.6 Conclusions References

2 3 4 9 14 15 15 16 21

21 22 26 30 37 38 38 45

46 46 48 51 56 58 59

vi

4.

5.

6.

7.

Contents

Sugar-based deep eutectic solvent-aqueous two-phase system Sophie Jing Nee Chai, Xiao-Qian Fu, Dong-Qiang Lin and Pau Loke Show 4.1 Introduction 4.2 Sugar-based deep eutectic solvent 4.3 Sugar-based deep eutectic solvent-aqueous two-phase system 4.4 Effect of parameters 4.5 Application of sugar-based deep eutectic solvent-aqueous two-phase system 4.6 Advancement of sugar-based deep eutectic solvent-aqueous two-phase system over the last 5 years 4.7 Recycling of sugar-based deep eutectic solvent 4.8 Conclusions References

64

Ionic liquid–salt interaction Wang Sze Kuan, Malcom S.Y. Tang, Wen Yi Chia and Kit Wayne Chew 5.1 Introduction 5.2 Fundamentals of ionic liquid–salt: thermodynamic and properties 5.3 Determination of solution concentration in both phases 5.4 Factors that influence the two-phase separation in ionic liquid/salt ATPS 5.5 Applications of Ionic liquid/salt ATPS 5.6 Conclusion References

81

T-butanol–salt three-phase interaction Yan Jer Ng, Yoong Kit Leong, Wen Yi Chia, Kit Wayne Chew and Pau Loke Show 6.1 Introduction 6.2 Process description 6.3 Principle of three-phase partitioning 6.4 Application of three-phase systems 6.5 Future perspectives and challenges 6.6 Conclusion References Green solvents for multiphase systems Jia Rhen Loo and Wai Yan Cheah 7.1 Introduction 7.2 Green extraction solvents, principles, and reasons for its use 7.3 Increase of usage and future trend 7.4 Economical factor 7.5 Conclusion References

64 65 67 69 72 74 75 76 77

81 83 85 85 87 93 93 95

95 96 97 98 104 106 106 111 111 112 125 128 128 129

Contents

8.

9.

vii

Recyclability and reusability of the solvents Heam Boon Quah, Xuwei Liu, Shir Reen Chia, Saifuddin Nomanbhay and Pau Loke Show 8.1 Introduction 8.2 Solvents for bioseparation 8.3 Benefits of recycling solvents 8.4 Requirements on solvent recycling 8.5 Solvent recycling 8.6 Methods for solvent recovery and recycling 8.7 Feasibility of solvent recovery process References

133

Conventional designs for multiphase liquid separation Apurav Krishna Koyande, Teoh Rui Hong, Kit Wayne Chew and Pau Loke Show 9.1 Introduction 9.2 Principles of three-phase partitioning 9.3 Variables that affect TPP 9.4 Types of assisted TPP 9.5 Applications of TPP 9.6 Future prospects 9.7 Conclusion References

171

10. Advancement in system designs for multiphase liquid separation Nguyen Minh Duc, Shir Reen Chia, Saifuddin Nomanbhay and Vishno Vardhan Devadas 10.1 Introduction 10.2 Liquid biphasic system 10.3 Liquid biphasic flotation 10.4 Ultrasound-assisted liquid biphasic system 10.5 Magnetic-assisted liquid biphasic system 10.6 Electricity-assisted liquid biphasic system 10.7 Microwave-assisted liquid biphasic system 10.8 Future prospects References 11. Economical sustainability of multiphase systems Kien Xiang Bong, Wai Siong Chai and Pau Loke Show 11.1 Economic sustainability 11.2 Advantages of liquid–liquid separation over conventional method 11.3 Three-phase interactions 11.4 Costing in liquid separation system 11.5 Value of end product from biochemical engineering separation 11.6 Cost–benefit analysis of ATPS and conventional separation method

133 135 139 140 142 143 154 155

171 172 175 178 180 182 183 183 187

187 188 191 194 196 199 201 202 203 211 211 212 214 214 222 224

viii

Contents

11.7 11.8

ATPS process cost/benefits evaluation—polymer–salt interaction Conventional protein A affinity chromatography cost/benefits analysis 11.9 Conclusion References

225

12. Environmental sustainability of multiphase systems Hock Chee Lu, Sze Shin Low, Shuet Fen Lai and Kuan Shiong Khoo 12.1 Introduction 12.2 Environmental impact caused by conventional extraction method 12.3 Nonconventional extraction method 12.4 Comparison between alternative extraction methods 12.5 Environmental sustainability-related industrial applications 12.6 Conclusion References

241

13. Potential upscaling of multiphase systems Jasmine Tiong Sie Ming, Chin Kui Cheng, Shuet Fen Lai, Kit Wayne Chew and Kuan Shiong Khoo 13.1 Introduction 13.2 Chromatography 13.3 Membrane 13.4 Aqueous two-phase system 13.5 Precipitation 13.6 Conclusion References 14. Integrated systems for multiphase development Kho Wan You, Shir Reen Chia and Saifuddin Nomanbhay 14.1 Introduction 14.2 Ultrasonic-assisted extraction 14.3 Microwave-assisted extraction 14.4 Enzyme-assisted extraction 14.5 Conclusion References 15. Precursors for promoting liquid–liquid phase separation Mei Yuen Siau, Shuet Fen Lai, Kuan Shiong Khoo and Pau Loke Show 15.1 Introduction 15.2 Fundamentals of aqueous two-phase systems and its application 15.3 Parameters affecting ATPS 15.4 Future prospects and challenges of ATPS 15.5 Conclusion References

230 235 236

241 242 245 250 252 256 256 259

259 260 265 270 280 283 284 289 289 290 295 303 309 309 317 317 318 321 325 327 327

Contents

16. Considerations in designing a multiphase separation system Sridaran Raguraman, Wen Yi Chia, Kit Wayne Chew and Pau Loke Show 16.1 Introduction 16.2 Basis of separation 16.3 Considerations for designing multiphase bioseparation system 16.4 Conclusion References

ix

331 331 332 335 342 342

17. Life-cycle environmental and economical assessment of multiphase systems Zhi Ting Ang, Shuet Fen Lai, Kuan Shiong Khoo and Pau Loke Show 17.1 Introduction 17.2 Introduction of liquid biphasic system/technologies 17.3 Application of multiphase systems 17.4 Life-cycle assessment of multiphase systems 17.5 Case studies 17.6 Challenges 17.7 Conclusion References

345 346 348 355 357 370 372 372

Index

375

345

Contributors

Zhi Ting Ang, Department of Chemical and Environmental Engineering, University of Nottingham Malaysia, Semenyih, Selangor Darul Ehsan, Malaysia Zatul Iffah Mohd Arshad, Faculty of Chemical and Natural Resources Engineering, Universiti Malaysia Pahang, Lebuhraya Tun Razak, Gambang, Kuantan, Pahang, Malaysia Krisya Nicole Garcia Aung, Department of Chemical and Environmental Engineering, Faculty of Science and Engineering, University of Nottingham Malaysia, Semenyih, Selangor, Malaysia Kien Xiang Bong, Department of Chemical and Environmental Engineering, Faculty of Science and Engineering, University of Nottingham Malaysia, Semenyih, Selangor Darul Ehsan, Malaysia Sophie Jing Nee Chai, Department of Chemical and Environmental Engineering, Faculty of Science and Engineering, University of Nottingham Malaysia, Semenyih, Selangor Darul Ehsan, Malaysia Wai Siong Chai, Department of Chemical and Environmental Engineering, Faculty of Science and Engineering, University of Nottingham Malaysia, Semenyih, Selangor, Malaysia; School of Mechanical Engineering and Automation, Harbin Institute of Technology, Shenzhen, Shenzhen, Guangdong, China Chih-Kai Chang, Department of Chemical Engineering and Materials Science, Yuan Ze University, Chungli, Taoyuan, Taiwan Wai Yan Cheah, Centre of Research in Development, Social and Environment (SEEDS), Faculty of Social Sciences and Humanities, Universiti Kebangsaan Malaysia, Bangi, Selangor Darul Ehsan, Malaysia Chin Kui Cheng, Department of Chemical Engineering, College of Engineering, Khalifa University of Science and Technology, Abu Dhabi, United Arab Emirates Kah Rong Chew, Department of Chemical and Environmental Engineering, Faculty of Science and Engineering, University of Nottingham Malaysia, Semenyih, Selangor Darul Ehsan, Malaysia

xii

Contributors

Kit Wayne Chew, School of Energy and Chemical Engineering, Xiamen University Malaysia, Sepang, Selangor Darul Ehsan, Malaysia; College of Chemistry and Chemical Engineering, Xiamen University, Xiamen, Fujian, China Shir Reen Chia, Institute of Sustainable Energy, Universiti Tenaga Nasional (UNITEN), Jalan IKRAM-UNITEN, Kajang, Selangor Darul Ehsan, Malaysia; AAIBE Chair of Renewable Energy, Institute of Sustainable Energy, Universiti Tenaga Nasional (UNITEN), Jalan IKRAM-UNITEN, Kajang, Selangor Darul Ehsan, Malaysia Wen Yi Chia, Department of Chemical and Environmental Engineering, Faculty of Science and Engineering, University of Nottingham Malaysia, Semenyih, Selangor, Malaysia Jun Wei Roy Chong, Department of Chemical and Environmental Engineering, Faculty of Science and Engineering, University of Nottingham Malaysia, Semenyih, Selangor, Malaysia Vishno Vardhan Devadas, Department of Chemical and Environmental Engineering, University of Nottingham Malaysia, Semenyih, Selangor Darul Ehsan, Malaysia Nguyen Minh Duc, School of Biosciences, Faculty of Science and Engineering, University of Nottingham Malaysia, Semenyih, Selangor Darul Ehsan, Malaysia Xiao-Qian Fu, Zhejiang Key Laboratory of Smart Biomaterials, Key Laboratory of Biomass Chemical Engineering of Ministry of Education, College of Chemical and Biological Engineering, Zhejiang University, Hangzhou, China Teoh Rui Hong, Department of Chemical Engineering, Faculty of Science and Engineering, University of Nottingham Malaysia, Semenyih, Selangor, Malaysia Kuan Shiong Khoo, Department of Chemical Engineering and Materials Science, Yuan Ze University, Taoyuan, Taiwan Apurav Krishna Koyande, Department of Chemical Engineering, Faculty of Science and Engineering, University of Nottingham Malaysia, Semenyih, Selangor, Malaysia Wang Sze Kuan, Department of Chemical and Environmental Engineering, Faculty of Science and Engineering, University of Nottingham Malaysia, Semenyih, Selangor Darul Ehsan, Malaysia Shuet Fen Lai, School of Energy and Chemical Engineering, Xiamen University Malaysia, Sepang, Selangor Darul Ehsan, Malaysia

Contributors

xiii

Hui Yi Leong, Zhejiang Key Laboratory of Smart Biomaterials, Key Laboratory of Biomass Chemical Engineering of Ministry of Education, College of Chemical and Biological Engineering, Zhejiang University, Hangzhou, China Yoong Kit Leong, Department of Chemical and Materials Engineering, Tunghai University, Taichung, Taiwan Dong-Qiang Lin, Zhejiang Key Laboratory of Smart Biomaterials, Key Laboratory of Biomass Chemical Engineering of Ministry of Education, College of Chemical and Biological Engineering, Zhejiang University, Hangzhou, China Xuwei Liu, Department of Chemical and Environmental Engineering, University of Nottingham Malaysia, Semenyih, Selangor Darul Ehsan, Malaysia Jia Rhen Loo, Department of Chemical and Environmental Engineering, Faculty of Science and Engineering, University of Nottingham Malaysia, Semenyih, Selangor Darul Ehsan, Malaysia Sze Shin Low, Department of Chemical and Environmental Engineering, Faculty of Engineering, University of Nottingham Malaysia Campus, Semenyih, Selangor Darul Ehsan, Malaysia Hock Chee Lu, Department of Chemical and Environmental Engineering, Faculty of Engineering, University of Nottingham Malaysia Campus, Semenyih, Selangor Darul Ehsan, Malaysia Jasmine Tiong Sie Ming, Department of Chemical and Environmental Engineering, University of Nottingham Malaysia Campus, Semenyih, Selangor Darul Ehsan, Malaysia Yan Jer Ng, Department of Chemical and Environmental Engineering, Faculty of Science and Engineering, University of Nottingham Malaysia, Semenyih, Selangor, Malaysia Saifuddin Nomanbhay, Institute of Sustainable Energy, Universiti Tenaga Nasional (UNITEN), Jalan IKRAM-UNITEN, Kajang, Selangor Darul Ehsan, Malaysia Heam Boon Quah, Department of Chemical and Environmental Engineering, University of Nottingham Malaysia, Semenyih, Selangor Darul Ehsan, Malaysia Sridaran Raguraman, Department of Chemical and Environmental Engineering, Faculty of Science and Engineering, University of Nottingham Malaysia, Semenyih, Selangor, Malaysia

xiv

Contributors

Revathy Sankaran, Research and Knowledge Exchange Hub, Graduate School, University of Nottingham Malaysia, Semenyih, Selangor Darul Ehsan, Malaysia Pau Loke Show, Department of Chemical and Environmental Engineering, Faculty of Science and Engineering, University of Nottingham Malaysia, Semenyih, Selangor, Malaysia Mei Yuen Siau, Department of Chemical and Environmental Engineering, University of Nottingham Malaysia, Semenyih, Selangor Darul Ehsan, Malaysia Malcom S.Y. Tang, Institute of Biological Sciences, Faculty of Science, University of Malaya, Kuala Lumpur, Malaysia Kho Wan You, Department of Chemical and Environmental Engineering, Jalan Broga, University of Nottingham Malaysia, Semenyih, Selangor Darul Ehsan, Malaysia

Polymer–polymer interaction Kah Rong Chew a, Revathy Sankaran b, Kit Wayne Chew c and Pau Loke Show a a Department of Chemical and Environmental Engineering, Faculty of Science and Engineering, University of Nottingham Malaysia, Semenyih, Selangor, Malaysia, b Research and Knowledge Exchange Hub, Graduate School, University of Nottingham Malaysia, Semenyih, Selangor, Malaysia, c School of Energy and Chemical Engineering, Xiamen University Malaysia, Sepang, Selangor Darul Ehsan, Malaysia

1.1

1

Introduction

Downstream processing or bioseparation represents the combinations and vast variety of processes involved in the purification of biomolecules from different sources. A typical downstream process is composed of four stages, including recovery, isolation, purification, and polishing. Among the four steps, the majority of the processing costs come from the purification step (Raja et al., 2012). The conventional method of purification of biomolecules usually requires multistep procedures which involve several unit operations, thus, resulting in a high operating cost. Moreover, the process may cause a low recovery yield of the target biomolecule, leading to a more significant drawback (Molino et al., 2013). Hence, aqueous two-phase system (ATPS) is proposed as an alternative method for the purification of biomolecules as this method is able to reduce the overall cost and prevent the use of multistep procedures. One of the most common ATPS is the polymer–polymer ATPS, which involves the mixing of two types of water-soluble polymers above a critical concentration pe (Castro et al., 2020; Pereira and Coutinho, 2020). Polymer–polymer ATPS have been widely used in the purification, recovery, and separation of biomolecules, such as proteins, enzymes, antibiotics, and DNA (Torres-Acosta et al., 2018). Typically, nonionic polymers such as polyethylene glycol (PEG) and dextran are used as the phaseforming components in the ATPS (Khoo et al., 2020). Besides, other combinations of water-soluble polymers were used to form ATPS, which include a mixture of an ionic and a nonionic polymer (e.g., PEG/poly(acrylic acid) (PAA) and PEG/dextran) as well as a mixture of two charged polyelectrolytes (e.g., polystyrene sulfonate/sodium dextran sulfate). Fig. 1.1 shows the examples of polymer–polymer ATPS reported in the literature (Sadeghi and Maali, 2016; Zaslavsky et al., 2016). Most studies conducted on polymer–polymer ATPS utilizes PEG and dextran as their components. Nevertheless, dextran is considered an expensive polymer, and research has been replacing dextran with less expensive polymers such as polypropylene glycol (PPG) (Salabat et al., 2011). Generally, both PEG and PPG have received the Food and Drug Administration (FDA) approval due to their low toxicity and can be used in oral, topical, and other formulations based on the FDA’s Inactive Ingredient Guide (D’souza and Shegokar, 2016). Although the cost of polymers may be expensive Principles of Multiple-Liquid Separation Systems: Interaction, Application and Advancement. DOI: https://doi.org/10.1016/B978-0-323-91728-5.00005-6 c 2023 Elsevier Inc. All rights reserved. Copyright 

2

Principles of Multiple-Liquid Separation Systems

Nonionic Polymer/Nonionic Polymer

• • • • • • • • • • • •

PEG/Polymer

Dextran/Polymer

PPG PVME PES PVA Dextran HP-Starch Pullulan Maltodextrin PVP Ficoll UCON PPGDME

• • • • • • • •

PEG PVP UCON PVA PPG Ficoll Natrosol HP-Starch

PPG/Polymer

• • • • • •

PEG PVA PEGME PEGDME Dextran Ficoll

Nonionic Polymer/Ionic Polymer • • • • • •

PEG/Dextran sulfate PEG/PAM PVP/RAM PEG/PAA PEG/Carboxymethyl dextran PVA/Acrylic polymers

Ionic Polymer/Ionic Polymer • Dextran sulfate/PSS • Dextran sulfate/DEAE-Dextran

• HP-Starch/Ficoli • Ficoli/UCON • HP-Starch/UCON

Figure 1.1 Examples of polymer–polymer ATPS reported in literature.

when compared with salts, cheap polymer alternatives, for example, xanthan, starch derivatives, and tree gum (Benavides et al., 2011), are being investigated. Besides, the recycling of polymers is also being studied as another alternative to reduce cost (Pereira et al., 2012). Polymer–polymer ATPS is typically conducted in batch mode, however, it was shown that the process overall costs can be greatly improved when conducted in continuous mode (Molino et al., 2013). In this book chapter, the principles of polymer–polymer ATPS and the partitioning behavior of biomolecules are discussed by using a phase diagram. Moreover, the parameters that affect the partitioning behavior of phase-forming components were evaluated. The chapter also reviewed the application of polymer–polymer ATPS in the pharmaceutical and biotechnological industries. Future perspectives and research gaps were also addressed at the end of this chapter.

1.2 Phase diagram The phase diagram can be described as a fingerprint to a system, which illustrates the working area of ATPS under specific pH and temperature. Based on the phase diagram, valuable information such as the concentration of components in the top and bottom phases as well as their concentration for phase formation can be obtained (Raja et al., 2012). The phase diagram is composed of a tie line, binodal curve, and critical point. The binodal curve is constructed by connecting the binodal points, forming a convex curve. The coordination of these points can be identified by using three methods, namely, cloud point method, turbidometric titration method, and node determination method (Iqbal et al., 2016).

Polymer–polymer interaction

3

Binodal Curve

S1

Top phase constituent % (w/w)

T

S2

S3

∆Y Biphasic region C Monophasic region

B

∆X Bottom phase constituent % (w/w)

Figure 1.2 Schematic representation of a phase diagram. This figure is reproduced from Iqbal et al. (2016).

Fig. 1.2 shows the binodal curve that separates the area of concentrations of each component. The curve splits the concentrations into two regions. The region above the curve indicates the biphasic region, which forms two aqueous phases that are immiscible, whereas the region below the curve indicates the monophasic region (Iqbal et al., 2016). The tie line is represented by line TB according to Fig. 1.2, which connects two nodes that lie on the binodal curve. On the same tie line, all potential systems are assumed to contain the same equilibrium composition in both the top and bottom phases. The critical point is denoted on point C. At the critical point, the value of tie line length (TLL) is equivalent to zero. Above this point, the volume of both phases is equal to zero. The estimation of TLL can be calculated using the weight ratio as shown in Eq. (1.1) (Iqbal et al., 2016). SB Vt ρt = Vb ρb ST where V is the volume of top (t) and bottom (b) phases, P is the density of top (t) and bottom (b) phases, and SB and ST are the lengths of segments on the binodal curve.

(1.1)

4

Principles of Multiple-Liquid Separation Systems

Alternatively, TLL can also be calculated using a more accurate method as shown in Eq. (1.2).  TLL = X 2 + Y 2 (1.2) Typically, the tie line is presented as a straight line and the slope of the tie line can be calculated using Eq. (1.3). STL =

Y X

(1.3)

The partition of biomolecules is determined by the relationship between the equilibrium of the top and bottom phases in ATPS. The partition coefficient, K can be represented using Eq. (1.4) (Raja et al., 2012). K=

ConcAT ConcAB

(1.4)

where conc. AT is the equilibrium concentration of A in the top phase, conc. AB is the equilibrium concentration of A in the bottom phase.

Based on the model, partition behavior can be influenced by various factors, that include hydrophobicity (where separation is determined by hydrophilic properties of polymer), size (surface area or molecular size of polymer), electrochemical (where partition behavior is driven by electrical potential), conformation-dependent (where the partition is determined by conformation of polymers), and biospecific affinity (when the target molecules binds to the polymer on a particular site). ln K = ln Kh f ob + ln Ksize + ln Kelec + ln Kconf + ln Kaffinity + ln K ◦

(1.5)

The expression of the factors of partition coefficient is shown in the form of a logarithm in Eq. (1.5) (Iqbal et al., 2016).

1.3 Parameters influencing phase diagram 1.3.1 Molecular weight In the polymer–polymer ATPS, the partitioning of biomolecules is greatly affected by the molecular weight of polymers. Generally, polymers with higher molecular weight require lower concentration for phase formation (Atefi et al., 2017). It was shown that the reduction of molecular weight of a phase polymer could result in an ATPS that favors a greater distribution of cells toward the aqueous phase that is enriched in that polymer. The possible mechanisms are due to the alteration of phase hydrophobicity or the increased steric exclusion of biomolecules from that phase, which demonstrated that polymers with higher molecular weight can decrease the hydrophobic area or

Polymer–polymer interaction

5

hydrophilic groups, causing an increase in hydrophobicity. Besides, the increased hydrophobicity due to the high molecular weight of the polymer can reduce the free volume by increasing the polymer chain length (Goja et al., 2013; Khoo et al., 2020). In contrast, polymers with low molecular weight can result in a reduction in purification factor due to the fact that the target biomolecules will be partitioned at the top phase with the other contaminant proteins (Khoo et al., 2020). Hence, in order to acquire the greatest recovery of target compounds, it is crucial to ensure that the hydrophobicity of polymers is maintained at an optimum condition. In fact, the effect of the molecular weight of polymer on phase diagrams has been discussed in several studies (Singh and Tavana, 2018). The studies have reported that an increase in the molecular weight of polymer resulted in decreased partition coefficient when the PEG concentration is kept constant (Ketnawa et al., 2017). Moreover, it was shown that better protein partitioning is observed in PEG with lower molecular weight when compared to that with higher molecular weight. Singh and Tavana (2018) studied the partitioning of collagen in PEG-dextran ATPS using different molecular weights of polymers. The study found that there was greater collagen being partitioned to the aqueous phase when the molecular weight of the corresponding phase polymer was reduced (Singh and Tavana, 2018). According to research, it was shown that PEG with generally low molecular weights between 1000 and 4000 can result in the highest partition parameters. Singh and Banik (2012) investigated the effect of different PEG molecular weights on the partitioning of L-glutaminase in PEG-dextran ATPS (Singh and Banik, 2012). It was shown that increasing PEG molecular weight from 2000 to 4000 kDa resulted in a higher partitioning value, whereas PEG with molecular weight of 6000 kDa led to a lower partitioning value (Singh and Banik, 2012). The authors suggested that PEG with high molecular weight contributed to the top phase with high hydrophobicity, forming an interaction between L-glutaminase with dextran, which explains the observation of top phase with low partition coefficient. Moreover, the study also concluded that PEG 4000-dextran T500 was the most appropriate ATPS to obtain the maximum partition coefficient value for L-glutaminase partitioning. Another study done by Mehrnoush et al. (2012) demonstrated that the molecular weight of PEG had a great influence on serine protease partitioning. Based on the study, increasing molecular weight of PEG reduces the partition coefficient due to the fact that PEG becomes more compact by forming hydrophobic bonds between molecules (Mehrnoush et al., 2012). This causes the target enzymes to deposit at the bottom phase as the migration of enzymes into the top phase was inhibited. Moreover, when PEG with high molecular weight of 10,000 g/mol was used, the yield and purification factor of serine protease were reduced. The possible mechanism is that the increased chain length of PEG can lead to a decreased available free volume in the top phase to fit the enzymes, causing the enzyme-specific activity to decrease. On the other hand, the purification factor of the enzyme was also reduced when PEG with molecular weight of 6000 g/mol was used. This phenomenon occurs as a result of the reduction in PEG exclusion effect, leading to the migration of target and contaminate enzyme from bottom to top phase, which causes the low enzyme yield and purity (Mehrnoush et al., 2012). To obtain the maximum partition coefficient, the study recommended that the

6

Principles of Multiple-Liquid Separation Systems

intermediate PEG molecular weight of 8000 g/mol was the most appropriate ATPS to purify serine protease.

1.3.2 Polymer concentration The effect of PEG concentration on the partitioning of polymers has been widely discussed in several studies (Benavides et al., 2011; Hemavathi and Raghavarao, 2011; Wu et al., 2013). In general, increasing PEG concentration results in a decreased partition coefficient of the target biomolecules. Besides, increased polymer concentration is associated with viscosity, refractive index, and high density of the phase properties (Ketnawa et al., 2017). In fact, the effect of concentration of polymers is related to the partition coefficients of enzymes and proteins. It was demonstrated that the concentration of phase-forming components had an influence on purity and partition coefficient of polyphenol oxidase and bromelain. The study found an increase in purity by 3.6–4.0-fold when the concentration of PEG 1500 was increased from 12% to 18% (w/w). This phenomenon occurs as a result of more total proteins being partitioned to the bottom phase. Besides, the study also found that increasing PEG concentration can reduce the partition coefficient of total proteins and polyphenol oxidase (Babu et al., 2008). It was suggested that the effect of volume exclusion plays a part in this outcome. Wu et al. (2013) suggested an alternative method to extract human immunoglobulin G (IgG) by using PEG-hydroxypropyl starch (HPS) ATPS (Wu et al., 2013). In the study, different concentrations of PEG and HPS on IgG partitioning were compared. It was found that an increase in PEG or HPS concentrations can lead to a reduction in the HSA partition coefficient but an increase in IgG partition coefficient. Moreover, increasing concentrations of both polymers can cause unequal protein partitioning by improving the differences in hydrophobicity of top and bottom phases (Wu et al., 2013). Therefore, it was suggested to use high PEG and HPS concentrations to ensure that the viscosity of both phases are within the normal range and all components can disintegrate in the system. The concentration of polymers ranges between 8% and 18% (w/w) for the formation of two phases alongside polymers with specific critical value of the phase diagram (Ketnawa et al., 2017). Generally, increased polymer concentration is associated with viscosity, refractive index, and high density of the phase properties. Therefore, high polymer concentration contributes to the huge variations in phase properties. In addition, the molecular weight of polymers is linked to its concentration to form ATPS. Similarly, molecular weight of polymers also influences phase viscosity as the viscosity is affected by concentration. An increase in polymer molecular weight can lead to a lower concentration needed for the formation of phase as viscosity at high levels may impact further process (Ketnawa et al., 2017). There are two mechanisms involved in explaining the concentration of polymers in water, which are the concentrated and diluted solutions regimes. The diluted solutions regime consists of a few solvent layers to keep polymers apart. When there is an increase in molecular weight, the range of concentration that is applicable in this regime will be limited (Grilo et al., 2016). Subsequently, the solutions can be explained using the concentrated regime. When the polymers have occupied high volumes and begin to

Polymer–polymer interaction

7

form a complex network or a mesh-like structure, the concentrated regime is considered sufficient. The crossover concentration is defined as the limit concentration when the regime switches in a polymer solution. It was found that polymers with high molecular weight are associated with lower crossover concentration and the converse is also true (Grilo et al., 2016). Other than that the study has demonstrated that in a diluted solution regime, polymers with low molecular weight act at higher weight fractions compared to polymers with high molecular weight.

1.3.3 Temperature The phase diagram is also affected by the temperature factor and this can influence the density and viscosity of partition (Iqbal et al., 2016; Khoo et al., 2020). In a polymer– polymer ATPS, phase separation can be achieved at lower temperature alongside the polymers with lower concentrations (Iqbal et al., 2016). Nevertheless, the effect was in contrast to the polymer-salt system (Chakraborty and Sen, 2016). Hence, experiments associated with ATPS are recommended to be conducted under strictly controlled temperatures. When there is an increase in temperature, the polymer–polymer binodal curve will shift away from the origin, which is toward phase-forming agents with higher concentrations (Meghna et al., 2010). This phenomenon occurs as a result of the reduced biphasic area and can be referred to as an upper critical solution temperature behavior (Pereira and Coutinho, 2020). For the greatest partitioning and recovery of target biomolecules, it was suggested to maintain the temperature between 20 and 40°C for optimal reaction (Khoo et al., 2020). Various studies have shown that temperature can greatly influence the behavior of phase diagrams in polymer–polymer ATPS (Chakraborty and Sen, 2016; Machado et al., 2012). An interesting outcome was observed in the study done by Machado et al. (2012), where the increase in temperature had a dual behavior in the PEG-maltodextrin ATPS with varying molecular weight (Machado et al., 2012). For the system consisting of PEG 8000 and maltodextrin with dextrose equivalent of 15, increasing the temperature can displace the phase separation area minimally. This shows that the enthalpy and heat capacity had undergone small changes during the phase segregation process. The study demonstrated that the displacement of the equilibrium curve at lower maltodextrin concentration and higher PEG concentration was most notable when the temperatures were at 298.2K and 308.2K. It was suggested that the formation of ATPS at lower polymer concentration takes place at lower temperatures, which shows that a reduction in temperature can increase the heterogeneous biphasic region in the phase diagram. In contrast, the ATPS consisting of PEG 6000 and maltodextrin with dextrose equivalent of 10.5 demonstrated an inverse behavior corresponding to increasing temperature. The authors believed that lower concentrations of PEG and maltodextrin with decreased molecular weight may present the same outcome as a polymer-salt system (Machado et al., 2012). According to Grilo et al. (2016), in the case of ATPS with the same compositions but varying temperatures, a reduction in temperature can lead to increased interfacial tension (Grilo et al., 2016). Nevertheless, this phenomenon does not apply in ATPS with similar phase separation behavior. In this case, an increase in temperature results

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Principles of Multiple-Liquid Separation Systems

in increased interfacial tension when the same TLL are compared (Grilo et al., 2016). Studies have demonstrated that the changes in interfacial tension are associated with TLL, which is the length between the critical point and the working composition (Grilo et al., 2016; Liu et al., 2012). The outcomes of the studies have concluded that higher interfacial tensions are linked to longer TLL (Grilo et al., 2016; Liu et al., 2012).

1.3.4 pH The pH factor plays the role of changing the surface properties and charge of solute, which can have an effect on phase separation (Iqbal et al., 2016; Raja et al., 2012). In general, the net charge of the protein is determined based on the values of pH and isoelectric point (Iqbal et al., 2016; Raja et al., 2012). For example, the net charge of protein is negative when pH is higher than isoelectric point; the net charge of protein is positive when pH is lower than isoelectric point; and the net charge of protein is zero when pH equals to isoelectric point (Raja et al., 2012). Research has demonstrated that biomolecules that are negatively charged increase the partition coefficient and prefer the top phase at higher pH (Raja et al., 2012). This occurs due to the PEG units and biomolecules that exert electrostatic interaction between each other (Raja et al., 2012). Moreover, due to positive dipole moment, proteins with pH greater than isoelectric point will be attracted toward the phase that is rich in PEG (Andrews et al., 2005; Olivera-Nappa et al., 2004). The effect of pH on the partitioning of biomolecules in the polymer–polymer ATPS has been scarcely studied. According to Yan and Cao (2014), it was shown that a reduction in pH can cause the polymers to shift toward the origin, which is the high biphasic area in the binodal curve (Yan and Cao, 2014). Saravanan et al. (2008) have evaluated the influence of pH on protein purification and partitioning using a poly(ethylene glycol)–poly(acrylic acid) (PEG-PAA) ATPS (Saravanan et al., 2008). The results showed increasing yield for PEG 6000 and PEG 10000 when the pH was increased. In general, pH efficacy on protein partitioning can be adjusted based on the alteration of either the ratio of the charged species or the charge of solute (Saravanan et al., 2008). Proteins at low pH will gain a net positive charge due to the extra proton acquired by the amine. On contrary, proteins at high pH will gain a net negative charge as the carboxyl loses its proton. The isoelectric point is defined as the pH at which protein acquires a zero net charge (Li et al., 2002). According to the study, an increase in pH can result in an increased partition coefficient, which explains the phenomenon where proteins are more negatively charged at higher pH (Saravanan et al., 2008). Various studies have demonstrated that proteins that are positively charged will partition to the bottom phase whereas proteins that are negatively charged to the PEG-rich top phase (Asenjo et al., 1994). In this study, the optimal pH for the partitioning of myoglobin and ovalbumin is at pH 8.0 as this pH value is greater than both protein isoelectric points (Saravanan et al., 2008). Therefore, better partitioning of protein toward the PEG-rich phase can be observed. As a result of increased partition coefficient, the outcome can be explained by hydrophobic interaction and net charge effect (Saravanan et al., 2008).

Polymer–polymer interaction

9

Based on Mehrnoush et al. (2012), it was demonstrated that serine protease had a purification factor of pH 6 due to the fact that the enzymes were denatured at lower pH (Mehrnoush et al., 2012). Besides, it was observed that the enzymes were partitioned to the bottom phase at pH 6, indicating that the change of partitioning behavior was due to the charge of the protein. When the pH is above 7–9, serine protease will gain a negative charge as the enzyme has an isoelectric point of 6, resulting in PEG to interact with the negative charge (Mehrnoush et al., 2012). According to the study, it was recommended to maintain the pH level of ATPS at 7.5 to obtain maximum purification of a serine protease (Mehrnoush et al., 2012). According to Ketnawa et al. (2017), protein partitioning can be affected by electrochemical interactions. Following the attraction between molecules of opposite charges, solutes, and proteins that have opposite charges will have a selective fractionation toward a specific phase. Research has demonstrated that pH can affect the concentration of phase-forming polymers and alter the targeted protein partitioning. This phenomenon causes the polymer to gain or lose charge from their functional groups, resulting in contraction and expansion of materials as well as attraction and repulsion (Saravanan et al., 2008).

1.3.5 Tie line length The partitioning of biomolecules can also be influenced by TLL, which involves the interfacial tension and hydrophobicity of ATPS (Goja et al., 2013). Generally, increased TLL results in an ATPS that is more hydrophobic (Liu et al., 2012). This phenomenon occurs because the water availability is decreased. Moreover, increasing TLL also increases the partition coefficient of proteins, causing more proteins to be partitioned toward the top phase (Mehrnoush et al., 2012). According to Mehrnoush et al. (2012), TLL significantly affects the yield and partition coefficient of serine protease in terms of the effect of interaction between molecular mass of PEG and TLL as well as the quadratic and main effects of TLL (Mehrnoush et al., 2012). Studies have demonstrated that increasing TLL can result in the increase of interfacial tension between top and bottom phases as well as the hydrophobicity of the top phase (Mehrnoush et al., 2012). The outcome of the study showed that the maximum yield and partition coefficient were observed at 17.4% of TLL. However, at an extremely high TLL of 25.6%, there was a decrease in yield and partition coefficient due to decreased volume ratio of top phase, causing the activity and yield of serine protease to reduce in this phase (Mehrnoush et al., 2012).

1.4

Application of aqueous two-phase system

Although the partitioning mechanisms of ATPS have not been fully understood, the purification and recovery of products widely recognize the use of these systems as an effective platform for downstream processing. The ATPS system is mostly used to substitute traditional liquid–liquid extraction processes that use organic solvents (Iqbal et al., 2016; Soares et al., 2015). Other than that these systems can also be used to stabilize biological structures that are delicate due to low interfacial tension

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Principles of Multiple-Liquid Separation Systems

(Teixeira et al., 2018). The application of ATPS has been widely discussed in various reviews and studies (Freire et al., 2012; Iqbal et al., 2016). The areas where ATPS is applied include the purification or separation of enzymes, pharmaceutical products like antibiotics and monoclonal antibodies, proteins such as nucleic acids and DNA, as well as metal extraction (Pereira and Coutinho, 2020). Table 1.1 describes the application of polymer–polymer ATPS that is available in the current literature.

1.4.1 Proteins Proteins have been widely used in the field of biotechnology as these biomolecules are versatile in terms of structure and functionality. The application of ATPS specializes in the recovery, purification, and separation of proteins, for example, therapeutic proteins, functional proteins, and enzymes. Various studies have been done on enzyme recovery for its application in medical and bioremediation treatments as well as detergent, food, and paper industries (Benavides et al., 2011; Phong et al., 2018). In the study done by Rosa et al. (2007), an experiment was carried out to recover human immunoglobulin gamma (IgG) using polymer–polymer ATPS in the presence of different variations of PEG like the diglutaric acid functionalized PEGs (PEGCOOH) (Rosa et al., 2007). It was found that the best purification of IgG was obtained in the ATPS of PEG-dextran with 20% (w/w) of PEG 150-COOH, where the purification factor was 1.9 and the yield of recovery was 93%. The outcome of this study showed an increase by 60-fold in IgG selectivity with the addition of PEGCOOH when compared to PEG-dextran alone (Rosa et al., 2007). Furthermore, back extraction can be implemented on thermoseparating polymers, which enables phase separation through increasing the temperature in the system (Molino et al., 2013). In the study done by Ferreira et al. (2008), the application of back extraction using ATPS consisting of ethylene oxide/propylene oxide (UCON) and dextran, alongside the addition of modified polymer such as triethylene glycol-diglutaric acid (TEG-COOH) was investigated (Ferreira et al., 2008). Following this method, the recovery yield and purity of IgG showed significant improvements as the IgG partition was shifted into the top phase. According to the result, the best performance was achieved by using ATPS which consists of 6% dextran, 8% UCON, and 20% TEG-COOH, which led to a 88% purity and 85% recovery of IgG (Ferreira et al., 2008). Ooi et al. (2011) demonstrated the use of ATPS consisting of PEG and dextran in the extractive fermentation of extracellular lipase from Burkholderia pseudomallei (Ooi et al., 2011). According to the study, it was found that the best condition for the production of lipase was at PEG 8000 and dextran T500, where 92.1% yield was obtained. During the process, lipase was partitioned in the upper phase whereas the biomass was deposited in the bottom phase (Ooi et al., 2011). According to Chen and Lee (1995), the production of chitinase from Serratia marcescens was studied via extractive bioconversion in PEG-dextran ATPS (Chen and Lee, 1995). Based on the results, the enzymes were recovered in the bottom phase while the cells partitioned in the upper phase. The maximum recovery yield of enzymes was obtained when the ATPS was composed of PEG 20,000 and dextran T500. The possible mechanisms that result in the improved chitinase production are not associated with increased

ATPS Protein PEG-dextran

Biomolecules

Yield/purity

Reference

Human immunoglobulin gamma (IgG)

Rosa et al. (2007)

Ethylene oxide/propylene oxide (EOPO)-dextran, with triethylene glycol-diglutaric acid (TEG-COOH) PEG-dextran

Human immunoglobulin gamma (IgG)

Diglutaric acid functionalized PEGs (PEG-COOH) showed great affinity to IgG.PEG150-COOH had the best purification of IgG with a purification factor of 1.9 and recovery yield of 93%. The best performance was achieve using ATPS with 8% EOPO, 6% dextran and 20% TEG-COOH with a purity of 88% and recovery yield of 85%. The best performance was at PEG8000 and dextran T500 with 92.1% yield.

Ooi et al. (2011)

The maximum recovery yield of enzyme was obtained in ATPS with PEG 20,000 and dextran T500.

Chen and Lee (1995)

PEG-dextran

Extractive fermentation of extracellular lipase from Burkholderia pseudomallei. Production of chitinase from Serratia marcescens

Polymer–polymer interaction

Table 1.1 Applications of polymer-polymer ATPS.

Ferreira et al. (2008)

(continued on next page)

11

12

Table 1.1 Applications of polymer-polymer ATPS—cont’d ATPS Pharmaceutical products PEG-dextran

PEG-dextran, with modified magnetic particles

PEG-dextran

Biomolecules

Yield/purity

Reference

Purification of monoclonal antibodies (anti-CD34) from hybridoma cells Human immunoglobulin (IgG)

Maximum yield was obtained in ATPS with PEG6000-dextran with ionic strength of 150mM sodium chloride. The combined process of PEG-dextran ATPS with magnetic particles showed > 98% purity and > 92% recovery yield. Total reaction time also for phase separation also decreased from 40 to 25 min. There was no selectivity in ATPS with PEG8000-dextran 500,000 and Ficoll 400,000-dextran 70,000.

Silva et al. (2014)

Potential recovery yield of lutein using PEG-dextran was >70% and of beta-carotene was >85%.

Chavez-Santoscoy et al. (2010)

Recovery yield of pDNA and RNA was 95% and 55%, respectively, whereas protein removal was 42%. Addition of affinity ligands like PEI demonstrated high affinity for pDNA. The use of PEG 3350-dextran 110 ATPS resulted in the desired outcome.

Kepka et al. (2004a)

Removal of cell debris from CD133+ stem cells

PEG-PEI and PEG-dextran

Purification of pDNA from alkaline lysates

GonzálezGonzález et al. (2016)

Duarte et al. (2007)

Principles of Multiple-Liquid Separation Systems

Low molecular weight compounds Recovery of cyanobacterial Recovery of cyanobacterial products, that is, lutein products, that is, lutein and and beta-carotene beta-carotene Genetic material PEG-dextran Extraction of plasmid DNA (pDNA) from E. coli lysates

Dhadge et al. (2014)

Polymer–polymer interaction

13

enzyme stability and activity, decreased production of protease, or decreased enzyme adsorption to chitin (Chen and Lee, 1995).

1.4.2 Pharmaceutical products There is growing concern about the possible consequences of disposal and intensive use of pharmaceutical products on ecological and human health. Studies have demonstrated that these products will be released directly into the environment following wastewater treatment (Freire et al., 2012). Due to the high mobility and persistence in the environment, antibiotics may leach into groundwater and be transported in soil, aquifers, and surface waters. Moreover, some of the pharmaceutical products can develop resistance toward degradation (Freire et al., 2012). Therefore, it is important to conduct a preconcentration step for the reliable quantitative determination of pharmaceutical products due to samples of environment that has intrinsic complexity and antibiotics that have low concentrations (Kümmerer, 2003). Typically, the preconcentration of antibiotics can be carried out using different methods of separation like solid-phase extraction, crystallization, ion-exchange, liquid–liquid extraction, chromatography, or a mixture of two or more methods (Freire et al., 2012). Moreover, pharmaceutical products that include therapeutic proteins such as blood or serum-related proteins as well as monoclonal and polyclonal antibodies are some of the examples that have been studied for their partitioning behavior in ATPS (Benavides et al., 2011; Phong et al., 2018). Silva et al. (2014) investigated the use of PEG-dextran ATPS for the purification of monoclonal antibodies (anti-CD34) from hybridoma cells (Silva et al., 2014). Based on the outcomes, it was observed that the soluble protein partitioned in the bottom phase that is rich in dextran, anti-CD34 monoclonal antibodies partitioned in the top phase that is rich in PEG and hybridoma cells deposited at the interface. The maximum yield was obtained when the reaction was conducted at pH 3, in an ATPS consisting of PEG 6000-dextran with the ionic strength of 150 mM sodium chloride (Silva et al., 2014). Dhadge et al. (2014) developed a selective nonchromatographic method to purify antibodies by combining magnetic separation with aqueous two-phase extraction (Dhadge et al., 2014). In the study, ATPS that consists of PEG and dextran with the addition of surface-modified magnetic particles were used. The combined process was successfully applied in the purification of antibodies and resulted in >98% purity and >92% recovery yield. Moreover, the addition of magnetic particles also sped up the time required for phase separation, leading to a total reaction time of 25 min as compared to the conventional method that requires a total of 40 min. Zhang et al. (2018) investigated the use of ATPS consisting PEG and dextran into pH-sensitive liposomes as a drug delivery system (Zhang et al., 2018). When carried out on soluble drugs like doxorubicin, it was found that the improved ATPS system resulted in better inhibition efficiency than the conventional pH-sensitive liposomes due to higher rate of uptake of doxorubicin and longer release time from hierarchical drug release. Considering that the new system is relatively stable, it can be used as an alternative approach in the pharmacologic therapy of cancer treatment (Zhang et al., 2018). González-González et al. (2016) investigated the use of ATPS to remove

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Principles of Multiple-Liquid Separation Systems

cell debris from CD133+ stem cells (González-González et al., 2016). It was shown that the ATPS that is composed of UCON-dextran resulted in all CD133+ stem cells partitioning to the bottom phase, whereas cell debris, nonviable cells, and other nonmononuclear cells partitioned substantially in the top phase. As compared to the other ATPS systems being studied, which is the PEG 8000-dextran 500,000 ATPS and Ficoll 400,000-dextran 70,000 ATPS, the results showed no selectivity. Therefore, this approach showed great potential in the application of cell-based technologies to reduce contamination of cell debris (González-González et al., 2016).

1.4.3 Low molecular weight compounds Biomolecules that have low molecular weight such as phytochemicals and secondary metabolites are highly valuable because they are widely applied in the pharmaceutical, food, nutraceutical, and cosmetic industry (Benavides et al., 2011). Typically, organicaqueous biphasic systems are being used to partition these biomolecules. However, this system may pose drawbacks such as the need to work with solvents that are flammable and toxic like dichloromethane, toluene, or hexane (Benavides et al., 2011; Iqbal et al., 2016). Therefore, the application of ATPS is proposed to reduce such risks. Several studies have been done on the recovery, purification, and separation of phytochemicals in the nutraceutical industry (Benavides et al., 2011; ChavezSantoscoy et al., 2010). Chavez-Santoscoy et al. (2010) evaluated the use of two ATPS, namely, PEG-phosphate and PEG-dextran, for the recovery of cyanobacterial products, which is lutein and beta-carotene (Chavez-Santoscoy et al., 2010). It was found that PEG-phosphate ATPS was not suitable to be used in this context as salt will cause negative impact toward cell growth. On contrary, PEG-dextran was able to promote the growth of cyanobacteria and lutein concentration in opposite phases. As for beta-carotene, it was observed to partition in the upper phase alongside biomass. The outcome of the study demonstrated that the extractive fermentation process with the use of PEG-dextran ATPS can be proposed to recover cyanobacterial products.

1.5 Genetic materials Besides, polymer–polymer ATPS is also widely used in the extraction of genetic material like pDNA, which is commonly composed of PEG-dextran with varying molecular weights. To improve further purification and extraction of plasmid DNA (pDNA), affinity ligands may be added into the process. According to the study done by Kepka , the extraction of pDNA from E. coli lysates was carried out by using ATPS that consists of thermal separating polymer and dextran (Kepka et al., 2004a, b). The outcome of this study showed pDNA recovery yield of 95%, 55% of RNA and protein removal of 42%. In another study, the authors attempted to further purify the pDNA by combining the use of an anion-exchange chromatography with extraction of ATP. This causes the complete removal of RNA from pDNA and the yield of recovery to decrease from 90% to 70% (Kepka et al., 2004a).

Polymer–polymer interaction

15

In another study done by Duarte et al. (2007), the authors investigated the addition of affinity ligands like polyethylenimine (PEI) to purify pDNA from alkaline lysates through different types of ATPS (Duarte et al., 2007). It was shown that PEI, in the form of free and PEGylated, demonstrated high affinity for pDNA. However, both of them could not alter the pDNA partitioning in PEG-salt ATPS due to the presence of high salt concentration that hinder the interaction between PEI and the plasmid. In contrast, the use of PEG 3350-dextran 110 ATPS resulted in the desired outcome with the addition of ammonium sulfate to avoid RNA partitioning in the same phase as the plasmid. The authors utilized the outcomes to develop a two-step ATPS extraction method to purify pDNA into polyplexes, which led to 100% plasmid yield with no RNA present in the final preparation and only traces of contaminant protein (Duarte et al., 2007).

1.6

Future perspective

There are some drawbacks of the polymer–polymer ATPS due to the limited information about the partitioning mechanisms. The drawbacks include the unpredictable behavior of target biomolecules in the ATPS system as well as the presence of high polymer concentration that affects the monitoring of proteins in the process. In particular, a disadvantage of the polymer–polymer ATPS include the slow segregation, high cost, and viscosity as compared to a novel alcohol-salt ATPS. Moreover, the isolation of extracted biomolecules from the polymer phase poses a challenge via reextraction, as well as the recycling of phase-forming polymers that causes pollution to the environment. Hence, the use of modifications such as copolymers, affinity ligands or a combination with other methods can result in increased recovery yield as well as improved efficiency of purification and separation of bioproducts. Recent studies have proposed the addition of carbohydrates, ionic liquids, surfactants, or amino acids into the polymer–polymer ATPS. The introduction of these modifications to purify biomolecules that are of good quality help to minimize the cost and is environmentalfriendly. However, although these components tend to increase the greenness of ATPS, however, there is still limited evidence to support the sustainability at an industrial scale. Hence, the development of approaches that are economical and novel is needed for the full reuse and recovery of these components.

1.7

Conclusion

This chapter addresses the application of polymer–polymer ATPS in terms of theoretical and practical aspects. This technique is commonly used for the purification, recovery and separation of various biomolecules. The practicality of ATPS is investigated in numerous studies, including laboratory and industrial scale, which gives excellent yield and purity. In fact, partitioning behavior of the polymer–polymer ATPS can be affected by various factors, which include polymer molecular weight, polymer concentration, temperature, pH, and tie-line length. This technique is versatile, selective, simple, and easily scalable, which is preferred by most industries to be used in downstream

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Principles of Multiple-Liquid Separation Systems

processing. In the near future, the application of ATPS is expected to increase as there is growing interest in the industry and academia fields. As studies have shown promising results from pharmaceutical products like monoclonal antibodies, enzymes, and human recombinant proteins, the extraction of biological products via ATPS showed a great potential. When more studies are done to investigate the partitioning mechanisms of ATPS, it may lead to a revolution in separation science.

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Mehrnoush, A., Mustafa, S., Sarker, M.Z.I., Yazid, A.M.M., 2012. Optimization of serine protease purification from mango (mangifera indica cv. chokanan) peel in polyethylene glycol/dextran aqueous two phase system. Int. J. Mol. Sci. 13 (3), 3636–3649. https://doi. org/10.3390/ijms13033636. Molino, J.V.D., Viana Marques, D.A., Júnior, A.P., Mazzola, P.G., Gatti, M.S.V., 2013. Different types of aqueous two-phase systems for biomolecule and bioparticle extraction and purification. Biotechnol. Progr. 29 (6), 1343–1353. https://doi.org/10.1002/btpr.1792. Olivera-Nappa, A., Lagomarsino, G., Andrews, B.A., Asenjo, J.A., 2004. Effect of electrostatic energy on partitioning of proteins in aqueous two-phase systems. J. Chromatogr. B: Anal. Technol. Biomed. Life Sci. 807 (1), 81–86. https://doi.org/10.1016/j.jchromb. 2004.03.033. Ooi, C.W., Hii, S.L., Kamal, S.M.M., Ariff, A., Ling, T.C., 2011. Extractive fermentation using aqueous two-phase systems for integrated production and purification of extracellular lipase derived from Burkholderia pseudomallei. Process Biochem. 46 (1), 68–73. https://doi.org/10.1016/j.procbio.2010.07.014. Pereira, J.F.B., Coutinho, J.A.P., 2020. Aqueous two-phase systems. Liquid-Phase Extraction. Elsevier, United States, pp. 157–182. https://doi.org/10.1016/B978-0-12-8169117.00005-0. Pereira, J.F.B., Santos, V.C., Johansson, H.O., Teixeira, J.A.C., Pessoa, A., 2012. A stable liquid-liquid extraction system for clavulanic acid using polymer-based aqueous two-phase systems. Sep. Purif. Technol. 98, 441–450. https://doi.org/10.1016/j.seppur.2012.08.008. Phong, W.N., Show, P.L., Chow, Y.H., Ling, T.C., 2018. Recovery of biotechnological products using aqueous two phase systems. J. Biosci. Bioeng. 126 (3), 273–281. https://doi.org/ 10.1016/j.jbiosc.2018.03.005. Raja, S., Murty, V.R., Thivaharan, V., Rajasekar, V., Ramesh, V., 2012. Aqueous two phase systems for the recovery of biomolecules – a review. Sci. Technol. 1 (1), 7–16. https://doi. org/10.5923/j.scit.20110101.02. Rosa, P.A.J., Azevedo, A.M., Ferreira, I.F., de Vries, J., Korporaal, R., Verhoef, H.J., Visser, T.J., Aires-Barros, M.R., 2007. Affinity partitioning of human antibodies in aqueous two-phase systems. J. Chromatogr. A 1162, 103–113. https://doi.org/10.1016/j.chroma.2007.03.067. Sadeghi, R., Maali, M., 2016. Toward an understanding of aqueous biphasic formation in polymer-polymer aqueous systems. Polymer 83, 1–11. https://doi.org/10.1016/ j.polymer.2015.11.032. Salabat, A., Sadeghi, R., Moghadam, S.T., Jamehbozorg, B., 2011. Partitioning of l-methionine in aqueous two-phase systems containing poly(propylene glycol) and sodium phosphate salts. J. Chem. Thermodyn. 43 (10), 1525–1529. https://doi.org/10.1016/j.jct.2011.05.001. Saravanan, S., Rao, J.R., Nair, B.U., Ramasami, T., 2008. Aqueous two-phase poly(ethylene glycol)-poly(acrylic acid) system for protein partitioning: Influence of molecular weight, pH and temperature. Process Biochem. 43 (9), 905–911. https://doi.org/10.1016/ j.procbio.2008.04.011. Silva, M.F.F., Fernandes-Platzgummer, A., Aires-Barros, M.R., Azevedo, A.M., 2014. Integrated purification of monoclonal antibodies directly from cell culture medium with aqueous two-phase systems. Sep. Purif. Technol. 132, 1–25. https://doi.org/10.1016/ j.seppur.2014.05.041. Singh, P., Banik, R.M., 2012. Partitioning studies of L-glutaminase production by Bacillus cereus MTCC 1305 in different PEG-salt/dextran. Bioresour. Technol. 114, 730–734. https://doi. org/10.1016/j.biortech.2012.03.046.

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Polymer–salt interaction Jun Wei Roy Chong a, Zatul Iffah Mohd Arshad b, Kit Wayne Chew c and Pau Loke Show a a Department of Chemical and Environmental Engineering, Faculty of Science and Engineering, University of Nottingham Malaysia, Semenyih, Selangor, Malaysia, b Faculty of Chemical and Natural Resources Engineering, Universiti Malaysia Pahang, Lebuhraya Tun Razak, Gambang, Kuantan, Pahang, Malaysia, c School of Energy and Chemical Engineering, Xiamen University Malaysia, Sepang, Selangor Darul Ehsan, Malaysia

2.1

2

Introduction

There are various conventional liquid–liquid extraction techniques such as Soxhlet extraction, microwave-assisted extraction, supercritical fluid extraction, and solvent extraction for the separation and purification of biomolecules and bioactive compounds. Nevertheless, the above-mentioned methods lead to several drawback limitations, such as the following: (1) solvent extraction uses a large quantity of solvents, long duration time, causes toxicity, and possibility of thermal degradation of bioactive ingredients (Kumar et al., 2017); (2) supercritical fluid extraction has a very complicated system and expensive (Arun et al., 2020); (3) microwave-assisted extraction has complex mass transfer affecting the chance of scaling up (Chan et al., 2015), and (4) Soxhlet extraction methods are time-consuming, causes thermal degradation, less extraction selectivity, costly solvents, and nonenvironment friendly (Arun et al., 2020). Recently, researchers have proposed another liquid–liquid separation method which is the aqueous two-phase system (ATPS). As mentioned by Mastiani et al. (2019), ATPS can be formed by dissolving two incompatible polymers or a polymer and salt such as polyethylene glycol (PEG)/dextran (DEX) and PEG/salt, respectively (Mastiani et al., 2019). Usually, phase separation occurs above the critical concentrations which result in two aqueous phases each enriched with one of the components, producing a polymer-rich, salt-poor top phase, and vice versa (Song et al., 2013; Hatti-Kaul, 2000). ATPS has sparked various attention due to its high potential for separation, extraction, enrichment, and purification of several biomolecules such as membranes, proteins, enzymes, viruses, and nucleic acids (Iqbal et al., 2016). The advancement of ATPS systems can provide low operational cost, low toxicity, large-scale up, good mass transfer, biocompatible, low energy requirement, and able to adhere to the economic and environmental protection (Shaker Shiran et al., 2020; Tang et al., 2014; Yau et al., 2015). The polymer–salt and two polymer ATPS are much more beneficial compared to conventional liquid–liquid fractionation due to some reasons: (1) water is used as the solvent instead of organic solvent which provides a mild environment to prevent denaturation of biomolecules; (2) ATPS is efficient and easy to operate; and (3) scaling Principles of Multiple-Liquid Separation Systems: Interaction, Application and Advancement. DOI: https://doi.org/10.1016/B978-0-323-91728-5.00009-3 c 2023 Elsevier Inc. All rights reserved. Copyright 

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Principles of Multiple-Liquid Separation Systems

up of equipment based on ATPS is relatively simple and easy (Berlo et al., 1998). Furthermore, polymer–salt ATPS is more favorable than that of polymer–polymer ATPS because salt is less costly as compared to dextran and lower viscosity between phases leading to less time consumption for phase separation (Berlo et al., 1998). There are some drawbacks of polymer–salt ATPS in which incompatible when dealing with high ionic strength environment. In contrast, polymer–polymer ATPSs poses low ionic strength, thus are more suitable for recovery, separation, and purification of solutes sensitive to ionic conditions (Albertsson, 1961). In most ATPS, PEG is the most commonly used polymer due to cost-efficient, greener, and noninflammable reagent (de Oliveira et al., 2008). The principle behind PEG polymer is as such (1) solubilization in water enables the hydrogen bonding process; (2) the decrease in solubility of polyethylene-oxide due to the addition of monovalent cations; and (3) the cloud point decreases due to the high concentration of salt ions which reduces the amount of free water required for the solubilization of polyethylene (Mazzola et al., 2008). Some of the commonly used polymer–salt ATPSs are PEG-citrate, PEG-phosphate, and PEG-sulfate. Phosphate and sulfate are considered to a lesser degree as compared to citrate, as citrate is more environmental friendly due to nontoxic and biodegradability when discharged into sewage or wastewater treatment plants (de Oliveira et al., 2008).

2.2 Mechanism and working principles The concept of ATPS started back in 1896 in The Netherlands, when a person named Martinus Willem Beijerinck accidently found and observed that the aqueous starch solution is immiscible in gelatine aqueous solution. Instead, the application of ATPS is discovered by Per-Åke Albertsson when attempting to purify chloroplast, and managed to publish his first studies on the application of ATPS for the purification of biological molecules (Grilo et al., 2014; Iqbal et al., 2016). Firstly, ATPS can be performed by mixing two incompatible aqueous solutions such as one polymer with a type of high concentration salt (Albertsson and Tjerneld, 1994). Unlike conventional liquid–liquid separation using organic solvents, as the term suggests “Aqueous” comprises high concentration of water content (80%–95%) in both phases, forming low interfacial tension systems, a nontoxic and gentle environment for the separation of biomolecules, such as proteins and enzymes, thus less chances of damage to biomolecules and enable polymers to stabilize their structure and biological activities (Albertsson and Tjerneld, 1994; Asenjo and Andrews, 2011; Hatti-Kaul, 2000). The basic graphical process pathway representing the basic working mechanism of a polymer–salt system for the partitioning and purification of desired protein molecules is shown in Fig. 2.1. In contrast to polymer–polymer ATPS, the interaction of physical properties between the two phases of polymer–salt systems has greater differences, thus partitioning of biomolecules is often unequal, favors one of the phases, and extraction process can be carried out at a faster rate. Moreover, molecules with increasing molecular weight such as proteins, nucleic acids, and peptides will tend to be more uneven and sensitive to phase compositions (Johansson, 1994).

Polymer–salt interaction

23

Figure 2.1 General process mechanism and working principle of polymer–salt ATPS system. ATPS, aqueous two-phase system.

Based on Fig. 2.1, the polymer–salt ATPS can be formed by mixture of PEG, salt, and water. Next, the soluble and particulate matters such as the crude proteins are mixed together with polymer–salt solution (Chew et al., 2019). The mixture is either accomplished by settling under gravity or by centrifugation. After some period of time, it can be observed that the mixture will be distributed into two aqueous phases named top (light) and bottom (heavy) phase. Furthermore, most soluble materials and impurities will partition at the lower, more polar phase, which also known as saltrich phase, while protein will partition at the top, nonpolar phase known as PEG-rich phase (Harris and Angal, 1989). There are several important parameters to improve the separation and purification of protein molecules from PEG such as the following: (1) manipulating polymer molecular weight to achieve desired partition coefficient; (2) selection of suitable ions; (3) addition of salt (e.g., NaCl) to increase the affinity partitioning toward the top phase; and (4) implementing affinity ligand to increase the degree of purification (Asenjo and Andrews, 2011). The aqueous extraction mostly can be achieved in a single-stage operation, otherwise the extraction process is to be repeated again in a chain or cascade of contacting and separation units. Promisingly, the concentration and recovery of ATPS in many cases can produce yields more than 90% using single-stage extraction procedure (Doran, 2013). The thermodynamics and partitioning of two-phase formation explained that the formation of two phases is due to the miscibility of solutions containing polymers, whereas similar incompatible phenomenon can be observed under the mixture of both high ionic strength salt and a polymer (Iqbal et al., 2016). The phase separation of polymer–polymer into two different phases by using the steric exclusion chromatography technique in which separation takes place solely on the basis of size. In the meantime, the same concept of exclusion technique can be applied in polymer– salt ATPS, whereas large amount of water can be absorbed using various salt type (Asenjo and Andrews, 2011; Iqbal et al., 2016). According to Mazzola et al. (2008),

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Principles of Multiple-Liquid Separation Systems

Figure 2.2 Schematic of Hofmeister series where cation & anion salts are arranged according to their salting-out ability.

a few parameters are affecting the phase separation in ATPS, for instance molecular weight and concentration of the polymer, composition, and concentration of salt (Mazzola et al., 2008). In regard to the de-mixing process of polymer–salt ATPS, the effectiveness of phase separation depends on the selection of salt type which follows the Hofmeister series, whereby salt ions (e.g., anion is much preferable due to higher effectiveness compared to cation in terms of phase separation) are ranked based on their salting-out ability (Ananthapadmanabhan and Goddard, 1987). Fig. 2.2 represents the sequence of cations and anions arranged in empirical ordering of salts which interprets the minimum salt concentration needed for the partitioning of biomolecules (Pereira et al., 2020). Besides, high charged ions and multivalent anions (e.g., H2 PO4– and SO42– ) are mostly favored due to their high effectiveness in inducing salt–polymer ATPS with PEG as compared to univalent salts (e.g., Cl– and Br – ) (Ananthapadmanabhan and Goddard, 1987). Thus far, the interaction of salt between the phase separation of polymer and aqueous solutions is still unclear, but it is clear that the principle of phase separation is relevant to the hydration interaction between salt and polymer (Huddleston et al., 1999). From the understanding of Ananthapadmanabhan and Goddard (1987), the effectiveness of triply charged anions is greater than doubly charged sulfate and followed by singly charged hydroxide in terms of polymer salting-out effect, mainly due to competition for water (Ananthapadmanabhan and Goddard, 1987). Furthermore, cations are more complex and contributes to unfavorable effects such as hydration and the tendency to form specific interaction of cation-polymer (e.g., interaction of lithium with the ethylene oxide units of PEG).

2.2.1 Binodal curve The binodal curve in Fig. 2.3 represents the phase diagram for polymer–salt ATPS under a specific set of parameters such as system pH, temperature, or salt concentration,

Polymer–salt interaction

25

Figure 2.3 Typical polymer–salt binodal curve. TCpB, representation of binodal curve; TB, tie-line; T, top phase composition; B, bottom phase composition; X, Y, and Z, total composition; Cp , critical point.

which is required to form a potential working area of aqueous two-phase system. Binodal curve can provide several information such as concentration of phase forming components needed for two-phase, concentration of phase components in the top and bottom phases, and the ratio of phase volumes. Referring to the figure, the binodal curve (TCpB) is divided into a region of two incompatible aqueous phase which can be observed above the curve (e.g., known as biphasic or two-phase region) from those below the curve (e.g., known as monophasic or one-phase region). Although same top phase equilibrium composition (Tpolymer , Tsalt ) and same bottom phase equilibrium composition (Bpolymer , Bsalt ), the three-system named X, Y, and Z are not fixed as the volume ratio and initial compositions are not constant. Moreover, the line (TB) is defined as the tie-line, which links two nodes lying on the binodal curve. The point Cp is the critical point and the volume of both phases is the same above the critical point. Similar to the component phases composition with the unit of % (w/w), the TTL is zero at point Cp . The mass of the phases represents the tie-line length (TLL) by the equation: Vt ρt XB = Vb ρb XT

(2.1)

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Principles of Multiple-Liquid Separation Systems

where V = volume ρ = density t = subscript of top phase b = subscript of bottom phase XB and XT = segment length of the tie-lines as shown in Fig. 2.3 The equilibrium phase composition is related to the (TLL) and slope of tie-line (STL), as shown in Eq. (2.2).  T LL =

2  (Bsalt − Tsalt )2 + Tpolymer − Bpolymer

(2.2)

The tie-lines are normally in parallel, thus (STL) can be further calculated using Eq. (2.3).  Tpolymer − Bpolymer polymer = ST L = (Bsalt − Tsalt ) salt 

(2.3)

The partition coefficient (K) defines the separation behavior between the top and bottom phase in salt–polymer ATPS: K=

ConcAT ConcAB

(2.4)

where ConcAT = concentration of component A at the top phase ConcBT = concentration of component A at the bottom phase The value of partition coefficient (K) will vary according to the concentrations. For an instant, if component A is more soluble or favor the top phase, then K value will be higher and vice versa.

2.3

Key process parameters

The basic understanding and principle of polymer–salt ATPS partitioning is yet to be clear. Therefore, physicochemical properties of the solute of biomolecules are considered in many studies of ATPS. The main factors that affect the partitioning behavior in salt–polymer ATPS are polymer molecular weight, pH system, concentration of polymers, effect of temperature, hydrophobicity, and addition of salt.

Polymer–salt interaction

27

2.3.1 Effect of polymer molecular weight (MW) Molecular weight of the polymer is one of the key factors to be considered in polymer–salt ATPS. The partitioning effectiveness is greatly influenced by the particle or molecular size of the polymer. Large biomolecules such as protein and DNA usually lead to one-sided distribution, either collected in the top phase or lower phase (Albertsson, 1970). In general, the increasing molecular weight of polymer decreases the extraction efficiency, in which more biomolecule distribution will tend to be attracted to the opposite phase (bottom/salt-rich phase). Thus, the repulsive interaction between polymer and biomolecule increases and decreases in partition coefficient (K) (Ratanapongleka, 2010). As the polymer molecular weight increases, a reduction in free volume is observed due to increase in chain length and exclusion effect. This interprets that the polymer has acquired a greater extend of compact structural formation and hydrophobic bonds which interferes the affinity of desired biomolecules to the top phase. Adding on, polymer molecular weight below the critical limit is not recommended due to reduction in exclusion effect, resulting in attracting all of the protein molecule including contaminants or impurities to the top phase. Therefore, it can be concluded that intermediate molecular weight of the polymer is the best option for best partitioning efficiency (Mohamadi et al., 2007). The purification of alcohol dehydrogenase (ADH) from Baker’s yeast has been performed by Madhusudhan et al. (2008) using potassium phosphate salt and PEG with various molecular weights (600, 1000, 1500, 4000, 6000, and 20,000) (Madhusudhan et al., 2008). The increase in PEG molecular weight from 600–20,000 reduces the free volume in the polymer (top) phase and hence, maximum affinity of desired ADH toward the bottom phase. A similar study conducted by Naganagouda and Mulimani (2008) stated that high recovery of α-galactosidase from Aspergillus oryzae is achieved using 12% (w/w) PEG 4000 and 11.9% (w/w) phosphate salt, where the partitioning of α-galactosidase toward the bottom phase due to the increase in PEG molecular weight (less space available) (Naganagouda and Mulimani, 2008). On the other hand, lower molecular weight of PEG 1550 was found desirable for the partitioning of enzyme and cell debris to the top and bottom phase, respectively, from a plant named Ipomea palmetta (Srinivas et al., 2002). As the molecular weight decreases, the interfacial tension decreases, whereby differential partitioning occurs below the critical value of the interfacial tension.

2.3.2 Effect of concentration of polymer Next, high polymer concentration correlates to an increase in refractive index, viscosity of the phase, and density while providing large difference in properties between top and bottom phases (Albertsson and Tjerneld, 1994; Ratanapongleka, 2010). In polymer– salt ATPS, the partitioning is affected by the salting-out effect (salt-rich) and volume exclusion effect (polymer-rich). More protein molecules will move toward the saltrich phase when the concentration of polymer increases (Grilo et al., 2014). There are several factors affecting the increase in partition coefficient (K) in polymer–salt ATPS such as the following: (1) low molecular weight of polymer leads to low interfacial

28

Principles of Multiple-Liquid Separation Systems

tension, followed by an increase in K value; (2) high salt concentration at the bottom phase will tend to partition biomolecules to the top phase due to high ionic strength; and (3) as polymer concentration increases, the number of polymer units involved increases leading to increase in biomolecules partitioning to the top phase (polymer-rich) due to hydrophobic interaction between polymer and biomolecule (Yusof, 2015). For an instant, the partitioning behavior of bromelain and polyphenol has been examined by Babu et al. (2008) by varying the concentration of PEG (Babu et al., 2008). Nonetheless, increase in the concentration of PEG 1500 from 12% to 18% (w/w) leads to increase from 3.6- to 4.0-fold of purity as the higher affinity of total proteins toward the bottom phase. The decrease in the partition coefficient of total proteins and polyphenol oxidase to the top (PEG) rich phase is caused by the volume exclusion effect, which increases as polymer concentration increases. Therefore, a low concentration of PEG would do the opposite where biomolecules will tend to partition to the top phase.

2.3.3 Effect of system pH The electrochemical interactions due to pH change affect the partitioning due to the change in ratio of charged solute and molecules, thus the pH of the system may be altered according to the selective separation (Asenjo and Andrews, 2011). Besides, pH of the system will affect some of the factors: (1) charge of the targeted biomolecule and ion composition; (2) surface character of the contaminating materials; and (3) partitioning affinity either toward to the bottom or top phase (Mohamadi et al., 2007). Supposing, if the pH is greater than the isoelectric point (pI) the protein net charge will be negative and vice versa (Yusof, 2015). However, zero or neutral protein net charge can be observed in the case where pH is equal to the pI (Iqbal et al., 2016). Firstly, the initial pH of the system must be higher than pI of the targeted biomolecule to generate electrochemical affinity toward polymer-rich phase due to the positive dipole moment. According to Benavides and Rito-Palomares (2008), the increase in pH value is not recommended due to the potential of contaminants achieving electrochemical affinity toward the recovery phase (Benavides and Rito-Palomares, 2008). Furthermore, due to salt solubility constraints, certain pH conditions are only suitable for each individual salt–polymer system. As proposed by, Hong Yang (2013) PEG-phosphate ATPS fit under pH condition above 7 and PEG-sulfate ATPS fit under pH condition below 6.5, but most biomolecules (e.g., protein and amylase) are suitable under neutral pH condition of 7 which is known as a stable condition for partitioning purposes (Hong Yang, 2013).

2.3.4 Effect of temperature The effect of temperature greatly depends on the type of polymer utilized for each phase system. In addition, change in temperature also affects the equilibrium composition of both top and bottom phases, hence a stringent control of temperature is required while dealing with ATPS (Grilo et al., 2014). A study deduced by Albertsson and Tjerneld (1994) polymer–salt ATPS is suitable in high-temperature condition because

Polymer–salt interaction

29

only small concentration of either salt or polymer is required for the formation of two-phase condition, while polymer–polymer systems an opposite effect is acquired (Albertsson and Tjerneld, 1994). The increase in temperature greatly improves the partition coefficient effectiveness of polymer and salt toward the top and bottom phase, respectively. Since salt concentration is high in the bottom phase, the amount of water molecules for solute salvation and the solubility of biomolecules decreases. Unfortunately, further increase in temperature could denature or rupture the bonds of the desired biomolecules. In addition, the structure of biomolecule became more flexible due to bond weakening which enables the water in the system to undergo reaction with the functional group of biomolecules while forming new hydrogen bonds. As the interaction between water molecule and biomolecule increases, the effective dielectric constant increases due to weakening of nearby hydrogen bonds. Furthermore, the weakened structure lead to losses in solubility of molecules due to the presence of hydrophobic groups out to the solution (Ratanapongleka, 2010). The effect of temperature on PEG 4000 and sodium citrate salt has been examined by de Oliveira et al. (2008) investigating the partitioning behavior by comparing various temperature from 10 to 45˚C (de Oliveira et al., 2008). When increasing the temperature, the biphasic area starts to expand in which increases the distance between the tie-lines and origin and hence, increase in the separation of PEG-sodium citrate more easily. Furthermore, an optimal temperature for the partitioning of α-galactosidase and total proteins using different temperatures from 25 to 55˚C (Naganagouda and Mulimani, 2008). As the temperature increases, the concentration of phosphate salt increases in the bottom phase which increases the affinity of both α-galactosidase and total proteins toward the top phase due to reduction in solubility or salting-out effect in the bottom phase. The same argument observed by Gautam and Simon (2006) states that increasing temperature from 25 to 35˚C greatly increases the salting-out effect which favours the partitioning of both total proteins and β-glucosidase to the top phase (Gautam and Simon, 2006). Thus far, the lowest and highest temperature for the extraction of biomolecules are said to be at room temperature (25˚C) and 55˚C, respectively.

2.3.5 Effect of hydrophobicity and addition of salt The partitioning of biomolecule is influenced by the hydrophobicity (e.g., mainly protein) in ATPS. Besides, salting-out effect and phase hydrophobicity are the two main terms affecting the hydrophobicity interactions (Andrews et al., 2005; Schmidt et al., 1995). Generally, the top (polymer-rich) phase is more hydrophobic which indeed favors the attraction of less hydrophilic and amphipathic substances toward that respective phase (Rito-Palomar and Benavides, 2017). Based on Iqbal et al. (2016), the degree of hydrophobicity effect can be altered by manipulating the TLL, molecular weight of the polymer, and addition of salt (e.g., NaCl) in polymer–salt ATPS (Iqbal et al., 2016). The reduction of water concentration will increase the TLL, which promotes the ATPS to be more hydrophobic (Asenjo and Andrews, 2011). Moreover, the increase in PEG molecular weight tends to increase the hydrophobicity interaction due to reduction in free volume. As deduced, PEG is most commonly used for the

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Principles of Multiple-Liquid Separation Systems

partitioning of biomolecules due to its hydrophobic nature. The functional group of PEG is hydrophilic, whereas the remaining of polymer chain is considered hydrophobic and hence, the rise in hydrophobicity can be accomplished by altering the ratio of hydrophilic to hydrophobic group (Asenjo and Andrews, 2011; Rito-Palomar and Benavides, 2017). As dictated by Andrews and Asenjo (2010), the polymer–salt ATPS is even more complicated compared to polymer–polymer ATPS because additional understanding is required when involving various type of salt (Andrews and Asenjo, 2010). By varying the concentration of NaCl, the ratio between ions and pH changes will affect the partitioning effectiveness of protein molecules. Positively, the presence of NaCl will not rigorously affect the equilibrium of both aqueous phases or the phase diagram, unless if a high salt concentration more than 1M is adopted (Andrews and Asenjo, 2010; Ratanapongleka, 2010). Next, salting-out effect can be observed in salt–polymer ATPS if one highly ionic phase is present. Such cases involved high water concentration for the dissolution of salts, making the solutes to be partially hydrated during partitioning. Subsequently, the solutes or biomolecules will be partitioned toward the less hydrophilic phase or the top phase (polymer-rich) (Rito-Palomar and Benavides, 2017). The addition of NaCl salt also affects the hydrophobicity whereby, concentration up to 10% will increase the difference of electrical potential and subsequently, increase in hydrophobic differences between both polymer and salt phase (Hong Yang, 2013). Interestingly, the rise in the hydrophobicity will decrease the quantity of bound water to keep the final composition of the system to be constant. Thus, the protein solvation will be less expose to water, whereby the presence of hydrophobic patches on the protein surface will develop which favors the hydrophobic interaction with polymer in the top phase and eventually cause precipitation by salting-out (Rosa et al., 2009). The saltingout effects tend to partition the desired biomolecules toward the polymer-rich phase from the salt-rich phase (Yusof, 2015). Nevertheless, too high salt concentration will cause denature of biomolecule, and hence low concentration of salt ranging from 0 to 1.0 M is much preferable for ATPS (Hong Yang, 2013).

2.4 Applications 2.4.1 Protein purification The high demand and growing need of biological macromolecule especially protein is an important source in food class, cosmetics industries, biotechnology, and pharmaceutical (Labrou, 2014). From the perspective of Kalyanpur (2002), most of the biotechnological proteins are in the form of complex mixtures of products and thus, protein purification requires many steps which makes it difficult (Kalyanpur, 2002). Adding on, conventional techniques for instance column chromatography and precipitation leads to high processing cost and low production rate, which leads to process bottlenecks (Gupta et al., 1999). Unfortunately, chromatography of protein is not appropriate for manufacturing up-scaling due to unfavorable large pressure drop

Polymer–salt interaction

31

and batch processes. On the other hand, the purification of protein molecule using the precipitation method is not efficient due to factors such as low solubility, toxic solvents, and high potential of disintegration (Carlson, 1988). In order to obtain high protein purification, contaminants such as viruses, pyrogens, cell culture media, and nucleic acids should be eliminated as well as the existence of various isoforms due to posttranslational modifications (Labrou, 2014). Therefore, much recent research has been directed toward the use of ATPS to overcome such limitations (Carlson, 1988). The idea of implementing ATPS can benefit the protein recovery from crude feedstocks in terms of scale-up potential, continuous operation, simple process integration, low toxicity, and biocompatibility (Asenjo and Andrews, 2011). According to Gupta et al. (1999), the basis of protein partitioning depends on the surface properties which include size, charge, and hydrophobicity (Gupta et al., 1999). Normally, protein molecules will end up in the top phase (PEG-rich), less polar, and hydrophobic phase. The lingering, menthol-like sweet taste of thaumatin is known as a protein sweetener which recently been cloned into yeast and Escherichia coli. The development of thaumatin is due to its distinct properties of flavor and aroma enhancement, thus proper purification and separation techniques are being employed (Smith, 1991). This study is being conducted by, Cascone et al. (1991) who are looking into the partitioning behavior of pure thaumatin in ATPS which comprises of PEG/dextran and PEG/phosphate (Cascone et al., 1991). Several factors such as molecular weight of PEG, pH, and concentration of salt (e.g., NaCl) are of the main influence on the partition coefficient (K). Unlike PEG/dextran system, PEG/phosphate ATPS imposed the greater effect on the partitioning behavior of thaumatin. Later in the study, it is observed that higher K values (up to K = 18) can be obtained at lower molecular weight of PEG and vice versa. Surprisingly, drastic increase in partition coefficient (K) by 62-fold can done by adding NaCl with concentration of 1.5 M into the PEG 6000-phosphate ATPS, which result in the increase of K from 0.53 to 33. Coincidently, up to a threshold of 40 g/L of thaumatin concentration, there is no effect of protein concentration being observed. The analysis of protein molecules in the top and bottom phase is determined using a single step purification coupled with high-performance liquid chromatography (HPLC) generating a recovery of 90%–95% along with 20-fold thaumatin purity (Cascone et al., 1991). Another protein purification study by Alves et al. (2000) determined the partitioning of proteins from bovine serum albumin, cheese whey, and porcine insulin using ATPS of PEG-phosphate, and PEG-citrate (Alves et al., 2000). In general, cheese whey contains high protein content, mainly α-lactalbumin (α-La) and β-lactoglobulin (β-Lg), in which β-Lg contain traces of allergenic compound in food substance for children (Kruse et al., 1973). Next, BSA also known as bovine serum albumin is one of the main constituent in whey proteins representing roughly 20% of the total protein in bovine milk and essential source of amino acids (Deeth et al., 2019). Throughout the experiment, high concentration of α-La and β-Lg are partitioned in the top phase (PEGrich) and bottom phase (salt-rich), respectively, using ATPS of 14% PEG 1500%–18% potassium phosphate. Lastly, porcine insulin has a greater affinity toward the PEG-rich phase with a partition coefficient of 10 and could be further increased by increasing the molecular weight of PEG (Alves et al., 2000).

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Principles of Multiple-Liquid Separation Systems

In recent times, virgin coconut oil (VCO) has been gaining commercial attention due to various health benefits showing high protein content of 33% w/w and low-fat content of 3% w/w (Naik et al., 2012). The “whey” that remains after the separation of VCO by wet or aqueous processing from the whole coconut milk by centrifugation involved a by-product called as “coconut skim milk.” This large volume of whey produced from the by-product is underutilized or disposed off to the environment (let to drain as effluent) will promote drastic environment pollution due to its high biological oxygen demand of 98,333 ppm compared to the standard discharge regulation of 100 ppm (Nur et al., 2015). Whey produced from coconut still retain the rich and edible source of proteins such as albumins, globulins, prolamines, glutelins-1, and glutelins-2 where the recovery and purification of these proteins will greatly improve the economical productivity (Kwon et al., 1996; Naik et al., 2012). Accordingly, Santhi et al. (2020) have conducted the differential partitioning of coconut whey proteins and fat using ATPS by working with several process parameters such as tie-line length (%TLL), volume ratio, polymer molecular weight, and system pH (Santhi et al., 2020). The highest protein separation or recovery from coconut whey is identified under the optimum condition such as (1) TLL of 19.16%; (2) volume ratio of 0.2, and (3) system pH of 8 using PEG 6000-potassium phosphate ATPS. Considering the best condition, the highest protein recovery of 92.65% is achieved in a single purification step, where both whey protein and fats successfully separate into bottom phase (salt-rich) and top phase (PEG-rich), respectively (Santhi et al., 2020). The partitioning optimization of proteins from Zea mays Malt has been conducted by, Ferreira et al. (2007) utilizing the response surface methodology (RSM) to determine the relationship between pH and compositions of PEG 6000-CaCl2 (Ferreira et al., 2007). Foremost, there is the negative response on the phase diagrams and composition of TLL when changing the pH value. Meanwhile, the highest partitioning coefficient k of 4.2 is obtained in a single purification step which corresponds to the theoretical maximum partitioning coefficient of k ranging from 4.1 to 4.3.

2.4.2 DNA and nucleic acids An increasing trend of gene vaccination has proved to overwhelmed the conventional clinical strategy due to increasing demand for pharmaceutical-grade plasmid DNA (Ferreira et al., 2000). These therapeutic methods can be applied to a widespread of application for the treatment of several health problems such as cystic fibrosis, cancer cells, autoimmune, and cardiovascular illnesses (Flotte, 2007; Kelly, 2003). According to Gardlík et al. (2005), the main purpose of gene therapy is to maintain a stable delivery of transgene to the desired tissue for a substantial period of time while preventing any form of side effect and toxicity (Gardlík et al., 2005). Generally, the delivery of gene is performed either viral or nonviral vectors. Nonetheless, nonviral vectors are much preferable due to lower toxicity, simple process, safer, and high transgene capacity as compared to viral vectors. In contrast, the downside of nonviral vectors is due to low efficiency in transfecting the target cells and hence, thousands of plasmid are required for higher success rate of gene transfer (Crystal, 1995). Commonly, plasmid DNA is isolated from E. coli by fermentation, followed by alkaline cell lysis and

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lastly purification, but contaminants such as genomic RNA and DNA, endotoxins, and proteins are the main interference causing limitations to the desired specifications for clinical applications (Prazeres et al., 1999). A more favorable approach coming from, Frerix et al. (2005) proposed the extraction and purification of plasmid DNA using ATPS as an alternative, as chromatographic processes have a very low capacity for large molecules (e.g., plasmid DNA), and high cost when scaling-up (Frerix et al., 2005). The recovery of plasmid DNA was carried out by Ribeiro et al. (2002) from cell lysate using the PEG-K2 HPO4 ATPS with varying molecular weight from 200 to 8000 (Ribeiro et al., 2002). The plasmid DNA partitioning activity is carried out carefully with cell lysate mass concentrations of 20%, 40%, and 60% w/w to determine the performance of the process to overcome the interference of impurities such as genomic DNA, RNA, and protein. First of all, the best recovery yield (>67%) can be obtained from PEG 1000 with 40% and 60% w/w of lysate load, whereby the ATPS system is manage to separate protein and genomic DNA but the presence of RNA can be found copartitioning with plasmid DNA. Next, PEG 300 in ATPS concentrates both plasmid DNA and protein in the top phase. Moreover, a successful partitioning of plasmid DNA and the subsequent impurities (i.e. genomic DNA and RNA) to the top and bottom phases can be observed, respectively (Ribeiro et al., 2002). An interesting investigation was carried out by Nazer et al. (2017) taking into account of affinity ligand for the separation of plasmid DNA using PEG-sodium sulfate in ATPS from alkaline bacterial cell lysate to further improve the partitioning effectiveness (Nazer et al., 2017). In the system containing 12% w/w PEG and 12% w/w sodium sulfate followed by a modified 20bp pyrimidine oligonucleotide acting as a triplex-forming oligonucleotide is utilized as an affinity ligand. Among the plasmid DNA, pUC118 plasmid is chosen for the purification and partitioning procedure by varying molecular weight of PEG, lysate load, and pH to determine the best result and condition. The addition of affinity ligand greatly enhanced the affinity of plasmid DNA toward the top phase without interfering the RNA and protein partitioning. The experiment by Nazer et al. (2017) resulted in 67% of plasmid DNA recovery and contaminants in the top phase (PEG-rich phase) and bottom phase, respectively, with system pH, PEG molecular weight, and lysate load of 6, 600 Da, and 60%, respectively (Nazer et al., 2017). Purification of plasmid DNA from alkaline cell lysate originating from E. coli cells was studied by Rahimpour et al. (2006) using PEG-sodium citrate ATPS (Rahimpour et al., 2006). The partitioning performance is conducted by evaluating the system pH, polymer molecular weight, TLL, lysate load, and phase volume ratio along with RSM as a tool to define the best condition for purification. Overall, the best-optimized condition has been achieved by producing 99% plasmid DNA recovery with PEG 400, system pH of 6.9, TLL of 38.7%, lysate load of 10% v/v, phase volume ratio of 1.5 and RNA removal rate of 68% (Rahimpour et al., 2006). Combination of PEG with potassium citrate or potassium phosphate ATPS for the purification of nucleic acids (e.g., plasmid DNA) tested by Frerix et al. (2005) shows strong partitioning of plasmid DNA toward the bottom phase, using configuration of 15% PEG 800, 20% potassium phosphate, and system pH of 7.0 (Frerix et al., 2005). In addition, the current

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Principles of Multiple-Liquid Separation Systems

experiment shows the depletion and disintegration of genomic DNA (impurities) more than 99%.

2.4.3 Virus, virus-like particles (VLPs) Virus-like particles (VLPs) inhibit great importance, representing a new class of bionanoparticles in the medical sector incorporating special features such as gene delivery vectors, in diagnosis or as vaccines (Enders et al., 2007; Vicente et al., 2008; Young et al., 2006). In general, a single or few of viral structural proteins are available within the VLPs which enable to spontaneously self-assembling into capsids, hence they can imitate the pathogens (Garcea and Gissmann, 2004), as well as promoting antigen epitopes of foreign pathogens (Lua et al., 2014), or tumor cells (Klamp et al., 2011). Apart from gene or biomolecule delivery tools, VLPs can be defined as attractive health care tool due to the following reasons: (1) The rapid demand for effective vaccine over the years due to the occurrence of infectious diseases; (2) tons of medicinal care do not meet the requirements in the case where vaccines play an essential role; and (3) the advances in microbiological, immunology and the better understanding of pathogenesis have now overcome the previously intractable targets, thus vaccination is within our reach (Smith et al., 2011). Nonetheless, the evolving infectious diseases causes the uprising need for high grade VLPs in both research and pharmaceutical sectors. According to Ladd Effio et al. (2015), the safety limit for VLP-human-based vaccines: (1) DNA concentrations below 10 mg/dose; and (2) protein purity more than 95% under the US Food and Drug Administration and the World Health Organization (Ladd Effio et al., 2015). Furthermore, ATPS has proven to be suitable for the separation of biomolecules from the solid particles due to rapid process, cost efficiency, and high chances for integrated biotechnology as compared to chromatography and filtration techniques. First of all, VLPs can be classified into two distinct category named intracellular and extracellular products. B19 parvovirus is obtained from the intercellular of VLPs (Kajigaya et al., 1991) and HIV-1 core from the extracellular of VLPs (Cruz et al., 2000). Due to the fact that, the recovery rate of B19 parvovirus using conventional method resulting in very low recovery of 6%, two processes have been carried out by, Luechau et al. (2011) in PEG 1000-magnesium sulfate ATPS to determine the best overall recovery (Luechau et al., 2011). The first process successfully isolate B19 particle from the bottom phase while 31% protein and cell debris are distributed across the top and interface region. After the recovery of VP1 (minor capsid protein with molecular weight of 83 kDa) and VP2 (major capsid protein with molecular weight of 58 kDa) capsid protein, B19 particles yields 92.8% and 85.7, respectively, in the bottom phase. Likewise, the other second process where B19 particles are recovered from the clarified cell disruptate by interfacial partition, resulting in 95.3% and 33.2% recovery with respect to VP1 and VP2. These processes concluded the effectiveness in separating both total bulk protein along with nonassembled VP2 proteins (Luechau et al., 2011). The work of the purification of a human immunodeficiency virus (HIV) VLP is carried out by, Jacinto et al. (2015) using PEG-potassium sulfate, ammonium sulfate,

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and trisodium citrate (Jacinto et al., 2015). Among these polymer–salt ATPS, PEGammonium sulfate system demonstrated the best result with the highest partition coefficient of K = 4.4. Among the many VLPs, Adenovirus is considered one of the most preferred platforms for vaccine production due to its high potency in delivering DNA to the targeted cells (Nestola et al., 2015). The recovery of adenoviral vectors from HEK 293 cells is performed by Negrete et al. (2007) in PEG 300-phosphate ATPS (Negrete et al., 2007). Prior to the recovery phase, different parameters such as harvesting time, addition of buffer (HEPES), and initial cell density are studied to enhance the production of adenoviral vectors in HEK 293 cells using a 2-liter bioreactor. Soundly, the infectivity of the adenoviral vectors did not have significant effect in the PEG-ammonium sulfate ATPS and thus, resulting a total recovery of 90%. Besides, PEG 400-phosphate ATPS with volume ratio of 13.0, TLL of 35%, and system pH of 7.0 provided a high recovery of 85% rotavirus-like particles (Benavides et al., 2006).

2.4.4 Drug residues in food and water Ever since, veterinary drugs are being introduced into the world partly for the animal cultivation and agro-industry, the significant amount of drug residues will greatly affect the food quality from animal products which is a great concern to the public health agencies (Rokka et al., 2005). As mentioned by Beyene (2015), the usage of drugs such as antibacterial and hormonal growth promoters are used in food-producing animals to increase the efficiency of weight gain, and feed while preventing diseases and other sickness (Beyene, 2015). The most known animal drugged foods are such: (1) meat; (2) milk; (3) honey; and (4) eggs and improper drug withdrawal or usage will pose health hazard to human consumption. Furthermore, drug residues or specifically known as pharmaceutical residues have also been detected (usually low mg/L) concentrations in drinking water sources which is part of human daily needs (Houeto et al., 2012). ATPS can also be applied in food and water applications for the extraction of drug residues, where it is regarded as an environmental friendly extracting strategy system that contains mostly water (negligible toxic volatile or organic solvent involved) (Li et al., 2009), cheap, simple, quick, easy, hygienic, and safe (Yang et al., 2014). An approach by Xie et al. (2011) utilizing poly (propylene glycol)400 -NaH2 PO4 to isolate the drug residue of sulfamethoxazole (SMX) in water samples. Various parameters such as the effect of salt, system pH, and temperature are optimized which ended with an average extraction efficiency of 99.2% due to the hydrophobicity and salting-out effect (Xie et al., 2011). Moreover, polyoxyethylene lauryl ether (POELE10)-NaH2 PO4 salt is used to extract the trace amount of chloramphenicol in shrimp from different waters, followed by HPLC for analytical evaluation (Lu et al., 2016). In addition, RSM has been coupled with the extraction of drug residue to improve process optimization. Thus, the final extraction efficiency of chloramphenicol increases significantly up to 99.42%. Similarly, HPLC coupled with poly (ethylene glycol-ran-propylene glycol) EOPOL31-K2 HPO4 salt for the separation of drug residue ciprofloxacin from milk, egg, and shrimp successfully reach 97.7% for shrimp and egg and subsequently 86.2% for milk (Chen et al., 2014). Table 2.1 represents various separations and

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Table 2.1 Separation and extraction of drug residues using polymer–salt aqueous two-phase system. Detection limit 0.1 μg/L

Drug type Sulfamethoxazole

Milk

Ciprofloxacin

Polymer–salt ATPS Poly (propylene glycol)400 -NaH2 PO4 EOPOL31-K2HPO4 6.8 ng/g

Egg

Ciprofloxacin

EOPOL31-K2HPO4

6.8 ng/g

Shrimp

Ciprofloxacin

EOPOL31-K2HPO4

6.8 ng/g

Shrimp

Chloramphenicol

(POELE10)-NaH2PO4 0.8 μg/kg

Recovery (%) 96.0–100.6

Linear range 2.5–250.0 μg/L

1st = 86.15 2nd = 85.3 1st = 92.05 2nd = 86.05 1st = 93.8 2nd = 86.1 99.42

1st = 85.9–86.4 2nd = 84.4–86.2 1st = 86.4–97.7 2nd = 85.6–86.5 1st = 89.9–97.7 2nd = 85.4–86.8 98–100.4

– – – 0.5–3.0 μg/kg

References Xie et al. (2011) Chen et al. (2014) Chen et al. (2014) Chen et al. (2014) Lu et al. (2016)

Principles of Multiple-Liquid Separation Systems

Source Water

Average extraction efficiency (%) 99.2

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extraction of drug residue from food and water using suitable salt–polymer ATPS to achieve maximum recovery in order to maintain the safety net limit prior to human’s consumption.

2.5

Limitation and future challenges

Previously in downstream manufacturing, conventional techniques such as filtration and centrifugation are applied for the separation of proteins from cells and cell debris, yet it poses difficulties when scaling-up due to small particles, viscous slurries, filter fouling, and compressible filter cakes (Schmidt et al., 1994). Adding on, conventional recovery techniques such as column chromatography and precipitation are expensive yet result in lower yields (Gupta et al., 1999) and especially chromatography leads to process bottlenecks in large-scale due to lacking in simplicity and slow mass transfer rate (causing unacceptable large pressure drops) (Carlson, 1988). A recent liquid–liquid separation technique has been a promising alternative to overcome these limitations as mentioned by Rosa et al. (2009) introducing ATPS which has proven to be a powerful, reliable and nonchromatographic unit operation to improve the downstream processing, reducing complex processes into simpler or single step by allowing simultaneous clarification, concentration, and partial purification of the targeted biomolecules (Rosa et al., 2009). As compared to the conventional methods, ATPS is indeed simple, selective, low cost, and easy scalable which makes it valid for downstream processing in many industries. Unfortunately, ATPS has not been widely recommended or utilized at commercial scale due to poor understanding of partitioning mechanism (Iqbal et al., 2016), poor understanding of the experimental design, operation, and installation of ATPSs are still limiting and interfering its application (Yau et al., 2015). Besides, several factors such as high cost of phase-forming polymers, poor selectivity in partitioning, and difficulty in recovery of partitioned biomolecules from phases are hindrance to the commercial application of ATPS (Banik et al., 2003). Although PEG-salt ATPS has shown good recovery and partitioning properties, yet still poses concerns to the environment whereby disposal of ammonium and phosphate salts can lead to eutrophication in lakes. Based on Hatti-Kaul (2001), problems can be solved by consider recycling of polymers and salts to reduce the load on wastewater treatment (Hatti-Kaul, 2001). Although recycling of salt is possible but no evidence on the economic feasibility when comparing with nonsalt recycling method (Greve and Kula, 1991). Both environmental and economic issues can be solved by utilizing biodegradable and volatile salts, such as citrate (Vernau and Kula, 1990) and ammonium carbamate (van Berlo et al., 1998), respectively. Firstly, addition of system affinity ligands to improve the partitioning properties as it has the potential to recognize and bind to the other molecules and domains. The ligand can be covalent bond to the polymer, as it will partitioned to the polymer-rich phase (preferable phase) concurrent replacing precipitation and clarification steps (Mazzola et al., 2008). Furthermore, incorporating novel polymers in ATPS such as derivatized starch and cellulose (low-cost polymers) can help to overcome economic struggles (Banik et al., 2003). ATPS in microfluidic platform can be indicated as promising and effective tool

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Principles of Multiple-Liquid Separation Systems

to accelerate the bioprocessing design and optimization (Silva et al., 2012). Other modifications such as usage of synthesized polymers, copolymers, reverse micelle solvent ATPS followed by coupling of HPLC to further improve the extraction process. In addition, combination of ATPS with other downstream extraction and separation methods such as ultrafiltration, precipitation, and gel filtration to enhance product purification (Ratanapongleka, 2010). Lastly, a novel approach of aqueous two-phase flotation system (ATPF) utilizes the integration principle of ATPS, and solvent sublation has been established for its wide application in the extraction and purification of bio-products. ATPF approach is more favorable as compared to ATPS due to high recovery, purity, environmental friendlier, economical feasible, and most importantly, easily scalable to large volumes according to industry requirements (Sankaran et al., 2019). The same agreement by Jiang et al. (2020) stated that the separation technique of ATPF is a step ahead of ATPS due to lesser amount of polymer required for separation, reduced in the dissolution losses of polymer, and better mode of mass transfer which leads to milder, lower cost, and higher potential for up-scaling (Jiang et al., 2019). Additionally, ATPF has the ability to significantly reduce the number of processing steps, excellent purification efficiency, high concentration coefficients, and low toxic solvent consumption will definitely attract more attention and gain more interest in the near future.

Conclusion The emerging green technology of polymer–salt ATPS has successfully strived through the separation and purification process of various biomolecules. Polymer–salt ATPS can be formed by dissolving two incompatible polymers (e.g., PEG) and salt (e.g., citrate, phosphate, sulfate). The partitioning of biomolecules is dependent on the properties of proteins, DNA and nucleic acids, VLPs, drug residues in food and water. The acknowledgment of polymer–salt ATPS has become more attractive than most conventional extraction process due to high potential for extraction, purification, enrichment, low cost, environmental friendlier, good mass transfer, less processing steps, simple, and potential scale-up. In most studies, PEG is well-known for its biodegradable and nontoxic properties, but the cost of polymers needs to be evaluated toward process economics. The disposal of salts such as phosphate and sulfate can cause pollution to the environment thus, recycling of polymers and salts is recommended or substituting with biodegradable and nontoxic salt (e.g., citrate). As more knowledge for a new type of ATPS and phase forming components will be a breakthrough for more advanced applications and potential for commercialization. Integration of ATPS with other downstream processing technology will also help to enhance the recovery of high value-added bioproducts. In the near future, ATPF will more attractive since lesser processing steps are followed by lesser overall production cost.

References Albertsson, P.Å., 1961. Fractionation of particles and macromolecules in aqueous two-phase systems. Biochem. Pharmacol. 5, 351–358. https://doi.org/10.1016/0006-2952(61)90028-4.

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Alcohol–salt interaction Hui Yi Leong a, Chih-Kai Chang b, Krisya Nicole Garcia Aung c, Dong-Qiang Lin a and Pau Loke Show c a Zhejiang Key Laboratory of Smart Biomaterials, Key Laboratory of Biomass Chemical Engineering of Ministry of Education, College of Chemical and Biological Engineering, Zhejiang University, Hangzhou, China, b Department of Chemical Engineering and Materials Science, Yuan Ze University, Chungli, Taoyuan, Taiwan, c Department of Chemical and Environmental Engineering, Faculty of Science and Engineering, University of Nottingham Malaysia, Semenyih, Selangor, Malaysia

3.1

3

Introduction

In recent years, the production of various bio-based materials using renewable sources as starting raw material has garnered widespread attention in the research of downstream biotechnology processing. The renewable sources include microalgae, fruit, and organic waste such as crop and food waste. One of the main challenges in the production of bio-based products is the extraction and separation of biomolecules due to the product specific precise operating conditions (e.g., temperature, pH, and concentration) requirement to avoid damaging the highly sensitive reactants and products as well as ensure high-value end-products (Khoo et al., 2020). Traditional developed separation techniques such as membrane separation, precipitation, and column chromatography usually involve complex scale-up and batch operating mode which is time-consuming and lead to high operating costs due to the multiple steps processes requiring high energy inputs (Phong et al., 2017). Hence, scientists and researchers are constantly developing new forms of purification techniques that could carry out an extraction process within one-step in a short amount of time to counter the limitations of traditional separation methods. To this date, researchers have successfully managed to develop a new bioseparation technology known as the liquid biphasic system (LBS) which has attracted other scientists of the same field as well as those interested in the recovery and purification of biomolecules. Through the usage of liquid biphasic separation, the issues linked to the conventional separation methods are overcome. Generally, this liquid biphasic technology separates two liquids through the formation of an interfacial layer by exploiting the mixture of the two liquids incompatibility when the conditions are beyond critical conditions. As a result of the phase-forming components’ characteristics, the created physic–chemical interaction can lead the desired targeted biomolecules to be partitioned to either at the top or bottom depending on the selectivity (Khoo et al., 2020). Due to its technology still being in the early stages, many researchers are still developing and improving the technology by experimenting with different combinations of solvents to obtain the best possible recovery and purification outcome Principles of Multiple-Liquid Separation Systems: Interaction, Application and Advancement. DOI: https://doi.org/10.1016/B978-0-323-91728-5.00010-X c 2023 Elsevier Inc. All rights reserved. Copyright 

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Principles of Multiple-Liquid Separation Systems

relating to specific products. With that being said, different types of LBSs have been developed such as organic solvent-based LBS, ionic liquid-based LBS, alcohol-based LBS as well as some technology assisted incorporated to LBS namely ultrasoundassisted LBS and electricity-assisted LBS. This chapter serves to give an overview of one of the many different types of LBSs which is the alcohol/salt-based LBS. Firstly, the basic and fundamental principles of the specific alcohol/salt-based LBS are presented along with illustrations to show and give a better understanding of the working mechanism of how the alcohol and salt with its targeted biomolecules and impurities are partitioned either at the top or bottom phase. This is then followed by the discussion of the effect of key factors such as the system’s temperature, pH, and the two main liquid constituents’ type and concentration on the separation efficiency through the use of related equations. Furthermore, examples of industrial applications that use this specific alcohol/ salt-based LBS are included to give a brief idea on which processes tend to use this type of system. In addition, the limitations imposed by the alcohol/salt system along with its technological modification for process optimization are also discussed extensively toward the end of the chapter.

3.2 Background and basic principle of alcohol/salt-based liquid biphasic system LBS, a type of liquid–liquid extraction technology in the downstream biotechnology processing or commonly known as aqueous two-phase system (ATPS), was discovered by Martinus Willem Beijerinck in 1896 when mixing an aqueous solution of starch and gelatin. Despite the fairly early discovery of the separation technology, the application involving LBS only started in the late 1950s and early 1960s when Albertsson demonstrated the technology’s technique and principle for the downstream processing of particles and macromolecules (Benavides et al., 2011). Ever since then, research relating to the recovery and purification of biomolecules using ATPS-based techniques were carried out extensively. Generally, two phases are formed when polymers, salts, alcohols of low molecular weight, surfactants, and/or ionic liquids are combined over critical concentrations. Theoretically, both phases are hydrophilic in nature; however, the top phase is more acclimatized to being hydrophobic in nature. The basic mechanisms of the LBS and the partitioning behavior of solutes and particles between phases are governed by the thermodynamic equilibrium of the system. As mentioned previously, there are different types of LBSs, hence, the specific chemical interactions responsible for the phase formation in LBS are highly dependent on the two main liquid constituents. Although the principles behind LBS are extremely complex regarding the formation and separation of aqueous phases, simplification solely focusing on the hydration enthalpy and the entropy of balance could be done. Therefore, an equilibrium curve typically known as a binodal curve is a significant tool when dealing with LBS due to each particular system having its own specific conditions (e.g., pH, temperature, and pressure). This

Alcohol–salt interaction

47

Figure 3.1 Schematic representation of binodal curve diagram.

binodal curve which is sometimes known as a phase diagram provides information regarding the two-phase formation as well as the top and bottom phases’ concentration requirements (Rosa et al., 2010). In addition, the tie line (TL) which represents the thermodynamic equilibrium between the two phases allows the determination of the TL length (TLL) value and coexisting phases’ respective compositions. In Fig. 3.1, line AB represents the binodal curve while the dotted lines represent the TL. As observed from the curve, concentrations above the curve line AB indicate a biphasic system. Specifically, as the name suggests, alcohol/salt LBSs are created when aliphatic alcohols of low molecular weights and highly concentrated salt solutions are mixed over certain conditions forming two immiscible phases (Fig. 3.2) (Greve and Kula, 1991). Despite the alcohol/salt LBS not being extensively explored as the polymer/polymer and polymer/salt systems, this type of LBS has been applied for the fractionation of various bio-based constituents such as phytochemicals, amino acids, and enzymes (Li et al., 2010; Ooi et al., 2009). Utilizing alcohol/salt system comes with its specific advantages which the major benefits include inexpensive cost as the alcohol and salt used can easily be recovered and reused unlike polymer-based LBS whereby polymers used are unrecovered (Zhi and Deng, 2006). Since the two main constituents utilized are alcohol and salt respectively, this specific system has low toxicity toward the environment as well as possesses low viscous properties. In addition, it is a fairly simple operation whereby the targeted biomolecules can easily be recovered and purified by evaporating the alcohol settled at the top phase (Guan et al., 1996; Tianwei et al., 2002).

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Principles of Multiple-Liquid Separation Systems

Figure 3.2 Illustration of the principle of alcohol/salt-based LBS. LBS, liquid biphasic system.

3.3 Influence of key parameters In general, the mixing of appropriate amounts of aqueous solutions of a salt and an alcohol above critical conditions can lead to the formation of LBS. The design, optimization, and scale-up of LBS are heavily influenced by the liquid–liquid equilibrium (LLE) data and the phase diagram properties of two constituents chosen for LBS (Albertsson, 1995). Due to various chain lengths and/or bonding structure of compounds, each compound used will exhibit different workability under specific conditions. For instance, isopropanol coupled with water as a system is utilized more frequently due to its low boiling point characteristics which often results in high yield separation through the evaporation process (Lin et al., 2013). Therefore, in order to obtain the highest separation and recovery efficiency, several LLE studies for alcohol/salt biphasic systems were carried out. The study and results based on

Alcohol–salt interaction

49

Arzideh et al. (2018) will be referenced to elaborate on the influence of key parameters to the alcohol/salt system. Although many different types of appropriate alcohols can be utilized, this study focuses on using alcohol 2-propanol mixed with various inorganic salts as more there are more publications and information on this specific type of solvent. Theoretically, a biphasic system’s phase forming ability for a selected alcohol is significantly depends on the salting-out effects of the salts (Wang et al., 2010). Hence, to describe the salting-out ability of an alcohol/salt LBS, the effective excluded volume (EEV) theory was applied. The EEV parameter value is taken as an index to indicate interaction of one alcohol molecule with water molecules in the presence of an electrolyte (Guan et al., 1993). Besides EEV, construction of the binodal curve and the determination of TLL and TL slope (TLS) was also carried out for obtaining results using the following appropriate equations:  TLL = TLS =

 t 2  2 wA − wbA + wbS − wtS

wtA − wbA wtS − wbS

whereby wA and wS are indications of the mass fraction of alcohol and salt, respectively, at top (t) and bottom (b) phases.

3.3.1 Effect of temperature As with most systems bounded by thermodynamic laws, temperature has a significant influence on the alcohol/salt-based LBS. From the previous research by Arzideh et al. (2018), it is indicated that the diminishing of the biphasic area was caused by the rise of the system’s temperature. When subjugating the mixture of 2-propanol + H3 PO4 + water at three different temperatures of increasing value, it was observed that more alcohol migrated to the top phase layer at lower temperatures than at higher temperatures, consequently decreasing the density of the top phase. The TLL and the mass fraction of alcohol also decline as the temperature increases. As a result of the TLL decrease, hydrophobicity and water content of both phases increase. To further emphasize, higher temperatures for alcohol/salt system are not favorable as it could decrease the system’s phase forming ability through large absolute values of TLS. At high TLS, the top phase becomes saltier producing a system with lower salt concentration gradient between the top and bottom phases respectively. The claims can further be supported by studying the relationship between the effect of EEV values and the biphasic region. In short, a more efficient phase separation is obtained when the temperature of the system decreases, making the biphasic area wider.

3.3.2 Effect of anion salt type The effect of the type of anion used on the alcohol/salt biphasic system is investigated using different types of inorganic salts as shown in Table 3.1 under fixed temperature for standard comparison.

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Principles of Multiple-Liquid Separation Systems

Table 3.1 List of inorganic salts. Chemical name Potassium sulfate Potassium sulfite Potassium phosphate Dipotassium phosphate Potassium citrate Potassium acetate Potassium carbonate Potassium tartrate

Compound formula K2 SO4 K2 SO3 K3 PO4 K2 HPO4 K3 C6 H5 O7 C2 H3 KO2 K2 CO3 K2 C4 H4 O6

Type of salt Sulfate Sulfite Phosphate Phosphate Citrate Acetate Carbonate Tartrate

Figure 3.3 Hofmeister series.

Hofmeister series (see Fig. 3.3) which shows a range of cations and anions properties is useful when dealing with investigating of various salts’ effects to the overall biphasic system. When comparing systems of 2-propanol + K3 PO4 + water and 2-propanol + K2 HPO4 + water, it is noted that the former system has a greater TLL as phosphate ion (PO4 –3 ) has a better capability to transfer water to the bottom phase than hydrogen phosphate ion (HPO4 –2 ). This reasoning is in line with the Hofmeister series which indicates that PO4 –3 has a higher salting-out ability than HPO4 –2 due to its nature of being more hydrophobic. The experiment is repeated however this time with salts listed in Table 3.1. The 2-propanol + either K3 C6 H5 O7 / K2 CO3 / K2 C4 H4 O6 + water shows similar binodal behavior as with the LBS that utilize phosphate salts. On the other hand, LBS that uses sulfate and sulfite salts creates a system with large biphasic region making it ideal for efficient separation. Among all the inorganic salts used, the alcohol/salt system which uses acetate salt has a very small biphasic area indicating it inability to form a strong biphasic system. Thus, it affirmed that the phase separation ability of the anion is in the order of SO4 −2 > SO3 −2 > PO4 −3 > C6 H5 O7 −3 > HPO4 −2 > CO3 −2 > C4 H4 O6 −2 > CH3 COO− (Arzideh et al., 2018).

Alcohol–salt interaction

51

Besides that, saturation solubility also plays an important role in the effect of type of salt selected for the LBS. Researchers have successfully correlate the relate between EEV values and saturation solubility of salts inevitably making saturation solubility a parameter to indicate if an LBS of chosen constituents can carry out phase forming. Kosmotropic ions, which is small ions with higher charge densities, are responsible for the salt’s solubility in an aqueous system. It is noted that as the surface charge density of anion increases, these kosmotropic ions break the hydrogen bonds and reconstruct the water molecules to induce a powerful biphasic system (Kumar and Venkatesu, 2014). Overall, the relationship and establishment of ion–water networks play a pivotal role in the phase forming of alcohol/salt-based LBS.

3.3.3 Effect of type of alcohol Theoretically, when dealing with the type of alcohol used, the number of carbon as well as the chains of the selected alcohol are the influencing factor for separation workability in LBS. Although this section focuses on the chemistry of the organic compound alcohol, temperature comes hand-in-hand with the structure of the alcohol molecules specifically the chain length. Based on the study of temperature effect, an alcohol/salt LBS could salt-out more at lower temperatures. Temperature has no significant effect on the phase formation ability when it comes to shorter chain length of alcohols. However, long-chain alcohols such as 2-propoanol are severely governed by the temperature variable whereby the phase forming of the system could decrease or increase depending on the condition. Moreover, shorter chain length alcohols maintain the shape of the water molecules’ structure while the longer chain alcohols show similar properties to kosmotropic ions by disrupting the hydrogen bonding leading to disorder of the water molecules. For better understanding, common alcohols such methanol (CH3 OH), ethanol (C2 H5 OH), propanol (C3 H7 OH), and 1-propanol (C3 H7 OH) will be used accordingly along with 2-propanol (C3 H7 OH) for comparison. As seen with the number of chains on the compounds’ formula, the salting-out ability of alcohols increases in the order of methanol < ethanol < propanol < 2-propanol < 1-propanol. Even though 2-propanol and 1-propanol have the same number of carbons, the former alcohol has a lower salting-out ability than the latter alcohol. The reason behind this phenomenon is due to the position of the hydroxyl group (–OH) in 1-propanol which creates a stronger alcohol–alcohol interaction leading to more transfer of water molecules to the bottom phase—dilute salt bottom phase and rich alcohol top phase (Arzideh et al., 2018).

3.4

Applications of alcohol/salt-based LBS

3.4.1 Enzymes Enzymes are active proteins (except RNAse) that can catalyze biochemical reactions within the mild conditions of temperature, pH, and pressure of the cells (Blanco and Blanco, 2017; Patel et al., 2017). Conventional methods involving liquid– liquid extraction are not suitable for the purification of enzymes as the conditions

52

Principles of Multiple-Liquid Separation Systems

required for the conventional techniques such as high pressure and temperature would lead to the irreversible loss of enzymatic activity (Abelson et al., 1994). In practice, chromatography techniques specifically affinity and ion exchange chromatograph are the attractive selection for the simultaneous extraction and purification of recombinant enzymes. However, these chromatography methods require a pretreatment step for the capture of target protein and desalting step containing yeast extract which makes the environment not suitable for enzymes (Iqbal et al., 2016). Hence, as discussed in the beginning, LBS was developed to overcome the limitations imposed by the conventional methods as well as to create a single-step downstream process. Lipase, an enzyme that hydrolyses esters in glycerol, is frequently used for biotechnological and industrial applications (Ooi et al., 2009). Lipase derived from Burkholderia pseudomallei is an extracellular lipolytic enzyme that contains an operon with lifts—lipase-specific foldases in which the operon is a necessary component for folding of lipase into an enzymatically active form (Yang et al., 2007). Due to the B. pseudomallei lipase having characteristics such as broad substrate specificity, tolerance toward organic solvents, and high thermal stability, these enzymes are often used as biocatalyst in industrial applications either at conditions of elevated temperature or in the presence of organic solvent (Ooi et al., 2009). Hence, alcohol/salt-based LBS was applied to recover the Burkholderia-strain lipase. In this study, nine combinations of alcohol/salt biphasic system compromising of alcohol-rich top phase (ethanol, 2-propanol, and 1-propanol) and a salt-rich bottom phase (ammonium sulfate, potassium phosphate, and sodium citrate) were explored to evaluate the effectiveness of the selected alcohol–salt pair to the recovery of lipase. The extraction and purification of lipase achieved a 99% yield as well as a purification factor of 13.5 by utilizing LBS of 16% (w/w) 2-propanol and 16% (w/w) potassium phosphate salt in the presence of 4.5% (w/v) sodium chloride, NaCl (Ooi et al., 2009). Besides that, alcohol/salt-based LBS was employed by Amid et al. (2012) for the recovery and purification of serine protease from mango (Mangifera Indica Cv. Chokanan). Plant-based proteases are widely utilized in food industries due to the protease’s characteristics of having good solubility, substrate specificity, efficient enzyme activity over a wide range of temperature and pH as well as high stability under extreme conditions (Caffini et al., 1988). In order to achieve the highest partition coefficient, influence of different parameters to the separation of serine protease such as concentration and type of alcohol (1-propanol, 2-propanol, and ethanol), type of salt (sodium citrate, potassium phosphate, and ammonium sulfate), pH and NaCl required were extensively studied in this experiment. It was demonstrated from the experiment that the highest partition coefficient and selectivity that could be achieved by the serine protease were 64.5 and 343.2, respectively, in LBS of 16% (w/w) 2-propanol, 19% (w/w) potassium sulfate, and 5% (w/v) NaCl at pH 7.5. Nonetheless, the recovery yield for the purification of serine protease was 96.7% while having a purification factor of 11.6 (Amid et al., 2012).

3.4.2 Proteins Similar to enzymes, conventional separation techniques such as chromatography are not an economical ideal solution for the purification of proteins due to the compound’s

Alcohol–salt interaction

53

complexity which requires many purification steps. The large pressure drops and batch processing mode also restricts the chromatography of proteins on larger scale. Furthermore, the low solubility of proteins in organic solvents results in these type of aqueous solutions to be rendered not suitable for the extraction of protein (Asenjo and Andrews, 2011; Carlson, 1988). Due to the limitations imposed by the conventional methods, extraction of protein using LBS has been an attractive alternative separation technique that greatly exploits the hydrophobicity of proteins. Although polymer/polymer or polymer/salt systems are the preferred choice for the recovery of proteins, there are several studies that employs the alcohol/salt system to obtain the desired proteins. One of the applications that uses alcohol/salt LBS for the purification of proteins includes the recovery of human interferon alpha-2b (IFN-α2b) from recombinant Escherichia coli (Lin et al., 2013). Interferons (IFNs) are glycoproteins produced by a variety of cells as the inflammatory response infections. The production of IFNs is triggered by the immune system in response to the pathogens or cytokines (Graber and Dhib-Jalbut, 2014). In this experiment, the influence of nine biphasic systems compromised of alcohol-abundant top phase (ethanol, 1-propanol, and 2-propanol) and salt-abundant bottom phase (ammonium sulfate, dipotassium hydrogen phosphate, and monosodium citrate) to the effectiveness of the IFN-α2b’s partitioning coefficient were investigated. It was found that the optimum purification of IFN-α2b and highest purification factor of 16.24 was achieved in a LBS consisting of 18% (w/w) 2-propanol and 22% (w/w) ammonium sulfate with the addition of 1% (w/v) NaCl. The purification of IFN-α2b also recorded a yield of 74.64% (Lin et al., 2013).

3.4.3 Medicinal plants Medicinal plants have been used in traditional medicine and worldwide ethnomedicine (Hao, 2019). Due to the high valuable natural compounds they contain, medicinal plants offer the most dynamic, polyvalent approach for the management of complex, multifactorial physiologic imbalances (Atar and Çölgeçen, 2020; Hedayat et al., 2020). Once again, LBS is a novel approach for the extraction and purification of drug residue in water (Li et al., 2009), food of animal origin (Han et al., 2011) as well as herbs (Cha et al., 2016). Alcohol/salt-based LBS was applied to extract and purify chlorogenic acid (CGA) from ramie (Boehmeria nivea L. Gaud) leaf (Tan et al., 2014). In this study, four types of organic alcohol (methanol, ethanol, 1-propanol, and 2-propanol) and four different inorganic salts (K3 PO4 , (NH4 )2 SO4 , NaH2 PO4 , K2 CO3 ) were considered as phase forming constituents. Other influencing parameters such as concentration of alcohol and salt as well as extraction temperature and pH were investigated to optimize the biphasic system’s phase forming ability. The extraction of CGA from ramie leaf was able to achieve maximum extraction of 95.76% under operating conditions of 25˚C and pH environment of 3.29 using a combination of 15% (w/w) ethanol and 28.1% (w/w) disodium phosphate, NaH2 PO4 (Tan et al., 2014). Besides that, an alcohol/salt-based LBS has been successfully applied by Reis et al. (2014) to extract rutin from acerola waste. Rutin is a citrus flavonoid glycoside which is technically a low molecular weight polyphenolic compound often synthesized in higher plants especially citrus fruits and berries (Patel and Patel, 2019). The aim

54

Principles of Multiple-Liquid Separation Systems

of the study was to recover rutin from acerola which is also known as West Indian cherry or Barbados cherry as acerola is clinically proven to have positive health effects due to its antioxidant properties. Acerola-based dietary supplements also protective effects against cancer, arteriosclerosis, neurodegenerative diseases, and aging (de Freitas et al., 2006; De Oliveira et al., 2009; Mercali et al., 2011). In this project, different LBS composed of alcohol (methanol, ethanol, 1-propanol, and 2-propanol) and salts (K3 PO4 , K2 HPO4 , and potassium phosphate buffer composed of K2 HPO4 / KH2 PO4 ) were experimented to obtain the optimum system for the highest rutin extraction. Moreover, this study also included the effects of the temperature (278.15– 308.15 K) and addition of the electrolyte NaCl to the biphasic system’s phase forming ability. From this work, it was concluded that using LBS compromising of 40% (w/w) 1-propanol, 20% (w/w) K2 HPO4 /KH2 PO4 , 40% (w/w) water and 2.5% (w/w) NaCl at 25˚C contributes to the highest partition coefficient of 51.47% and rutin extraction efficiency of 98.64% (Reis et al., 2014).

3.4.4 Other applications Alcohol/salt system could nonetheless be applied for the extraction of valuable compounds or components from plants that are not for medicinal purposes (Table 3.2). As discussed before, the thermodynamics that drives the components to be partitioned at the top or bottom phase not only depends on the extraction conditions but on the alcohol/salt biphasic system selected as well. By having prior knowledge of the physical and especially the chemical properties of the compound to be recovered, appropriate alcohol/salt biphasic system could be chosen for high separation efficiency. Hence, although not as popular as its polymer/salt counterpart, alcohol/salt system could still be used to recover a wide range of components. With that being said, a study by Tan et al. (2013) has successfully used alcohol/ salt-based LBS to extract and purify anthraquinones (AQs) derivatives from Aloe vera L. One of the main objectives of this particular study was to develop a simple and efficient technique to recover AQs which not only possessed pharmacological properties but were also used as dyes in fabric coloring and bird repellent from seeds and crops depending on the AQs derivative type. Similar to all the previous studies discussed, main parameters that influence the biphasic system partitioning ability such as concentration and type of alcohol (ethanol, 1-propanol, 2-propanol), concentration, and type of salt (ammonium sulfate, disodium phosphate) as well as the extraction temperature and pH were investigated thoroughly in this study. In this experiment, it was concluded that 95.56% of AQs were able to be extracted into the alcohol-rich top phase in LBS consisting of 17.84 % (w/w) 1-propanol and 26.66 % (w/w) (NH4 )2 SO4 at 25˚C with the pH not being adjusted. The partition coefficient of the extraction of AQs from the aloe biomass was found to be 64.35 (Tan et al., 2013). A recent study covering the topic of sustainable approaches for recovery of phlorotannin from microalgae specifically Padina australis and Sargassum binderi species was conducted by Chia et al. (2018) by utilizing alcohol/salt LBS. In this research, the effect of the four combinations of biphasic system consisting of alcohol

Constituents 16% (w/w) 2-propanol, 16% (w/w) potassium phosphate 16% (w/w) 2-propanol, 19% (w/w) potassium sulfate 18% (w/w) 2-propanol, 22% (w/w) ammonium sulfate 33.5% (w/w) of 2-propanol, 10% (w/w) ammonium sulfate 25% (w/w) of 2-propanol, 12.5% (w/w) ammonium sulfate 15% (w/w) ethanol, 28.1% (w/w) NaH2 PO4 40% (w/w) 1-propanol, 20% (w/w) K2 HPO4 /KH2 PO4 17.84 % (w/w) 1-propanol, 26.66 % (w/w) (NH4 )2 SO4

Feedstock Burkholderia pseudomallei

Biomolecule Lipase

Partition Coefficient, Selectivity K 287.5 –

Purification Recovery Factor, Yield, % Reference PFT 13.5 99.3 Ooi et al. (2009)

mango (Mangifera Indica Serine protease 343.2 Cv. Chokanan) peel Escherichia coli Interferon (IFN) –

64.5

11.6

96.7

0.82

16.24

74.64

Padina australis

Phlorotannin

–

–

2.49

76.1

Sargassum binderi

Phlorotannin

–

–

1.59

91.67

–

38.72

–

95.76

–

51.47

–

98.64

Anthraquinones –

64.35

–

95.56

Ramie (Boehmeria nivea Chlorogenic L. Gaud) leaf acid (CGA) Acerola waste Rutin Crude Aloe vera L.

Alcohol–salt interaction

Table 3.2. Applications of Alcohol/Salt-based LBS.

Amid et al. (2012) Lin et al. (2013) Chia et al. (2018) Chia et al. (2018) Tan et al. (2014) Reis et al. (2014) Tan et al. (2013)

55

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Principles of Multiple-Liquid Separation Systems

(methanol, ethanol, 1-propanol, and 2-propanol) and ammonium sulfate salt, system pH, crude sample volume ratio to the system’s phase forming ability were studied thoroughly. The highest recovery of phlorotannin were 76.1% and 91.67% with purification factors of 2.49 and 1.59 from P. australis and S. binderi, respectively, in LBS of 2propanol and ammonium sulfate. The study was able to demonstrate an environmentalfriendly and good recyclability system through the consistent phlorotannin recovery results after conducting two cycles of the system (Chia et al., 2018).

3.5 Limitations and advancements to the alcohol/salt-based liquid biphasic system Despite the advantages possessed by the alcohol/salt-based LBS, this system has received lesser attention in the field of purification studies as compared to polymer/ polymer and polymer/salt systems. One of the major drawbacks of the alcohol/salt system includes the incompatibility of many targeted protein-based biomolecules with the organic solvent phase which could lead to denaturation or inactivation of biomolecules (Ooi et al., 2009). In addition, high concentration of salt is required for salting-out targeted proteins. This is due to the fact that salt solubility and polarity change depending on the concentration of the salt in the system, which will either lead to “salting-in” or “salting-out” of biomolecules. The former term is not desirable as it would promote the targeted proteins to be more soluble in water which results in low separation. However, the latter problem does not bring any serious concerns to those using the alcohol/salt-based system as salt is inexpensive and the shortcoming could easily be avoided by having prior knowledge of the targeted protein’s solubility. Modifications to the LBS were also conducted to further improve the shortcomings of the current system as well as to adjust to certain types of applications. The most recent development of modified LBS is the liquid biphasic flotation (LBF) which integrates both the principle of aqueous two-phase extraction (ATPE) (or known as LBS as well) and solvent sublation (SS). This LBF technology was developed by a group of bioseparation researchers from the University of Nottingham Malaysia in which the system was successfully applied to purify proteins from microalgae (Phong et al., 2017), Burkholderia cepacia (Sankaran et al., 2018), etc. Generally, LBS has been a well-favored alternative for the recovery and purification of natural as well as bio-based products such as proteins and enzymes—intracellular and extracellular (Naganagouda and Mulimani, 2008). Meanwhile, SS is somewhat of an adsorptive bubble separation technique whereby the compounds are adsorbed on the bubbles’ surfaces that ascend from one liquid phase to another liquid phase (Bi et al., 2010). Due to the fact that LBF is a combination of LBS and SS, this bioseparation method inherits both the advantages of the aforementioned individual separation techniques resulting in the technology having high separation efficiency, high concentration coefficient, simple operation, and environmental friendly (Leong et al., 2018). The newly developed LBF has been successfully applied for the recovery of protein from dairy milk waste products by using alcohol/salt system (Tham et al., 2019).

Alcohol–salt interaction

57

Expired dairy products are usually disposed due to the potential health hazard it imposes on the surrounding living organisms. In addition, expired dairy products are often disposed due to the lack of current proper methods for recovery of valuable constituents. Generally, milk encompasses several different components in which 3.4% are proteins with 80% of the proteins being Casein while the remaining 20% are Whey protein (Tham et al., 2019). The results of the experiment recorded a protein recovery yield and separation efficiency of 94.97% and 86.289%, respectively. The efficient separation of the protein from the expired milk products was obtained in an optimum condition consisting 80% ethanol concentration partitioned at the top phase, 150 g/L dipotassium hydrogen phosphate with 10% (w/v) milk partitioned at the bottom phase, and 7.5 min floatation time. When scaled by 40 times, the recovery yield of the protein and separation efficiency were 78.92% and 85.62%, respectively. In the recovery of proteins from wet microalgae Chlorella sorokiniana species by Phong et al. (2017), it was proposed to use alcohol/salt LBF with the aid of ultrasonication for cell rupturing. This study aimed to extract microalgae protein from algal biomass, whereby upon further processing could eliminate technical difficulties such as powder-like texture, dark green color, and slightly fish smell for incorporation of the recovered proteins into palatable food (Becker, 2007). The effect of different parameters such as varying crude feedstock concentration, flotation time, concentration and type of salt, concentration, and type of alcohol, initial volumes of salt and alcohol were investigated. From this study, it was found that the highest amount of protein recovered at the top phase was achieved utilizing LBF with 250 g/L ammonium sulfate, 60% (v/v) 2-propanol, 1.0 VR,initial , 20 g/L crude biomass load, 4 mm3 /min air flowrate and flotation time of 10 minutes after optimization process. Moreover, recycling the alcohol partitioned at the top phase also increases the separation performance through three consecutive recycling runs (Phong et al., 2017). Besides that, researchers have also come up with technology-assisted LBS for process optimization, namely microwave-assisted LBS which integrates the conventional microwave separation method with the general LBS. Microwave-assisted extraction (MAE) is an automated green technique for the extraction of active compounds from medicinal plants by using microwave energy to heat solvents containing feedstock resulting in the partitioning of the targeted components from the feedstock into the solvent (Kataoka, 2019). The major advantage of MAE over conventional extraction methods is the enhancement of feedstock residual water evaporation which breaks the cell walls of plant cells promoting extraction through internal diffusion (Reddy et al., 2020). Furthermore, MAE’s ability to heat samples/feedstock rapidly leads to the reduction of solvent consumption and extraction time (Llompart et al., 2019). A study by Zhang et al. (2015) shows the development of a novel separation technique known as microwave-assisted aqueous two-phase extraction (MAATPE) which combines both the principles of ATPE and MAE. This was done by exploiting the high efficiency of MAE and demixing effect of ATPE. MAATPE has been successfully applied for the simultaneous extraction and purification of alkaloids from Sophora flavescens Ait. using ethanol-ammonium sulfate as the biphasic system. Seven biphasic systems consisting of ethanol-rich top phase and salt-rich bottom phase ((NH4 )2 SO4 , K2 HPO4 , Na2 CO3 , Na2 SO4 , CaCl2 , KH2 PO4 , NaCl) along with its properties were studied in

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detail. Nonetheless, key parameters such as the composition of the selected LBS, solvent-to-material ratio and extraction temperature were also included in this study to obtain the optimum extraction criteria. From this work, it was demonstrated that the extraction of alkaloids from S. flavescens Ait. achieved a recovery yield of 92% in a LBS containing 28% (w/w) ethanol, 18% (w/w) (NH4 )2 SO4 , solvent-to-material ratio of 60:1, temperature 90°C, 5 min extraction time, and microwave power 780 W (Zhang et al., 2015). Another technological advancement made to the general LBS system includes ultrasound-assisted LBS which incorporates the principle of ultrasound extraction. Ultrasound-assisted extraction (UAE) is an innovative technique based on using ultrasonic waves which has gained much attention for the extraction of various compounds from a diversity of samples such as microbes and plants (Roohinejad et al., 2017). UAE also offers many advantages with the technique being simple, effective, clean extraction as well as environmentally friendly. The most significant advantage is the UAE method ability to increase the extraction yield through the acceleration of the targeted molecules kinetics. Recently, another novel method was developed by (Zhang et al., 2018) known as ultrasonic-assisted aqueous two-phase extraction (UA-ATPE) for the extraction of polysaccharides from Lilium davidiivar. unicolor Salisb (LPS). As the name suggests, this effective method is the combination of the general LBS and UAE techniques. For this project, four different combinations of biphasic system consisting of ethanol-rich top phase and salt-rich bottom phase ((NH4 )2 SO4 , K2 HPO4 , Na2 CO3 , NaH2 PO4 ) along with their compositions were investigated in this study. Additionally, influencing key parameters for the extraction efficiency such as the solvent-to-solid ratio, extraction time, temperature, pH, and the ultrasound frequency were extensively studied. After optimization, the extraction of polysaccharides achieved a partition coefficient of 14.17 and recovery yield of approximately 94.45% in LBS of 16.54% (w/w) ethanol and 31.45% (w/w) K2 HPO4 , solvent-to-solid ratio of 25:1 (g/g), extraction time of 10 minutes, 60°C extraction temperature, pH of 11, ultrasound power of 190W and 22 KHz of ultrasound frequency (Zhang et al., 2018).

3.6 Conclusions In summary, alcohol/salt-based LBS can be applied for various separation/ extraction/ purification of biomolecules. Since every molecule has specific conditions at which it is extracted, studies regarding the different factors affecting the partitioning coefficient are exploited as soon as the LBS reached equilibrium conditions. Studies on the effect of temperature, pH, type, and concentration of phase-forming salt as well as concentration of alcohol are conducted to obtain the optimum conditions for highest separation efficiency. For a general alcohol/salt biphasic system, to obtain the most efficient separation (large biphasic area), one should use salts of anion with high surface charge density and low solubility in water and alcohol with long carbon chain at low temperatures. The low temperatures not only serve to avoid alcohol partitioned at the top phase to evaporate but also acts as a suitable medium for thermosensitive biomolecules. In cases whereby the biomolecules are acidic in nature, pH

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is an important factor in providing an acidic environment for the acidic molecules’ stability. Although alcohol/salt LBS might not be suitable for most proteins due to incompatibility with the organic solvent, this challenge could overcome by having prior knowledge of the protein’s solubility. Technological advancements made to the general alcohol/salt LBS such as LBF, MAATPE, and UA-ATPE are just the beginning. As with other biphasic systems such as polymer/polymer, polymer/salt, and even ionic liquids/salt, researchers are exploring more into integrating conventional available technologies with LBS to overcome the shortcomings of the general LBS as well as to compensate for the different types of recovery applications.

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aqueous two-phase system. Anal. Chim. Acta 653 (2), 178–183. https://doi.org/ 10.1016/j.aca.2009.09.011. Li, Z., Teng, H., Xiu, Z., 2010. Aqueous two-phase extraction of 2, 3-butanediol from fermentation broths using an ethanol/ammonium sulfate system. Process Biochem. 45 (5), 731–737. Lin, Y.K., Ooi, C.W., Tan, J.S., Show, P.L., Ariff, A., Ling, T.C., 2013. Recovery of human interferon alpha-2b from recombinant Escherichia coli using alcohol/salt-based aqueous two-phase systems. Sep. Purif. Technol. 120, 362–366. https://doi.org/10.1016/ j.seppur.2013.09.038. Llompart, M., Garcia-Jares, C., Celeiro, M., Dagnac, T., 2019. Extraction | Microwave-Assisted Extraction. Encyclopedia of Analytical Science, Third Edition. Elsevier, Oxford, pp. 67–77. https://doi.org/10.1016/B978-0-12-409547-2.14442-7. Mercali, G.D., Sarkis, J.R., Jaeschke, D.P., Tessaro, I.C., Marczak, L.D.F., 2011. Physical properties of acerola and blueberry pulps. J. Food Eng. 106 (4), 283–289. Naganagouda, K., Mulimani, V.H., 2008. Aqueous two-phase extraction (ATPE): An attractive and economically viable technology for downstream processing of Aspergillus oryzae α-galactosidase. Process Biochem. 43 (11), 1293–1299. https://doi.org/ 10.1016/j.procbio.2008.07.016. Ooi, C.W., Tey, B.T., Hii, S.L., Kamal, S.M.M., Lan, J.C.W., Ariff, A., Ling, T.C., 2009. Purification of lipase derived from Burkholderia pseudomallei with alcohol/salt-based aqueous two-phase systems. Process Biochem. 44 (10), 1083–1087. https://doi.org/ 10.1016/j.procbio.2009.05.008. Patel, A.K., Singhania, R.R., Pandey, A., 2017. Production, Purification, and Application of Microbial Enzymes. Biotechnology of Microbial Enzymes: Production, Biocatalysis and Industrial Applications. Academic Press, San Diego, pp. 13–41. https://doi.org/ 10.1016/B978-0-12-803725-6.00002-9. Patel, K., Patel, D.K., 2019. The Beneficial Role of Rutin, A Naturally Occurring Flavonoid in Health Promotion and Disease Prevention: A Systematic Review and Update. Bioactive Food as Dietary Interventions for Arthritis and Related Inflammatory Diseases, Second Edition. Academic Press, San Diego, pp. 457–479. https://doi.org/10.1016/ B978-0-12-813820-5.00026-X. Phong, W.N., Show, P.L., Teh, W.H., Teh, T.X., Lim, H.M.Y., binti Nazri, N.S., Tan, C.H., Chang, J.S., Ling, T.C., 2017. Proteins recovery from wet microalgae using liquid biphasic flotation (LBF). Bioresour. Technol. 244, 1329–1336. https://doi.org/10.1016/ j.biortech.2017.05.165. Reddy, A.V.B., Moniruzzaman, M., Madhavi, V., Jaafar, J., 2020. Chapter 8 - Recent improvements in the extraction, cleanup and quantification of bioactive flavonoids. Bioactive Nat. Prod., Vol. 66. Elsevier, pp. 197–223. https://doi.org/10.1016/B978-0-12817907-9.00008-8. Reis, I.A.O., Santos, S.B., Pereira, F.D.S., Sobral, C.R.S., Freire, M.G., Freitas, L.S., Soares, C.M.F., Lima, Á.S., 2014. Extraction and recovery of rutin from acerola waste using alcohol-salt-based aqueous two-phase systems. Sep. Sci. Technol. 49 (5), 656–663. https://doi.org/10.1080/01496395.2013.860461. Roohinejad, S., Nikmaram, N., Brahim, M., Koubaa, M., Khelfa, A., Greiner, R., 2017. Potential of Novel Technologies for Aqueous Extraction of Plant Bioactives. Water Extraction of Bioactive Compounds: From Plants to Drug Development. Elsevier, Oxford, pp. 399–419 Chapter 16 https://doi.org/10.1016/B978-0-12-809380-1.00016-4 . Rosa, P.A.J., Ferreira, I.F., Azevedo, A.M., Aires-Barros, M.R., 2010. Aqueous two-phase systems: a viable platform in the manufacturing of biopharmaceuticals. J. Chromatogr. A. 10.1016/j.chroma.2009.11.034.

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Sophie Jing Nee Chai a, Xiao-Qian Fu b, Dong-Qiang Lin b and Pau Loke Show a a Department of Chemical and Environmental Engineering, Faculty of Science and Engineering, University of Nottingham Malaysia, Semenyih, Selangor, Malaysia, b Zhejiang Key Laboratory of Smart Biomaterials, Key Laboratory of Biomass Chemical Engineering of Ministry of Education, College of Chemical and Biological Engineering, Zhejiang University, Hangzhou, China

4.1

Introduction

In the 1990s to late 2000s, ionic liquids (ILs) were rigorously studied (Abbott et al., 2007) in the hopes of replacing traditional solvents that were known to have detrimental effects on the environment (Chiappe and Pieraccini, 2005; Welton, 1999). ILs were found to possess advantageous properties that made them the more apparent option when compared to traditional solvents, especially volatile organic solvents (Chiappe and Pieraccini, 2005). For example, ILs are suitable solvents for a variety of inorganic, organic, and polymeric materials. Furthermore, ILs have low vapor pressure and high thermal stability, making them an ideal solvent under vacuum conditions. However, ILs are far from perfect because their potential value does not justify the cost at this point in time, this is mainly because of the high cost incurred from the raw materials required and the complexity of the fabrication process of ILs (Li et al., 2020). Secondly, ILs are considered toxic when compared to deep eutectic solvent (DES). For instance, imidazolium and pyridinium based ILs decrease the viability of human cells significantly down to 50% after 48 h of direct exposure (Gouveia et al., 2014). Furthermore, there are concerns regarding the “green” status that ILs were given in the 1990s because ILs are said to have poor biodegradability and biocompatibility (Craveiro et al., 2016). DES was first discovered by Abbott when he mixed choline chloride and urea at ambient conditions. Choline chloride was defined as the hydrogen bond donor (HBD), while urea was the hydrogen bond acceptor (HBA). At a specific ratio of HBD and HBA, also known as the eutectic composition, the mixture experiences a deep melting point depression, causing the mixture to remain as a liquid at low temperatures (Abbott et al., 2003). DESs are often compared to ILs because they have similar properties; both have high thermal stability, low vapor pressure, and good solubility for a diverse portfolio of substances (Hayyan et al., 2012). As advancement and application of green technology receive growing attention, DESs have become an attractive alternative in academia and industry due to being more cost-effective, as well as possessing Principles of Multiple-Liquid Separation Systems: Interaction, Application and Advancement. DOI: https://doi.org/10.1016/B978-0-323-91728-5.00016-0 c 2023 Elsevier Inc. All rights reserved. Copyright 

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lower toxicity, better biodegradability, and biocompatibility when compared to ILs (Benvenutti et al., 2019; Halder and Cordeiro, 2019; Juneidi et al., 2016; Khandelwal et al., 2016; Radoševi´c et al., 2015; Xu et al., 2017). DESs have many applications, one example is gas capture, where DESs could be used as an absorbent for ammonia in industrial tail gas (Li et al., 2020). DESs are also deemed as a good solvent/cosolvent in biocatalytic reactions such as ester hydrolysis reactions and they can be employed in the extraction/separation of biomolecules or products from biochemical reactions (Xu et al., 2017). There are several types of DESs that can be classified systematically according to their composition into five types. Type I DESs are mixtures composed of a quaternary ammonium salt and a metal chloride. When metal chloride hydrate is used instead, Type II DESs are synthesized. Type III DESs are made up of quaternary ammonium salt and HBD. Some examples of common HBDs are carbohydrates, carboxylic acid, and amides. Type IV DESs consist of metal chloride hydrate, and HBD and Type V DESs are relatively new and are different from the other four types in the sense that they are nonionic (Abbott et al., 2007; Abranches et al., 2019). Aqueous two-phase system (ATPS) is a liquid–liquid fractionation method that can potentially extract, separate, and purify biomolecules like proteins, enzymes, nucleic acids, viruses, and many more (Iqbal et al., 2016). Typically, ATPS is composed of polymer and salt or two types of polymer that are immiscible with respect to each other. In the 1958s, Per-Ake Albertsson discovered and used ATPS to isolate protein molecules for the first time (Albertsson, 1958). From then onwards, new applications of ATPS have emerged and gained interest from various industries and fields of study. ILs-ATPS can be used to extract proteins, but when DES-ATPS was discovered, it quickly becomes the more favorable alternative in protein extraction due to its lower cost and being more environmentally friendly (Zeng et al., 2013). In 2014, Zeng and coworkers pioneered the application of DES-ATPS in protein separation. They had studied four kinds of DES, i.e., choline chloride–urea, tetramethylammonium chloride–urea, tetrapropylammonium bromide-urea, and choline chloride–methylurea based DESs, and choline chloride–urea based DES was determined to provide the highest extraction efficiency for bovine serum albumin (Zeng et al., 2014). In the following year, Xu and colleagues proposed using choline chloride based DES-ATPS to extract bovine serum albumin (Xu et al., 2015). Moreover, Li and the group used green betaine based DES-ATPS to extract proteins (Li et al., 2016). From these studies, temperature, pH, concentration of salt, mass of DES, separation time, agitation time, and mass of proteins are parameters that can affect the extraction efficiency. As DES-ATPS technology steadily progresses and evolves, the research field is filled with new and exciting discoveries. With this much information out there, there comes a need for a comprehensive guide for those interested in entering this research field, and this chapter aims to be just the resource for that purpose. This chapter mainly covers sugar-based DES (Type III DES)-ATPS, sugar/carbohydrate based DES-ATPS, and their significant roles in the biomolecules (i.e., proteins, lipids, carbohydrates, and nucleic acids) separation, extraction, and purification. Additionally, how parameters like temperature, mass of DES, type of HBA and HBD, and type of phase forming agent affect phase formation and extraction efficiencies are critically analyzed and reviewed.

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Heat

Stir

HBD

HBA

DES

Figure 4.1 DES synthesis using heating and stirring method.

Furthermore, this chapter also provides an overview of recent research progress and breakthroughs made over the last five years for sugar/carbohydrate based DES-ATPS. Being a greener alternative compared to most other biomolecule extraction methods, the recycling, environmental considerations, and impact of sugar/carbohydrate based DES-ATPS are discussed as well.

4.2

Sugar-based deep eutectic solvent

4.2.1 Synthesis of deep eutectic solvent When compared to ILs, DESs are generally easier to prepare/synthesize. Multiple preparation methods or synthesis routes exist, depending on the available equipment, personal preference, and ability to keep moisture content to the minimum. DESs are a mixture of HBD and HBA, a common HBA used is choline chloride because of its low-cost advantage, and it can be combined with different types of HBD to form different kinds of DES. Some examples of HBDs are glycerol, glucose, urea, ethylene glycol, and more (Zhang et al., 2019). This section will be focused on the synthesis of sugar-based DES. The general preparation method of sugar-based DESs is simple and can be summarized as follows. For the heating and stirring method, the raw materials, the researcher’s choice of HBA, and sugar/carbohydrates at different ratios, are first dried to remove excess moisture using an oven or vacuum drying oven when heat-sensitive compounds are present. This is because moisture may affect the physical properties of DES (Hayyan et al., 2012). Then, the HBA and HBD are heated and stirred continuously for a predetermined period if agitation time is a parameter to be studied. This can be done using a hot plate or a hot oil bath depending on the design of DF-101S, which is equipped with a magnetic stirrer to produce a homogenous and transparent mixture (Li et al., 2016; Xu et al., 2015; Zhang et al., 2019). The synthesis process is similar for sugar-based DESs, as shown in Fig. 4.1. For example, Li and coworkers synthesized sorbitol-betaine and glucose-betaine-based DES using the heating and stirring method (Li et al., 2016). Also, Xu and the group synthesized sorbitol-choline chloride and glucose-choline chloride based DESs by stirring the HBA and HBD together at 80°C (Xu et al., 2015).

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Some of the advantages of sugar-based DESs are that no additives are required during synthesis. The process does not involve any chemical reactions; hence, the prepared DESs can be used directly without being purified, keeping the application cost low, and having the advantage of being relatively environmentally friendly as there are no undesirable by-products and no waste is generated (Hansen et al., 2020).

4.2.2 Characterization of deep eutectic solvent The determination of the physicochemical properties of DESs is essential because these properties can affect the biomolecule extraction capability of DES. Some examples of these properties are polarity, melting points, density, viscosity, conductivity, pH, thermal stability, and many more. Therefore, the characterization of DES can be instrumental in the development and application of DES. Some notable characterization methods and process for DESs will be covered here. Craveiro and the group attempted to study the thermal stability of three types of choline chloride–sugar based DESs and two types of citric acid-sugar-based DESs using differential scanning calorimetry analysis under an inert atmosphere in the range of -40°C to 250°C (Craveiro et al., 2016). The results were consistent for the different types of DESs, where all of them are susceptible to degradation at temperatures higher than 120°C. Besides that, the group discovered that when the water content of the DESs is larger than 4 weight%, any further addition of water will result in the increase of the melting point of the mixture, which is undesirable in biomolecule separation-related applications (Craveiro et al., 2016). This increase in melting point is induced by the increase of hydrogen bond interactions between the water molecules and the DES. Ultraviolet spectrometry was used to determine the polarity of DESs. The resulting wavelength of maximum absorbance (λmax ) of Nile red can be used to calculate ENR using this relationship: ENR = 28591/λmax

(4.1)

where ENR is in kcal/mole and λmax is in nm. More polar DESs give higher λmax values and smaller ENR values. This method was postulated by Deye et al. to determine the polarity in solvents (Deye et al., 1990). It is worth noting that high water content (larger than 50 weight%) has significant effects on the polarity of DES. The additional water molecules can disrupt the hydrogen bonds formed between the HBA and HBD (Dai et al., 2013). Silva and colleagues attempted to characterize the viscosity of six different types of choline chloride–sugar based DESs at increasing temperatures, from 25°C to 100°C, using a viscometer-densimeter. It was found that the viscosity of the DESs decreases as the temperature increases (Silva et al., 2018). When the viscosities of the different DESs were compared at a constant temperature, the group observed that choline chloride–mannose based DES has the highest viscosity followed closely by choline chloride–fructose based DES, then choline chloride–glucose based DES and the least viscous one was choline chloride–xylose based DES (Silva et al., 2018).

Sugar-based deep eutectic solvent-aqueous two-phase system

Phase forming salt

DES

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Biomolecules

DES-ATPS

DES-ATPS

Figure 4.2 Synthesis of DES-ATPS and partition of biomolecules to DES phase.

4.3

Sugar-based deep eutectic solvent-aqueous two-phase system

ATPS is composed of two immiscible aqueous phases, and it has been used in the separation and purification process for biomolecules like proteins, enzymes, nucleic acids, cells, and many more. Conventionally, ATPS is formed by two types of polymer or polymer and inorganic salts like phosphates, citrate or sulfates (van Berlo et al., 1998). As time progressed, alcohol/salt ATPS was proposed as an alternative to polymer/polymer ATPS to lower the cost. However, this type of ATPS is mostly incompatible with protein separation applications (Jiang et al., 2009). As the popularity of ILs increases, researchers developed ILs based ATPS (Berthod et al., 2008). However, due to the expensive preparation route for ILs and their inherent toxicity risks, ILs-based ATPS become less desirable in biomolecules separation applications, where the biomolecules are most likely going to be used in food and health related industries. Most DESs synthesized are hydrophilic in nature, though hydrophobic DESs exist and recently received attention (Florindo et al., 2019). Thus, hydrophilic DESs can be used to form ATPS, which can be used to extract, separate, and purify a wide array of biomolecules (Marchel et al., 2020). Since the raw materials of DESs are more accessible than that of ILs, and the synthesis of DESs is cheap and straightforward, DESs-based ATPS quickly gained popularity and became a better alternative than ILsbased ATPS in biomolecule separation. Sugar-based DES is a mixture of HBD and HBA, making it possible for DES to form hydrogen bonds from donating and accepting protons and electrons. This phenomenon promotes the dissolution strength of the solvent (Zhang et al., 2012). As a result, sugarbased DESs can effectively act as an extraction agent while keeping the cost low and have minimal impact on human health and the environment. Generally, DES needs to combine with another component, either an inorganic salt, most used are phosphates, or polymer, to drive them into the biphasic region, which is ATPS, as shown in Fig. 4.2. Primarily, researchers analyze the binodal curve of the phase diagram to obtain the phase formation ability and properties of DES-ATPS. The binodal curve shows at which mass fraction of DES and concentration of phase forming agent that the DESATPS can be achieved. This indicates that when the DES to phase forming agent ratio

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is above the binodal curve, a two-phase system can be formed. The experimental data can be fitted to the following expression.     Y = A exp BX0.5 − CX3

(4.2)

where Y and X are the phase forming agent and DES mass fraction percentages, respectively. A, B, and C are constants obtained by the regression of the experimental data (Farias et al., 2017). As such, the effect of HBA to HBD mass/molar ratio, type of phase forming agents, and type of HBA and HBD of DES on the phase formation ability of the mixture can be illustrated clearly. After the sugar-based DES-ATPS has been established, the target biomolecules like proteins, amino acids, DNA, organic acids, and more will partition preferentially toward either the top or bottom phase depending on their interactions with the DES. For example, Li and group reported that three types of proteins (i.e., bovine serum albumin, trypsin, and ovalbumin) were mainly partitioned to the top phase, consisting of either betaine-urea, betaine-methylurea, betaine-glucose, betaine-sorbitol, betaineglycerol, or betaine-ethylene glycol based DES. This is because the salt-rich bottom phase is highly hydrophilic and caused a hydrophobic interaction between the proteins and the DES. The difference in the degree of hydrophobicity/hydrophilicity in sugarbased DES-ATPS or DES-ATPS, in general, allows it to be used as a tool to separate, extract, recover, or even purify biomolecules produced from upstream processes (Li et al., 2016). Xu and colleagues pioneered the protein extraction method using DES-based ATPS, and the work is highly regarded within the field. In the study, four types of choline chloride based DESs, two of which were carbohydrate-based (glucose and sorbitol), were synthesized to extract bovine serum albumin with ATPS using potassium hydrogen phosphate (K2 HPO4 )as the phase separation salt (Xu et al., 2015). It was found that the phase forming ability of DES of choline chloride–glucose DES is higher than choline chloride–sorbitol DES. It is postulated that choline chloride–glucose DES is less hydrophilic, and thus less K2 HPO4 is required to achieve phase separation. On another note, choline chloride–sorbitol ATPS achieved the second-highest extraction efficiency, up to 74.07% among the DESs studied. The study also illuminated the effects of several parameters such as concentration of DES, concentration of salt solution, mass of protein, shaking time (initial mixing, not phase separation), temperature, and pH value. Some of these parameters will be covered in more detailed in the next section of this chapter. As conventional ATPS present limitations in terms of product purity, Marchel and the group synthesized carbohydrates based DES-ATPSs containing polypropylene glycol 425 and elucidated their potential in the purification of virus-like particles (VLPs) which are used mainly in creating vaccines, where product purity is of priority (e.g., bovine serum albumin is a major contaminant). Fructose, sucrose, and glucose are the three carbohydrates studied in the work (Marchel et al., 2020). It was observed that sucrose-based DES forms ATPS the easiest, followed by glucose-based DES and finally fructose-based DES. The study also highlighted the importance of the selectivity of the carbohydrate-based DES ATPS toward VLPs from bovine serum albumin. The

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results of this study and the implications will be covered in more detail in the next section of this chapter.

4.4

Effect of parameters

The extraction performance and efficiency of DESs for biomolecules are dependent on the physicochemical properties of the mixture, such as the degree of hydrogen bonding interactions, viscosity, pH, and polarity (Duan et al., 2016). The effect of mass fraction of DES, type of HBA and HBD, HBA to HBD mass/molar ratio, temperature, and type of phase forming agent on the extraction efficiencies of sugar-based DES-ATPSs will be discussed.

4.4.1 Mass fraction of deep eutectic solvent Jafari and Jouyban investigated the separation effectiveness of a sugar-based DESATPS containing polyethene glycol dimethyl ether 250 (PEGDME250), where choline chloride and sucrose were the HBA and HBD, respectively. The concentration of DES in the range of 25 weight% to 29 weight% was studied, and they have concluded that the partition coefficient and extraction efficiency of caffeine and three other anti-epileptic drugs (i.e., lamotrigine, clonazepam, and oxcarbazepine) increase when the ATPS is composed of more DES. This is because the increase in the amount of DES in the ATPS increases the difference of hydrophobicity between the PEGDME250 and DES phases, promoting more caffeine or antiepileptic drugs to partition to the PEGDME250 top phase (Jafari and Jouyban, 2021). Li and group studied betaine’s potential as choline chloride’s cheaper and natural alternative in DES-ATPS application. They experimented on six DESs, all using betaine as HBA, two of the DESs are sugar based, and each of them used glucose and sorbitol as HBD. They noted that the increase in the amount of DES would slightly increase the volume of DES at the top phase and decrease the volume of the bottom phase. It is postulated that water molecules move toward the top phase preferentially due to hydrogen bonding with DES (Li et al., 2016).

4.4.2 Type of hydrogen bond acceptor and hydrogen bond donor Wang and colleagues were the first to study the potential of eight types of DES-ATPS containing K2 HPO4 in separating and extracting chlorogenic acid, which has health benefits like anti-inflammatory, antitumor, antihypertensive, antioxidative, and many more, from blueberry leaves, which are an agricultural by-product. All eight types of DES had choline chloride as the HBA, while the HBD used for each type are ethylene glycol, glycerin, 1,3-butanediol, citric acid, oxalic acid, glucose, maltose, and sucrose. Preliminary studies showed that the three sugars-based DES-ATPSs underperformed in terms of extraction yields compared to the most promising DES-ATPS, which used 1,3-butanediol as the HBD. The glucose-based DES-ATPS was the best choice among

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the three sugar-based DES-ATPS, in which it provided an extraction yield of around 38 mg of chlorogenic acid per gram of dried blueberry leaves (Wang et al., 2017). Wang et al. (2020) reported the potential of five types of DES-ATPS containing K2 HPO4 in separating and purifying a protein found in cyanobacteria, C-phycocyanin. All five types of DES are comprised of choline chloride as the HBA, while the HBD used for each type are glucose, fructose, glycerol, urea, and ethylene glycol. Preliminary studies showed that the two sugars-based DES-ATPSs underperformed compared to the most promising DES-ATPS, which used urea as the HBD. The fructose-based ATPS has comparable recovery to the urea-based ATPS at around 67%. However, the purity of the extracted C-phycocyanin was only 0.825, the lowest among all 5 DES-ATPS, which is lower than the initial purity of crude C-phycocyanin of 1.275. Meanwhile, the glucose-based ATPS came in third in terms of recovery and purity of C-phycocyanin, recovery of C-phycocyanin was slightly lower than that of urea-based ATPS, but the purity of the extracted C-phycocyanin was significantly lower at around 2 when compared to that of urea-based ATPS which achieved the highest purity at 3.307 (Wang et al., 2020). Farius and coworkers investigated three sugars based DES-ATPS with different hydrophilicity to extract gallic acid. Sucrose is the most hydrophilic among the three, while fructose is the least hydrophilic. It was found that the sucrose-based DES-ATPS has the highest extraction efficiency for gallic acid at around 70%, 75%, and 85% when the gallic acid concentration was 250 mg/L, 500 mg/L, and 750 mg/L, respectively. Meanwhile, the extraction efficiency for gallic acid was around 55%, 65%, and 75% when the gallic acid concentration was 250 mg/L, 500 mg/L, and 750 mg/L, respectively, where fructose was used as the HBD (Farias et al., 2017). This suggests that gallic acid preferentially partitions to the more hydrophilic DES phase as stronger hydrogen bond interactions can be achieved. Similarly, Li and the group found that sugar-based DESs showed weaker phase forming ability than the other DESs studied, like betaine-methylurea, which had the best phase forming ability among the six DESs studied (Li et al., 2016). This is primarily attributed to the high hydrophilicity of the sugar compounds involved, which in turn require more phase-forming agent like K2 HPO4 to induce the DES into the two-phase region. These DESs were tested to extract three different proteins (i.e., bovine serum albumin, trypsin, and ovalbumin). Betaine-sorbitol and betaine-glucose DES-ATPSs came in last and fourth in terms of extraction efficiency for bovine serum albumin; came in last and third in terms of extraction efficiency for trypsin; came in fifth and last for ovalbumin. Betaine-urea DES-ATPS produced the best result in bovine serum albumin extraction with an extraction efficiency of 93.95%. At the same time, betaine-methylurea DES-ATPS demonstrated the best overall performance in all three protein extraction attempts (Li et al., 2016). Marchel and the group pioneered the research in the extraction and purification of VLPs using DES-ATPS. Three single sugar-based DES-ATPSs involving fructose, glucose, sucrose, and four combinations of sugars-based DES-ATPSs, fructose–glucose, fructose–sucrose, glucose–sucrose, and fructose–glucose–sucrose, were studied. A polymer phase forming agent was used, polypropylene glycol 425 (PPG 425). In terms of phase forming ability, sucrose-based DES was the best,

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followed by glucose–sucrose DES, fructose–sucrose DES, glucose–fructose–sucrose DES, glucose DES, fructose–glucose DES and lastly, fructose DES (Marchel et al., 2020). Like the reasonings mentioned previously, sucrose is the most hydrophilic out of all the sugars or combinations of sugars experimented with; therefore, it has the strongest phase forming ability. Most of the DES-ATPSs performed well in extracting VLPs. Notably, fructose–sucrose DES achieved the highest extraction efficiency for VLPs at 99.6%. In contrast, the extraction efficiency of glucose–sucrose DES, glucose–fructose–sucrose DES, and fructose–glucose DES are 93.1%, 93.2%, and 81.9%, respectively. Furthermore, bovine serum albumin, a major contaminant from the upstream production of VLP, was introduced to DES-ATPS to further study the purification potentials of the separation systems. It was found that fructose–sucrose DES has the lowest selectivity toward VLPs. This makes it a less attractive DES-ATPS than fructose–glucose DES, which has the highest selectivity of 46.5 (Marchel et al., 2020).

4.4.3 Hydrogen bond acceptor to hydrogen bond donor mass/molar ratio Jafari and Jouyban studied the HBA to HBD (choline chloride to sucrose) molar ratio of 1:1, 2:1, and 4:1, and it was observed that the increase of choline chloride improves phase separation efficiency. This implies that choline chloride is the salting-out agent in this mixture. From another perspective, sucrose is said to hinder or slow down the phase separation process. Additionally, it was observed that at a higher HBA to HBD molar ratio, the partition coefficient and extraction efficiency of caffeine and three other anti-epileptic drugs (i.e., lamotrigine, clonazepam, and oxcarbazepine) are increased. For example, the extraction efficiency for clonazepam saw an increase from 92.09% to 95.14% when HBA to HBD molar ratio is increased from 1:1 to 4:1 (Jafari and Jouyban, 2021). Therefore, the higher the amount of choline chloride in the DES, the more caffeine or antiepileptic drugs will move to the hydrophobic PEGDME250 top phase. Farias and coworkers investigated three sugars based DES-ATPS involving glucose, fructose, and sucrose while containing choline chloride as HBA and K2 HPO4 as phase forming salt. Each type of DES-ATPS is further breakdown into three HBA to HBD mass ratio (i.e., 2:1, 1:1, and 1:2). It was observed that a higher amount of HBA promotes phase formation of all DES studied. The amount of phase forming salt quantifies this; the smaller the amount of K2 HPO4 required for the same amount of DES to go into the biphasic region, the easier it is to form ATPS (Farias et al., 2017).

4.4.4 Temperature Jafari and Jouyban investigated the phase separation characteristics of sugar-based DES-ATPS at three different temperatures (i.e., 25°C, 35°C, and 45°C). Higher temperatures were observed to improve two-phase formation efficiency. It is postulated that the hydrophobic interactions between the hydrocarbon chains in PEGDME250 are increased at elevated temperatures. At the same time, the hydrogen bonding between

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water molecules and ether groups of PEGDME250 are diminished. This increase in hydrophilicity between choline chloride and PEGDME250 leads to easier formation of ATPS. Similarly, the hydrophobic interactions between PEGDME250 and caffeine or antiepileptic drug molecules are enhanced at higher temperature. Therefore, they observed an increase in the extraction efficiency for clonazepam from 92.09% to 96.84%, for caffeine from 63.33% to 68.64% when the temperature is increased from 25°C to 45°C (Jafari and Jouyban, 2021). Marchel and the group presented their findings on the effect of temperature on phase formation efficiency for seven single, binary, and ternary sugar-based DESATPSs at two temperatures, 25°C and 37°C. They discovered that at 37°C, lower amounts of PPG 425 and sugar-based DES are needed for ATPS formation. Thus, DESATPSs are highly sensitive to temperature and have the potential for back extraction by manipulating the temperature (Marchel et al., 2020).

4.4.5 Type of phase forming component Li and the group studied three-phase forming salts, disodium phosphate (Na2 HPO4 ), potassium hydrogen phosphate (K2 HPO4 ), and potassium dihydrogen phosphate (KH2 PO4 ) were used to induce DES-ATPS formation. It was discovered that only K2 HPO4 induced two-phase separation in all six DESs studied, two of which are sugarbased DESs (Li et al., 2016). K2 HPO4 is known to have high solubility; therefore, it possesses strong phase forming ability. Zhang, Teng, and Zhang utilized three-phase forming salts, ammonium sulfate [(NH4 )2 SO4 ], potassium hydrogen phosphate (K2 HPO4 ), and potassium dihydrogen phosphate (KH2 PO4 ), in inducing four different choline chloride-based DES to form an ATPS. It was discovered that only K2 HPO4 induced two-phase separation in the DESs studied, one of which is glucose-based DESs (Zhang et al., 2019). This outcome is similar to Li and coworkers’ findings, although different types of DESs were studied (Li et al., 2016). This also explains why K2 HPO4 is the commonly used phase-forming salt in DES-ATPSs.

4.5 Application of sugar-based deep eutectic solvent-aqueous two-phase system This section summarizes the potential applications of sugar-based DES-ATPS demonstrated in the recent research activities. Biomolecule extraction applications in general and exciting potential applications in the medical and pharmaceutical fields are included here. Farias and the group attempted to analyze the properties and behavior of three sugarbased (i.e., fructose, glucose, and sucrose) DES-ATPS. At the same time, their potential to extract gallic acid, a phenolic compound derived from plants with anticancer properties was studied (Farias et al., 2017; Locatelli et al., 2013). The results from this research elucidated that sucrose based DES-ATPS has the potential to be used in gallic acid extraction, these authors also hinted that the sugar based DES-ATPS studied

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could very well be used to separate other biomolecules. Since only one target molecule is experimented with, researchers may study different HBA and HBD, HBA:HBD mass/molar ratios, and also the extraction efficiency for other types of biomolecules like proteins, enzymes, amino acids, cells, DNA, RNA, and many more. Jafari and Jouyban synthesized a biodegradable choline chloride–sucrose based DES-ATPS containing PEGDME250 to extract caffeine and antiepileptic drugs such as lamotrigine, clonazepam, and oxcarbazepine. The encouraging results from this research meant that the DES-ATPS has the potential to be used in therapeutic drug monitoring of patient’s blood and urine samples, providing a single step, nontoxic, and environmentally friendly alternative to conventional testing methods (Jafari and Jouyban, 2021). Researchers may continue this work to further enhance the application scope and optimize the separation process by experimenting with different HBA and HBD, HBA:HBD mass/molar ratios, and also other types of drugs that require therapeutic monitoring, such as carbamazepine, valproate, digoxin, etc. Currently, no detailed economic comparisons between the application of DES-ATPS and conventional methods in therapeutic drug monitoring is available. An economic feasibility study would be the next step to promote the adoption of this new technology in the medical field. Marchel and coworkers proposed seven single and combination of sugar (i.e., glucose, sucrose, and fructose) based DES-ATPSs containing PPG 425 to extract and purify VLPs, which could be used in producing vaccines for certain diseases. These DES-ATPSs are potential alternatives to existing recovery methods such as ultrafiltration, chromatography, and ultracentrifugation, which can be more expensive and less efficient in terms of recovery yield. The research outcome was promising; it demonstrated that fructose–glucose-based DES-ATPS could extract VLPs efficiently while purifying the VLPs by removing bovine serum albumin due to its high selectivity toward VLPs (Marchel et al., 2020). This means that certain type of DES-ATPS can be and is suited to extract and purify VLPs for pharmaceutical applications due to its scalable, cost-effective, stable, green, and sustainable properties. Most importantly, the DES-ATPSs studied were tested at concentrations of 0 mM to 50 mM on human kidney and liver cells to ensure these substances are biocompatible and safe for the human body. Some of the DESs tested are slightly more toxic than the others, especially for human liver cells. However, the researchers concluded that the sugar-based DESs are generally compatible with biotechnological applications. Similarly, researchers may explore other types of HBA:HBD, HBA:HBD mass/molar ratios, ways to minimize the toxicity of DES toward human cells, and attempt to scale up the separation process in future works to convince the industry that this is a viable and profitable separation method. Currently, the application of sugar-based DES-ATPS in downstream bioprocessing in the industry is not prevalent. One of the main reasons could be that there is not enough evidence that sugar-based DES-ATPSs are superior to the currently employed separation method. Additionally, there have not been many attempts to scaling up the extraction process involving sugar-based DES-ATPS, making it difficult to assess its effectiveness, process efficiency, stability, and consistency at a larger scale. Also, it would be a substantial financial commitment for companies to fund research for

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the potential application of sugar-based DES-ATPS for their processes. It also takes time to optimize the separation process. Therefore, even if some companies are using the technology for their downstream bioprocessing purposes, they would not publicly publish their formula (e.g., type of HBA and HBD, HBA to HBD mass/molar ratio, temperature, pressure, mass fraction of DES, mixing and separation duration, and many more) unless a patent is filed. Overall, the latest research results have proven that sugar-based DES-ATPS could be a simple, economical, efficient, safe, nontoxic, environmentally friendly, and scalable solutions for downstream bioprocesses. For now, the bioprocessing industry is not under direct pressure from the public and stakeholders to embrace green technology. Thus, governments and policymakers must provide incentives for companies to promote the wide adoption of this novel separation method.

4.6 Advancement of sugar-based deep eutectic solvent-aqueous two-phase system over the last 5 years This section of the chapter illustrates how sugar based DES-ATPS has evolved by providing several exciting and innovative examples such as ultrasonically assisted extraction (UAE), microwave assisted extraction (MAE), and continuous separation process using DES-ATPS. Rathnasamy and group proposed a novel idea of combining MAE with sugar-based DES-ATPS containing phase forming salt to recover phycocyanin while reducing the amount of solvent needed. Interestingly, glycerol is used as the HBD, and five sugars (i.e., glucose, fructose, sucrose, maltose, and xylose) are used as the HBA in the DES. Xylose-glycerol DES was deemed to have the best phase forming ability and was solely studied in the later stages of the research. The optimum microwave temperature and extraction time for the highest phycocyanin extraction yield at 86% is 75°C and 5 min, respectively (Rathnasamy et al., 2019). Zhang and group combined UAE and several types of choline chloride based DESATPSs containing phase-forming salt, one of which is glucose-based, to extract ursolic acid, which has antitumor, antibacterial, and antioxidant properties, from natural sources. Existing methods of ursolic acid extraction require longer extraction time, vast amounts of solvents, and are energy-intensive. The glucose-based DES-ATPS has the second-highest phase forming efficiency. At the same time, it has the highest extraction efficiency and partition coefficient for ursolic acid at around 82% and 8%, respectively. After some preliminary study, the optimized mass fraction of glucosebased DES-ATPS and K2 HPO4 were 36% and 25%, respectively (Zhang et al., 2019). Generally, the yield of ursolic acid can be increased by increasing the ultrasound power density. However, there is an upper limit as the ursolic acid can be damaged due to heat at extreme power density. Therefore, the optimum ultrasound power density range is 3–5 W/mL. Besides that, this UAE-DES-ATPS method was compared to the conventional extraction method, which is UAE-ethanol. The latter yielded almost two times more extract. However, the effective yield of ursolic acid is slightly lower, and the purity of ursolic acid is two times lower than that obtained from the UAE-DES-ATPS

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method. With these results, UAE-glucose-based DES-ATPS has been proven to be a promising separation method for ursolic acid while providing all the benefits of sugarbased DES. This study is a prime example that sugar-based DES-ATPS has the potential to be combined with other technology to improve extraction efficiencies, extraction time, and purity of target molecules (Zhang et al., 2019). Based on the positive results from their previous work, Miliˇcevi´c and coworkers decided to study four choline chloride based DES-ATPSs containing K2 HPO4 in polyphenols extraction in a continuous microextractor where one of the studied DES-ATPS is glucose-based. Gallic acid was chosen to represent polyphenols in this study. During the preliminary tests, choline chloride–ethylene glycol DES-ATPS presented the highest extraction yield of 97.26%, followed by choline chloride–glucose DES-ATPS (Miliˇcevi´c et al., 2020). Unfortunately, the authors only performed further analysis on the choline chloride–ethylene glycol DES-ATPS in the continuous microextractor. Nevertheless, the experimental data and the results obtained from the proposed 2D mathematical model adapted from Žnidaršiˇc-Plazl and Plazl are in agreement (Žnidaršiˇc-Plazl and Plazl, 2009). This achievement means that the proposed model can be used to describe the continuous extraction process with high accuracy. Additionally, the extraction efficiency of the continuous setup improved by 6% and the residence time was reduced by at least 14 min compared to that of the batch process (Miliˇcevi´c et al., 2020). This sets a precedent for operating DES-ATPS continuously to extract a target molecule which will encourage other researchers to study the reproducibility of this study for sugar DES-ATPSs and other biomolecules in the hopes that sugar DES-ATPSs can be used to separate, recover, extract, and purify biomolecules at a scale that is comparable to that of the downstream bioprocessing industry. Currently, the sugar-based DES-ATPS technology is still in its infancy stage after its discovery half a decade ago. Various researchers have taken strong strides from all over the world to promote, advance, and bring attention to the potential applications of sugar-based DES-ATPSs in the downstream bioprocessing field. However, time and financial investment are required to reach its fullest potential and achieve industrial maturity.

4.7

Recycling of sugar-based deep eutectic solvent

There is no doubt that DES-ATPS is a greener and more environmentally friendly alternative for traditional organic solvent in bioseparation processes. However, it is still crucial to minimize waste which can be achieved by recycling used solvents for secondtime extraction and so forth. This will not only lower operating cost and improve process efficiency but also minimize the impact that DES has on the environment. In this section, some studies relating to the recycling of DES will be presented and discussed. Additionally, insights on the recycling process and the extraction efficiency of recycled DES will be provided. Jeong and coworkers were the first to report the recycling of DES used to extract natural products. The group proposed to use DES-ATPS to extract ginsenosides, a bioactive compound in ginseng, a popular herbal medicine. A wide variety of DESATPSs was experimented with, there were five citric acids based DES-ATPSs, six

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choline chloride based DES-ATPS, and seven glycerol-based DES-ATPSs, some of which involves sugars such as glucose, galactose, fructose, and sucrose (Jeong et al., 2015). From preliminary testing results, citric acid based DES-ATPSs are not suitable for the extraction of ginsenosides due to their acidic nature, while glycerol and choline chloride based DESs-ATPSs are more suitable in this application, especially for glycerol–sucrose DES-ATPSs as evident by its relatively higher extraction efficiency for ginsenosides. Based on that, the authors further synthesized three sucrose-based DESs-ATPSs with different molar ratios to determine the best-optimized combinations of HBA and HBD for ginsenosides extraction. This led to the conclusion that glycerolsucrose DES-ATPS is superior to betaine-sucrose and choline chloride–sucrose DES-ATPSs (Jeong et al., 2015). Nevertheless, proline–sucrose DES-ATPSs were shown to have the highest extraction efficiency, at least 10% more efficient than the other tested DES-ATPSs, in the second screening. To further tailor the DES, the author synthesized three more ternary glycerol–sucrose DES-ATPSs with proline, betaine, and choline chloride. The extraction efficiency of the ternary DES-ATPSs was found to be higher than that of their binary counterparts. For instance, glycerol–proline–sucrose DES-ATPS saw at least a 10% improvement in terms of extraction yield when compared to proline–sucrose DES-ATPS (Jeong et al., 2015). Following the recovery of ginsenosides, the group attempted to recycle the glycerol–proline–sucrose DES-ATPS by addition of water to dilute the DES then it was freeze-dried until a constant weight was achieved to ensure removal of excess water, resulting in a viscous liquid which was reused to extract ginsenosides under the optimized conditions, this was repeated for another two rounds. It was found that the extraction efficiency of the recycled DES decreased from 91.9% to 82.6% after being reused for the third time (Jeong et al., 2015). To summarize, the glycerol–proline–sucrose DES-ATPS was the best combination for ginsenosides extraction. This also means that DES can be tailored and customized according to the requirements of the process and the target molecule. Also, the glycerol–proline– sucrose DES-ATPS can be recycled through the freeze-drying method at least three times.

4.8 Conclusions With attributes like cost-effectiveness, ease to synthesize, recyclable, high extraction efficiency, and nontoxic, sugar-based DES-ATPS has demonstrated immense potential for downstream bioprocessing applications. The standard synthesis methods and working principle of sugar-based DES-ATPS were presented to explained for general knowledge. Five parameters affecting the phase forming ability and extraction efficiency of DES-ATPS were analyzed and discussed comprehensively to illuminate their influence in downstream bioprocessing applications. Several recent applications and advancement in sugar DES-ATPS technology have been discussed. Future work and improvements have also been suggested to promote research in this area. Furthermore, the recyclability of sugar-based DES and its process were presented to enforce its environmental friendly claims. The tuneability of DES to better suit a specific application was also highlighted in this chapter. Overall, sugar-based DES-ATPS has

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the potential to be adopted in the industry for bioseparation processes. However, it still requires much work to prove its feasibility at a larger scale.

References Abbott, A.P., Barron, J.C., Ryder, K.S., Wilson, D., 2007. Eutectic-based ionic liquids with metal-containing anions and cations. Chemistry 13, 6495–6501. Abbott, A.P., Capper, G., Davies, D.L., Rasheed, R.K., Tambyrajah, V., 2003. Novel solvent properties of choline chloride/urea mixtures. Chem. Commun. 1, 70–71. Abranches, D.O., Martins, M.A., Silva, L.P., Schaeffer, N., Pinho, S.P., Coutinho, J.A., 2019. Phenolic hydrogen bond donors in the formation of non-ionic deep eutectic solvents: the quest for type V DES. Chem. Commun. 55, 10253–10256. Albertsson, P.-Å., 1958. Partition of proteins in liquid polymer–polymer two-phase systems. Nature 182, 709–711. Benvenutti, L., Zielinski, A.A.F., Ferreira, S.R.S., 2019. Which is the best food emerging solvent: IL, DES or NADES? Trends Food Sci. Technol. 90, 133–146. Berthod, A., Ruiz-Angel, M., Carda-Broch, S., 2008. Ionic liquids in separation techniques. J. Chromatogr. A 1184, 6–18. Chiappe, C., Pieraccini, D., 2005. Ionic liquids: solvent properties and organic reactivity. J. Phys. Org. Chem. 18, 275–297. Craveiro, R., Aroso, I., Flammia, V., Carvalho, T., Viciosa, M., Dionísio, M., Barreiros, S., Reis, R., Duarte, A.R.C., Paiva, A., 2016. Properties and thermal behavior of natural deep eutectic solvents. J. Mol. Liq. 215, 534–540. Dai, Y., van Spronsen, J., Witkamp, G.-J., Verpoorte, R., Choi, Y.H., 2013. Natural deep eutectic solvents as new potential media for green technology. Anal. Chim. Acta 766, 61–68. Deye, J.F., Berger, T.A., Anderson, A.G., 1990. Nile Red as a solvatochromic dye for measuring solvent strength in normal liquids and mixtures of normal liquids with supercritical and near critical fluids. Anal. Chem. 62, 615–622. Duan, L., Dou, L.-L., Guo, L., Li, P., Liu, E.-H., 2016. Comprehensive evaluation of deep eutectic solvents in extraction of bioactive natural products. ACS Sustain. Chem. Eng. 4, 2405–2411. Farias, F.O., Sosa, F.H.B., Igarashi-Mafra, L., Coutinho, J.A.P., Mafra, M.R., 2017. Study of the pseudo-ternary aqueous two-phase systems of deep eutectic solvent (choline chloride: sugars)+ K2HPO4+ water. Fluid Phase Equilib. 448, 143–151. Florindo, C., Branco, L.C., Marrucho, I.M., 2019. Quest for green-solvent design: from hydrophilic to hydrophobic (deep) eutectic solvents. ChemSusChem 12, 1549–1559. Gouveia, W., Jorge, T., Martins, S., Meireles, M., Carolino, M., Cruz, C., Almeida, T., Araújo, M., 2014. Toxicity of ionic liquids prepared from biomaterials. Chemosphere 104, 51–56. Halder, A.K., Cordeiro, M.N.D., 2019. Probing the environmental toxicity of deep eutectic solvents and their components: an in silico modeling approach. ACS Sustain. Chem. Eng. 7, 10649–10660. Hansen, B.B., Spittle, S., Chen, B., Poe, D., Zhang, Y., Klein, J.M., Horton, A., Adhikari, L., Zelovich, T., Doherty, B.W., 2020. Deep eutectic solvents: a review of fundamentals and applications. Chem. Rev. 121 (3), 1232–1285. Hayyan, A., Mjalli, F.S., AlNashef, I.M., Al-Wahaibi, T., Al-Wahaibi, Y.M., Hashim, M.A., 2012. Fruit sugar-based deep eutectic solvents and their physical properties. Thermochim. Acta 541, 70–75.

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Iqbal, M., Tao, Y., Xie, S., Zhu, Y., Chen, D., Wang, X., Huang, L., Peng, D., Sattar, A., Shabbir, M.A.B., 2016. Aqueous two-phase system (ATPS): an overview and advances in its applications. Biol. Proced. Online 18, 1–18. Jafari, P., Jouyban, A., 2021. Partitioning behavior of caffeine, lamotrigine, clonazepam and oxcarbazepine in a biodegradable aqueous two-phase system comprising of polyethylene glycol dimethyl ether 250 and choline chloride/saccharose deep eutectic solvent. J. Mol. Liq. 323, 115055. Jeong, K.M., Lee, M.S., Nam, M.W., Zhao, J., Jin, Y., Lee, D.-K., Kwon, S.W., Jeong, J.H., Lee, J., 2015. Tailoring and recycling of deep eutectic solvents as sustainable and efficient extraction media. J. Chromatogr. A 1424, 10–17. Jiang, B., Li, Z.-G., Dai, J.-Y., Zhang, D.-J., Xiu, Z.-L., 2009. Aqueous two-phase extraction of 2, 3-butanediol from fermentation broths using an ethanol/phosphate system. Process Biochem. 44, 112–117. Juneidi, I., Hayyan, M., Ali, O.M., 2016. Toxicity profile of choline chloride-based deep eutectic solvents for fungi and Cyprinus carpio fish. Environ. Sci. Pollution Res. 23, 7648–7659. Khandelwal, S., Tailor, Y.K., Kumar, M., 2016. Deep eutectic solvents (DESs) as eco-friendly and sustainable solvent/catalyst systems in organic transformations. J. Mol. Liq. 215, 345–386. Li, N., Wang, Y., Xu, K., Huang, Y., Wen, Q., Ding, X., 2016. Development of green betainebased deep eutectic solvent aqueous two-phase system for the extraction of protein. Talanta 152, 23–32. Li, Z.-L., Zhong, F.-Y., Huang, J.-Y., Peng, H.-L., Huang, K., 2020. Sugar-based natural deep eutectic solvents as potential absorbents for NH3 capture at elevated temperatures and reduced pressures. J. Mol. Liq. 317, 113992. Locatelli, C., Filippin-Monteiro, F.B., Creczynski-Pasa, T.B., 2013. Alkyl esters of gallic acid as anticancer agents: a review. Eur. J. Med. Chem. 60, 233–239. Marchel, M., Niewisiewicz, J., Coroadinha, A.S., Marrucho, I.M., 2020. Purification of viruslike particles using aqueous biphasic systems composed of natural deep eutectic solvents. Sep. Purif. Technol. 252, 117480. Miliˇcevi´c, N., Pani´c, M., Valinger, D., Bubalo, M.C., Benkovi´c, M., Jurina, T., Kljusuri´c, J.G., Redovnikovi´c, I.R., Tušek, A.J., 2020. Development of continuously operated aqueous twophase microextraction process using natural deep eutectic solvents. Sep. Purif. Technol. 244, 116746. Radoševi´c, K., Bubalo, M.C., Srˇcek, V.G., Grgas, D., Dragiˇcevi´c, T.L., Redovnikovi´c, I.R., 2015. Evaluation of toxicity and biodegradability of choline chloride based deep eutectic solvents. Ecotoxicol. Environ. Saf. 112, 46–53. Rathnasamy, S.K., sri Rajendran, D., Balaraman, H.B., Viswanathan, G., 2019. Functional deep eutectic solvent-based chaotic extraction of phycobiliprotein using microwave-assisted liquid-liquid micro-extraction from Spirulina (Arthrospira platensis) and its biological activity determination. Algal Res. 44, 101709. Silva, L.P., Fernandez, L., Conceição, J.H., Martins, M.A., Sosa, A., Ortega, J., Pinho, S.P., Coutinho, J.A., 2018. Design and characterization of sugar-based deep eutectic solvents using conductor-like screening model for real solvents. ACS Sustain. Chem. Eng. 6, 10724– 10734. van Berlo, M., Luyben, K.C.A., van der Wielen, L.A., 1998. Poly (ethylene glycol)–salt aqueous two-phase systems with easily recyclable volatile salts. J. Chromatography B: Biomed. Sci. Appl. 711, 61–68. Wang, Q., Wei, N., Wang, Y., Hou, Y., Ren, X., Wei, Q., 2020. Single-step purification of C-phycocyanin from Arthrospira platensis using aqueous two-phase system based on natural deep eutectic solvents. J. Appl. Phycol. 32, 3873–3883.

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Ionic liquid–salt interaction Wang Sze Kuan a, Malcom S.Y. Tang b, Wen Yi Chia a and Kit Wayne Chew c a Department of Chemical and Environmental Engineering, Faculty of Science and Engineering, University of Nottingham Malaysia, Semenyih, Selangor Darul Ehsan, Malaysia, b Institute of Biological Sciences, Faculty of Science, University of Malaya, Kuala Lumpur, Malaysia, c School of Energy and Chemical Engineering, Xiamen University Malaysia, Sepang, Selangor Darul Ehsan, Malaysia

5.1

5

Introduction

Aqueous two-phase system (ATPS) is an example of liquid–liquid extraction (LLE) process for the separation of biological products. It is a biphasic system where two water-soluble components such as polymer, surfactant, or salts are mixed (Pereira et al., 2020). Upon mixing, the components separate into two phases with one phase being richer in one of the two components. ATPS can be used to achieve selective extraction, eliminate impurities, and most importantly concentrate dilute solutions (Rogers and Voth, 2007). ATPS is biocompatible with most biomolecules, simple, cost effective, consumes low energy, and easy to scale-up compared to other types of LLE. ATPS has been extensively used in the extraction and purification of biomolecules, such as protein, antibiotic, and enzyme.

5.1.1 Types of ATPS There are numerous types of ATPS. In the section below, we will discuss several types of commonly used ATPS.

5.1.1.1

Polymer/polymer ATPS

When a pair of water-soluble polymers are mixed at a critical concentration, the polymer–polymer ATPS is formed (Pereira et al., 2020). Thermodynamically, polymer/polymer ATPS phase separation can be attributed to two plausible theories: 1. The first point of view is the Flory–Huggins theory. It is based on the energetically unfavorable segment interactions of polymers overcoming the entropy increase. 2. The second theory is based on the water structure. The ordered polymeric water structures is the main element involved in two phases separation (Pereira et al., 2020).

5.1.1.2

Polymer/salt ATPS

Polymer/salt ATPS can be produced by mixing a water-soluble polymer and an inorganic or organic salt above a certain critical concentration, creating a polymer-rich Principles of Multiple-Liquid Separation Systems: Interaction, Application and Advancement. DOI: https://doi.org/10.1016/B978-0-323-91728-5.00013-5 c 2023 Elsevier Inc. All rights reserved. Copyright 

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IL salt

Phase separation Analyte

Figure 5.1 Extraction of biological components using ATPS technique. Adapted from Escudero et al. (2013). Bioanalytical separation and preconcentration using ionic liquids. Analytical Bioanalytical Chem., 405(24), 7597–7613. https://doi.org/10.1007/s00216013-6950-x.Ionicliquid/saltATPS.

phase at the top and a salt-rich phase at the bottom (Pereira et al., 2020). ATPS can be formed by mixing polymers such as polyalkylene glycols (UCON) or glycol dimethyl ether (PEGDME) with nitrate or carbonate salts. Organic salts like acetate, formate, and citrate salts are used as they are environmental friendly (Pereira et al., 2020). The most common used polymer phase agent is PEG polymer while sulfate-based salts are used as ionic compound.

5.1.1.3

Ionic liquid/salt ATPS

Ionic liquid (IL) is defined as liquids that consists of ions that are fluid around or below 100°C (Rogers and Voth, 2007). IL has distinctive characteristics like low volatility, high thermal and chemical stability, adjustable chemical and physical properties. The IL/salt ATPS is formed by mixing high charge density salt ion with low charge density salt ion. The most commonly used IL for producing IL/salt ATPS is imidazolium, mixed with high charge density inorganic salts such as sulfate and phosphate salts as the second component (Rogers and Voth, 2007). Since both components in an IL/salt ATPS are ionic, the distribution of the constituent ions between the phases is affected by the electroneutrality of the overall system and individual phase (Pereira et al., 2020). Fig. 5.1 shows the extraction of biological components using ATPS technique. The effectiveness of different salts in forming the IL/salt ATPS obeys the Hofmeister series where salt ions are sorted based on the salting-out ability (Pereira et al., 2020). Hofmeister series can be described by simple hydration phenomenon (Freire et al., 2012). In this phenomenon, the order of bulk water is related with the ability of the ions to decrease or increase the structure of water. The mechanism of salting-out ability of salt ions is driven by entropic process (Freire et al., 2012). This results in

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83

100 Binodal curve

Component 1 / wt%

80 T (T1, T2) Biphasic region

60

a b

40

Tie-lines CP c

20

B (B1, B2) Monophasic region

0 0

20

40 60 Component 2 / wt%

80

100

Figure 5.2 Scheme of an orthogonal ternary phase diagram composed of two components and water. Adapted from Pereira et al. (2020). Aqueous two-phase systems. In Liquid-phase extraction (pp. 157–182). Elsevier. Table 5.1 Hofmeister series. Anions: CO3 2− > SO4 2− > S2 O3 2− > H2 PO4 − > F− > Cl– > Br– > NO3 − > I– > CIO4 − > SCN– Cations: (CH3 )4 N+ > Cs+ > Rb+ > NH4 + > K+ > Na+ > Li+ > Mg2+ > Ca2+

solute dehydration and increase in the surface tension of cavity (water-ion complexes formation) (Freire et al., 2012). Fig. 5.2 shows the combination of IL/Salt ATPS. The empirical order of salts needed for precipitation is shown in Table 5.1.

5.2

Fundamentals of ionic liquid–salt: thermodynamic and properties

ATPS can only generate a biphasic regime when the concentrations of the constituent solutions fall within a certain region. The dynamic of the solution concentration may be represented by a phase diagram. The phase diagram could provide information about the concentration of phase-forming components required to form an ATPS. Fig. 5.3 shows a ternary phase diagram of two-phase forming agents (components 1 and 2) and water (Pereira et al., 2020). The thickest line—(the binodal curve)— shows the separation between the two-phase region, (above the curve) and single-phase region, (below the curve) (Pereira et al., 2020). The lines that connect the dots on the binodal curve is the tie line (TL). The TL actually represents the composition of the phases at equilibrium (Pereira et al., 2020). Lastly, when the concentration of one of the constituents reaches a maximum, combined with a minimum concentration of the other component, the ATPS will become a monophasic solution, as shown by the monophasic region label in Fig. 5.3.

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0.5

0.4 Tie lines

w1

0.3 Two-phase region 0.2 Binodal curve 0.1 One-phase region 0.0 0.00

0.05

0.10

0.15

0.20 w2

0.25

0.30

0.35

Figure 5.3 Graph of TLL against IL concentration difference. From Li et al. (2010). Ionic liquid-based aqueous two-phase systems and their applications in green separation processes. BioCop – Monitoring Chemical Contaminants in Foods, 29(11), 1336–1346. https://doi.org/10.1016/j.trac.2010.07.014.

Merchuk discovered a three-parameter equation to fit the binodal curve using empirical equations, as shown in Eq. (5.1).   Y = A exp B X 0.5 − C X 3

(5.1)

where A, B, and C are adjusted parameters. The TL can be determined by using the gravimetric method where the compositions of top and bottom phases are determined mathematically:   YT = A exp B X 0.5 − CXT 3   YB = A exp B X 0.5 − C XB 3 1−α YM − YB α α 1−α XM − XB = α α

(5.2) (5.3)

YT =

(5.4)

XT

(5.5)

Where YT = weight fraction of component 1 in top phase. XT = weight fraction of component 2 in top phase. YB = weight fraction of component 1 in the bottom phase.

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85

XB = weight fraction of component 2 in the bottom phase. YM = weight fraction of component 1 in both phases. XM = weight fraction of component 2 in both phases. α = mass ratio at top phase to total mass of mixture.

5.3

Determination of solution concentration in both phases

The partition coefficient, K, can be calculated as the ratio of solution distribution between two phases. K=

CT CB

(5.6)

where CT and CB are equilibrium concentrations of the solution at top and bottom phase. Phase volume ratio, R is calculated as the volume ratio between two phases. R=

VT VB

(5.7)

where VT and VB are the volume of IL-rich top phase and salt-rich bottom phase. Extraction efficiency, E, can be calculated using the following equation: E=

CT VT KR = CT VT + CBVB 1 + KR

(5.8)

Change of entropy, TS◦ T and change of enthalpy, H◦ T is calculated as below: ln K =

−H0T ST0 + RT R

(5.9)

where R and T are universal gas constant and temperature of the system.

5.4

Factors that influence the two-phase separation in ionic liquid/salt ATPS

There are various factors that affect the phase equilibria of the IL-ATPS. In the section below, we will discuss the factors.

5.4.1 Effect of type of inorganic salt Inorganic salt is one of the main components in the formation of IL/salt ATPS. Kosmotropic ions such as CO3 2− , OH– , SO4 2− , and HPO4 2− are advantageous in

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forming two-phase system (Li et al., 2010). This is because these anions exhibit a stronger interaction with hydrogen bond present in the water molecule than those between water molecules. Due to the nature of water structure, when water molecules move closer to the kosmotropic ions, they will form an electro-constriction state which eventually results in improving the separation process. Conversely, the interaction between chaotropic ion like CI– , K+ , H2 PO4 − , and NH4 + ions and water molecules are weaker. Thus, they are not able to form into two phases. With regards to the mechanisms of IL/salt ATPS, the phase separation efficiency can be affected by the Gibbs’ free energy of hydration (Ghyd ), whereby kosmotropic ions with large and very negative Ghyd is preferentially solvated (Pereira et al., 2020). The low Ghyd salts that have the capability to salt out are ranked according to the Hofmeister series.

5.4.2 Effect of inorganic salt concentration Inorganic salt is one of the main components for ILATPS. Kosmotropic ions such as HPO4 2− , SO4 2− , OH– , CO3 2− , and PO4 3− can contribute positively to the formation of ATPS due to the strong interactions between these anions and water molecules. On the other hand, chaotropic ions such as CI– , NH4 + , K+ , and H2 PO4 − show weaker interaction with water. This makes them less ideal to be used as components to form ATPSs with ILs. By increasing the concentration of inorganic salt, the concentration of IL at the top phase increases. This is because increasing the salt concentration results in enhancing kosmotropic ions in the bottom phase. This will cause more chaotropic ions to move to the top phase (Li et al., 2010). This can be proved by plotting TLL against the concentrations difference of IL between the top and bottom phase, [IL] (Li et al., 2010). Fig. 5.3 shows the relationship of TLL and the concentrations difference of IL.

5.4.3 Effect of temperature Temperature is another factor that influences the solution partitioning in ATPS. Temperature can change the shape of the binodal curve (Dreyer et al., 2009). When the temperature increase, the binodal curves of the IL/salt and polymer/polymer move further away from the origin. This indicates that the phase-forming agent concentration is high which results in the reduction of biphasic region (Pereira et al., 2020). The shifting temperature phenomenon known as upper critical solution temperature (UCST) (Pereira et al., 2020). However, the binodal curve for polymer/salt moves to lower concentration when the temperature increase. This result in increasing the biphasic region hence, more polymer or salt is needed to favor the phase separation process (Pereira et al., 2020). During extraction of protein, the range of temperature that can be used is between 25 and 50°C. Temperature above 50°C will result in protein molecules to denature and bring a change in the structure of the protein as protein is highly sensitive to temperature (Dreyer et al., 2009). The calculation of G◦ T , TS◦ T , and H◦ T is important to ensure the extraction is a spontaneous process and determine which is the driving force during the reaction (Li et al., 2010). Table 5.2 shows the temperature effect on different types of ATPS.

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87

Table 5.2 Effect of temperature on different types of ATPS. Increase of the temperature Biphasic region (binodal curve) Type of critical solution temperature behavior

Temperature effect Polymer-polymer Polymer–salt ATPS ATPS Decrease (shift to Increase (shift to higher polymer smaller polymer-salt concentrations) concentrations) UCST LCST

Salt–salt ATPS Decrease (shift to higher salt concentrations) UCST

From Pereira et al. (2020). Aqueous two-phase systems: towards novel and more disruptive applications. Fluid Phase Equilibria, 505, 112341. https://doi.org/10.1016/j.fluid.2019.112341.

OH

OH Cl

O2N

HN Cl O

Figure 5.4 Chemical structure of chloramphenicol.

5.5

Applications of Ionic liquid/salt ATPS

IL-based ATPS are promising candidates for the separation of small organic biomolecules and biochemicals because of their unique properties. In the section below, we will discuss several types of applications using IL/salt ATPS.

5.5.1 Separation and concentration of chloramphenicol using Ionic liquid/salt two-phase flotation system (IL-ATPF) Chloramphenicol (CAP) is a type of effective antibiotics which functions to halt bacteria growth. It works against a range of Gram-positive and Gram-negative bacteria in animal and human (Bregni, 2021). However, this antibiotic is only effective against bacterial eye infections. Besides, typhoid fever and plague can also be treated using CAP through vein injection (2021). In EU countries, CAP is not allowed to be used in food products due to its high toxicity to human (Han et al., 2011). Fig. 5.4 shows the chemical structure of chloramphenicol which contains a nitrobenzene ring, two alcohol functions, as well as an amide bond. Han et al. published a work that aimed to investigate the factors that influence the separation of CAP using IL-based ATPS (Han et al., 2011). ILs that were used in the experiment were [C4 mim]BF4 , [C4 mim]Cl, and [C8 mim]Cl while the inorganic salts used were K3 PO4 , K2 CO3 , K2 HPO4 , and KOH (Han et al., 2011). The factors that were taken into consideration were the type of IL and salts, K2PO4 concentration, pH, and amount of [C4 mim]Cl.

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Based on the results obtained, [C4 mim]BF4 is not a good choice as it has high density and viscosity which could weaken the mass transfer of CAP to the water-IL interface. During the experiment involving [C8 mim]Cl, bubbles formed at the top phase which were difficult to disintegrate (Han et al., 2011). [C4 mim]Cl was the most suitable IL to be used as it has low viscosity and surface activity compared to [C4 mim]BF4 and [C8 mim]Cl as well as good phase separation ability (Han et al., 2011). The mixture of [C4 mim]Cl-inorganic salt ATPS can be influenced by the polar interaction between the two constituent components (Han et al., 2011). The higher valence anion is easier to salt out compared to lower valence anion. This is because large amount of water molecules can be hydrated by anion with a higher valence, resulting in less water molecules that are available to hydrate ILs (Han et al., 2011). Thus, K2 HPO4 was selected in this study. As the concentration of K2 HPO4 increased, the efficiency of sublation increased up to 98.5% (Han et al., 2011). Further increasing the concentration would no longer result in increasing efficiency. Conversely, it will decrease the efficiency sharply. By increasing the K2 HPO4 concentration, the mass transfer of CAP to water–air interface will eventually slow down due to high viscosity of the salt solution (Han et al., 2011). It was found out that pH less than 7.0 was not suitable to be used in this study as K2 HPO4 dominated at acidic pH value (Han et al., 2011). The CAP separation efficiency reduced when the pH was above 11.02. The strong alkaline solution affected and broke the molecular structure of CAP (Han et al., 2011). Thus, pH 10.0 was chosen as the optimum pH in this study. As the [C4 mim]Cl initial volume increased, the CAP sublation efficiency slowly increased. The molecular diffusion of CAP is weak because of the penetration of gas bubbles into IL layer causing the small water droplets to flow back to the aqueous phase by gravity (Han et al., 2011). Hence, the CAP sublation efficiency does not depend on the initial volume of IL but based on the adsorption process.

5.5.2 Extraction of protein using Ionic liquid Protein is a type of macronutrient, which the body needs in larger quantities (Szalay, 2015). Protein consists of long chain of amino acids, which are organic compound made of carbon, nitrogen, oxygen, hydrogen, and sulfur (Brazier, 2020). Protein plays an important role in metabolism, gene expression, maintaining cellular structures as well as other diagnostic applications (Pei et al., 2009). The separation of contaminated proteins requires high cost. Hence, purification and recovery of protein is crucial in modern biotechnology application. The traditional method for protein purification such as ionic and affinity chromatography is expensive, requires a lot of time and difficult to scale-up. Thus, ATPS is extensively used in the purification of protein as it offers a low-energy consumption, easily to scale up and environmental-friendly methodology (Pei et al., 2009). Fig. 5.5 shows the chemical structure of protein. Pei et al. (2009) published a work which aimed to investigate the factors that influence the protein extraction of albumin (BSA), cytochrome c, trypsin, and ɣglobulins. using ionic-liquid based ATPS. ILs that were used involved in the experiment were ([C4 mim]Br), ([C6 mim]Br) and ([C8 mim]Br), while the inorganic salt used was K2 HPO4 . The factors that were taken into consideration were pH, temperature of the

Ionic liquid–salt interaction

89

R´

H N H

O C

C

OH

H

Figure 5.5 Building block of protein.

100

E%

90

80

70

60

50 7

8

9

10

11

12

pH

Figure 5.6 Graph of extraction efficiency of cytochrome against pH. From Pei et al. (2009). Ionic liquid-based aqueous two-phase extraction of selected proteins. Separation and Purification Technology, 64(3), 288–295. https://doi.org/10.1016/ j.seppur.2008.10.010.

system and the concentration of IL. Based on the results obtained, as the pH value increased, the extraction efficiency of proteins decreased. This shows that the isoelectric point of proteins influences the proteins’ charged state (Pei et al., 2009). Therefore, the optimum pH for all the proteins is capped at 9.3, as protein denature at extreme high pH. Fig. 5.6 shows the influence of pH in the cytochrome’s extraction efficiency. The isoelectric points of trypsin, cytochrome c, BSA, and ɣ-globulins protein were found to be 9.4, 10.3, 4.6, and 5.6, respectively. Since the pH of 9.3 was used, the net charge of trypsin approximate to zero, while the cytochrome remained positively charged and the BSA and ɣ-globulins were negatively charged (Pei et al., 2009). As the pH slowly increased to the isoelectric point of protein, a strong hydrophobic interaction was formed. This resulted in trypsin extraction efficiency to be the highest. The strong hydrophobic interaction can be explained by the presence of aromatic π rings in the protein structures and the Imidazolium cation of IL where π–π interactions took places.

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N

O

H3C H3C

O

OCH3

CH3

OH

OH OH

H3C CH3 HO O

H3C O CH3 O

O CH3 O H3C

N O

CH3 CH3

OCH3 CH3 OH

Figure 5.7 Chemical structure of Roxithromycin.

Thus, π–π interactions between the cation and proteins are the key factor for extraction of proteins. The greater the size of protein, the more difficult it is to separate the protein in the solution. This is because more energy is required to transfer the protein from the salt-rich bottom phase to the IL-rich top phase (Pei et al., 2009). On the other hand, the effect of temperature on the extraction of protein was also studied. Protein extraction is an endothermic reaction which means higher temperature favors the extraction process. Nonetheless, the system temperature should remain below 60◦C as protein denatures at high temperature. The results show that the value of G◦ T was negative in contrast with TS◦ T and H◦ T which showed positive values (Pei et al., 2009). The negative value of G◦ T proved that the extraction of protein is a spontaneous process whereas the higher positive value of TS◦ T indicated that the separation of protein is governed by change of entropy (Pei et al., 2009).

5.5.3 Purification of roxithromycin using IL-salt ATPS Roxithromycin (ROX) is a semisynthetic antibiotic that is used to treat infections on the skin or chest (Li et al., 2009) by stopping the bacterial growth. The main advantage of using ROX in medical field is that it can deliver high performance at low dosage (Han et al., 2011). However, ROX cannot be used to treat infections that are caused by virus, and the residue from ROX can have adverse effect on human health (Li et al., 2009). Thus, it is important to examine the residue of ROX using a reliable pretreatment technique. Fig. 5.7 shows the structure of ROX. Li (2009) published a work that aimed to determine the factors that influence the extraction of ROX trace using ionic-liquid-based ATPS (Li et al., 2009). The author used IL [Bmim]BF4 in combination with salts like cation Na+ and anions CO3 2− , SO4 2− , OH– and PO4 2− were (Li et al., 2009). The author examined multiple parameters such as the type of salts used, and the extraction temperature. CO3 2− , SO4 2− , OH– , and PO4 2− are kosmotropic ions. Based on their solubility, CO3 2− produced the strongest interactions with water molecules compared to other salts (Li et al., 2009).

Ionic liquid–salt interaction

91

0.20 0.16

8 Top phase Bottom phase Vt /Vb

6

0.12

4

0.08

2

Volume ratio, Vt /Vb

Phase volume/mL

10

0.04

0 0.00 0.5

1.0

1.5 2.0 2.5 Mass of Na2CO3/g

3.0

3.5

Figure 5.8 Effect of the mass of Na2 CO3 required for the formation of [Bmim]BF4 − Na2 CO3 ATPS. From Li et al. (2009). Extraction and mechanism investigation of trace roxithromycin in real water samples by use of ionic liquid–salt aqueous two-phase system. Analytica Chimica Acta, 653(2), 178–183. https://doi.org/10.1016/j.aca.2009.09.011.

This indicated that the higher valence anion is a better salting-out agent compared to lower valence anion (Li et al., 2009). Hence, Na2 CO3 was chosen as the main inorganic salt. The amount of Na2 CO3 used was manipulated in the range of 2.3–3.3 g. It was found out that 90% of extraction efficiency can be obtained when more than 2.9 g of Na2 CO3 was used (Li et al., 2009). Further increasing the mass of Na2 CO3 will no longer result in ROX decomposition. Conversely, it would lead to the production of ROX impurity (Li et al., 2009). Thus, 2.9 g of Na2 CO3 was used for the extraction process. Fig. 5.8 shows the influence on the amount of Na2 CO3 of the [Bmim]BF4 - Na2 CO3 ATPS formation. The extraction efficiency of ROX is determined by the extraction temperature of the system which took place between 25 and 60°C. It was found out that below 50°C, the extraction efficiency of ROX was well maintained at 90% which indicated that temperature did not affect the extraction process significantly (Li et al., 2009). Above 50°C, the formation of two-phase was difficult and the separation efficiency decreased because the antibiotic denatured at high temperature.

5.5.4 Extraction of anthraquinones using ILATPS Anthraquinones (AQ) are aromatic compounds that exists in plants such as Aloe vera, Rhubarb, or Senna. The two main active ingredients that can be found in aloe vera are aloe anthraquinones and aloe polysaccharides (Tan et al., 2012). In the medical field, the bioactive ingredient in the aloe vera can cure various diseases such as diabetes and malaria (Adams et al., 2014). Aloe vera can be used in the cosmetic product as well (Tan et al., 2012). Fig. 5.9 shows the chemical structure of aloe AQs.

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OH

O

R1 emodin aloe-emodin rhein chrysophanol physcion

OH

OH

O

CH2OH

R2 O R1=OH R1=H R1=H R1=CH3 R1=CH3

OH

O R2=CH3 R2=CH2OH R2=COOH R2=H R2=OCH3

OH

OH OH

OH Aloin

Figure 5.9 Structure of anthraquinones. From Tan et al. (2012). Isolation and purification of aloe anthraquinones based on an ionic liquid/salt aqueous two-phase system. Separation and Purification Technology, 98, 150–157. https://doi.org/10.1016/j.seppur.2012.06.021.

Figure 5.10 The purification of Aloe AQ in IL/Salt ATPS. From Tan et al. (2012). Isolation and purification of aloe anthraquinones based on an ionic liquid/salt aqueous two-phase system. Separation and Purification Technology, 98, 150–157. https://doi.org/10.1016/j.seppur.2012.06.021.

Tan et al. published a work which aimed to achieve high AQ extraction efficiency and to determine the factors that influence the AQ extraction using ionic-liquidbased ATPS (Tan et al., 2012). Six types of IL were used :[C2 mim]BF4 , [C2 mim]Br, [C4 mim]BF4 , [C4 mim]N(CN)2 , [C4 mim]Br and [C6 mim]BF4, while (NH4 )2 SO4 , MgSO4 , NaH2 PO4 and Na2 SO4 were used as inorganic salts (Tan et al., 2012). The factors that took into consideration were the type of IL and salts used, extraction temperature, time required for centrifugation and pH. Fig. 5.10 shows the diagram of Aloe AQ purification. It was found out that [C4 mim]BF4 has the satisfactory AQ extraction efficiency among others (Tan et al., 2012). When BF– approaches water molecules, they form stronger interaction with hydrogen bond in water (Pereira et al., 2020). However, BF4 − is more chaotropic than Br– (Tan et al., 2012). According to Li et al. (2010), chaotropic ions show weaker interaction with water molecules, which do not favor the ATPS formation compared to kosmotropic ions. Thus, BF4 − shows only satisfactory

Ionic liquid–salt interaction

93

extraction efficiency. The ability of two phases formation is greatly depend on anion of the inorganic salts. The Ghyd of sulfate ion is lower than the Ghyd of phosphate ion. Hence, the sulfate ion has greater salting-out ability compared to the phosphate ion (Tan et al., 2012). Although NH4 + , Mg2+ , Na+ share a similar anion, but their ability for phase formation is not stable (Tan et al., 2012). Na2 SO4 was chosen for further experimentation, as only 1g of Na2 SO4 was required to achieve the highest extraction efficiency which was 88.54% (Tan et al., 2012). Besides, the effect of extraction temperature was conducted in the range of 25–50°C. As the temperature increased, the phase ratio decreased insignificantly. The extraction of AQ is an exothermic reaction which means lower temperature favors the extraction process (Tan et al., 2012). The G◦ T , TS◦ T , and H◦ T calculation showed that the value of G◦ T , TS◦ T , and H◦ T were negative. The negative value of G◦ T proved that the extraction of AQ is a spontaneous process. The H◦ T is greater that TS◦ T also indicated that the separation of protein was governed by the change of enthalpy. The experiment was conducted at room temperature. The extraction of AQ was performed within 3–30 min. It was found out that two-phase separation could be obtained when the centrifugation time reached 5 min (Tan et al., 2012). Further increasing the time varied the result insignificantly (Lin et al., n.d.). This shows that extraction equilibrium can be achieved in a short time. The pH of AQ extraction was examined between pH 2 to 7. An ionization equilibrium of HA to H+ and A– occurred at different pH values (Tan et al., 2012). Under the acidic circumstance, the hydrophobic part of AQ would remain at IL rich phase (Tan et al., 2012). pH 4 was selected as it produced the highest extraction efficiency.

5.6

Conclusion

IL/ATPS is an economical and efficient processing method. It is form by mixing high charge density salt ion with low charge density salt ion. IL/ATPS is a promising technology to be used for pretreatment, purification, and separation, mainly on protein. The benefits of using IL/ATPS are simple experimental procedure, consumes low energy, and easy to scale-up compare to other LLE. The extraordinary properties that make IL stand out compared to organic solvent are low volatility, high thermal and chemical stability, tunable chemical, and physical properties. The both bulk phases only consist of water and without the use of organic solvent make the IL/ATPS environmental friendly. In this chapter, the applications of IL–salt interaction are extensively discussed.

References Adams, K., Eliot, T., Gerald, A., 2014. Extent of use of aloe vera locally extracted products for management of ailments in communities of Kitagata sub-county in Sheema District, Western Uganda. Int. J. Sci. Basic Appl. Res. 15 (1), 1–15. Brazier. (2020). Protein: sources, deficiency, and requirements. Medical News Today. Bregni, C., 2021. Bacteriostatic antimicrobial agents of chloramphenicol. J. Molecular Pharmaceutics Organic Process Res. 9 (2), 1000215.

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Dreyer, S., Salim, P., Kragl, U., 2009. Driving forces of protein partitioning in an ionic liquid-based aqueous two-phase system. Biochem. Eng. J. 46 (2), 176–185. https://doi.org/ 10.1016/j.bej.2009.05.005. Freire, M.G., Cláudio, A.F.M., Araújo, J.M.M., Coutinho, J.A.P., Marrucho, I.M., Lopes, J.N.C., Rebelo, L.P.N., 2012. Aqueous biphasic systems: a boost brought about by using ionic liquids. Chem. Soc. Rev. 41 (14), 4966–4995. https://doi.org/10.1039/C2CS35151J. Han, J., Wang, Y., Yu, C., Li, C., Yan, Y., Liu, Y., Wang, L., 2011. Separation, concentration and determination of chloramphenicol in environment and food using an ionic liquid/salt aqueous two-phase flotation system coupled with high-performance liquid chromatography. Anal. Chim. Acta 685 (2), 138–145. https://doi.org/10.1016/j.aca.2010.11.033. Li, C.-X., Han, J., Wang, Y., Yan, Y.-S., Xu, X.-H., Pan, J.-M., 2009. Extraction and mechanism investigation of trace roxithromycin in real water samples by use of ionic liquid–salt aqueous two-phase system. Anal. Chim. Acta 653 (2), 178–183. https://doi.org/10.1016/ j.aca.2009.09.011. Li, Z., Pei, Y., Wang, H., Fan, J., Wang, J., 2010. Ionic liquid-based aqueous two-phase systems and their applications in green separation processes. BioCop – Monitoring Chemical Contaminants in Foods, 29 (11), 1336–1346. https://doi.org/10.1016/j.trac.2010.07.014. Lin, H., Yan, H., & Luo, M. (n.d.). Enrichment of nicotine in human plasma and urine with ionic liquid based liquid phase microextraction. In 2010 3rd International Conference on Biomedical Engineering and Informatics (Vol. 5, pp. 2038–2040). 10.1109/BMEI.2010.5639652 Pei, Y., Wang, J., Wu, K., Xuan, X., Lu, X., 2009. Ionic liquid-based aqueous two-phase extraction of selected proteins. Sep. Purif. Technol. 64 (3), 288–295. https://doi.org/10.1016/ j.seppur.2008.10.010. Pereira, J.F.B., Freire, M.G., Coutinho, J.A.P., 2020. Aqueous two-phase systems: Towards novel and more disruptive applications. Fluid Phase Equilib. 505, 112341. https://doi.org/ 10.1016/j.fluid.2019.112341. Rogers, R.D., Voth, G.A., 2007. Ionic Liquids. Acc. Chem. Res. 40 (11), 1077–1078. https://doi. org/10.1021/ar700221n. Szalay, J. (2015). What Is Protein? | Live Science. Live Science Contributor. Tan, Z., Li, F., Xu, X., 2012. Isolation and purification of aloe anthraquinones based on an ionic liquid/salt aqueous two-phase system. Sep. Purif. Technol. 98, 150–157. https://doi. org/10.1016/j.seppur.2012.06.021.

T-butanol–salt three-phase interaction

6

Yan Jer Ng a, Yoong Kit Leong b, Wen Yi Chia a, Kit Wayne Chew c and Pau Loke Show a a Department of Chemical and Environmental Engineering, Faculty of Science and Engineering, University of Nottingham Malaysia, Semenyih, Selangor, Malaysia, b Department of Chemical and Materials Engineering, Tunghai University, Taichung, Taiwan, c School of Energy and Chemical Engineering, Xiamen University Malaysia, Sepang, Selangor Darul Ehsan, Malaysia

6.1

Introduction

In recent years, the human population has been growing exponentially, which means that the food requirement to feed the whole human population would increase exponentially as well. To cope with this exponential increase, scientists have been developing new methods for food production every year. For these food production methods, most of the cost would be allocated for separation processes as separation processes where they took up 70% of the total processing costs (Raja et al., 2012). Therefore, a more economically friendly separation method must be proposed and implemented to reduce the cost of food production. Three-phase partitioning (TPP) is a separation process that was first discovered by Tan and Lovrien in year 1972 (Tan and Lovrien, 1972). This method was first used only to purify several specific macromolecules. However, in recent years it has developed into a process that is able to separate various biomolecules such as proteins, polysaccharides, and oil (Ketnawa et al., 2017; Ravindran and Jose, 2014). As there are already many different separation techniques in the industry for biomolecules purification. However, TPP holds its own advantages over these other techniques. First, since the chemicals used for TPP system is inexpensive, this process can be carried out at a very low operating cost which makes this process economically friendly. This is because TPP only involves two chemicals, one is an organic solvent and the other being a salt. As TPP involves only mixing, the operating cost for the process would be low (Ketnawa et al., 2017; Ravindran and Jose, 2014). The operating cost for this process is also reduced as this process can be carried out under ambient room temperature conditions, thus there is no heating and cooling needed. Also, as the chemicals used for this process is mild chemicals, the chemicals do not denature the proteins, thus there would be less loss for the process. The time cost for this process is also very low as this process does not require a long operating time for the whole process to be completed. The time cost for the whole process is further reduced as there is no need of pretreatments for this method (Ketnawa et al., 2017; Kiss and Borbás, 2003; Ravindran and Jose, 2014; Shah et al., 2004). For extraction feed that contains Principles of Multiple-Liquid Separation Systems: Interaction, Application and Advancement. DOI: https://doi.org/10.1016/B978-0-323-91728-5.00012-3 c 2023 Elsevier Inc. All rights reserved. Copyright 

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Principles of Multiple-Liquid Separation Systems

Figure 6.1 Schematic diagram for three-phase partitioning.

undesired molecules such as impure lipid molecules, TPP can remove those molecules as well as the t-butanol will dissolve these unwanted molecules. The chemicals that are used in this system can also be recycled as well as the chemicals are not used up in the process, further reducing the overall operating cost of the process (Ketnawa et al., 2017). The process is also easily scalable to large-scale separation or scaled down to microlevel, thus increasing the flexibility of this process (Ketnawa et al., 2017; Ravindran and Jose, 2014). TPP also poses an advantage over other separation methods as the selectivity of this process is high (Sanglard et al., 2018). In this chapter, the basic process description, principles, applications, future perspectives, and challenges of TPP will be thoroughly discussed.

6.2 Process description 6.2.1 Process overview For a TPP system, aqueous solutions of salts such as ammonium sulfate and sodium citrate are mixed with the sample and t-butanol. First, samples that require separation are mixed with ammonium sulfate and pure t-butanol where the ratio of the volume of ammonium sulfate solution and t-butanol used can be optimized. The mixture will be stirred and the mixture will be allowed to sit for a period of time at room temperature for separation. After 30 min, a clear three-phase solution will be observed where the top phase of the mixture will consist of mainly t-butanol while the bottom phase will consist of an aqueous mixture of proteins and salt for which the protein solubility will be affected by salt concentration. Protein molecules will selectively precipitate into a middle phase in between the top and bottom phase of the solution, which forms the third phase of the whole system (Chia et al., 2019). Fig. 6.1 shows the TPP of proteins by the use of t-butanol.

6.2.2 About tert-butanol and ammonium sulfate Tert-Butanol (t-butanol or 2-propanol, 2-methyl) is an alcohol that has the formula of C4 H10 O and it has a shape of t-like structure. Fig. 6.2 shows the 2D shape of t-butanol. The melting point of t-butanol is 25.7°C and the boiling point of t-butanol is 82.41°C.

T-butanol–salt three-phase interaction

97

CH3 HO

C

CH3

CH3

Figure 6.2 Structure of t-butanol.

At a temperature of 20°C, the density of t-butanol is recorded to be at 0.79 g/mL, whereas the flash point of t-butanol is at 11.1°C. This means that at 11.1°C, t-butanol will release enough flammable vapor to be ignited even when in solid form. At room temperature, t-butanol will exist as a colorless liquid that gives off a camphor-like odor (Clark, 2001). Although t-butanol is an organic solvent, t-butanol increases the stability of protein molecules (Dennison et al., 2000). This is quite an unusual behavior for organic solvents as organic solvents such as toluene and chloroform usually denature proteins (Asakura et al., 1978). This behavior is resulted from the size of t-butanol molecules being too large to permeate into the 3D-structure of the proteins (Gagaoua and Hafid, 2016). It was also observed that t-butanol inhibits the enzymatic activities as well as interactions between proteins. This property holds an important role in the purification process of proteins, as proteins must be present in aqueous state to be separated and purified (Dennison, 1999). Conventional protein purification processes usually start with the homogenization of cells but the homogenization process causes unwanted interactions between protein molecules. These unwanted interactions will result in the formation of homogenization artifacts. As t-butanol is able to prevent the interactions between protein molecules, the formation of unwanted homogenization artifacts will be reduced (Dennison et al., 2000). This results in the usage of t-butanol in a three-phase separation system for proteins with the aid of ammonium sulfate. Although there are many other salts that can be used for the TPP of proteins, ammonium sulfate is mostly used for this purpose. This is because ammonium sulfate is a very common salt in the industry, thus it can be easily obtained at relatively low prices compared with other salts. The chemical properties of ammonium sulfate are also one of the reasons to why ammonium sulfate is chosen for this application. Similarly as t-butanol, ammonium sulfate does not denature proteins and for some protein molecules, it actually increases their stabilities (Gagaoua et al., 2014; Gagaoua and Hafid, 2016).

6.3

Principle of three-phase partitioning

In this subsection, the principle and the interactions occur between the phases will be discussed. In Section 6.2.2, it was mentioned that proteins were stabilized by t-butanol with simultaneous inhibition of the interaction between protein molecules. This cause the protein molecules to clump together and precipitate with the aid of ammonium sulfate.

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Principles of Multiple-Liquid Separation Systems

The role of ammonium sulfate in TPP is to induce a special mechanism that was referred as push on the left and pull from the right mechanism. In this mechanism, pushing and pulling hold true both thermodynamically and physically. For the precipitation to occur, the macromolecules must be closed and tightened. This means that water molecules trapped within the macromolecules must be removed. Under the pushing mechanism, a large concentration of ions will be needed for it to work. Salts such as ammonium sulfate induces a reaction named as “exclusion crowding mechanism” where a conformation tightening force is produced (Jacobsen et al., 1996; Timasheff, 1992). Protein hydration changes were induced due to this tightening force where changes in the physical properties of the molecule may be observed. The same goes for the pulling mechanism, with the difference being that the reason for producing the conformation tightening force is different and that much lower concentrations of SO4 2– ions are needed to achieve the purpose. Under the pulling mechanism, the sulfate ions will attach themselves onto the cation groups of protein molecules via electrostatic interactions. This results in the structure of the protein being drawn inwards and further reducing net protein hydration. This interaction between the protein molecules and sulfate ions will result in the protein molecules being changed to a form of sulfate salt where the overall structure of the protein molecule will be more stable compared to the normal protein molecules, which are relatively unstable and fragile. These changes can also be observed via different physicochemical tools such as observing the intrinsic viscosities of the protein (Matulis et al., 1996) or via spectroscopic means (Fink, 1995). It is believed that with this push and pull mechanism, the water molecules within the protein molecules can be squeezed out from the protein molecules, thus causing the protein molecules to precipitate out as a middle layer in the TPP system (Ketnawa et al., 2017; Ravindran and Jose, 2014; Yan et al., 2018).

6.4 Application of three-phase systems 6.4.1 Extraction of proteins Three-phase partition can be applied in the separation process of many biomolecules, with the most common as protein molecules. In this section, the applications of TPP on the separation and purification of protein molecules from different samples were discussed. Vetal has succeeded in the extraction and purification of peroxidase molecules from orange peels by applying TPP with a recovery yield of 93.96% at the optimized conditions of 1:1.5 broth to t-butanol ratio, 80 min stir time at 200RPM, 50% ammonium sulfate concentration, 30°C and pH 6 (Vetal and Rathod, 2014). Following that, Liu has reported that laccase that is produced from Coriolopsis trogii can also be purified by using TPP. The process was also optimized so that the recovery yield reached up to 75%. The optimum operating conditions where the process should be carried out is at an ammonium sulfate concentration of 40%, t-butanol concentration of 50%, 20°C, and pH of 5 (Liu et al., 2015). Niphadkar reported that polyphenol oxidase can be extracted from potato peels as well via TPP. A 70% recovery yield was

T-butanol–salt three-phase interaction

99

obtained and according to Niphadkar, the best operating condition for this process is at 40% ammonium sulfate concentration, 1:1 ratio of broth to t-butanol, and carrying out the process at 30°C for 40 min at a pH of 7. The importance of optimizing the ratio of broth to t-butanol is discussed. It is important to optimize the ratio because when insufficient t-butanol is used, the t-bunaol molecules are unable to efficiently synergize with ammonium sulfate whereas when excess t-butanol is used, the excess t-butanol molecules will start to denature the protein molecules resulting in a reduced yield of proteins extracted (Niphadkar and Rathod, 2015). Research carried out by Gagaoua suggested that TPP system can purify proteolytic enzymes as well (Gagaoua and Hafid, 2016). TPP can also be used in the extraction of lipase from pacific white shrimp hepatopancreas. This was proven to be true by Kuepethkaew where the recovery yield for that process was recorded to be 87.41% after the process is optimized. The parameters that were optimized include broth to t-butanol ratio in volume and concentration of ammonium sulfate where the optimum values are 1:1 and 50%, respectively (Kuepethkaew et al., 2017). In this paper, the effects of using other salts were tested as well. The salts tested were ammonium sulfate, dipotassium hydrogen phosphate, and trisodium citrate. The results still show that ammonium sulfate gave the best results among the three salts tested. The effects of using different organic solvent were also tested where the solvents tested are 1-propanol, 2-propanol, 1-butanol, and t-butanol. Results proved that t-butanol yields the best separation effects. The effects of pH were discussed in this paper, where it was proposed that when pH is increased, the two main effects that drive protein partition, the electrostatic component, and hydrophobic component, would be affected. It was proposed that when the pH of the system was raised from 3.0 to 7.0, the hydrophobic component of the protein partition driving force would be greater than the electrostatic forces, resulting in the increase in partition coefficient of proteins (Kuepethkaew et al., 2017). Lipase was also extracted from Rhizopus arrhizus which was proven by a study that was carried out by Dobreva where the best recovery yield was calculated to be at 71%. According to Dobreva, this single-step process is best carried out at conditions of 30% ammonium sulfate concentration, 1:0.5 ratio and at a pH of 7 (Dobreva et al., 2019). Chia reported that by using TPP assisted with ultrasonication on Chlorella vulgaris FSP-E, microalgal proteins can be extracted. A separation yield of 56.57% can be obtained with the best conditions where this process should be carried out is at salt saturation of 50%, ratio of 1:2 for crude extract to t-butanol, duty cycle of 80% and the biomass loading at 0.75 wt %. The ultrasound parameters that best suits the process is at a sonication power of 100% and a frequency of 35 kHz for 10 min (Chia et al., 2019). It is proven in the same research that incorporating sonication pretreatment into the TPP system will result in a better extraction of proteins. This is due to the cavitation bubbles that are created by the ultrasonic energy would result in the breaking of cell walls of the microalgae, releasing the proteins into the system thus making the extraction yield higher (Chia et al., 2019). The most recent development for proteins extraction by TPP was conducted by Gruyter. In this study, polyphenol oxidase was extracted from rosemary. The reported recovery was at 230% where the optimized parameters are 50% (w/v) ammonium

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sulfate, 1:1 ratio for broth to t-butanol, and carrying out the process at room temperature under a pH of 6.5 (Karakus et al., 2020). A summary for the applications on proteins extraction by three-phase partition is included in Table 6.1.

6.4.2 Oil extraction As mentioned in the previous subsection, biomolecules other than proteins can be extracted by TPP. This subsection will be discussing on oil extraction via TPP. Dutta reported the possibility of extracting oil from Crotalaria juncea via TPP. This method was compared with Soxhlet-based extraction for which TPP can obtain a higher yield at a shorter time. For the traditional Soxhlet-based extraction, the time needed for the process is 4 h to obtain a yield of 13% whereas for TPP, the time needed to obtain a yield of 37% is only 2 h. The ratio of ammonium sulfate used for the optimized process is 6.74% and the ratio used for t-butanol is 97.78% (Dutta et al., 2015). According to Dutta, this difference in efficiency is due to TPP being a purely batch equilibriumdriven process, which means that TPP will offer a longer contact time between the molecules. This results in the equilibrium to be reached within a shorter time compared with the Soxhlet-based extraction as the traditional Soxhlet-based extraction process will require a higher solvent to solid ratio to reach the same equilibrium (Dutta et al., 2014). The optimized process is to be carried out at a temperature of 29.45°C and a pH of 4.587 (Dutta et al., 2015). A study carried out by Tan has reported that oil can be extracted from flax seed via enzyme-assisted TPP. The concentration of t-butanol used for the optimized process was at 49.29%, whereas the concentration of ammonium sulfate used was 30.43% and the process was carried out at a temperature of 35°C to yield oil at 71.68%. The enzymes used in this research are cellulase, proteinase, and pectinase. When the enzymes are tested individually, the results show that cellulase has the highest enzymolytic effects on the sample. However, when the enzymes are mixed, the extraction yield of oil from the sample is the highest. The reason behind this is that when the enzymes are mixed, the enzymes will produce synergetic actions which will hydrolyze all three types of molecules and thus disrupt the structural integrity of the cell wall of the sample. The reason behind optimizing temperature for TPP is also discussed, where it was proposed that when under high temperature, not only do more protein molecules denature but more protein molecules will redissolve to the top and bottom phase of the TPP. When the system is slightly heated, the solubility of triglycerides in organic solvents will be increased, thus the oil separation will be carried out under a slightly heated environment rather than at ambient room temperature (Tan et al., 2016). Custard apple seed oil can also be extracted by the use of TPP. This was proven by two studies carried out by Panadare et al., (2019) and Panadare and Rathod (2020). A maximum oil yield of 33.23% was obtained when Panadare incorporated microwave pretreatment into the TPP system. Salt concentration, broth to t-butanol ratio, salt solution to t-butanol ratio were optimized where the optimized values are 45%, 1:10 and 1:2, respectively. Proteins were also recovered in this process where the yield was 18.46% (Panadare and Rathod, 2020).

Target Application molecules Protein extraction Laccase Lipase

Microalgal proteins

Oil extraction

Sample Coriolopsis trogii

Recovery yield 75%

Broth to t-butanol ratio –

Pacific white shrimp hepatopancreas Phizopus arrhizus Chlorella vulgaris FSP-E

87.41%

1.1

71% 56.57%

– –

Peroxidase

Orange peels

93.96%

1:1.5

Polyphenol oxidase

Potato peels

70%

1:1

Rosemary Crotalaria juncea

230% 13%

1:1 –

Custard apple seed

33.23%

1:10

Flax seed

71.68%

–

Sesame

86.12%

1:1

Optimized Conditions Ammonium sulfate pH Other conditions Reference concentration 40% 5 ∼T-butanol concentration: Liu et al. (2015) 50% ∼Temperature: 20°C 50% – – Kuepethkaew et al. (2017) 30% – 7 Dobreva et al. (2019) 50% – ∼Duty cycle: 80% ∼Biomass loading: 0.75wt% ∼Sonication Chia et al. (2019) power: 100% ∼Frequency: 35 kHz ∼Sonication time: 10 min 50% 6 ∼Stir time: 80 min ∼Stir Vetal and Rathod speed: 200 RPM (2014) ∼Temperature: 30°C 40% 7 ∼Temperature: 30°C Niphadkar and Rathod (2015) 50% 6.5 – Karakus et al. (2020) 6.74% 4.587 ∼Ratio of T-butanol: Dutta et al. (2015) 97.78% ∼Temperature: 29.45°C ∼Time: 2 h 45% – ∼Salt to t-butanol ratio: 1:2 Panadare et al. (2019), Panadare and Rathod (2020) 30.43% – ∼T-butanol concentration: Tan et al. (2016) 49.29% ∼Temperature: 35°C 40% 5 – Juvvi and Debnath (2020)

T-butanol–salt three-phase interaction

Table 6.1 Application of TPP on proteins and oil extraction, multimolecule separations, and separation of other biomolecules.

(continued on next page) 101

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Table 6.1 Application of TPP on proteins and oil extraction, multimolecule separations, and separation of other biomolecules—cont’d

Application Multimolecule separation

Target molecules

Sample Aloe powder

Optimized Conditions Ammonium sulfate pH Other conditions concentration 26.35% 6.5 ∼T-butanol concentration: 20.82% ∼Temperature: 30°C

Phellinus baumii

Mangiferin

Mangifera indica

54 mg/g

–

Sodium citrate concentration: 19% 40%

Polysaccharide

Corbucula fluminea

9.32%

-

20%

6

Inonotus obliquus

2.20%

–

20%

8

Remazol Brilliant Blue R

97.20%

–

–

–

Schizochytrium limacinum

Textile dye

Reference Tan et al., 2015)

28%

5.1

∼Temperature: 40°C ∼Time: 1 h

Wang et al. (2020)

34%

–

Chen et al. (2020)

4

–

∼Enzyme used: Protamex ∼Temperature: 40°C ∼Time: 41 min ∼Broth to dimethyl carbonate ratio: 1:2 ∼Temperature: 30°C ∼Pretreatment time: 5 min ∼Microwave power: 272W ∼Solute to solvent ratio: 1:20 ∼Broth to T-butanol ratio: 1:1 ∼Soaking time: 5 min ∼Duty cycle: 50% ∼Temperature: 25.3°C ∼Time: 30 min ∼Solid to liquid ratio: 1 g to 12 mL ∼Temperature: 30°C ∼Time: 30 min ∼Temperature: 25°C ∼Time: 12 h

Wang et al. (2019)

Kulkarni and Rathod (2015)

Yan et al. (2017) Liu et al. (2019)

Sanglard et al. (2018)

Principles of Multiple-Liquid Separation Systems

Exopolysaccharides

Recovery yield ∼Aloe polysaccharide: 92.26% ∼Proteins: 92.78% ∼Oil: 17.28% – ∼Proteins: 6.81% ∼Polysaccharides: 2.09% ∼Oil: 35.69% – ∼Polysaccharide: 5.16% 71.02% –

Rice bran

Separation of other biomolecules

Broth to t-butanol ratio –

T-butanol–salt three-phase interaction

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Juvvi reported that sesame oil can also be extracted from TPP as well, which the highest sesame oil recovery can reach up to 86.12%. The optimum conditions where the process should be carried out are at an ammonium sulfate concentration of 40%, 1:1 ratio for broth to t-butanol and a pH of 5 (Juvvi and Debnath, 2020). These applications are summarized in Table 6.1 for easy referencing.

6.4.3 Multimolecule separation Other than the separation of proteins individually, TPP can also be used to separate multiple biomolecules simultaneously. This was reported by Tan for which proteins and aloe polysaccharide are recovered. The TPP system was created by mixing ammonium sulfate and t-butanol with aloe powder in the form of crude slurry. The middle phase of this system consists of the usual precipitated proteins, whereas the lower phase of this system holds the aloe polysaccharide extracted. Extraction parameters such as concentration of t-butanol and ammonium sulfate was optimized, as well as the operating conditions such as the temperature and pH. The final extraction efficiency of the products are calculated to be at 92.26% and 92.78% for aloe polysaccharide and proteins respectively. Post TPP treatments include evaporation for t-butanol recovery and dialysis for salt removal from aloe polysaccharide. The extract were tested with UV-V is spectroscopy for protein molecules and FT-IR spectroscopy for aloe polysaccharide molecules. The results show that both molecules are well extracted and the structure of the molecules is not disrupted by the process (Chia et al., 2019; Tan et al., 2015). Other than the study carried out by Tan, Wang also reported another multimolecule separation system via TPP where oil, protein and polysaccharide were extracted from rice bran. The oil extracted is mainly present at the top phase, whereas the polysaccharides extracted are mainly present in the bottom phase. As usual, the middle phase of the system consists of the precipitated proteins. Yields of oil, protein, and polysaccharides were calculated to be 17.28%, 6.81%, and 2.09%, respectively, after optimization of the process. The optimum operating parameters are found to be 28% for the concentration of ammonium sulfate, 1:1.1 for ratio of broth to t-butanol and operating conditions at pH 5.1, 40°C and 1 h extraction time (Wang et al., 2020). A multimolecule system was developed by Chen to simultaneously extract oil and polysaccharides from Schizochytrium limacinum via enzyme-assisted TPP. The oil yield and polysaccharide yield from this microalga that can be obtained are at 35.69% and 5.16% respectively. Protamex enzymes were used for this system and the pretreatment process was optimized to be at 45°C and pH 3 for 2 h at an enzyme concentration of 2%. The process parameters that were optimized for the TPP were 34% concentration ammonium sulfate, 1:2.4 broth to t-butanol ratio and for 41 min at a temperature of 40°C (Chen et al., 2020). Table 6.1 shows the summarized applications for multimolecule separation systems.

6.4.4 Other molecules Other than the biomolecules mentioned in the previous sections, there are also several different molecules that TPP can purify.

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As mentioned in the previous sections, polysaccharides can be purified via TPP. However, in Section 6.4.3 only separation of polysaccharides in multimolecule separation systems was mentioned. Polysaccharides can also be isolated and separated out individually via TPP. This was proven to be true by Yan, with the feed being Corbucula fluminea and the highest yield of polysaccharide is reported to be at 9.32%. The optimized process consists of parameters such as 20% mass fraction of ammonium sulfate, 9.8 mL of t-butanol used per extraction, and an operating condition of 25.3°C at pH 6 for 30 min (Yan et al., 2017). Other polysaccharides extracted are from immunomodulatory medicinal mushroom Inonotus obliquus. This study was carried out by Liu where the maximum yield of polysaccharides obtained was at 2.2%. The optimized parameters for this process include a solid to liquid ratio of 1g to 12 mL, 20 wt % ammonium sulfate and 11 mL of t-butanol used for each extraction. The operating conditions for this process is at a pH of 8 under 30°C for 30 min. The polysaccharides extracted undergo testing via high-performance size exclusion chromatography multiangle laser light scattering, where the molecular weight and conformation of the polysaccharides are characterized whereas the antioxidant properties of these polysaccharides are tested by DPPH radical scavenging activity, TEAC and FRAP (Liu et al., 2019). Exopolysaccharides were extracted from Phellinus baumii by Wang with the highest extraction yield of 71.02%. In this TPP system, dimethyl carbonate was used as organic solvent and sodium citrate was used as the salt. The optimum conditions for this process are salt concentration of 19% and sample to solvent ratio of 1:2 at a pH 4 and 30°C (Wang et al., 2019). Mangiferin was successfully extracted from Mangifera indica by Kulkarni for which the yield obtained was 54 mg/g via TPP assisted with microwave extraction. The reference method used for this process is Soxhlet extraction for which the extraction yield for that method was found to be at 57 mg/g with a 5 h process. The optimized parameters for the TPP process were 5 min microwave time, ammonium sulfate concentration of 40 wt %, a power of 272 W for the microwave, solute to solvent ratio of 1:20, broth to t-butanol ratio of 1:1, soaking time of 5 min, and a duty cycle of 50%. The duty cycle of microwave used was optimized as continuous molecular friction will occur when the microwave is operated under continuous mode where the temperature is increased consistently thus posing a possibility of degradation for the biomolecules (Kulkarni and Rathod, 2015). Textile dye was also successfully partitioned by Sanglard, with the partition coefficients above 26.4 and an extraction efficiency of 97.2% (Sanglard et al., 2018). These applications are summarized in Table 6.1.

6.5 Future perspectives and challenges TPP has been proven for its potential to be used in the separation of many biomolecules. Due to their similarities with the liquid biphasic system (LBS), it can be predicted that the future of TPP would show a similar trend when compared with the LBS. It was predicted that the total cost of the overall process would be reduced when the process is more well known to the industry as different, less expensive salts and organic

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solvents can be used for the separation process when research are being carried out (Show et al., 2015). For the case of TPP, it can be safely assumed that different salt and organic solvent can be used as well for the same purpose but with lower cost. Although there is only one different combination of solvent and salt (t-butanol and ammonium sulfate) that was proven to be effective for TPP, more research can be carried out to find other combinations, and eventually the cheapest possible combination can be found. The only known combination currently is substituting ammonium sulfate with sodium citrate and substituting tert-butanol with dimethyl carbonate (Wang et al., 2019). It was also proposed that LBS be paired with other separation methods such as bubbling, ultrasonication, and microwave to achieve a higher efficiency and yield. It was suggested that LBS can be carried out as a pretreatment method or as a posttreatment method. This holds true for TPP as well as it has already been proven true with incorporating a microwave pretreatment step before TPP, which will significantly enhance the performance of the TPP system (Panadare and Rathod, 2020). Other than pairing TPP with another method, TPP itself can also be used for different applications as well. It was suggested that LBS can be used simultaneously with fermentation methods to produce an extractive fermentation method. It is also suggested that this process can be further incorporated with other designs to form an in situ product bioconversion process where the converted products may be separated from the reaction mixture after every process cycle (Show et al., 2015). This concept can also be used in TPP as well as TPP is also a batch process. TPP has the potential to extract proteins while simultaneously producing other biomolecules for extraction. In one paper, it was suggested that LBS can be used to recover biomolecules from wastewaters (Selvaraj et al., 2011), where it can be predicted that TPP can be used for the same application as well. For example, LBS has been used to recover proteins in milk waste (Tham et al., 2019) and TPP may also be used for the same application as well. Although TPP provides a very promising future in separation technology, this method still has some challenges that it needs to be overcome before it can be widely commercialized. The most significant challenge faced by TPP is that TPP is less widely understood. It was stated that one of the challenges faced by LBS is that the partition mechanism for LBS is not widely understood. This directly affects the availability of tools for the modeling and optimization of the process. The design of the system can be further perfected if modeling tools were available as the cost for optimization would be greatly reduced. This would further attract the interests of investors to invest money into this process thus further spreading this process (Grilo et al., 2016; Iqbal et al., 2016). As TPP is an even more uncommon technique when compared to LBS, this would be a more difficult challenge to overcome for TPP. Furthermore, TPP is a relatively new technique so even if TPP is able to achieve a same yield with the traditional methods at the same cost, the driving force for companies to switch to using TPP is not high. This is because there might still be unknown hazards or side effects for TPP and not many companies would be willing to be the first to test out this new method. However, this challenge can be overcome by altering the application of TPP. For example, rather than replacing a safe and effective method like chromatographic separation, TPP can replace centrifugation as it is more

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dangerous and has the potential to destroy protein molecules. This replacement would be to remove the potential damages that might occur on the product molecules and it might push the companies to try out new methods (Grilo et al., 2016). Recycling used chemicals for TPP is also a big challenge. Currently, there is limited research that is conducted on the recycling of the chemicals used (Aguilar et al., 2006; Selvaraj et al., 2011). This will pose a big issue on both environmental aspects and economical aspects as this would produce large quantities of wastewater and the treatment process for the wastewater would be costly (Show et al., 2015).

6.6 Conclusion As conclusion, TPP has proven to be a separation method that is very effective in both time and cost. Although there are several challenges that awaits TPP before it can be widely commercialized, TPP has a very promising future. Many more research are being carried out to improve TPP as well as developing new applications for TPP. Salts similar to ammonium sulfate can be tested with tert-butanol for combinations with better separation or lower cost and other organic solvents should also be tested with ammonium sulfate as well. Research on converting TPP to a continuous process should also be carried out to further improve the safety of TPP. It would be a waste if TPP is unable to be commercialized since TPP has such high potential to become the future leading separation method for biomolecules.

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Liu, Z., Yu, D., Li, L., Liu, X., Zhang, H., Sun, W., Lin, C.-C., Chen, J., Chen, Z., Wang, W., Jia, W., 2019. Three-Phase Partitioning for the Extraction and Purification of Polysaccharides from the Immunomodulatory Medicinal Mushroom Inonotus obliquus. Molecules 24 (3), 403. https://doi.org/10.3390/molecules24030403. Matulis, D., Lovrien, R., Richardson, T.I., 1996. Coprecipitation of proteins with matrix ligands: scaleable protein isolation. J. Mol. Recog. 9 (5–6), 433–443. https://doi.org/ 10.1002/(sici)1099-1352(199634/12)9:5/63.0.co;2-p. Niphadkar, S.S., Rathod, V.K., 2015. Ultrasound-assisted three-phase partitioning of polyphenol oxidase from potato peel (Solanum tuberosum). Biotechnol. Progr. 31 (5), 1340–1347. https://doi.org/10.1002/btpr.2139. Panadare, D., Gondaliya, A., Rathod, V., 2019. Comparative study of ultrasonic pretreatment and ultrasound assisted three phase partitioning for extraction of custard apple seed oil. Ultrason. Sonochem. 61, 104821. https://doi.org/10.1016/j.ultsonch.2019.104821. Panadare, D.C., Rathod, V.K., 2020. Process intensification of three phase partition for extraction of custard apple seed oil using microwave pretreatment. Chemical Eng. Processing— Process Intensification 157, 108095. https://doi.org/10.1016/j.cep.2020.108095. Raja, S., Murty, V.R., Thivaharan, V., Rajasekar, V., Ramesh, V., 2012. Aqueous two phase systems for the recovery of biomolecules—a review. Sci. Technol. 1 (1), 7–16. https://doi.org/ 10.5923/j.scit.20110101.02. Ravindran, R., Jose, L., 2014. Three phase partitioning—a novel protein purification method. Int. J. ChemTech Research 6, 3467–3472. Sanglard, M.G., Farias, F.O., Sosa, F.H.B., dos Santos, T.P.M., Igarashi-Mafra, L., Mafra, M.R., 2018. Measurement and correlation of aqueous biphasic systems composed of alcohol (1propanol/2-propanol/tert-butanol) + (NH4)2SO4 + H2O at 298 K and a textile dye partition. Fluid Phase Equilib. 466, 7–13. https://doi.org/10.1016/j.fluid.2018.03.009. Selvaraj, R., Murty, V., Varadavenkatesan, T., Sekar, R., Ramesh, V., 2011. Aqueous two phase systems for the recovery of biomolecules—a review. Sci. Technol. 1, 7–16. https://doi.org/10.5923/j.scit.20110101.02. Shah, S., Sharma, A., Gupta, M.N., 2004. Extraction of oil from Jatropha curcas L. seed kernels by enzyme assisted three phase partitioning. Ind. Crops Prod. 20 (3), 275–279. https://doi. org/10.1016/j.indcrop.2003.10.010. Show, P.-L., Ling, T., Lan, J., Tey, B.T., Ramanan, R.N., Yong, S.-T., Chien Wei, O., 2015. Review of microbial lipase purification using aqueous two-phase systems. Curr. Org. Chem. 19, 19–29. https://doi.org/10.2174/1385272819666141107225446. Tan, K.H., Lovrien, R., 1972. Enzymology in aqueous-organic cosolvent binary mixtures. J. Biol. Chem. 247 (10), 3278–3285. Tan, Z.-J., Wang, C.-Y., Yi, Y.-J., Wang, H.-Y., Zhou, W.-L., Tan, S.-Y., Li, F.-F., 2015. Three phase partitioning for simultaneous purification of aloe polysaccharide and protein using a single-step extraction. Process Biochem. 50 (3), 482–486. https://doi.org/10.1016/ j.procbio.2015.01.004. Tan, Z.-J., Yang, Z.-Z., Yi, Y.-J., Wang, H.-Y., Zhou, W.-L., Li, F.-F., Wang, C.-Y., 2016. Extraction of oil from Flaxseed (Linum usitatissimum L.) using enzyme-assisted three-phase partitioning. Appl. Biochem. Biotechnol. 179 (8), 1325–1335. https://doi.org/10.1007/ s12010-016-2068-x. Tham, P.E., Ng, Y.J., Sankaran, R., Khoo, K.S., Chew, K.W., Yap, Y.J., Malahubban, M., Aziz Zakry, F.A., Show, P.L., 2019. Recovery of protein from dairy milk waste product using alcohol-salt liquid biphasic flotation. Processes 7 (12). https://doi.org/10.3390/pr7120875. Timasheff, S.N., 1992. Water as ligand: preferential binding and exclusion of denaturants in protein unfolding. Biochemistry 31 (41), 9857–9864. https://doi.org/10.1021/bi00156a001.

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Green solvents for multiphase systems

7

Jia Rhen Loo a and Wai Yan Cheah b a Department of Chemical and Environmental Engineering, Faculty of Science and Engineering, University of Nottingham Malaysia, Semenyih, Selangor Darul Ehsan, Malaysia, b Centre of Research in Development, Social and Environment (SEEDS), Faculty of Social Sciences and Humanities, Universiti Kebangsaan Malaysia, Bangi, Selangor Darul Ehsan, Malaysia

7.1

Introduction

As time passes, the term sustainability becomes more vital point in chemical processing, and as the solvent quantities are used in large quantities in fine-chemical and pharmaceutical production, large amounts are used for the separation of higher purity final products. Therefore, solvent plays a very significant role in environmental performance of a process and impact on cost, safety, and health issues. As time passes solvents used in industries that cannot be properly recovered or reused or saturated (to a point where regeneration is not possible) are treated as waste chemicals and sold to companies which professionally handles the waste solvent. Not only does this increase the annual cost of the company, but it also poses a treat the environment if not handled correctly; therefore, studies have been carried out to increase the findings on renewable or green solvents. Green solvents are eco-friendly solvents, or biosolvents, which are derived from renewable resources, and it takes into consideration different factors such as the amount of waste produced, toxicity, and environmental burden of all chemicals used and waste generated; energy and electricity used in the extraction processes, as well as the production of chemicals and safety of the extraction (Turner, 2013). As green solvents show various similar characteristics compared to commercial solvents, the multiphase systems created/formed are similar to that of commercial solvent. Except for DES and NADES solvent, as their viscosity is high compared to commercial solvents, this limitation can be surmounted through increasing temperature of the system. Not only does green solvents help the environment and reduce the costings, some of the studied solvents also increases the rate and effectiveness of extraction of certain products in recent studies. This book chapter discusses about the recent findings, working principles, and reasons of usage of green solvents and its future trends.

Principles of Multiple-Liquid Separation Systems: Interaction, Application and Advancement. DOI: https://doi.org/10.1016/B978-0-323-91728-5.00015-9 c 2023 Elsevier Inc. All rights reserved. Copyright 

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H

O

H

C

O

C

R

H

C

O

C

R

H

C

O

C

R

H

O

Figure 7.1 Simple representation of a triglyceride molecule.

7.2 Green extraction solvents, principles, and reasons for its use 7.2.1 Vegetable oil 7.2.1.1

Properties

With high unstable petroleum prices and depleting natural resources, there is a need to discover new sources which can replace petroleum. With the understanding of the metabolic pathways in the synthesis of fatty acids, studies have been carried out in manufacturing desired or preferred fatty acids. Vegetable oil is normally extracted from the seed of plants, which can be extracted via an oil mill or chemical extraction using a solvent. One of the examples of oils used in industries is castor oil. As the building blocks of fats and oil is relatively small, it is an ester of fatty acids and glycerol; and extraction of the solvent (oil) can be easily performed, such as mechanical pressing and/ or solvent extraction or sub- or super-critical water extraction, which depends on the volume of the industry. The main components in vegetable oil are mainly triglycerides (Fig. 7.1), which are composed of three fatty acid molecules which are esterified to 1 glycerol molecule, making up the main building blocks of vegetable oils (95%–98%) (Yara-Varón et al., 2017). They are considered nonpolar and lipophilic solvent where it varies depending on its nature, production method, and species. The characteristics and properties of vegetable oil are determined by the types, proportion, and structure of fatty acids on the glycerol backbone. As vegetable oil is obtained from plant seeds or fruits its fatty acid composition of triglycerides varies depending on cultivations, climate conditions, and species of seedlings used. The other percentage which makes up the vegetable oil are considered minor components ( 95 > 99.5 (frequent regeneration) i) Possible treatment for diluted mixtures ii) Various operating data iii) Adjustable and lower outlet concentration i) Requires pre-treatment of gas ii) Requires high energy for regeneration process

bed adsorber is shown in Fig. 8.4 and the adsorption system can be altered by fixing both adsorption and desorption cycles. Lastly, the general characteristics of fixed bed adsorber are tabulated in Table 8.5. The adsorption process is generally used and studied for solid-solid interface and there are important factors that affects the surface adsorption of the biomolecules which

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is the surface, liquid medium, and the physicochemical property of biomolecules. Apart from that, there are also some parameters identified in characterizing the adsorption process of the biomolecules such as the aggregation state, the amount adsorbed on surface, adsorption reversibility, and configuration change during the adsorption process (Migliorini et al., 2018). However, to have control over the biomolecules in both liquid or solid interface as the fundamental significance in various fields like diagnostics, pharmaceutics, drug delivery, or tissue engineering. It is crucial to characterize and understand how biomolecules link with the surfaces and measured parameters such as the adsorption and desorption kinetics, aggregation, orientation, and more. Hence, only by having a good understanding of the interfacial phenomena will aid in conserving the occupation of the biomolecules, having optimized engineering of the biofunctional surfaces as well as preventing side effects (Jena and Hore, 2010; Ojea-Jiménez et al., 2018).

8.6.3 Membrane separation Concentration, separation, and purification of every compound present in a chemical mixture is a vital issue in the processing industries. Conventional separation techniques including crystallization, distillation, solvent extraction, and more have been completed by the use of semipermeable membranes as the barrier of separation (Chen et al., 2018; Li et al., 2018a; Mat Aron et al., 2021; Zou et al., 2020). Aside from that, membrane processes are reported to be more efficient, economical, and faster than the existing commercial techniques in separating the components (Lin et al., 2020; Scholes et al., 2017; Yang et al., 2019). In the last few years, membrane separation processes have been developing from laboratory scale to utilizing essential industrial operations with notable commercial and technical impact (Cui and Chung, 2018; Handojo et al., 2019; Li et al., 2019; Lively and Sholl, 2017). Besides, membrane separation processes have also gained a noticeable growth on account of their outstanding characteristics such as the gratuitous in phase transformation, low energy consumption, and smaller footprint (Buonomenna and Bae, 2015; Pusch and Walch, 1982). The idea of a classic semipermeable membrane being able to perform the separation process between two species has been fully utilized above 150 years. However, practical separation process did not launch until 1900, until the first ion exchange membrane was launched in 1930 by Teorell, Meyer, and Sievers in developing the theory of ion transportation (Teorell, 1953). A working principle of pervaporation membrane is illustrated in Fig. 8.5. In order to subdivide solutes with higher purity and yield, it is necessary to reject approximately 100% of the solutes (Szekely et al., 2014). Yet, obtaining sharp separation in fractionating two or more solutes still remain a challenge. Additionally, in order to fractionate the solutes via membrane separation, a weighty amount of solutes must be used in achieving the desired purity. Other than that, the bottleneck of screening process in searching for a “right” membrane must be done during the development stage as the performance of the membranes is unpredictable based on the requirements of each specific separation. Therefore, an extensive range of membranes must be screened during the selection process. However, since the screening process of each membranes are rather time-consuming and may not guarantee the overall

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Figure 8.5 The principle of a pervaporation membrane.

efficiency of the membrane separation as compared to other conventional processes like flash chromatography and recrystallization, the development stage on membrane screening is advised to be reduced or eliminated as process development plays a huge role in contributing more than 30% of the cost in bringing a product to the market. Further, an alternative in replacing the screening of the “right” membrane is to address the challenge in the separation process using a perspective on engineering. For instance, to employ membrane cascades with existing membranes in achieving a desired separation outcome in reducing the membrane screening process. Remarkably, the potential in utilizing membrane cascades has been acknowledged. However, some minor drawbacks and challenges such as difficulty in control have also been reported (Mayani et al., 2010). Among different membrane technologies in the separation process, the organic solvent nanofiltration (NF) has been reported to be one of the common techniques in recovering organic solvents and concentrations of pharmaceutical products (Ferreira et al., 2017; Geens et al., 2007; Rundquist et al., 2012). Nevertheless, the major drawbacks of the membrane separation technology such as having a higher maintenance and operating costs still remain a challenge in operating at high pressure (Quek et al., 2021). On the other hand, forward osmosis (FO) has also obtained substantial attention in reusing water and desalination process for seawater (Cath et al., 2006; Chung et al., 2015; Valladares Linares et al., 2014; Zhao et al., 2012). FO process stood out among the normal pressure-driven membranes such as NF and RO as it takes full advantage of the chemical gradient principle across the semipermeable membrane in which the medium flows from lower concentration to higher concentration side

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Figure 8.6 Categories of various separation technologies and their respective cost as stated by their elemental principles. Modified from Keller et al. (2001).

instead of utilizing the external hydraulic pressure. For instance, FO has a low foul tendency while transporting similar feed solution as compared to the direct filter process. In addition, in comparison with the direct filter process, FO also has a relatively high recovery flux which may greatly decrease both maintenance and operational costs (Shen et al., 2017; Valladares Linares et al., 2014, 2016). In the contrary, as proposed by Lively and Sholl (2017), the organic solvent forward osmosis (OSFO) also utilizes similar principle which can simultaneously increase the feed concentration of pharmaceutical products during the transportation of organic solvent into the draw solution. After, the water-down draw solution can be revived by distillation, evaporation and direct filtration (Achilli et al., 2010; Ge and Chung, 2015). Besides, OSFO is normally used in demanding separation processes where the mother liquor is relatively high in osmotic pressure to take part in a pressure-driven process (Lively and Sholl, 2017). Nevertheless, parameters of OSFO such as cost, energy consumption, fouling, regeneration of draw solution, and the hybrid systems should be further investigated. To sum up, the separation technologies are summarized under four different categories comprising mechanical, physical, thermal and chemical according to each of their fundamental principles and their costs, as shown in Fig. 8.6. As described in the Bioseparation Handbooks by Ahuja (2000), both chemical and thermal separation techniques require higher effort in operating as compared to that in mechanical techniques. However, the engineering challenges faced in each type of bioparticles will continue to play a key role in mechanical separation process based on the unique characteristics of each bioparticles (Antfolk and Laurell, 2017).

8.7 Feasibility of solvent recovery process Selective industrial case studies have successfully demonstrated the practicable method for spent solvent recycling. ChemGenes has an overall recycled spent solvent yield of

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97.7% and recycled approximately 1340 liters of spent solvent in 4 months which has led to a savings of $3500 in the preventable purchase of solvents. Additionally, the payback period of both the solvent recycling process and equipment are also being reported to be approximately 1.8 years to a maximum of 3 years (2021). Apart from that, LymTal International Inc. recycles approximately 150 gallons of organic solvents where almost 99% are being recovered, namely mineral spirit every week, and has achieved an annual savings of $167k solely on spent solvent recovery process instead of buying new solvents. It is expected to reach a payback period of fewer than 6 months (Maratek, 2021). Protea Chemicals has also saved at least $8.4k in characterization and testing costs of virgin material and another $530 for solvent disposal and transportation (Green, 2016). Ho et al. (2008) reported that by using vacuum desorption in carbon capturing process, the capturing cost has been greatly reduced from $57 to $51 per ton of carbon dioxide captured in comparison with the conventional MEA absorption method. Besides, according to Valladares Linares et al. (2016), a hybrid FO lowpressure RO has a higher capital expenditure of 21% and a lower operating expenditure of 56% which caused a total cost reduction of 16% as compared to seawater reverse osmosis by reason of the savings from both fouling control and energy consumption. Hence, it has been proven that recycling spent solvent can not only save cost but also benefits the environment.

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Apurav Krishna Koyande a, Teoh Rui Hong a, Kit Wayne Chew b,c and Pau Loke Show a a Department of Chemical Engineering, Faculty of Science and Engineering, University of Nottingham Malaysia, Semenyih, Selangor, Malaysia, b School of Energy and Chemical Engineering, Xiamen University Malaysia, Sepang, Selangor Darul Ehsan, Malaysia, c College of Chemistry and Chemical Engineering, Xiamen University, Xiamen, Fujian, China

9.1

Introduction

The use of more efficient separation methods in the biochemical industry is a key area of development, due to the increased demand for various bioactive compounds. A key example is the increased demand for proteins such as industrial enzymes, due to their potential to catalyze reactions in many industries. The application of enzymes in manufacturing has proliferated into many industries such as the manufacture of food, pharmaceuticals, and biofuels, along with the treatment of solid and liquid wastes (Homaei et al., 2013). Furthermore, enzymes are favorable in many industries as they are highly specific in catalyzing reactions, reducing the formation of undesirable byproducts, along with the ability of enzymes to operate under milder conditions compared to traditional catalysts (Nadar et al., 2017). As a result, enzymatic catalysts in industrial processes can reduce the amount of raw material required for the manufacturing of a product, while also minimizing the environmental impact of the manufacturing process, through a reduction in energy consumption and waste generation (Jegannathan and Nielsen, 2013). However, the separation of proteins requires multiple processes such as precipitation, filtration, and chromatography. This results in high equipment costs and long processing times, along with large amounts of energy required to obtain the desired proteins (Nadar et al., 2017). Due to these factors, the cost of downstream processing of proteins can be responsible for 80% or more of the total cost of the production system (Aguilar and Rito-Palomares, 2010). A reduction in the number of separation processes and energy required can significantly reduce the cost of production of the desired proteins and reduce the environmental impact of these production processes. The production of other bioactive compounds such as oil, carbohydrates, and other small organic molecules also requires various methods of extraction, such as solvent extraction, supercritical fluid extraction, enzyme-assisted extraction, and ultrasonicassisted extraction (Gil-Chávez et al., 2013). These are typically followed by purification processes such as ultrafiltration and nanofiltration (Akin et al., 2012). The downstream processing of these bioactive compounds is also a large factor in the Principles of Multiple-Liquid Separation Systems: Interaction, Application and Advancement. DOI: https://doi.org/10.1016/B978-0-323-91728-5.00008-1 c 2023 Elsevier Inc. All rights reserved. Copyright 

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overall cost of these compounds, as large amounts of chemicals are required, along with the loss and degradation of the target compounds at each processing stage. As a result, an optimal method for the separation of these bioactive ingredients is desired, which requires less processing time, reduces the amount of energy used, minimizes environmental impact, and is cost-effective. The three-phase partitioning (TPP) is a method of bioseparation that can be used for the extraction, concentration, and purification of various biological compounds, such as proteins, saccharides, and oils, along with small organic molecules. The process of TPP begins with the addition of a salt, such as ammonium sulfate, to an aqueous solution that contains proteins. Tertiary butanol or t-butanol is then introduced to the aqueous solution, causing the solution to form three phases, with an t-butanol phase on top, an aqueous salt phase below, and an protein phase in the middle that has precipitated out of the solution (Dennison and Lovrien, 1997). As TPP consists of a polar aqueous phase and a nonpolar t-butanol phase, separation of proteins along with other molecules can be done simultaneously. Nonpolar compounds such as oils and lipids are separated into the t-butanol phase, and the aqueous salt phase will contain saccharides and other polar compounds (Tan et al., 2015). TPP has been used for upstream and downstream separation of biomolecules, along with use as an independent purification process. Various aspects of TPP are covered in this chapter, such as the fundamental principles of TPP, the design of a TPP system, and variants of TPP. Furthermore, the advantages and limitations of TPP are discussed along with the applications of TPP.

9.2 Principles of three-phase partitioning TPP has been used as a method of multiphase bio separation in both upstream and downstream processes. This is as it is a simple and efficient method of separating various proteins and other biomolecules from crude extracts. TPP was initially developed as an upstream separation method to precipitate enzymes such as cellulase. It has since been developed into an effective method of downstream separation for the isolation and purification of many other products. The initial step in the TPP process is the addition of an antichaotropic salt to the extract or suspension containing the desired product. Ammonium sulfate is typically used in this step. Next, an alcohol is added to the aqueous salt solution, with t-butanol being the solvent that is typically used. Following the addition of the solvent, the mixture is agitated and separated into three phases, as aqueous solutions of ammonium sulfate are not miscible with t-butanol (Yan et al., 2018). As t-butanol has a lower density than the aqueous solution of ammonium sulfate, the upper phase consists of t-butanol and the lower phase consists of aqueous ammonium sulfate. The precipitation of protein from the aqueous solution causes the third phase to form between the t-butanol and aqueous phases. The precipitation of the protein is caused by the binding of the alcohol to the hydrophobic section of the protein (Choonia and Lele, 2013). This reduces the density of the protein, causing the protein molecules to float and accumulate above the aqueous phase. Simultaneously, other molecules can be separated from the aqueous solution based on their polarity. Saccharides and other polar molecules recovered in the polar aqueous phase. Nonpolar

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Figure 9.1 Process of three-phase partitioning (TPP). Credit: Own figure.

molecules such as lipids and oils are recovered in the nonpolar t-butanol phase (Tan et al., 2015). This allows the simultaneous separation of different biomolecules from the same source using TPP, such as the extraction of oil and proteins from a single source (Panadare and Rathod, 2017). The TPP process is shown in Fig. 9.1. TPP can be compared to similar methods of separation, such as alcoholic precipitation and salting out. However, there are distinct differences between TPP and these methods of separation. TPP can be carried out at room temperature, decreasing the energy consumption, and equipment required. Furthermore, the addition of polymers to the system is not necessary, reducing the amount of separation required (Ketnawa et al., 2017). The mechanism for the precipitation of proteins in TPP is believed to be based on various principles that contribute to the separation of proteins from the aqueous solution. These include iso-ionic precipitation, salting out, conformation tightening, and kosmotropic precipitation (Dennison and Lovrien, 1997). The ability of TPP to precipitate proteins comes from the behavior of the alcohol and salt anion and their effect on the equilibrium of two reactions that precipitate the proteins from the aqueous solution. In the first reaction, the protein molecule is initially solubilized in water and in the free solution. The molecule is then conformationally tightened by the displacement of water molecules out of the protein. The macromolecule is squeezed shut and the extent of the molecules penetration by water is reduced. In the second reaction, the protein molecule is then precipitated or coprecipitated with bound sulfate ions and subsequently has the lowest penetration by water molecules. The sulfate anion and t-butanol have the effect of pushing and pulling on the two main reactions that cause the precipitation of the protein from the solution (Dennison and Lovrien, 1997). This pushing and pulling describe the effect on both the thermodynamics of the system and the physical aspect of the system. The pushing of the system represents the increase in the chemical potential of proteins that are present in the solution at the left side of the first reaction. The pulling represents the decrease in the chemical potential of products on the right of the first and second reactions. Pushing of the reaction due to the sulfate anion is caused by the thermodynamic effect of exclusion-crowding, producing conformation tightening along with changes in the hydration of protein in the solution. Pulling of the reaction is also caused by the sulfate anion, although lower concentrations of the anion are required. The sulfate anions

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Figure 9.2 Phase diagram of ammonium sulfate/t-butanol/water system. From Kiss, É., Szamos, J., Tamás, B. & Borbás, R. (1998). Interfacial behavior of proteins in three-phase partitioning using salt-containing water/tert-butanol systems. Colloids and Surfaces A: Physicochemical and Engineering Aspects, 142(2), 295–302. https://doi.org/10.1016/S0927-7757(98)00361-6.

are attracted to the cationic groups of the protein molecule and bind with these groups. This increases the conformational tightening and reduces the hydration of the protein (Yan et al., 2018). Subsequently, the proteins form sulfate salts after binding with the sulfate anions. The stability of proteins that have formed sulfate salts is higher compared with those that have not formed into salts. Some versions of TPP may be hybrids, with both the pushing and pulling of the reaction occurring in these versions of TPP. The concentration of t-butanol, water, and ammonium sulfate in the system has a significant effect on the effectiveness of TPP. The phase diagram of this threecomponent system is shown in Fig. 9.2. Below the tie line toward the water corner of the phase diagram, the system forms one miscible liquid phase. Two immiscible phases form within the area with tie lines, while above the tie lines, ammonium sulfate is present in excess as solid salt. As a result, the area with tie lines within the phase diagram represents the range of compositions that TPP can be conducted with. As ammonium sulfate has a higher solubility relative to other salts such as sodium sulfate, the area in the phase diagram where partitioning can be conducted is large. The length of each tie line indicates the difference in composition between the equilibrium liquid phases, with longer tie lines indicating a larger difference between the equilibrium compositions. The interfacial tension between the two liquid phases is also indicated by the length of the tie lines, with longer tie lines having higher values of interfacial tension. The length of the tie line is a good indicator of the effectiveness of the system, as the yield of systems with shorter tie lines was between 70% and 80%, whereas systems with longer tie lines had higher yields between 95% and 100% (Kiss et al., 1998). The interfacial tension

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between the two liquid phases is a determining factor in the amount of protein that forms in the intermediate layer during TPP. The composition that should be used for the TPP system should be based on the desired effectiveness of the system, with various compositions that are on the same tie line resulting in the same equilibrium composition and the same yield. Furthermore, identical accumulation ratios can be achieved with different compositions on the same tie line, with the suitable composition chosen based on additional aspects such as economic considerations. An important observation in the use of TPP is the concentration of protein within the precipitated layer is independent of the concentration of protein initially observed in the system. This is different from the salting-out process, where the precipitation of the protein from a salt solution is determined by its solubility (Rachana and Jose, 2014).

9.3

Variables that affect TPP

9.3.1 Solvent concentration Various solvents have been studied for use in TPP, such as t-butanol, ethanol, isopropanol, and 1-octanol. Although many solvents can be used for the TPP process, t-butanol is the most commonly used. This is due to the higher yield typically achieved with the use of t-butanol compared to other solvents. A reason for this is that t-butanol has a large, branched structure that prevents it from entering the protein molecules. As a result, the proteins are stabilized instead of denatured and their structure remains intact (Harald and Dennison, 2011). Furthermore, t-butanol can inhibit enzyme activity reversibly for many enzymes, which may contribute to its stabilizing effect on many proteins. The ratio of t-butanol to crude extract for the purification of milk clotting proteases from Wrightia tinctoria R. Br. was studied by Rajagopalan and Sukumaran (2018). The ratios of crude extract to t-butanol studied were from 1:0.5 to 1:2.0, with the highest activity recovery at 90% and fold purification of 23 obtained at a 1:1 ratio. An increase in the ratio to 1:1.5 caused the activity recovery to drop significantly to approximately 10% and the fold purification dropped to 8. This is as at high concentrations of tbutanol, the denaturation of protein is likely and the activity of the proteins is reduced as a result (Chaiwut et al., 2010). At a concentration of 1:0.5 of crude extract to t-butanol, the activity recovery was also low at 10%, with a purification fold of 18. This can be attributed to the reduction in the amount of t-butanol available, preventing effective synergy between the t-butanol and ammonium sulfate, causing the precipitation of the proteins to be reduced. Similar results were found by Garg and Thorat (2014), who studied the extraction of nattokinase from fermentation broth. The yield at a crude extract to t-butanol ratio of 1:0.5 was approximately 80%, with a fold purity of 4. At a ratio of 1:1 and 1:1.5, the yield and fold purity were approximately the same at 130% and 5.5, respectively. The yield dropped to 90% at a ratio of 1:2, with a fold purity of 4. As different concentrations of t-butanol significantly impact the recovery of the TPP process, the optimal concentration of t-butanol and salt to aqueous solution for industrial use would be the mixture that allows for the highest recovery at the lowest cost, which would be dependent on the characteristics of each system.

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9.3.2 Concentration of salt The effect of salt is significant in the TPP process, as the interaction between protein molecules and solution is highly influenced by the concentration of salt present in the solution. This is due to interactions between charged groups on the protein molecules and salt ions. At low concentrations, salts are attracted to the solution by the charged groups on the protein molecules, causing the proteins to be more easily dissolved. This is due to the disruption of the interaction between protein molecules. As the salt concentration is increased, the solubility of the proteins reaches a maximum. If the concentration of salt is increased past this point, the interaction between proteins is strengthened due to the kosmotropic properties of the salt (Zhang, 2012). This results in the decrease in the solubility of the proteins and the precipitation of the protein as a result. The effect of salt on the extraction of cellular material is also a significant factor in the use of TPP. The suspension and mixing of cells in a solution with a high concentration of salt lead to the cells experiencing large osmotic shocks, causing the cells to rupture (Li et al., 2015). This rupture increases the permeability of the cell and results in the release of material from the cell cytosol into the solution. As a result, molecules such as proteins and oil bodies are released into the solution, leading to the precipitation of protein and extraction of oils from these cells (Dutta et al., 2015). The amount of salt required is also dependent on the component that is to be recovered, such as in the recovery of oil from cells, which requires precipitation of the proteins present in lipid bodies to release the oils present. An excess of salt can lead to the denaturation of proteins and a reduction in the yield of oil obtained (Panadare and Rathod, 2017). Salt concentration and its effect on the TPP process used to extract milk clotting proteases from Wrightia tinctoria R. Br. was studied by Rajagopalan and Sukumaran (2018). The concentration of ammonium sulfate varied from 30% to 70%, with a ratio of t-butanol to crude extract of 1:1. The maximum recovery of 90% and purification fold of 22 were obtained at a concentration of 60% ammonium sulfate. The recovery percentage was below 9% and purification fold was below 12, from 30% to 50% concentration of ammonium sulfate. At 70% ammonium sulfate, the recovery was reduced to 80% and the purification fold was reduced to 15, indicating that other proteins are precipitated in addition to protease, due to the lower specific activity of the sample. As a result, the concentration of 60% ammonium sulfate was found to be optimal for this process. The effect of salt concentration on the recovery of fat from the Garcinia indica kernel using TPP was studied by Vidhate and Singhal (2013). The range of concentration of ammonium sulfate studied was from 10% (w/v) to 60% (w/v), with a constant ratio of 1:1 of t-butanol to crude extract. The recovery of fat increased from 10% (w/v) to 50% (w/v). The maximum recovery of fat was obtained at 50% (w/v) concentration of ammonium sulfate, with a recovery of 94.6% (w/w). Above this concentration, there was no significant change in the recovery. Another study by Varakumar et al. (2017) was conducted on the recovery of oleoresins from Zingiber officinale rhizome powder using TPP. A constant ratio of 0.5:1 of t-butanol to crude extract was used, while the range of concentration of ammonium sulfate studied was from 10% (w/v) to 50% (w/v). The maximum yield of gingerols was obtained at a

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concentration of 10% (w/v) ammonium sulfate. Above this concentration, the yield of gingerols was reduced. The large difference between the concentration of ammonium sulfate required for extraction of oils from different sources may be explained by differences in the protein network that contains oils within lipid bodies (Panadare and Rathod, 2017).

9.3.3 Temperature The rate of reaction of enzymes increases as the temperature of the medium increases. However, after the temperature reaches a certain point, the enzymes start to denature, causing the rate of reaction to decrease. As a result, TPP is usually conducted at low temperatures in order to dissipate any heat that is generated by the reaction, in order to prevent denaturation of the proteins (Garg and Thorat, 2014). The TPP of fibrinolytic enzyme from Bacillus sphaericus was studied with changes in temperature between 10 and 50 °C (Avhad et al., 2014). A maximum of 45% recovery and 9.98-fold purity was achieved at 30°C, with a decrease at lower temperatures and higher temperatures. This is as the rate of reaction of the TPP process is reduced at lower temperatures, along with the denaturation of the enzymes at higher temperatures. A study on the activity of ficin extracted from latex at temperatures between 20-90°C found that the optimum temperature was at 60°C (Gagaoua et al., 2014). Above this temperature, the enzyme activity and stability decreased due to denaturation. Therefore, conducting the TPP process at high temperatures would not be suitable due to damage to protein molecules. For industrial applications, the use of temperatures above room temperature would require energy consumption for heating, along with additional controls to ensure the TPP system does not overheat and denature the proteins.

9.3.4 pH The pH of the medium has a strong effect on the behavior of proteins, as changes in pH affect the ionization of amino acids, causing the interaction between the charged proteins and the solution to change according to the pH of the solution. These changes in interaction cause the partitioning and solubility of proteins to be heavily influenced by the pH of the solution. The net charge of the protein is dependent on the pH of the system and the pl (isoelectric point) of the protein. If the pl of the protein is above the pH of the solution, the protein obtains a positive net charge, causing it to precipitate out of the aqueous solution. If the pl of the protein is below the pH of the solution, the protein will obtain a negative net charge and it will soluble in the solution (Vetal and Rathod, 2015). A study on the extraction of peroxidase from Citrus sinenses peel found that the optimum pH for TPP was at pH 6 (Vetal and Rathod, 2015). As the peroxidase recovery and fold purification was found to be high in the aqueous phase compared to the top and intermediate phase, the bottom phase was chosen for analysis. As the pl for peroxidase was previously found to be between 4.5 and 5.2, at pH 6, the net charge on the protein was negative, increasing its partitioning into the aqueous phase while other undesired proteins were precipitated into the intermediate phase. In another study of the recovery

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of catalase from sweet potatoes, the optimum pH was found to be 7 (Duman and Kaya, 2013). In this study, catalase was recovered in the intermediate phase. Five pH values between pH 4–9 were studied and at pH 7, the recovery was found to be at 262%, with a fold purification of 14.1. Above pH 7, the recovery and fold purification dropped significantly, indicating that catalase did not precipitate out of the aqueous phase and instead remained solubilized. The optimum conditions for the extraction of various compounds using TPP are shown in Table 9.1.

9.4 Types of assisted TPP 9.4.1 Microwave-assisted TPP (MATPP) Microwave-assisted TPP is based on microwave-assisted extraction, which requires the exposure of the extract and solvent to microwave energy. This microwave energy heats the system and allows the desired compounds to separate from the extract into the solvent. This reduces the time required for extraction and the amount of solvent that is used for the extraction (Dahmoune et al., 2015). The performance of this process is dependent on the temperature of the system along with the polarity of the solvent. The use of microwave energy results in an increase of temperature and pressure in plant cells, due to the heating of moisture within the cell environment. As a result, the desired compounds are separated from the cell and spread into the solvent at a higher rate compared with conventional extraction (Rahath Kubra et al., 2013). A study was conducted on the performance of microwave-assisted TPP for the purification of mangiferin (Kulkarni and Rathod, 2015). The amount of time the system is exposed to microwaves was found to be significant, as the evaporation of solvent was affected by the duration of exposure to the microwaves. The optimum time was found to be 5 min, with longer exposure causing solvent evaporation, charring, and reducing the yield of mangiferin. The duty cycle of the microwave system also had a significant effect on the performance of the system. This is as the pulsed operation of microwaves can prevent excess heat and subsequent degradation of the compound. The optimum duty cycle was found to be 50%, as the maximum yield was obtained at this setting. A reduced energy consumption and minimal evaporation of the solvent were also observed at this duty cycle. A microwave power of 272 W was found to be optimal, as this power allows the maximum heating in the sample, causing the plant matrix to be destroyed and allowing the solvent to penetrate the plant cell. At a higher power, charring of the particles was observed, reducing the yield obtained. The use of MATPP compared to conventional TPP for the purification of laccase was studied (Patil and Yadav, 2018) and found that the use of MATPP resulted in an increase of fold purity from 2.4 to 13.9, and an increase of yield from 53.9% to 59.8% when compared to conventional TPP. This is in addition to a reduction in processing time from 60 min to 7 min. The use of MATPP to extract protein from microalgae was studied by Chew et al. (2019). The addition of MATPP increased the yield of protein from 24.9% to 63.2%. This is an increase of approximately 2.5 times, and is attributed to the ability of microwaves to disrupt the cell structure of the microalgae. As a result, the use of

Source Abrus precatorius Aloe vera Bacillus sphaericus Bacillus natto Citrus sinenses peel Ficus caricaL. Latex Garcinia indica Serratia marcescens Sweet potato Wrightia tinctoria

Product 49.5 Aloe protein Aloe polysaccharide Fibrinolytic enzyme Nattokinase Peroxidase Ficin Fat Serratiopeptidase Catlase Proteases

Extract to solvent ratio 1:1.15 1:0.25

Ammonium sulfate (%) 49.5 26.4

Temp (°C) 70 30

pH 5.2 6.5

1:0.5 1:1.5 1:1.5 1:0.75 1:1 1.5:1 1:1 1:1

80 30 50 40 50 30 40 60

30 37 30 25 45 25 35 50

9 8 6 7 2 7 7 7.5

Recovery (%) 156.2 92.3 92.78 65 129.5 94 167 95 96 262 90

Reference Sagu et al. (2015) Tan et al. (2015) Avhad et al. (2014) Garg and Thorat (2014) Vetal and Rathod (2015) Gagaoua et al. (2014) Vidhate and Singhal (2013) Pakhale and Bhagwat (2016) Duman and Kaya (2013) Rajagopalan and Sukumaran (2018)

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Table 9.1 Optimum conditions for extraction using three phase partitioning.

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MATPP should be considered in order to improve the purity and yield of a process along with reducing the processing time required.

9.4.2 Ultrasound-assisted TPP (UATPP) The use of ultrasound has been shown to increase the efficiency of many bioprocesses, such as fermentation, biocatalysis, and extraction. This is as ultrasound can increase the yield of these processes by improving the mass transfer characteristics of the system (Pakhale and Bhagwat, 2016). Therefore, the use of ultrasound to assist the TPP process can greatly increase the efficiency of the process, as the mass transfer characteristics of the system affect TPP significantly. The use of ultrasonic waves causes cavitation in the solution, where bubbles filled with gas from within the solution, which subsequently collapse. When these bubbles collapse, a shock wave is formed, and mechanical shear is imparted onto the surrounding solution. As a result of this mechanical shear, along with the change in energy density within the local environment, the mass transfer across different phases is increased (Avhad et al., 2014). Furthermore, this promotes the disruption of cell walls, resulting in better penetration of solvent into the cells and allowing the surface area in contact between the compound embedded within the cell and the solvent to be increased. Various parameters affect the cavitation of the solution, such as the solvent used, the ratio of solvent to solute, power used, along with other parameters (Kulkarni and Rathod, 2014). The optimization of these parameters can increase the mass transfer caused by the cavitation of the solution. In a study of the purification of serratiopeptidase using UATPP, a fold purification of 4.2 was achieved with unassisted TPP, along with an 83% recovery (Pakhale and Bhagwat, 2016). The use of ultrasound to assist the TPP process resulted in an increased purification fold of 9.4 and a recovery of 96%. This is coupled with a significantly lower process time of 5 min, compared with a processing time of 60 min for unassisted TPP. Another study was conducted to compare the use of UATPP, TPP, and solvent extraction for the extraction of oleanolic acid (Vetal et al., 2014). UATPP had the highest extraction yield at 85.6%, followed by TPP with an extraction yield of 80.7% and solvent extraction had the lowest extraction yield at 60.3%. The extraction time was the longest for TPP at 120 min, followed by solvent extraction at 40 min and UATPP had the shortest time at 14 min. The use of UATPP can increase recovery and purity, along with a reduction in process time. These advantages make UATPP a promising way to improve the conventional TPP process.

9.5 Applications of TPP 9.5.1 Proteins The use of TPP to recover and purify proteins at a higher purity and yield can be applied to various proteins that are used extensively in industries across the world. A significant protein that is used in industry is amylase, an enzyme that is used in many industries to depolymerize starch. This enzyme is prevalent in many industries, such as in the

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production of food, pharmaceuticals, textiles, and paper. As a result, amylase is the most produced enzyme worldwide. However, the production of amylase is dependent on specific strains of bacteria and fungi, limiting the use of amylase to specialized processes (Amid and Manap, 2014). The extraction and purification of beta-amylase from the stems of Abrus precatorius using TPP was studied as an alternative method of producing amylase (Sagu et al., 2015). The activity recovery was found to be 158% at optimal conditions with a purification factor of 10.1. The pH stability and thermal stability of the enzyme obtained from the TPP process were found to be similar to values that were obtained from previous studies of beta-amylase. This indicates that the beta-amylase extracted using the TPP process had similar characteristics to betaamylase from other sources. Furthermore, the stability of the enzyme allows it to be used under industrial processing conditions without a reduction in activity. The use of TPP for the extraction of amylase is a promising application of TPP, especially as the raw material used does not require microorganisms.

9.5.2 Polysaccharides The partitioning behavior of TPP causes polar components such as polysaccharides to accumulate in the bottom aqueous layer, allowing TPP to be used for the recovery of these polysaccharides from crude extracts. The extraction of polysaccharides from Corbicula fluminea using TPP has been studied (Yan et al., 2017). An extraction yield of 9.32% was achieved at the optimum process conditions, with the extracted polysaccharide requiring purification through dialysis, followed by concentration and lyophilization. The bioactive properties of these polysaccharides were then analyzed, with antioxidant activity and the capacity to scavenge free radicals being present. This preservation of bioactive properties allows TPP to be used as a method of separation for various bioactive polysaccharides for pharmaceutical and nutrition industries, whereas conventional separation methods such as enzyme extraction require large volumes of solvent and lengthy processing times. TPP can also be used for the simultaneous purification of both proteins and polysaccharides, as they are separated into two different phases. This has been studied for the purification of aloe polysaccharide and protein (Tan et al., 2015). Under optimized conditions, the extraction efficiency of aloe polysaccharide was 92.3% and the extraction efficiency of aloe protein was 92.78%. TPP is favorable compared to other methods of purification, as methods such as ionic liquid based aqueous two-phase system (IL-ATPS) require pretreatment using alcohol precipitation. Furthermore, protein is discarded as an impurity when the polysaccharide is recovered. As a result, TPP can be used as a more efficient and cost-effective method of purifying these polysaccharides.

9.5.3 Lipids The demand for lipids and oils has been increasing worldwide, as they are required as raw materials for many products such as paints, waxes and cosmetics. The use of solvent extraction with hexane for the recovery of oils and lipids is widespread, as it is convenient, cost-effective, and noncorrosive. However, the use of hexane as a solvent is

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Figure 9.3 Biorefinery of microalgae using liquid triphasic flotation (LTF) system. Credit: From Koyande, A. K., Chew, K. W., Lim, J.-W., Lam, M.-K., Ho, Y.-C. & Show, P.-L. (2020). Biorefinery of Chlorella sorokiniana using ultra sonication assisted liquid triphasic flotation system. Bioresource Technology, 303, 122931. https://doi.org/10.1016/j.biortech.2020.122931.

a safety hazard, as it is highly flammable and is classified as an air pollutant. Therefore, the use of other methods of extraction that are less harmful to the environment and are less hazardous is favorable. A study of the extraction of oil extraction from Spirogyra sp. using ultrasound-assisted TPP found that the yield with UATPP was favorable compared to conventional solvent extraction. The yield obtained with UATPP was 12.5%, compared with conventional extraction which had a yield of 4%. The use of TPP for the extraction of oil has good potential, as the yield is shown to be high, along with being more environmentally friendly and less hazardous compared to conventional solvent extraction.

9.6 Future prospects The use of TPP in the industry is promising, although certain aspects of TPP such as obtaining effective mass transfer within the system can affect the processing of extracts using TPP. As a result, novel methods of improving TPP such as the liquid

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triphasic flotation (LTF) have been studied. LTF combines the TPP process with flotation through the introduction of air bubbles to the TPP system, as illustrated in Fig. 9.3. These air bubbles cause the extraction of protein to be accelerated. The use of LTF to simultaneously obtain lipids and proteins from microalgae was studied by Koyande et al. (2020). The use of LTF increased the protein recovery from 70.9% to 82.1%, along with increasing the lipid recovery from 56% to 63.9%. The addition of ultrasonication to the LTF process increased the protein recovery to 97.4% and the lipid recovery to 69.5%. The use of LTF shows a significant improvement compared to conventional TPP, which can be coupled with ultrasonication in order to increase the recovery significantly. The implementation of TPP in the industry requires additional study, as the use of TPP at industrial scales is not widely reported (Gautam et al., 2012). However, the high purity and recovery of bioactive compounds using TPP is highly desirable for future bioprocesses.

9.7

Conclusion

TPP is a promising separation process for bioprocessing, which requires lower capital costs and higher efficiency compared to conventional separation processes. Optimization of the variables affecting the TPP process can result in high recovery and yield through a single-step TPP process. This is a significant advantage compared to conventional separation processes that require multiple processes. Furthermore, the use of assisted TPP allows the recovery and yield to be further improved, increasing the efficiency of the TPP process. The extraction of multiple bioactive compounds can also be conducted simultaneously using TPP, reducing the number of processes required along with the associated costs. Although the use of TPP is relatively limited, the development and implementation of TPP at industrial scales is desirable due to the high yield and recovery that can be obtained using TPP.

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Kulkarni, V.M., Rathod, V.K., 2014. Extraction of mangiferin from Mangifera indica leaves using three phase partitioning coupled with ultrasound. Ind. Crops Prod. 52, 292–297. https://doi.org/10.1016/j.indcrop.2013.10.032. Kulkarni, V.M., Rathod, V.K., 2015. A novel method to augment extraction of mangiferin by application of microwave on three phase partitioning. Biotechnology Reports 6, 8–12. https://doi.org/10.1016/j.btre.2014.12.009. Li, Z., Li, Y., Zhang, X., Tan, T., 2015. Lipid extraction from non-broken and high water content microalgae Chlorella spp. by three-phase partitioning. Algal Research 10, 218–223. https://doi.org/10.1016/j.algal.2015.04.021. Nadar, S.S., Pawar, R.G., Rathod, V.K., 2017. Recent advances in enzyme extraction strategies: a comprehensive review. Int. J. Biol. Macromol. 101, 931–957. https://doi.org/ 10.1016/j.ijbiomac.2017.03.055. Pakhale, S.V., Bhagwat, S.S., 2016. Purification of serratiopeptidase from Serratia marcescens NRRL B 23112 using ultrasound assisted three phase partitioning. Ultrason. Sonochem. 31, 532–538. https://doi.org/10.1016/j.ultsonch.2016.01.037. Panadare, D.C., Rathod, V.K., 2017. Three phase partitioning for extraction of oil: a review. Trends Food Sci. Technol. 68, 145–151. https://doi.org/10.1016/j.tifs.2017.08.004. Patil, P.D., Yadav, G.D., 2018. Application of microwave assisted three phase partitioning method for purification of laccase from Trametes hirsuta. Process Biochem. 65, 220–227. https://doi.org/10.1016/j.procbio.2017.10.006. Rachana, C., Jose, V., 2014. Three phase partitioning-a novel protein purification method. Int. J. Chem. Tech. Res 6 (7), 3467–3472. Rahath Kubra, I., Kumar, D., Rao, L.J.M., 2013. Effect of microwave-assisted extraction on the release of polyphenols from ginger (Zingiber officinale). International Journal of Food Science & Technology 48 (9), 1828–1833. https://doi.org/10.1111/ijfs.12157. Rajagopalan, A., Sukumaran, B.O., 2018. Three phase partitioning to concentrate milk clotting proteases from Wrightia tinctoria R. Br and its characterization. Int. J. Biol. Macromol. 118, 279–288. https://doi.org/10.1016/j.ijbiomac.2018.06.042. Sagu, S.T., Nso, E.J., Homann, T., Kapseu, C., Rawel, H.M., 2015. Extraction and purification of beta-amylase from stems of Abrus precatorius by three phase partitioning. Food Chem. 183, 144–153. https://doi.org/10.1016/j.foodchem.2015.03.028. Tan, Z.-J., Wang, C.-Y., Yi, Y.-J., Wang, H.-Y., Zhou, W.-L., Tan, S.-Y., Li, F.-F., 2015. Three phase partitioning for simultaneous purification of aloe polysaccharide and protein using a single-step extraction. Process Biochem. 50 (3), 482–486. https://doi.org/ 10.1016/j.procbio.2015.01.004. Varakumar, S., Umesh, K.V., Singhal, R.S., 2017. Enhanced extraction of oleoresin from ginger (Zingiber officinale) rhizome powder using enzyme-assisted three phase partitioning. Food Chem. 216, 27–36. https://doi.org/10.1016/j.foodchem.2016.07.180. Vetal, M.D., Rathod, V.K., 2015. Three phase partitioning a novel technique for purification of peroxidase from orange peels (Citrus sinenses). Food Bioprod. Process. 94, 284–289. https://doi.org/10.1016/j.fbp.2014.03.007. Vetal, M.D., Shirpurkar, N.D., Rathod, V.K., 2014. Three phase partitioning coupled with ultrasound for the extraction of ursolic acid and oleanolic acid from Ocimum sanctum. Food Bioprod. Process. 92 (4), 402–408. https://doi.org/10.1016/j.fbp.2013.09.002. Vidhate, G.S., Singhal, R.S., 2013. Extraction of cocoa butter alternative from kokum (Garcinia indica) kernel by three phase partitioning. J. Food Eng. 117 (4), 464–466. https://doi.org/10.1016/j.jfoodeng.2012.10.051. Yan, J.-K., Wang, Y.-Y., Qiu, W.-Y., Shao, N., 2017. Three-phase partitioning for efficient extraction and separation of polysaccharides from Corbicula fluminea. Carbohydr. Polym. 163, 10–19. https://doi.org/10.1016/j.carbpol.2017.01.021.

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Yan, J.-K., Wang, Y.-Y., Qiu, W.-Y., Ma, H., Wang, Z.-B., Wu, J.-Y., 2018. Three-phase partitioning as an elegant and versatile platform applied to nonchromatographic bioseparation processes. Crit. Rev. Food Sci. Nutr. 58 (14), 2416–2431. https://doi.org/ 10.1080/10408398.2017.1327418. Zhang, J., 2012. Protein-protein interactions in salt solutions. Protein-Protein InteractionsComputational and Experimental Tools 1, 359–376.

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Nguyen Minh Duc a, Shir Reen Chia b,c, Saifuddin Nomanbhay b and Vishno Vardhan Devadas d a School of Biosciences, Faculty of Science and Engineering, University of Nottingham Malaysia, Semenyih, Selangor Darul Ehsan, Malaysia, b Institute of Sustainable Energy, Universiti Tenaga Nasional (UNITEN), Jalan IKRAM-UNITEN, Kajang, Selangor Darul Ehsan, Malaysia, c AAIBE Chair of Renewable Energy, Institute of Sustainable Energy, Universiti Tenaga Nasional (UNITEN), Jalan IKRAM-UNITEN, Kajang, Selangor Darul Ehsan, Malaysia, d Department of Chemical and Environmental Engineering, University of Nottingham Malaysia, Semenyih, Selangor Darul Ehsan, Malaysia

10.1

Introduction

With the rapid growth of the population and the field of biotechnology, the demand for biological molecules such as enzymes to serve in various industries such as food and pharmaceutical manufacturing has also increased significantly. However, the production of these molecules is often limited by downstream processing which can account for more than half of the production cost (Kim et al., 2013; Sankaran et al., 2019). During the separation and purification steps, the most extensively utilized techniques include organic solvent, chromatography-based methods, centrifugation, and filtration despite their disadvantages of high cost, time-consuming due to multiple steps, high energy input, and low biocompatibility. Therefore, alternative methods based on multiphase liquid systems have been growing in popularity due to their relatively lower cost, ease of upscaling, high biocompatibility, and flexibility (Khoo et al., 2020; Sankaran et al., 2019). Multiphase liquid systems are based on the principle that several aqueous solutions (polymer, salt, alcohol, etc.) mixed together will form different phases beyond a certain critical condition, and this interaction can be used to separate different components of a mixture by partitioning of those molecules to the more favorable phase (Iqbal et al., 2016; Khoo et al., 2020). Although over 300 different systems with as much as six phases have been proposed (Mace et al., 2012), the majority of research and applications in the field of bioprocessing have been focusing on utilizing only two phases; until recent years, when several three-phase systems were investigated (Morandeira et al., 2019). Known by many names such as liquid biphasic system (LBS), aqueous biphasic system, or aqueous two-phase system (ATPS), it has shown tremendous potential as a highly efficient separation and purification method for valuable biological materials (Freire et al., 2012; Khoo et al., 2020; Raja et al., 2011). Current trends in the development of LBS include: looking for alternative solvents Principles of Multiple-Liquid Separation Systems: Interaction, Application and Advancement. DOI: https://doi.org/10.1016/B978-0-323-91728-5.00017-2 c 2023 Elsevier Inc. All rights reserved. Copyright 

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Figure 10.1 Schematic diagram of liquid biphasic systems (LBS).

with better cost, efficiency, and recyclability (Freire et al., 2012; Show et al., 2013; Xu et al., 2015); incorporating other systems to aid with partitioning and reduce the overall number of steps during downstream processing (Khoo et al., 2020; Sankaran et al., 2019) or designing a continuous system for industrial applications instead of the traditional laboratory batch-scale (Espitia-Saloma et al., 2014; Ferreira-Faria et al., 2020; Pereira et al., 2020). This chapter is dedicated to the overall design and mechanism of LBS as well as the advance designs developed in recent years. Furthermore, the key factors, advantages, and limitations of such systems will also be evaluated.

10.2

Liquid biphasic system

10.2.1 Overall mechanism LBS was first observed by Martinus W. Beijerinck back in 1896 when he mixed gelatin with starch in an aqueous solution and noticed the formation of two immiscible phases. More than half a century later, it was rediscovered by Per-Åke Albertsson, who described the phenomenon in detail and kick-started the development of LBS as a separation method (Albertsson, 1970; Grilo et al., 2016). Fig. 10.1 describes the mechanism of a typical LBS for the purpose of separating a valuable biomolecule from a crude extract. The crude extract is thoroughly mixed with the two–phase-forming components in an aqueous solution by mild mechanical methods such as shaking and agitation. Then, partitioning of the target biomolecule (usually to the top phase) and the contaminants (usually to the bottom phase) occurs along the formation of two immiscible phases, either through gradual gravitational

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settling or rapid centrifugation within the container. Finally, the two phases can be easily separated for further purification and polishing. The exact driving forces for the partitioning effect are still not fully understood and are still debated among the scientific community. Several notable models have been proposed, including the excluded volume effect (Luechau et al., 2009), hydrophobic interaction (Andrews and Asenjo, 2010), and special affinity between the target molecule and the phase-forming solute (Rocha and Nerli, 2013) but none have fully explained or making predictions that match with real experimental results (Pirdashti et al., 2015). Nevertheless, it is usually agreed that a combination of all of the mentioned interactions and several others such as Van der Waal force, electrochemical, ionic, and conformation interaction are responsible for the partitioning effect (Grilo et al., 2016; Iqbal et al., 2016). The most common phase-forming combinations are two polymers (e.g., polyethylene glycol [PEG] and dextran), a polymer with a salt (e.g., ammonium phosphate), and an alcohol (e.g., ethanol) with a salt. Other phase-forming components such as ionic liquids (IL), sugars, and surfactants/detergents have also been considered as suitable alternatives (Khoo et al., 2020).

10.2.2 Key factors Since the LBS component is essentially unchanged throughout most of the advance systems discussed later, the common key factors like phase-forming component, pH, and temperature should have similar effect regardless of design.

10.2.2.1

Phase-forming component

For polymers such as PEG, phase formation occurs more easily and at lower concentration when the molecular weight of the polymer increases. However, partitioning toward the polymer-rich phase decreases with the increase in molecular weight and concentration due to an increase in hydrophobic groups and reduce in free volume (Goja et al., 2013; Grilo et al., 2016; Iqbal et al., 2016). Salt phases usually promote negatively charged molecules such as proteins to partitioning away from the salt phase and high salt concentration has shown to increase both partition and phase-forming efficiency due to the “salting-out effect” (Khoo et al., 2020; Mohammadi and Omidinia, 2013; Wang et al., 2010). However, too much salt content will result in the decrease of efficiency instead (Phong et al., 2017a). The type of salt phase is also crucial for the efficiency of LBS; however, it varies greatly between different systems and target molecule. Although phosphate, sulfate, and citrate are among the most commonly used and well-studied (Goja et al., 2013), systems using other types of salt such as carbonate may achieve better efficiency (Mohammadi and Omidinia, 2013) and is case-to-case dependent. Similarly, both alcohol type and concentration are crucial factors affecting LBS. Methanol and ethanol have shown undesirable properties such as high volatility and low hydrophobicity despite being the cheapest and the most readily available (Mathiazakan et al., 2016). 1-Propanol was found to be ideal for use in LBS instead

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since an increase in carbon chain length increases hydrophobicity, therefore increases phase-formation capability and partitioning efficiency (Mathiazakan et al., 2016; Tan et al., 2013). Furthermore, t-butanol is also being utilized in a variation of LBS called three-phase partitioning, where the target molecules precipitate in the interface instead of partitioning to either, thus creating the “third phase” (Gagaoua et al., 2014).

10.2.2.2

pH

pH is a very important for the partition of biomolecules, especially proteins due to their isoelectric point (pI). A protein can be negatively charged if the pH is higher than its pI, or positively charged if the pH is lower. Moreover, negatively charged molecules tend to partition toward the PEG phase and positively charged molecules tend to favor the salt-rich phase in polymer–salt LBS (Iqbal et al., 2016). As a result, the target molecules can be directed to either phase by simply changing the pH of the system. In addition, most biomolecules are stable at neutral pH which is also the favorable condition for the operation of LBS (Goja et al., 2013). However, optimal partitioning of several proteins has been shown to occur at pH 8 (Iqbal et al., 2016) and pH 9 (Chow et al., 2015) despite the protein degradation risk at high pH. Hence, the effect of pH can vary greatly and must be investigated.

10.2.2.3

Temperature

Most LBS systems operate at 20–40°C to maintain the bioactivity of the target molecules which are heat-sensitive. Polymer–salt systems usually reach optimum at higher temperature while polymer–polymer systems prefer lower temperatures (Grilo et al., 2016; Khoo et al., 2020). This is due to phase-forming components react differently to the change in temperature, which usually results in the alteration of their viscosity, density, and partition efficiency.

10.2.3 Advantages and limitations Conventional liquid–liquid separation methods often involve the use of organic solvents or extreme conditions such as high pressure/temperature, which could damage the biological product. Moreover, organic solvents are recognized as a hazard to the environment. In contrast, LBS provides a mild environment comprises of mostly water (80% or more) and biocompatible compounds (Lee et al., 2016) that mimic the natural environment inside the cells that preserves the bioactivity of the products. In addition, most of the phase-forming components are regarded as nontoxic, with some of them can even be recycled or safely biodegraded (Grilo et al., 2016; Iqbal et al., 2016; Khoo et al., 2020; Phong et al., 2018). Chromatography, filtration, and centrifugation are still currently the most used methods for production of valuable biological compounds (Espitia-Saloma et al., 2014). However, these methods all share the disadvantages of laborious operation, costly equipment/consumables, require high energy input, and difficulties in upscaling and process integration. In addition, these methods are unable to handle large input load

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while requires multiple operation steps, which limits the production rate and increases costs (Lee et al., 2016). In that regard, LBS can perform separation, concentration, and purification during the same processing step (Phong et al., 2018) and upscaling is achievable as seen in several pilot experiments (de Araújo Padilha et al., 2018; Mathiazakan et al., 2016). Moreover, LBS requires relatively cheap phase-forming components with potential recyclability, low energy input, and simple operation with no complex apparatus setups. Finally, advance LBS equipped with flotation (bubble), ultrasound, microwave, electric or magnetic systems can both improve efficiency and provide addition functionality to further shorten the production steps (Khoo et al., 2020; Phong et al., 2018). Another advantage of LBS is that it has the capability to extract, separate, and purify a wide variety of molecules with high efficiency. The list of products includes proteins (Phong et al., 2017a, 2017b; Song et al., 2018), antibodies (Wu et al., 2014), enzymes (Jong et al., 2017; Kavakcıo˘glu et al., 2017), antibiotics (Liu and Cao, 2016), cells (Luby et al., 2018), many organic molecules such as phytochemicals (Tan et al., 2013), isoflavones (Bi et al., 2010b), polymer esters (Leong et al., 2017), and even inorganic molecules like metal ions (Zheng et al., 2015) and graphene (Godoy et al., 2019). The most significant limitation of LBS is that no theory or model currently exists that can thoroughly explain the partition mechanism or accurately predict how the system and the target molecule will interact. Hence, development and optimization of LBS depend entirely on empirical data collected from mostly one-at-a-time experiments which are time-consuming and complicated (Grilo et al., 2016; Pirdashti et al., 2015). Another disadvantage of LBS is that polymer-based systems often utilize expensive (dextran) or unrecyclable (PEG) phase-forming component. Compensation trends include switching to alcohol–salt systems which is considerably more cost effective and environmentally friendly or development of reusable polymers like ethylene oxide– propylene oxide copolymer (Goja et al., 2013; Leong et al., 2017). Finally, settling of the system is usually performed by gravitational force which can take a significant amount of time. Centrifugation can be employed to speed up the process, however much care must be taken in order to not damage the target molecule (Lee et al., 2016).

10.3

Liquid biphasic flotation

10.3.1 Mechanism Liquid biphasic flotation (LBF), aqueous two-phase flotation, flotation-assisted LBS, or bubble-assisted LBS is a novel separation method in downstream processing of biological products. First introduced in 2009, LBF is the integration of solvent sublation (SS) principles into LBS (Bi et al., 2009). Surfactants, which often consists of a hydrophilic head and a hydrophobic tail, and other hydrophobic compounds in an aqueous solution can adsorb onto the surface of rising bubbles and get carried to the top of the solution. Conventional SS utilizes this mechanism with the addition of an organic phase above the aqueous phase to effectively collect these molecules (Bi et al., 2010a).

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Figure 10.2 Schematic diagram of liquid biphasic flotation (LBF).

Fig. 10.2 outlines the basic setup of a LBF for the separation of a target molecule. The two aqueous phases serve as the medium for the mass transfer process, with the crude extract usually being mixed with the bottom phase prior to the addition of the “collector” top phase (Lee et al., 2016; Phong et al., 2017b). The two-phase system is then subjected to an upward bubble flow generated by pressurized gas flowing through a sintered glass disk or a membrane placed at the bottom of the apparatus. During this process, the hydrophobic tail of the biomolecules tends to adsorb to the bubble surface and migrate into the internal gas phase away from the aqueous environment. As the bubble travel through the aqueous phase, the number of adsorbed biomolecules gradually increases. Finally, the adsorbed biomolecules will dissolve directly into the top phase due to hydrophobic interaction or be released when the bubble bursts at the atmosphere air–aqueous interphase. This active mass transfer mechanism of SS can result in better separation efficiency and higher concentration of target molecule in the top phase (Lee et al., 2016; Sankaran et al., 2019). Proteins usually consist of both hydrophilic and hydrophobic regions in their folded state, therefore LBF should also be able to the separate and purify protein as shown in various studies (Mathiazakan et al., 2016; Phong et al., 2017b; Show et al., 2013, 2011).

10.3.2 Key factors 10.3.2.1

Phase-forming component

Similar to LBS, common phase-forming component includes polymer, salt, and alcohol. Interestingly, salt is the most popular component choice for the bottom aqueous phase (Sankaran et al., 2019). This can be attributed to the fact that the required amount of top phase in LBF can be very small (i.e., a thin layer covering the surface; Show et al., 2011), hence the necessity of the salting-out effect for the sustenance of the two immiscible phases (Lee et al., 2016). In addition, increasing the salt concentration

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should also enhance the salting-out effect and the hydrophobic interactions of the target molecule which leads to better yield (Phong et al., 2017b).

10.3.2.2

pH

The effect of pH can be more significant for LBF than LBS since the mass transfer mechanism depends heavily on the availability of hydrophobic sites on the target molecules. A change in pH may result in the change of protein net charge, leading to different folding and alteration of hydrophobicity. However, the change in pH can also affect the properties of the phase-forming components that may a homogenous solution instead. A good example was demonstrated by Show et al. through the extraction of lipase by alcohol–salt LBF, where low pH (9) leads to the total denaturation of the target enzyme, while the optimal condition was at pH 8.5 (Show et al., 2013).

10.3.2.3

Bubble type, size, flow rate, and flotation time

To not cause unwanted chemical reactions, inert gas is a must when it comes to application in LBF. Therefore, there is hardly a better choice than nitrogen gas (N2 ) as it is also the most readily available. As a rule of thumb, an increase in bubble size will decrease the surface area to volume ratio, which leads to a reduce in adsorption capacity and mass transfer rate (Parmar and Majumder, 2013). However, if the bubble is too small, it cannot overcome the interfacial tension and has to aggregate with others to increase to the appropriate size. Bubbles generated from a G4 sintered glass disk have shown to be at just the right size for optimal performance of LBF (Show et al., 2013). An increased flow rate means a lower bubble retention time, which leads to poor adsorption of target molecules and inefficient mass transfer. In addition, high flow rate may induce turbulent mixing and disrupt the two phases alongside the formation of foam due to rapid aggregation of bubbles at the atmospheric air–aqueous interphase (Lee et al., 2016). Generally, flotation time and separation efficiency are directly proportionate to each other until a plateau value is acquired. After that, the maximum separation capability of the system has been reached and a continuation of flotation is a waste of resources, especially in large-scale processes (Show et al., 2013).

10.3.3 Advantages and limitations As an advance LBS, LBF also inherits the advantages like mild aqueous phases, high biocompatibility, simple operation, and ease of scaling up. The application of recyclable phase-forming components combined with the reduction in top phase volume requirement should further ensure that LBF is both environmentally friendly and costeffective when compared with conventional methods (Lee et al., 2016; Sankaran et al., 2019).

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With the integration of SS, LBF also acquires a rapid and effective mean of mass transfer that results in high efficiency and product concentration. In the first ever comparative study of LBF with the two parent methods in separation of Penicillin G (Bi et al., 2009), the efficiency of LBF (95%) only loses to LBS (97%) while that of SS is the lowest (48%). However, the slight lost in efficiency of LBF is compensated by an overwhelming high concentration coefficient of 18 while that of LBS and SS were only 2.9 and 9.6, respectively. As a result, LBF has shown to be able to perform both separation and concentration of the target molecule in the same processing step at high efficiency. Unfortunately, LBF also inherits the largest limitation of LBS which is the lack of theory and prediction models. Hence, recent studies on the optimization of LBF still rely heavily on complex and time-consuming one-of-a-time experiments (Chia et al., 2020; Ng et al., 2020). Another limitation came from the inherent mechanism of SS which is LBF requires additional equipment, energy input, and budget to supply the gas bubbles when compared to normal LBS. However, all of this could be offset by the advantages mentioned above.

10.4

Ultrasound-assisted liquid biphasic system

10.4.1 Mechanism Ultrasound-assisted LBS (UA-LBS) is a combination of LBS and ultrasonication to perform cell disruption, extraction and separation/purification in a singular step with great efficiency, yield and purity of target biomolecule (Yan et al., 2021). Conventional cellular disruptive methods such as maceration in organic solvents, mechanical bead milling, or enzymatic extraction are costly, time-consuming and may result in low yields due to degradation of the target molecule. Hence, a more efficient disruptive method such as ultrasonication is much needed (Wang et al., 2014). Fig. 10.3A describes the basic principles of this method. When applied to a solution, the ultrasound creates a pressure wave and mechanically vibrates the liquid medium. This vibration also induce compression or expansion of dissolved gas bubbles (Chen et al., 2011). These bubbles can grow in size due to either coalescence of multiple smaller bubbles or rectified diffusion (Ashokkumar, 2011; Leong et al., 2016). When the bubble reach critical size and resonance with the ultrasound, it can rapidly and violently disintegrate into smaller bubbles and the process can start over again. These implosions can result in very short-lived extreme conditions such as local temperature of 5000 K, pressure of up to 1000 atm, and generation of a microjet of water that can breakdown or erode nearby materials (Chemat et al., 2017). This mechanism can be used to disrupt even the hardiest of cells such as plants or microalgae (Guo et al., 2013; Wang et al., 2014). Fig. 10.3B illustrates a simple UA-LBS setup. Cell suspension is usually added and stays in the interphase between the two immiscible aqueous phases. Under the vibrational and cavitation effects of ultrasonication, the two phases may briefly mix together resulting in an emulsion while the cell walls are disrupted or broken down to

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Figure 10.3 (A) Acoustic cavitation effect of ultrasound. (B) Schematic diagram of ultrasound-assisted liquid biphasic system (UA-LBS).

release the intracellular content. Finally, the system undergoes a settling step to allow the formation of two immiscible phases like normal LBS. Finally, the target molecule will partition to the top phase while the cell debris and contaminants are separated in the bottom phase (Guo et al., 2013; Yan et al., 2021).

10.4.2 Key factors 10.4.2.1

Sonication power and duration

During the extraction of schizandrin (SA), schisantherin A (SAA), and deoxyschizandrin (DSA) from the seeds of Schisandra chinensis Baill, it was reported that high sonication power and long duration result in a better extraction yield (Guo et al., 2013). An increase in power and duration of ultrasound directly causes more cavitation, resulting in more throughout cell disruption and extraction. The ultrasound may also directly enhance the diffusion coefficient of the target molecules from inside the cells

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to the outside aqueous phase. In addition, the constant generation of unstable bubbles by cavitation may also increase mass transfer rate and help with extraction. However, after a certain exposure time at high power, the yield of SAA started to reduce instead due to degradation caused by excessive cavitation. Similar results were also observed in the extraction of resveratrol (Zhou et al., 2019), and polyphenols (Wang et al., 2021) where yield and efficiency initially increase with the raise in power and duration, then decrease after reaching a peak value. Hence, these two factors must be heavily considered since unnecessary exposure can result in loss of product and waste of input power.

10.4.3 Advantages and limitations UA-LBS retains the advantages of both individual system. Ultrasound provides a simple and quick method to disrupt even the most durable of cells with a relatively low power input. Furthermore, ultrasound does not require any extra solvent and is environmental-friendly, which results in a huge economic advantage over other methods (Guo et al., 2013). The extreme effects of ultrasound only last for a fraction of a second at microscopic scale and will not affect bioactivity of the product unless under prolonged exposure. On the other hand, the LBS part is unchanged and still provides all of its benefits with additional mass transfer help due to cavitation effects. A comparative study between UA-LBS, ultrasound-assisted extraction (UAE), and conventional solvent extraction was performed on resveratrol extraction (Zhou et al., 2019). Out of the three, UA-LBS performed with the best extraction yield followed by UAE and finally solvent extraction. In addition, UAE method causes the highest level of contamination while that of UA-LBS was the lowest, proving that UA-LBS is the superior method out of the three. Limitations of UA-LBS include the risk of biomolecule degradation due to extended exposure to high ultrasound power (Guo et al., 2013; Yan et al., 2021; Zhou et al., 2019) and requiring power input which can further increase the costs and complexity of the system. In addition, designing and optimization of UA-LBS must also rely on one-at-a-time experiments which in the inherent downside to all LBS.

10.5

Magnetic-assisted liquid biphasic system

10.5.1 Mechanism Magnetic separation is a popular method in metal processing industries such as mining and metallurgy, only until recently it was applied to bioprocessing (Borlido et al., 2013). The mechanism of this method is described in Fig. 10.4A. First, the surface of nanoparticles with magnetic properties is modified with various compounds to increase biocompatibility, prevent metal leeching, and contamination; and provide specific affinity ligands (Borlido et al., 2013; Dhadge et al., 2014). Then, these modified particles are added into the crude solution and allowed to bind with the target molecules through the aforementioned ligands. Finally, a simple magnet or magnetic

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Figure 10.4 (A) Mechanism of magnetic separation. (B) Schematic diagram of magnetic-assisted liquid biphasic system. (C) Schematic diagram of magnetic ionic liquid aqueous two-phase system (MIL-ATPS).

field is applied to collect the magnetic complex, and the target molecule can be easily eluted and purified while the magnetic particles can be reused. Magnetic-assisted LBS (MG-LBS) aims to utilize the mechanism of magnetic separation to further improve separation and purification efficiency of conventional LBS (Dhadge et al., 2014). Fig. 10.4B illustrates a setup where both phase-forming components, crude extract and the modified magnetic particles, are mixed together. The magnetic particles will partition into the droplets of the more favorable phase, and under the effect of a magnetic field, will pull the droplets together to accelerate the settling process by a factor of at least 10 (Flygare et al., 1990; Larsson, 1994). In addition, the available affinity ligands should bind with the target molecule to further enhance the separation and purification efficiency. Recently, a novel system illustrated in Fig. 10.4C also incorporates the help of magnetism by utilizing magnetic ionic liquid (MIL) as a phase-forming component (Yao et al., 2016), hence the name MIL-ATPS was coined. The operating procedure starts out with MIL being thoroughly mixed with the crude solution before the addition of the second phase-forming component. A strong neodymium magnet is then used to attract the MIL droplets, leading to extremely swift phase formation and partitioning (Yao and Yao, 2017).

10.5.2 Key factors 10.5.2.1

Magnetic particle

The characteristics of the magnetic particles decide how effective they can be at binding to target molecules for separation and purification purposes. A review published by Borlido et al. in 2013 has described in detail the desirable properties of these particles and how to synthesize them (Borlido et al., 2013). Several key characteristics include

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that magnetic particles are usually iron oxides with the most common being Fe2 O3 and γ-Fe2 O3 (magnetite and maghemite, respectively) since they are oxidation resistant and biocompatible. A reduction in particles size leads to an increase in binding capacity and a reduction of magnetic affinity, with nano sized particles usually being the most optimal for both. In addition, particles should not have large size variation to ensure uniform and predictable behavior when subjected to magnetic fields. The optimal shape would be spherical to achieve the best hydrodynamics and stress resistance. Surface modification can drastically change how these particles bind with target molecules. For example, the addition of carboxyl groups results in selective binding to positively charged proteins while repelling negatively charged ones (Gai et al., 2011). Another study has shown that the addition of aminophenyl boronic acids enhances the particles’ ability to bind with cis-Diol functional group of glycoproteins, leading to effective binding and purification of monoclonal IgG (Dhadge et al., 2014).

10.5.2.2

Salt concentration

Interestingly, salt concentration has totally opposite effects on the performance of the two following systems. In the extraction of different proteins using carboxyl modified magnetic particles in a PEG/sulfate system (Gai et al., 2011), high salt concentration leads to poor binding of the particles to the target protein. The authors mentioned that increasing salt contents causes an increase in ionic strength of the system, which will negate the carboxyl–protein electrostatic interaction. Hence, the system was designed differently, with the addition of magnetic particles and magnetic field being performed before the addition of the sulfate phase or after diluting the salt concentration. On the other hand, in the purification of monoclonal IgG using magnetic particles coated with polymer and aminophenyl boronic acids in a PEG/dextran system (Dhadge et al., 2014), an increase in salt concentration improves both the binding of the particles to IgG and partition of IgG to the PEG-rich phase due to salting-out effect. It can be concluded that the effect of salt concentration varies greatly between different systems, particle modification, target molecule, and their interactions.

10.5.2.3

pH

Similarly, the effect of pH on system performance is totally dependent on each specific system, particle modification, and interaction toward the target. A change in pH can lead to different conformations and net charge of proteins, which proves to be a simple and effective in “aiming” for the target molecule. As described in the system utilizing carboxyl modified particle (Gai et al., 2011), these particles have high affinity for positively charged proteins, hence proteins with pI > pH will bind more strongly and vice versa.

10.5.2.4

Regarding MIL-ATPS

Similar to conventional ATPS utilizing IL, MIL-ATPS is also affected by factors such as pH, temperature, and phase-forming components; and the effect is highly dependent

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on the type of the target molecule and system. In addition, there is still lack of research dedicated to MIL-ATPS leading to even poorer understanding when compared to other types of ATPS.

10.5.3 Advantages and limitations MG-LBS has proven to be extremely versatile due to the various modifications of the magnetic particles that are available (Borlido et al., 2013). In addition, these particles can be easily recycled (Dhadge et al., 2014) while also reducing operation time significantly by speeding up the phase settling process (Flygare et al., 1990; Larsson, 1994), which can help save costs and increase production capacity. When compared to conventional LBS and magnetic extraction (also called “magnetic fishing”; Dhadge et al., 2014), MG-LBS achieved the best yield and purity of the target molecule while also took less time during phase formation and required less magnetic particle than magnetic fishing. Hence, MG-LBS can be a powerful alternative for the recovery of various biological molecules. Meanwhile, the most significant advantage of MIL-ATPS is the extremely rapid formation of the two immiscible phases under a magnetic field without the need for magnetic particles as shown in the processing of chloramphenicol (Yao and Yao, 2017) and berberine hydrochloride (Nie et al., 2018). In addition, MIL retains the benefits of other ILs as “green solvents” due their biocompatibility and environmentally friendliness. The largest limitation of both MG-LBS and MIL-ATPS is the severe lack in research paper dedicated to these systems, which lead to poor understanding regarding the exact mechanisms, interactions as well as possible applications. This can be attributed to the fact that MILs are just recently developed (Yao et al., 2018) and the modification process of magnetic particles is highly complex (Borlido et al., 2013).

10.6

Electricity-assisted liquid biphasic system

10.6.1 Mechanism Similar to UA-LBS, electricity-assisted LBS (EA-LBS) incorporate the electrolysis method of cell disruption into LBS for the purpose of performing extraction, separation, and purification in the same operation step with improved efficiency (Sankaran et al., 2019) as described in Fig. 10.5A. The crude extract is mixed with a preformed LBS before the continuous application of direct electric current to the whole system through the two electrodes (Leong et al., 2019). The electric current can directly rupture the cell membrane (Koyande et al., 2019) to release the intracellular contents which also include the target molecule. The released molecules are immediately subjected to partitioning in LBS, essentially both extraction and separation can be performed by this system at the same time. Electrolysis was further combined with LBF to create a novel system called liquid biphasic electric flotation (LBEF; Sankaran et al., 2018). The overall design of the

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Figure 10.5 (A) Schematic diagram of electricity-assisted liquid biphasic system (EA-LBS). (B) Schematic diagram of liquid biphasic electric flotation system (LBEFS).

system is illustrated in Fig. 10.5B. The crude extract is mixed with the bottom phase before the addition of the top phase, then continuous electric current and flotation were applied. Similar to EA-LBS, extraction and separation (in this case, through SS) can be performed simultaneously. This system is a proof of concept that multiple advance LBS systems can be further combined together to achieve even better performance.

10.6.2 Key factors 10.6.2.1

Voltage and position of electrodes

It is reported that an increase in voltage also increases separation efficiency, however, voltage that is too high will damage the target molecules leading to poor efficiency and waste of power (Azmi et al., 2020). In addition, high voltage can damage the electrodes through oxidation, causing system contamination and poor performance (Leong et al., 2019). The placement of electrodes is also an important factor that determines how well the system will operate. In the extraction of betacyanins from pitaya using EA-LBS (Leong et al., 2019) and protein from Chlorella vulgaris microalgae using LBEF (Koyande et al., 2019), the best separation efficiency was achieved when the electrode tips were placed inside the bottom phase. On the other hand, for the extraction of protein from Chlorella sorokiniana using LBEF (Sankaran et al., 2018), electrode tips being placed inside the top phase gave the best performance. There is still a lack of explanation for this seemingly contradicting behavior, and more research is required.

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10.6.3 Advantages and limitations A major advantage of LBS/LBF combining with electrical system is that electrolysis is the simplest cell disruption method which does not require extensive equipment like ultrasound or microwave and is not costly like enzymatic methods. Furthermore, it is also much gentler than mechanical methods and relatively does not generate heat (Leong et al., 2019), hence the target molecule is better preserved. Both EA-LBS and LBEF have been shown to be highly efficient and able to perform both extraction and separation within the same operation step (Azmi et al., 2020; Leong et al., 2019; Sankaran et al., 2018), therefore the application of these two systems will help reduce costs and simplify downstream processing of biological products. The limitations of being novel systems are that there is still a significant lack of knowledge regarding EA-LBS and LBE. More research is required to fully understand the mechanisms and how to optimize them for upscaling as well as potential application for recovery of other molecules.

10.7

Microwave-assisted liquid biphasic system

10.7.1 Mechanism Microwave-assisted extraction is the process where microwaves are used to rapidly generate heat and pressure, through which mass transfer is facilitate (Belwal et al., 2020). The setup only requires a microwave oven and a microwave-compatible vessel containing the raw material suspended by an organic solvent, which makes it a simple, rapid, and effective extraction method. When combined with LBS to produce microwave-assisted LBS (MA-LBS), the organic solvent is simply replaced by the two immiscible aqueous phases (Dang et al., 2014). As shown in Fig. 10.6, the raw material is usually suspended in the interphase of the LBS (Lin et al., 2019) before the microwave treatment of the whole system. Next, a filtration step is usually performed to remove any insoluble residues. Finally, the two phases are allowed to reform to facilitate partitioning of the target molecule.

10.7.2 Key factors 10.7.2.1

Microwave power and temperature

In the extraction of phenolics (Dang et al., 2014), flavonoids (Ma et al., 2013; Xie et al., 2017), and alkaloids (Zhang et al., 2015), it was reported that microwave power has insignificant direct effect on the efficiency of the system. Meanwhile, an increase in temperature leads to better yield of the target products and excess heat will decrease yield instead. A simple explanation is that higher heat means quicker degeneration of the cell walls and mass transfer, but extreme heat will lead to degradation of the target molecule. Another study regarding the extraction of polysaccharides (Cao et al., 2018) reported that both microwave power and temperature have positive correlation

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Figure 10.6 Schematic diagram of microwave-assisted liquid biphasic system (MA-LBS).

with system efficiency up until a certain point, where the target molecule starts to get damaged.

10.7.2.2

Irradiation time

Similarly, increasing processing time can lead to more throughout extraction of the target molecule leading to better yield (Ma et al., 2013; Zhang et al., 2015) while excessive exposure duration will have detrimental effects on the target molecule.

10.7.2.3

Advantages and limitations

MA-LBS combines the simple and rapid extraction principles of microwave-assisted extraction and the excellent separation capacity of LBS into a singular operation step. In a comparison study with regular LBS, conventional heat reflux and Soxhlet extraction (Dang et al., 2014), MA-LBS has the best yields while requiring shortest operation time and solvent. Therefore, implementing MA-LBS in downstream processing can reduce cost and the amount of operation steps leading to simpler production design. However, much care must be taken when using microwave as excessive heating can degrade the target molecules and all equipment must be microwave-safe to avoid accidents. The advance LBS, the added system, and function of the respective system are summarized in Table 10.1.

10.8

Future prospects

Currently, the largest obstacles preventing LBS from replacing conventional methods in industrial applications are the lack of knowledge regarding the exact mass transfer mechanism and models to predict how the system will function with different target molecules, phase-forming components, scale, etc. without prior experimentation (Phong et al., 2018). Even so, lab-scale LBS is highly dependent on one-at-a-time

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Table 10.1 Summary of various advance liquid biphasic systems. LBS type Flotation-assisted

Added system Solvent sublation

Ultrasound-assisted

Ultrasonication

Magnetic-assisted

Magnetic particles

Electricity-assisted Microwave-assisted

Electrolysis Microwave extraction

Function Generating bubbles to adsorb target molecules and carry them to the top phase. Acoustic cavitation to disrupt cells and mix components. Magnetic particles with affinity coating to bind with target molecules. Direct electric current disrupt cells. Microwave generates heat and pressure to facilitate mass transfer and disrupt cells.

experiments as there are a large number of factors to consider. Therefore, future research should be dedicated to finding out the mechanism of LBS or designing an approximation model for prediction of system behavior. In addition, designing largescale LBS system that can directly translate between experimental data and industrial application is also highly prioritized. Another development trend is to explore novel phase-forming components with desirable characteristics like lower costs, eco-friendly, recyclable, and nontoxic. One such component is the copolymers that can separate into phases or rejoin depending on the system conditions like temperature or pH (Leong et al., 2017; Liu and Cao, 2016). Recyclable ionic liquids (Freire et al., 2012; Yao and Yao, 2017) and biodegradable saccharides (Koyande et al., 2019) have been found to be suitable replacement for traditional salts. However, little research has been done to assess how these new components would perform in combination with the advance LBS in both laboratory as well as industrial scale. It can be said that the true potential of LBS has not been reached yet as long as these possibilities are still not explored. Furthermore, continuous mode or industrial-scale LBS is limited to reusing old designs from conventional liquid–liquid extraction using organic solvents in which the system might behave differently from lab-scale experimentations (Espitia-Saloma et al., 2014). Efforts have been made to develop large-scale systems which can faithfully replicate laboratory batch results (Luo et al., 2016; Ruiz-Ruiz et al., 2019; Vázquez-Villegas et al., 2011); however, the complex mass transfer mechanism still prevents these systems from being implemented. Once again, this knowledge gap must be filled as fast as possible in order to truly unleash the potential of LBS in commercial production.

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Yao, T., Zang, H., Yao, S., Dai, X., Song, H., 2018. Measurement and correlation of phase equilibria in aqueous two-phase systems containing functionalized magnetic ionic liquids and potassium phosphate at different temperatures. J. Mol. Liq. 263, 72–80. https://doi.org/10.1016/j.molliq.2018.04.131. Zhang, W., Zhu, D., Fan, H., Liu, X., Wan, Q., Wu, X., Liu, P., Tang, J.Z., 2015. Simultaneous extraction and purification of alkaloids from Sophora flavescens Ait. by microwave-assisted aqueous two-phase extraction with ethanol/ammonia sulfate system. Sep. Purif. Technol. 141, 113–123. https://doi.org/10.1016/j.seppur.2014.11.014. Zheng, Y., Tong, Y., Wang, S., Zhang, H., Yang, Y., 2015. Mechanism of gold (III) extraction using a novel ionic liquid-based aqueous two phase system without additional extractants. Sep. Purif. Technol. 154, 123–127. https://doi.org/10.1016/j.seppur.2015.09.014. Zhou, L., Jiang, B., Zhang, T., Li, S., 2019. Ultrasound-assisted aqueous two-phase extraction of resveratrol from the enzymatic hydrolysates of Polygonum cuspidatum. Food Bioscience 31, 100442. https://doi.org/10.1016/j.fbio.2019.100442.

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Kien Xiang Bong a, Wai Siong Chai a,b and Pau Loke Show a a Department of Chemical and Environmental Engineering, Faculty of Science and Engineering, University of Nottingham Malaysia, Semenyih, Selangor, Malaysia, b School of Mechanical Engineering and Automation, Harbin Institute of Technology, Shenzhen, Shenzhen, Guangdong, China

11.1

Economic sustainability

Economic sustainability is an integrated part of sustainability and must be used, safeguard, and sustain resources to create long-term sustainable values whether it can bring profits or prevent from losses (Basiago, 1998). The general definition of economic sustainability is the ability of an economy to support a defined level of economic production indefinitely. Economical sustainability is encircling around financial costs and benefits (Singh et al., 2009). In a chemical separation process, the main subject on this topic is usually evaluated with economic sustainability indicators of capital costs and operating costs (Saavalainen et al., 2015). Capital cost is the cost needed to set up the process which mainly includes the equipment cost needed for the process. The general definition of economic sustainability is the ability of an economy to support a defined level of economic production indefinitely. Many factors contribute to the economic sustainability in the chemical engineering field. Economic feasibility is the most important factor to ensure whether a process is economically sustainable. Economic feasibility of a process can be analyzed by using the cost–benefits analysis (Koopmans and Mouter, 2020). Besides that, some of the financial techniques can also be used to assist the findings such as break–even analysis and time value of money.

11.1.1 Economic feasibility The purpose of economic feasibility is to determine whether the process is economical sustainable or viable. It requires a preliminary study undertaken on a specific project to determine its viability. The outcome of the economic feasibility analysis is very important in decision making on whether the project should be proceeded or not. This analytical tool is used during the project planning phase that shows how a project can bring profits or even prevent from lost that are operating under a set of assumptions such as equipment and raw material used (Bause et al., 2014). Project that can generate enough amount of cash flow and profit to cover the risks that might be encountered and remain a stable economical sustainability in long term is a project that is considered feasible. In this section, economic feasibility liquid–liquid separation will be analyzed which includes polymer–polymer separation, polymer–salt separation, alcohol–salt Principles of Multiple-Liquid Separation Systems: Interaction, Application and Advancement. DOI: https://doi.org/10.1016/B978-0-323-91728-5.00007-X c 2023 Elsevier Inc. All rights reserved. Copyright 

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separation, sugar–solvent separation, ionic liquid–salt separation, T-butanol–salt threephase separation, and green solvents (Chai et al., 2016, 2019a, 2019b; Leong et al., 2017; Vashist and Beckmann, 1968). These are all the types of processes that are part of liquid–liquid separation process. All of the separation that has two-phase can be done by aqueous two-phase system (ATPS), whereas T-butanol–salt can be done by using three-phase partitioning (TPP) system (Vashist and Beckmann, 1968). In order to assess the economic feasibility, first the costs and benefits of the project have to be evaluated. Capital cost of each project is major factor that can affect the economic evaluation. The final outcome of each project can be predicted with cost estimation. However, exact value of a project is difficult to be evaluated by estimation only. There are few different methods to make cost estimations on a project (Loutatidou et al., 2014). One of the techniques for cost estimation is expert judgments where experts are used in both software development and application domain to predict the software cost (Datta and Roy, 2010). Besides that, estimation by analogy is also one of the common methods where using the cost of a similar project that are in the same application domain (Datta and Roy, 2010). On the other hand, the Parkinson’s law is by using available systems online to predict the cost (Parkinson, 1955). Lastly, algorithmic cost modeling uses mathematical functions derived from a study of historical costing data to estimate the costs. These are some of the methods that can be used when it comes to cost estimation for economic feasibility analysis.

11.1.2 Cost–benefit analysis Before conducting a process or taking on a new project, a cost–benefit analysis should be undergone to evaluate all the potential costs and revenues that can be generated by the company or person in charge (Koopmans and Mouter, 2020). The outcome of this analysis will determine whether a project is able to be financially independent or if changes should be made in order for it to be financially feasible. In this case, the cost–benefit analysis of the ATPS will be analyzed as it is the main process for the separation interaction listed above. Costs that are considered are the equipment cost, operating cost, and maintenance cost. Cost–benefit analysis can be done by using economic evaluation method such as net present value (NPV) and internal rate of return (IRR), which implement the time value of money to determine whether a project is feasible in a long run (McAuliffe, 2015; Shultz, 2005). Projects in which NPV greater than zero or IRR higher than the discounted rate are acceptable (Shultz, 2005). Besides that, break–even analysis is also a very important tool to determine when will the initial investment be break–even by the revenue gain from the project (Herman and Zabloski, 1979).

11.2

Advantages of liquid–liquid separation over conventional method

11.2.1 Water content Liquid–liquid separation is more economical compared to the conventional process of separation. One of the main advantages of liquid–liquid separation in the downstream

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processing of biomolecules is the usage of high-water content which is around 80% w/w (Hatti-Kaul, 2000), which is very beneficial in terms of economic point of view. With water being a cheap material, it is one of the pros for liquid–liquid separation to be more economical compared to the conventional methods (Goja et al., 2013).

11.2.2 Interfacial tension Utilizing liquid–liquid separation process is able to achieve low interfacial tension between conjugated phases that are being separated (Norde, 2011). A low interfacial tension means that the work needed to break the force of attraction between both of the molecules at the interface of two liquid is lower. This not only can provide a secure separation and purification technique for biomolecules, but also able to decrease the cost of operation and steps required in order to lower down the interfacial tension as it is already at a suitable rate for separation (Zaslavsky et al., 2019). With the elimination of extra steps and workload of interfacial tension, the cost for equipment and utility needed for this purpose on the conventional method can also be eliminated, which makes liquid–liquid separation more economical compared to the conventional method.

11.2.3 Energy consumption The energy consumption for liquid–liquid separation particularly ATPS is lower than the conventional method. This is because the mechanism of liquid–liquid separation is much simpler compared to the conventional method of separation such as protein A capture process which requires process such as equilibration that require high amount of energy (Goja et al., 2013). Process that requires less energy consumption tend to have lower operating cost, which is considered to be more economical (Chai et al., 2020; Gregorio et al., 2020).

11.2.4 Equipment requirement Liquid–liquid separation such as ATPS is a separation process with simple mechanism. This means that the full process flow of the liquid–liquid separation process requires less equipment and chemical unit to perform biomolecule separation. In that sense, having lesser process equipment and unit meaning lower capital cost to construct the process plant (Torres-Acosta et al., 2019). This means that liquid–liquid separation is more economically sustainable due to the lower capital cost compared to the conventional method. Moreover, lower complexity of the equipment used in ATPS process means lower capital cost for the plant (Torres-Acosta et al., 2020). This is due to the fact that equipment with more complexity such as equilibration column is much costlier compared to a normal mixer and settler tank.

11.2.5 Solvents The solvents needed for the separation system play an important role in economical sustainability. This is because various separation approaches require different types of

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solvent for separation. The variation of solvents will end up in different prices of the solvent and the amount needed in order to perform the separation (Todd, 2014). In this case most of the solvents needed for liquid–liquid separation are relatively cheaper than the ones needed for conventional use (Zhao et al., 2020). However, the costing is very dependent on the process being dealt and the solvents that are required for the process.

11.3

Three-phase interactions

The three-phase interaction for liquid–liquid separation is very similar to the twophase interaction. Three-phase interaction is also known as TPP, is a simple and fast purification technique (Ward, 2009). The TPP systems are targeted by the market due to their economical sustainability mainly by the cheap setup cost due to simpler mechanism that does not require many equipment (Dennison and Lovrien, 1997). The TPP system is similar to the ATPS because it is also a one step process. The number of phases in the liquid–liquid separation does not much of an impact to the economical point of view as the mechanism is the same. It is just that there are one more phase to be sorted out. However, the capital and operating cost for TPP system will be higher compared to the two-phase system due to the extra phase that is needed to be separated from the column. The economic advantages for the TPP system are similar as any other liquid–liquid separation stated in the previous section.

11.4

Costing in liquid separation system

11.4.1 Technology for liquid separation system Liquid–liquid separation can be categorized into many different interactions due to the various type of material that are being separated. In biochemical engineering field, liquid separation system is to assist in clarification, partitioning, and partial purification of biomolecules and bioproducts to obtain the end products such as antibody and other pharmaceutical products from cell culture supernatant (Vashist and Beckmann, 1968), where antibody has been used in various detections (Cheng et al., 2021; Li et al., 2019; Liu et al., 2020; Low et al., 2016a 2016b). The liquid separation interactions covered in this book are polymer–polymer interactions, polymer–salt interactions, alcohol– salt interactions, ionic liquid–salt interactions, etc. These kind of liquid separation systems can be done by the ATPS. For the conventional methods, liquid separation is done by chromatography method, precipitation method, and normal liquid–liquid separation method. These conventional methods are very costly due to the complication of process which requires large amount of equipment to run the process. Also, for the conventional method, organic solvents and other types of toxic substance are required for the separation which is very costly in terms of maintenance cost (Coskun, 2016). On the other hand, ATPS is a liquid–liquid fractional method which is based on the incompatibility of two aqueous solution that will tend to separate the mixture

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(Hatti-Kaul, 2000). Systems that are available for ATPS are polymer–salt system, which uses polyethylene glycol (PEG) and potassium phosphate. Besides that, polymer–polymer system uses PEG and dextran. Moreover, systems such as ionic liquid–salt and alcohol–salt system are also feasible for ATPS (Goja et al., 2013). The usability of this system is capable to run all the interactions stated above with two phases due to the similar mechanism of separation system. An ATPS is able to provide fast and large scale of purification compared to the conventional process (Azevedo et al., 2009). Also, ATPS is an isolation process which has lower operating cost compared to the conventional method such as chromatography. Another advantage of ATPS is economically as it does not contain toxic waste which is good for maintenance cost (Hatti-Kaul, 2000). ATPS is a process that can be done as a one-step process that allows the removal of contaminant components and also separating the desired product at the same time (Rito-Palomares, 2004). The separation is carried out by partitioning of the desired products between the two phases. ATPS is a system that is manipulated from the aspects of complexity of process and the chemical interactions between the separation process in order to recover the biopharmaceutical products (Rito-Palomares, 2004). Experiments are often undergone to obtain the optimal purification conditions of the process. As different types of separation products require different types of ATPSs, a four-stage strategy is usually implemented to obtain the optimal results. Stage one is to characterize the feedstock of raw material and the desired product. In the second and third stage, the correct type of ATPS is being analyzed and chosen with correct parameters, whether it is polymer–polymer, polymer–salt, or etc. Lastly, the final stage is to evaluate the effect of the process on the purity and the recovery of the process (Rosa et al., 2011). ATPS is a very promising technology as it can combine high biocompatibility and selectivity with an easy and reliable scale-up and capability for continuous operation (Albertsson, 1970). With ATPS, some of the conventional process drawbacks can be eliminated such as batch operation, low productivities, packing problems, diffusional limitations, and low chemical stability which are directly affecting the quality of final products (Shukla et al., 2007).

11.4.2 Equipment cost for ATPS liquid separation system To evaluate the equipment cost for ATPS, the process equipment has to be estimated based on some combined studies due to limited large-scale ATPS process. This is because the partitioning behavior of biomolecules in ATPS is complex, many laborious trials have been performed to optimize whether the system is feasible. Thus, largescale process of ATPS has to be estimated in order to get the overall equipment cost for the separation process to calculate the economic feasibility. A scale-up model of the ATPS had been developed and validated in a pilot scale pump mixer–settler battery. This pilot scale process was developed under aqueous two-phase separation concept to capture the human immunoglobulin G from cell culture media (Rosa et al., 2009). The downstream aqueous two-phase separation process is based on a pilot scale validation reported (Rosa et al., 2011). The process flow diagram of a scale-up process to the size

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Figure 11.1 Process flow diagram of continuous aqueous two-phase separation of human antibodies from cell culture supernatant. Credit: From Rosa, P.A.J., Azevedo, A.M., Sommerfeld, S., Bäcker, W., Aires-Barros, M.R. (2011). Aqueous two-phase extraction as a platform in the biomanufacturing industry: economical and environmental sustainability. Biotechnol. Adv., 29 (6), 559–567. https://doi.org/10.1016/j.biotechadv.2011.03.006.

of conventional industry for continuous aqueous two-phase separation on capturing human antibodies from cell culture supernatant is shown in Fig. 11.1. The aqueous two-phase separation process of human antibodies from cell culture supernatant process above consists of three main sections. The first section is where separation occurs, whereas the second section is back separation, followed by washing. In the separation section, high molecular weight contaminants will be removed whereas in the washing section, the purpose is for further purification and also separation of immunoglobulin G from the lower molecular weight contaminants and polymerrich phase (Rosa et al., 2011). A mixer–settler is included before the separation step due to validation trials performed in pack column. This addition of mixer–settler can increase the efficiency of mixing at the top phase inlet solutions to ensure that the phase components would reach equilibrium concentration before entering the column (Pietsch and Eggers, 1999). As this is a polymer–salt system, PEG/phosphate ATPS is assumed for all the steps. The dimension of the separation and washing column were estimated based on the experimental results according to a scale-up factor from the pilot scale aqueous twophase separation system (Pilhofer and Schröter, 1984). The scale-up factor was calculated based on the operating specific throughput which is at 70% of the throughput. The column dimensions and the mixer–settler dimensions are obtained by scaling up 70% of the operating throughput of the pump–mixer–settlers used in the pilot scale experiment (Rosa et al., 2011). Diameter of the mixing vessel is assumed to be the same as its height. To obtain the minimum volume of settler, the dynamic separation time had to be calculated. The dynamic separation can be assumed based on the density difference and volume ratio from the data in reference (Reissinger et al., 1981).

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Table 11.1 Equipment for scaled-up ATPS process. Equipment Description Quantity Mixer–settler separation Diameter = 0.5 m, height = 0.5 m, length = 1.5 m 1 Diameter = 0.3 m, height = 7.0 m 1 Separation Mixer–settler back separation Diameter = 0.5 m, height = 0.5 m, length = 1.5 m 1 Diameter = 0.45 m, height = 4.0 m 1 Washing column Volume = 3 m3 10 Storage tank Flowrate = 0.6 m3 /h 7 Pump

The diameter and height of mixer are set to be 0.40 m and the length is 1.42 m based on the minimum volume of the settler. Table 11.1 lists equipment in an ATPS process based on scale-up factor. Thus, the price is calculated based on conventional equipment and some scale-up factor. The full equipment cost estimation is set to be USD$ 4,966,000 as found in previous study (Rosa et al., 2011). As for the unlisted equipment cost, an estimation of 10% of the total equipment cost is estimated with USD$ 496,000. As for the installation and engineering cost, it is estimated to be USD$ 6,828,250 (Rosa et al., 2011). The total investment sum for the large-scale ATPS is USD$ 12,290,250.

11.4.3 Operating cost Operating cost of a process plant is usually divided into three categories which are variable cost, fixed cost, and plant overhead cost. The equation to calculate plant operating cost: Operating Cost = Variable Cost + Fixed Cost + Plant Overhead Cost

Variable cost is usually manipulated by the number of products that are being produced, whereas fixed cost is dependent on the fixed capital invested (Mery and Kawecki, 2004). In this aqueous two-phase separation design, utility cost is assumed to be negligible as there is no heat exchanger. However, the electricity utility of the equipment used cannot be estimated due to lack of information on the load of equipment on a large-scale process.

11.4.4 Variable cost Variable cost is one of the heaviest costs among other costs in terms of operating cost. Variable cost includes raw material cost, consumables, labor-dependent, laboratory, quality and control assurance, utilities, waste treatment, and disposal and royalty expenses (Mery and Kawecki, 2004). However, the utilities and royalty cost are set to be negligible in this design as it is a scaled-up design in which the cost is unable to be estimated.

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Table 11.2 Estimated pricing for raw materials for different type of interaction in ATPS process. Interaction Polymer–polymer interaction Polymer–salt interaction Alcohol–salt interaction

Raw material PEG Potassium sulfate PEG Dextran Ethanol Ammonium sulfate

Price (USD$/ton) 1750 630 1750 359,389 9751 230

Table 11.3 Main raw material cost for production of monoclonal antibodies. Raw materials Sodium phosphate Potassium phosphate PEG

11.4.4.1

Cost (USD $/ton) 1000 1000 1750

Raw materials and consumables

The raw materials and consumables of a process are the commodities used as an input at the initial stage of a production process. It includes everything that is needed to manufacture the desired end product. For an aqueous two-phase separation process, the raw materials required are more than the normal process because it requires two or more raw materials compared to conventional process (Hatti-Kaul, 2000). The raw materials of an aqueous two-phase separation process are based on the desired end product to be determined. Different type of aqueous two-phase separation process requires different raw material to perform separation. However, the price of raw materials for each type of aqueous two-phase separation process is varying. The example comparison of raw material for each different type of aqueous two-phase separation process is shown in Table 11.2, which shows the common raw materials use for each type of interaction in aqueous two-phase separation. It is shown that some of the interaction has highly expensive raw materials. However, the ratio of mixture is varied so it cannot be compared to a 1–1 ratio basis. Polymer salt separation interaction is the suitable process to extract monoclonal antibodies from cell culture supernatant with ATPS (Platis and Labrou, 2009). Thus, in this case raw materials of PEG and phosphate are used throughout the process. Also, cell culture being one of the main raw materials to produce monoclonal antibody. The cost of cell culture is hard to be estimated as it is mostly labor and time for laboratory scientist. However, the cell culture raw material is not considered in this section as the only focus is the aqueous two-phase separation process with PEG and phosphate. Table 11.3 shows the costs of raw materials that are used for production of monoclonal antibodies. The cost of each raw material is taken from the global chemical sales website (Chembid, 2021). The production of aqueous two-phase separation is set to process 500 m3 of cell culture annually which is around 176.57 tons. The raw material cost to process 500 m3 of cell culture is around $ 5,000,000 annually (Rosa et al., 2011).

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Labor cost

Labor cost is the sum of all wages paid to the employees that are working for the plant. Labor cost not only includes the payout for the actual work but also to cover the expenses so that the cost of employee benefits and payroll taxes paid by an employee. There are many expenses in the part of labor cost as it includes payroll taxes, overtime, health care, bonuses, sick days, vacation days, insurance, benefits, meals, supplies, and training costs (Novikov and Sazonov, 2020).

11.4.4.3

Gross pay

To calculate labor cost, first the gross pay needs to be computed. The plant runs for 133 hours per week. As it is a continuous running plant, shifts have to be done for the workers to get enough rest. Assuming a shift of two periods, an average employee will work for 50 hours per week. In a year of 365 days in a year, there are 52 weeks. To calculate the gross hours per year: Gross hours per year = 50 hours × 52 weeks = 2600 hours The pay for an hour of work is set to be USD$ 10/hour, so the gross pay for an employee is calculated by: Gross pay per year = 2600 hours × $ 10 = USD$ 26,000 per year

The annual gross pay is calculated to be USD$ 26,000.

11.4.4.4

Net hours work in a year

Estimation of net hours of an employee’s work in a year can be done by knowing how many days an employee is absent in a year. An assumption was made that each worker had an absent day of 15 days annually due to holiday or illness. The absent hours of work can be calculated: Hours not worked = 15 days × 10 hours = 150 hours The calculation was done assuming each worker worked 10 hours a day. With the data of hours not worked annually for an employee, the net hours worked can be calculated by: Net hours worked = Gross hours – Hours not worked Net hours worked = 2600 – 150 = 2450 hours

11.4.4.5

Additional annual cost

To calculate the real labor cost, additional expenses such as taxes, insurance, benefits overtime, and supplies need to be included to the cost, estimation of additional annual costs are listed in Table 11.4. This cost is estimated as there is no conventional design that fits the criteria of the project that are being researched: Other annual costs = taxes + insurance + benefits + overtime + supplies Other annual costs = 900 + 600 + 1200 + 800 + 400 n USD$ 3900

The total additional annual costs per employee are USD$ 3900.

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Table 11.4 Additional annual cost. Additional expenses Taxes Insurance Benefits Overtime Supplies

11.4.4.6

Cost (USD $) 900 600 1200 800 400

Annual payroll labor cost

In this section, the cost for an employee per year can be calculated: Annual payroll labor cost = Gross pay + Other costs

Thus, with the gross pay and other costs calculated above. The annual labor costs can be calculated. Annual payroll labor cost = 26,000 + 3900 = USD$ 29,900

11.4.4.7

Actual hourly labor cost

The actual hourly labor cost can be calculated with the equation: Actual hourly labor cost = Annual payroll labor cost/Net hours worked Actual hourly labor cost = 29,900/2450 = $ 12.20

These labor costs are inclusive of the labor dependent and also the quality control and assurance team. Assuming that the number of total workers in the plant is at least 30 employees. The total labor costs can be calculated by: Total annual labor costs = Number of employees × Annual payroll labor costs Total annual labor costs = 30 × 29,900 = USD$ 897,000 The total annual labor costs for the whole plant is USD$ 897,000.

11.4.4.8

Waste treatment and disposal

Waste treatment in a plant is also costly depending on the waste that is produced during the process. In this aqueous two-phase separation process, there are several waste streams. Some of the waste streams contain high concentration of PEG. A normal waste treatment costs are based on the range of $ 0.08–$ 1.60 per kg of wastewater (Evangelista et al., 1998; Keim and Ladisch, 2000). With the high number of raw materials used, the amount of waste generated will also be increased. Thus, the waste treatment costs are estimated to be around USD$ 520,000 annually. Sum of total variable costs: Total variable costs = 5000,000 + 897,000 + 520,000 = USD$ 6,417,000

The annual variable cost is summed up to be USD$ 6,417,000.

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11.4.5 Fixed cost The fixed costs of a process plant are usually inclusive of depreciation, equipment maintenance, insurance and local taxes. The taxes for this process plant are not estimated as the location of the plant are not specified, and the local taxes are not verified.

11.4.5.1

Depreciation cost

Equipment that are being used in the plant has a depreciation costs which means that how much the equipment had depreciated within the amount of years. A common depreciation equation is used to calculate the annual depreciation costs on the equipment: Annual depreciation cost = (Equipment cost – Salvaged value)/Useful Lifespan. For the equipment for the process, the salvaged value is set to be USD$ 2000 and the useful lifespan of an equipment is estimated to have a lifespan of at least 10 years (Kádárová et al., 2015). Pumps and storage tanks are set to be negligible in the depreciation costs. Annual depreciation costs = (4966,000 – 2000)/10 = USD$ 469,400

The annual depreciation costs include for the mixer–settler separation column, separation column, mixer–settler back separation column, and washing column. The total depreciation cost is estimated to be $ 469,400.

11.4.5.2

Equipment maintenance

Equipment maintenance is mandatory for all the plants as the safety of an equipment process is very important. Regular maintenance of equipment not only can ensure the quality of product but also the safety of employees and the lifespan of the equipment. To estimate the costs needed for maintenance of a plant, 2–5% of the total replacement asset value (RAV) should be assumed. RAV can be calculated by the proportion of the equipment spending, as this is being practiced in the industry (Sarı, 2020). In this case, the process plant is set to undergo maintenance for checking once annually. In this case, the common maintenance cost is calculated with %RAV equipment of 5%. Maintenance cost = Equipment cost × 5% RAV Maintenance cost = 4,966,000 × 5% = USD$ 248,300

The total fixed costs for the plant are: Total fixed cost = 469,400 + 248,300 = USD$ 717,700

The annual fixed cost is summed up to be USD$ 717,700.

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11.4.6 Plant overhead cost Plant overhead cost is the sum of indirect costs throughout the plant. It will be added to the variable costs and fixed costs to obtain the final operating cost. It includes indirect labor, indirect materials, and plant location rental fees.

11.4.6.1

Indirect labor

Indirect labor is the costs for the employees that are not directly involved into the production process. Labor fees for employees such as security guards, janitor, machine repairman, supervisors, and sales team are in the category of indirect labor. The labor costs of these position are based on activity-based costing. The salaries of the indirect labor are based on the amount of time spent on their job. A rough estimation is made that the indirect labor cost is set to be $ 150,000 annually (Chiang, 2013).

11.4.6.2

Indirect materials

Indirect materials are the materials that are being used during the process, but it is not assigned to any product. Materials such as janitorial supplies, light bulbs, gloves, and goggles are counted as indirect materials. The cost estimation for indirect materials is usually estimated based on the inventory as it is hard to track individually. The indirect labor cost is estimated to be USD$ 100,000 annually (Chiang, 2013).

11.4.6.3

Plant location rental fees

Most of the production plants rent a location to build their plant for production. Fees for rental or installment fees for the plant location are also considered as a plant overhead cost. For this project design, the plant location rental fees are estimated to be around USD$ 184,850 annually. The total plant overhead for the aqueous two-phase production is calculated to be: Total plant overhead = 150,000 + 100,000 + 184,850 = USD$ 434,850

With the variable cost, fixed cost and plant overhead cost calculated, the operating cost for the aqueous two-phase separation process plant can be calculated by: Operating cost = Variable cost + Fixed cost + Plant overhead cost Operating cost = 6417,000 + 717,000 + 434,850 = USD$ 7,569,550

The total annual operating cost is summed up to be USD$ 7,69,550.

11.5

Value of end product from biochemical engineering separation

Biochemical engineering products are very wide including large amount of biopharmaceutical, specialty chemicals, and reagents. The scope that is being focused on this

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Table 11.5 Some examples of approved biotechnological drugs and vaccines. Product name Epogen TM Epoetin Alfa Neupogen granulocyte colony stimulating factor G-CSF Humantrope somatotropin rDNA origin for injection Humulin human insulin rDNA origin Actimmune interferon gamma 1-b Activase Alteplase, rDNA origin Proptropin somatrem for injection Roferon A interferon alfa-2a Procrit erythropoietin Intron A interferon alpha2b Energix-B hepatitis B vaccines

Indication Dialysis anemia Chemotherapy effects Human growth hormone deficiency in children Diabetes Infection/chronic granulomatous disease Acute myocardial infarction Human growth hormone deficiency in children Hairy cell leukemia AIDS-related Kaposi’s sarcoma AIDS-related anemia predialysis anemia Hepatitis C Hepatitis B

chapter is the biopharmaceutical products. Biopharmaceuticals are substances produced using raw material of living organisms such as animal cells and microorganism (Strohl, 2001), which have high-therapeutic value. Most of the biochemical products are being utilized as drugs and vaccines for the need in the pharmaceutical field. The pharmaceutical products that are produced are primarily protein products such as insulin, growth hormone, monoclonal antibody, tissue plasminogen activator, hepatitis vaccine, and erythropoietin (Castilho, 2017). These products are highly valuable as the annual sales range in 1991 is from USD$ 3–5 billion (Bailey, 1991). In recent years, people in all walks of life are more focused on maintaining a healthy life. This will greatly boost the field for biotherapeutics products as many vaccines and drugs are being discovered. Table 11.5 lists some of the products and their indication that had been approved, with many are still under development for safety consumption, which are mainly for health care purposes. According to the market trend, health care products market value is increasing. As the years prolong, the global biopharmaceutical market value is at approximately USD$ 325.17 billion by the year 2006 and is expected to increase to USD$ 496.71 billion by 2026, with a CAGR of 7.32% over the forecast period (Danzon and Furukawa, 2006). The key market trend of biopharmaceutical product is monoclonal antibodies, which is expected to hold a high market share compared to the other products. This is because that the therapeutic applications of monoclonal antibodies include in the areas of cancer, rheumatoid arthritis, multiple sclerosis, cardiovascular diseases, etc. (Klug et al., 2020). This product has become even more relevant due to the new cases of cancers globally in the year 2020. The estimated number of new cases of cancers in 2020 worldwide are shown for both male and female (Fig. 11.2). This shows that the total number of cancers in a year can go up to as high as 19 million cases adding up for both male and females. This indicates that the demand for monoclonal antibodies is relatively important in order to fight the cancer cases in a year long.

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Figure 11.2 Number of (A) new cancer cases and (B) cancer-caused deaths in 2020. Credit: Source from Sung, H., Ferlay, J., Siegel, R.L., Laversanne, M., Soerjomataram, I., Jemal, A., Bray, F. (2021). Global cancer statistics 2020: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA: Cancer J. Clin., 71 (3), 209–249. https://doi.org/10.3322/caac.21660.

The global biopharmaceuticals market is mainly altered by the health care problems such as increase in elderly population that will surge in prevalence of chronic diseases such as cancer and diabetes. This will in fact increase in adoption of biopharmaceuticals for vaccines and drugs to fight diseases as such. One of the most common and wellknown drugs products is the monoclonal antibodies. Monoclonal is also claimed to be one of the costliest therapeutic options in the market due to its capability to fight cancer cells (How et al., 2021). The rising prices for this type of drugs are skyrocketing for the past decade. Price of monoclonal antibodies has been doubling throughout the decade. The price of monoclonal antibodies in the present is USD$ 202 for 100 μg (Klug et al., 2020). However, the price of monoclonal antibodies is predicted to be higher in the near future due to high demand of the antibody for the vaccines needed for Covid-19 (Chia et al., 2021). A graph of the frequency and price of the monoclonal antibody is shown (Fig. 11.3). It can be seen in the chart that the sale price of USD$ 7000/g is the most popular price as it has the most frequency compared to the other prices (Kelley, 2009). Thus, the price of USD$ 7000/g will be used as reference to calculate the revenue.

11.6

Cost–benefit analysis of ATPS and conventional separation method

In this following section, a cost–benefit analysis will be done on aqueous two-phase separation system and the conventional separation with chromatography method to

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Figure 11.3 The frequency of monoclonal antibody sold in $/gram. Credit: From Kelley, B. (2009). Industrialization of mAb production technology: the bioprocessing industry at a crossroads. mAbs, 1 (5), 443–452. https://doi.org/10.4161/mabs.1.5.9448.

evaluate its economic feasibility of both pathways. In present days, many of the antibody’s manufacturers are still utilizing the use of protein A chromatography as the capture step of choice due to the high degree of clearance capability for the host cell proteins. However, aqueous two-phase separation process has also shown some high capability for the downstream processing of biopharmaceuticals (Platis and Labrou, 2006). Aqueous two-phase separation technology can combine a high biocompatibility and selectivity which can be scaled up easily for a continuous operation system. An aqueous two-phase separation system is also able to solve some technical drawbacks of conventional separation system which include batch operation, low productivities, scale-up problems, diffusional limitations, and proteolytic stability. However, with all the advantages discussed above, the economical sustainability is still one of the most important aspects in order to ensure this system is feasible in a long run. Thus, an economic feasibility evaluation on the cost/benefits of a developed aqueous two-phase separation process for capture of human antibodies and conventional method of protein A affinity chromatography will be compared.

11.7

ATPS process cost/benefits evaluation—polymer–salt interaction

The type of aqueous two-phase separation process for capturing human antibodies developed in this section is polymer–salt interaction. In the continuous process, PEG and phosphate are used for each steps of the process (Ferreira et al., 2008), in which the aqueous two-phase separation process was reported by Rosa et al. (2011). To evaluate the cost/benefits of the two processes, first the cost of the project needs to be clarified. For the large-scale aqueous two-phase separation process, the equipment cost was calculated earlier in the section above that involved three main steps which are separation, back separation, and washing.

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Table 11.6 Quantity of equipment in the process (Rosa et al., 2011). Equipment Ms separation Separation Ms back separation Washing Storage tank Pumps

Description Mixer–settler Differential extractor Mixer–settler Differential extractor Store Flow

Quantity 1 1 1 1 2 3

Table 11.7 Annual operating cost. Type of cost Raw material Labor cost Waste treatment cost Depreciation cost Equipment maintenance cost Indirect labor cost Indirect material costs Plant location rental cost Total operating cost

Cost (USD$) 5,000,000 897,000 520,000 69,800 736,900 150,000 100,000 184,850 7,658,550

11.7.1 Capital/operating cost for ATPS process 11.7.1.1

Equipment cost

Table 11.6 shows the equipment and the quantity needed for the ATPS process. Equipment cost for the large-scale aqueous two-phase separation process for capture of human antibodies is estimated to be around USD$ 4,966,000 with a mixer–settler separation column, a differential extractor, a back separation column, a washing column, two units of storage tanks, and three units of pump (Rosa et al., 2011). As for the unlisted equipment cost, it is estimated to be around 10% of the total equipment costs which is USD$ 496,000. The cost for installation and engineering is estimated to be USD$ 6,000,000. Thus, the total capital investment of the plant can be calculated: Total capital investment = 4,966,000 + 4,96,000 + 6,000,000 = USD$ 11,462,000

11.7.1.2

Operating cost

The cost of raw material was covered in the previous section for polymer–salt interaction process on aqueous two-phase separation to capture human antibodies. Raw materials that are used are sodium phosphate, potassium phosphate, and PEG. To compare the process, an annual processing rate of cell culture of 500 m3 is being set, with the raw material cost is estimated to be USD$ 5,000,000 annually. The sum of operating cost shown in Table 11.7 for aqueous two-phase separation process is discussed in the section above including raw material cost, labor cost, waste

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treatment cost, depreciation cost, equipment maintenance cost, indirect labor cost, indirect material cost, and plant location rental cost. The annual cost of each parameter discussed above to process 500 m3 of cell culture annually will be finalized in the table for the total operating costs: Thus, the total operating cost of the plant is calculated to be USD$ 7,658,550.

11.7.2 Total revenue for ATPS process In this process flow, a total amount of 500 m3 /year of cell culture supernatant will be processed annually. The highest concentration of antibody in the cells is approximately 50 μg/mL (Kimiz-Gebologlu et al., 2018). Below calculation shows the monoclonal antibodies produced annually with the aqueous two-phase separation process: Calculation of Monoclonal Antibody Produced in 500 m3 /year: 500 m3 = 500,000 L 50 μg/mL = 0.05 g/L

Antibody produced = 500,000 × 0.05 = 25,000 g The aqueous two-phase process can produce 25,000 g of monoclonal antibody annually. To calculate the total revenue, the sales price of monoclonal antibodies needs to be identified. An average sales price of monoclonal antibodies discussed in above section is around USD$ 7000/g (Hernandez et al., 2018). Below calculation is to calculate the total revenue of the aqueous two-phase separation for 25,000 g annually. Total revenue = 25,000 × 7000 = USD$ 175,000,000

11.7.3 Time value of money Time value of money is the concept that money is more in the future because of its potential earning value. Time value of money is the primary step in basic finance concept with the theory of “A dollar today is worth more than a dollar in the future because a dollar today can be invested and earn a return” (Țilic˘a and Ciobanu, 2020). This principle of finance is known as present discounted value as money can be used to earn interest, which is very important to help in making investment decision. The purpose of time value of money serves as a central concept underlying discounted cashflow analysis (Țilic˘a and Ciobanu, 2020). It is an essential step before calculating the NPV and IRR. The formula to calculate the time value of money is shown below. PV =

FV (1 + r)n

(11.1)

where PV is the Present Value of the amount, FV is the Future Value of the amount n periods from now, I is the discounted rate or the expected rate of return, and n is the year that the amount occurs.

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Table 11.8 Net present value for ATPS process. Year Revenue (USD$) Capital cost Operating cost Free cash flow Present value

1 N/A 11,462,000 N/A −11,462,000 −10,420,000

2 175,000,000 N/A −7,658,550 167,341,450 138,298,719

3 175,000,000 N/A −7,658,550 167,341,450 125,726,108

4 175,000,000 N/A −7,658,550 167,341,450 114,296,462

5 175,000,000 N/A −7,658,550 167,341,450 103,905,874

11.7.4 Net present value NPV is the difference between the present value of cash inflows and the present value of cash outflows over a period of time. The profitability of a project can be analyzed with NPV calculation. NPV gives the present value of all payments, the larger the net present worth, the more favorable the investment. It is a widely used method as it captures the time value of money (McAuliffe, 2015). The period calculated for NPV for the aqueous two-phase separation process is 5 years. Below is the calculation to obtain the NPV of the project. Table 11.8 shows the summary of the present value calculated with the revenue, capital cost, operating cost, and free cash flow. Sample calculation for Present Value with discounted rate of 10%: Year 1:

PV =

−11, 462, 000 = −10, 420, 000 (1 + 0.1)1

(11.2)

Net present value = −10,420,000 + 138,298,719 + 125,726,108 + 114,296,462 + 103,905,874 = USD$ 471,807,163 It shows that the project is acceptable as the NPV is larger than zero. As the market price for monoclonal antibodies is high and the aqueous two-phase separation being continuous process that can produce large amount of cell culture in a year, the NPV will tend to be very high and being very profitable. Also, some of the costs are not included in the calculations due to uncertain information, therefore with a high NPV, it is still able to cover the unaccounted costs.

11.7.5 Internal rate of return IRR is defined as any discount rate that results in a NPV of zero and is usually interpreted as the expected return generated by the investment. It is a metric used in financial analysis to estimate the profitability of potential investments. If the internal return rate is greater than the project’s cost of capital rate, the project will add value for the company (Shultz, 2005). IRR analysis will be done in conjunction with a view of company’s capital cost and the NPV calculations. Table 11.9 tabulates the cash flow calculating the IRR.

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Table 11.9 Cash flow in period of 5 years for ATPS process. Year 1 2 3 4 5 Internal rate of return

Cash flow (USD$) −11,462,000 167,341,450 167,341,450 167,341,450 167,341,450 1460%

Equation to calculate IRR: NPV =

N 

Cn (1 + IRR)−n = 0

(11.3)

n=1

It shows the calculated IRR to be 1460%, because the revenue gained from this process is very high which in return gain high profit. This project is acceptable as it has higher IRR (1460%) than the discounted rate which is this case (10%).

11.7.6 Break–even analysis Break–even analysis is a type of cost–benefit analysis to identify the point where the benefits of the project are equal to the cost. Break–even point is a point that is expresses as the amount of revenue that must be realized where a process does not have a loss or profit (Herman and Zabloski, 1979). It is the central point where the cost of the project will be fully covered by the revenue. The break–even point can be calculated with the break–even ratio equation: Break − Even Ratio =

Yearly NPV Cash flow − Overall NPV Cash flow Yearly NPV Cash flow (11.4)

The break–even ratio was evaluated on a graph with the capital cost, operating cost, and revenue in terms of years. The break–even analysis will analyze, at which point of the year the break–even point will exist, that is, when the revenue totally cuts of the capital investment to be at zero. In Table 11.10, FC is the capital cost whereas VC is the operating cost at the point of the year. TC is the total cost at the point of the year and Reve is the revenue gain at the point of the year. BEP will be the break–even analysis. The break–even analysis of the ATPS of cell culture supernatant to extract monoclonal antibody is conducted (Fig. 11.4). The break–even point happens where the yellow line touches the gray line. In this case, the break–even point is at 0.068 years. The high revenue of the end product in this process causes the rapid break–even, which is a great advantage as the sooner the break–even point occurs, the sooner the profit is being gained.

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Table 11.10 Calculation for break-even analysis for ATPS process. Break–even analysis Years FC 0 11462000 0.005 11462000 11462000 0.01 11462000 0.015 11462000 0.02 11462000 0.025 0.03 11462000 0.035 11462000 0.04 11462000 0.045 11462000 0.05 11462000 0.055 11462000 0.06 11462000 0.065 11462000 0.07 11462000 0.075 11462000 0.08 11462000

11.8

VC 0 38292.75 76585.5 114878.25 153171 191463.75 229756.5 268049.25 306342 344634.75 382927.5 421220.25 459513 497805.75 536098.5 574391.25 612684

TC 11462000 11500292.8 11538585.5 11576878.3 11615171 11653463.8 11691756.5 11730049.3 11768342 11806634.8 11844927.5 11883220.3 11921513 11959805.8 11998098.5 12036391.3 12074684

Rev 0 875000 1750000 2625000 3500000 4375000 5250000 6125000 7000000 7875000 8750000 9625000 10500000 11375000 12250000 13125000 14000000

VC SP FC BEP

7658550 175000000 11462000 0.068494686

Conventional protein A affinity chromatography cost/benefits analysis

Protein A affinity chromatography is a conventional method of capturing human antibodies in many manufacturing plants. The capture process that are being evaluated are based on these literatures (Heinzle et al., 2006; Ishihara and Kadoya, 2007; Tugcu et al., 2008). There are several steps in the process consisting equilibration, loading of cell culture, washing, elution of purified monoclonal antibodies, column regeneration, cleaning, and washing, which are done under batch process. The size of column is set to a smaller size due to the high cost of protein A resin. To make a comparison on cost/benefit analysis, the annually process of cell culture supernatant for this method is also 500 m3 /year.

11.8.1 Capital/operating costs for protein A affinity chromatography According to the literature, the main equipment that is used in the protein A capture process is chromatography column, mixing tanks, and binary gradient pump. Table 11.11 shows the quantity of equipment for protein A chromatography process. The total equipment costs for the equipment listed above are USD$ 5,300,000 (Rosa et al., 2011). For the unlisted equipment, the same assumption is made by assuming 10% of the total equipment costs. The unlisted equipment costs are USD$ 530,000 and the total installation and engineering costs is set to be USD$ 7,500,000 due to the

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Figure 11.4 Break–even analysis of ATPS process. Table 11.11 The quantity of equipment for protein A chromatography process. Equipment Chromatography column Mixing tank Binary gradient pump Storage tank

Quantity 1 5 1 3

complexity of the chromatography column. Total capital investment for the protein A capture process can be calculated by: Total capital investment = 5300,000 + 7500,000 + 530,000 = USD$ 13,330,000

11.8.2 Operating costs for protein A capture process The conventional protein A capture process has an annual operating cost of USD$ 14,400,000 (Brämer et al., 2019). Operating condition of this process is much higher than the aqueous two-phase separation process because of the cost of raw material namely protein A resin. Protein A is seen as the gold standard in monoclonal antibody capture with a high price tag of USD$ 12,000 per L (Brämer et al., 2019). It took 79% of the overall annual operating costs remaining 6% labor dependent, 1% quality control, 10% plant overhead, and 4% consumables. The total operating cost of each parameter is listed in Table 11.12.

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Table 11.12 Annual operating costs for protein A capture process. Parameter Raw material Labor dependent Quality control Plant overhead Consumables Total operating costs

Cost (USD$) 11,376,000 864,000 144,000 1,440,000 576,000 14,400,000

Table 11.13 Net present value for protein A capture process. Year Revenue (USD$) Capital cost Operating cost Free cash flow Present value

1 N/A 13,330,000 N/A −13,330,000 −12,118,181

2 175,000,000 N/A −14,400,000 160,600,600 132,727,272

3 175,000,000 N/A −14,400,000 160,600,600 120,661,757

4 175,000,000 N/A −14,400,000 160,600,600 109,692,960

5 175,000,000 N/A −14,400,000 160,600,600 99,719,964

11.8.3 Total revenue Same as the aqueous two-phase separation process, the plant is assumed to process 500 m3 /year of cell culture supernatant annually, resulting in annual production of 25,000 g and same revenue of USD$ 175,000,000.

11.8.4 Net present value With the total capital costs, annual operating costs and annual revenue data, the NPV of protein A capture process can be evaluated. Table 11.13 is the present value calculation table with the information of revenue, capital cost, operating cost, free cash flow, and the present value. Calculation for present value with discounted rate of 10%: Net present value = −12,118,181 + 132,727,272 + 120,661,757 + 109,692,960 + 99,719,964 = USD$ 470,765,163

The NPV calculated for the protein A capture process is USD$ 470,765,163, which shows that this project is highly acceptable due to the high NPV. However, the NPV of protein A capture process is slightly lower than the aqueous two-phase separation process. This shows that aqueous two-phase separation process is more recommended because it has higher NPV.

11.8.5 Internal rate of return Same as for aqueous two-phase separation process, the IRR for protein A capture process is also evaluated as part of the economic feasibility evaluation. Table 11.14 tabulates the cash flow of protein A capture process for 5 years and the IRR calculated

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Table 11.14 Cash flow in period of 5 years for protein A capture process. Year 1 2 3 4 5 Internal rate of return

Cash flow (USD$) −13,330,000 160,600,000 160,600,000 160,600,000 160,600,000 1205%

Table 11.15 Calculation of break-even point for protein A capture process. Break–even analysis Years FC 0 13330000 0.005 13330000 13330000 0.01 13330000 0.015 13330000 0.02 13330000 0.025 0.03 13330000 0.035 13330000 0.04 13330000 0.045 13330000 0.05 13330000 0.055 13330000 0.06 13330000 0.065 13330000 0.07 13330000 0.075 13330000 0.08 13330000 0.085 13330000 0.09 13330000

VC 0 72000 144000 216000 288000 360000 432000 504000 576000 648000 720000 792000 864000 936000 1008000 1080000 1152000 1224000 1296000

TC 13330000 13402000 13474000 13546000 13618000 13690000 13762000 13834000 13906000 13978000 14050000 14122000 14194000 14266000 14338000 14410000 14482000 14554000 14626000

Rev 0 875000 1750000 2625000 3500000 4375000 5250000 6125000 7000000 7875000 8750000 9625000 10500000 11375000 12250000 13125000 14000000 14875000 15750000

VC SP FC BEP

14400000 175000000 13330000 0.083001245

to be is 1205%. Same as aqueous two-phase separation, the IRR is very high due to the high value of the end product (monoclonal antibodies), indicating that this process is acceptable.

11.8.6 Break–even analysis Similar to ATPS, break–even analysis of protein A capture process will be performed in this section. Break–even analysis can be performed with the data of protein A capture process including operating costs, capital costs, and revenue. Table 11.15 shows the break–even analysis of protein A capture process in terms of years where the revenue is break–even with the total cost. BEP of 0.083 years is the break–even point of this process.

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Figure 11.5 Break–even analysis for protein A capture process.

The analysis of the break–even point is shown (Fig. 11.5). Break–even point happens where the yellow line meets the gray line which is at 0.083 years period. This is a good process because the revenue is high enough to break–even at an early stage.

11.8.7 Comparison of ATPS and protein A chromatography Based on the cost–benefits evaluated above, the economic feasibility of both processes is acceptable with high NPV and high internal return rate. However, there are some differences between the NPV, IRR, and break–even point of the process. This happens due to the manipulating variable of the costs. Below is the comparison of capital costs, operating costs, and the revenue for both processes. Table 11.16 shows the costs and revenue and economic feasibility comparison for both processes. The operating costs for protein A chromatography process are almost doubled that of the ATPS due to the high cost of protein A resin and the complication of the process. Also, the capital costs for protein A chromatography are also higher than the capital costs for aqueous two-phase separation process because of the chemical equipment that has a more complex modification and tougher mechanism to run the process. Operating costs and capital costs are a manipulating variable for economic feasibility analysis. The difference in these variables will directly affect the economic analysis. The process with higher NPV indicates higher profitability of the process over a period of time, with time value of money included. In this case the NPV is calculated

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Table 11.16 Costs and revenue of ATPS and protein A capture process and the comparison of economic feasibilility. Process Operating cost (USD$) Capital cost (USD$) Revenue (USD$) Net present value (USD$) Internal rate of return (%) Break–even point (years)

ATPS 7,658,550 11,462,000 175,000,000 471,807,163 1460 0.068

Protein A affinity chromatography 14,400,000 13,330,000 175,000,000 470,765,163 1205 0.083

for a period of 5 years. Thus, ATPS with the higher NPV is better comparing to protein A affinity chromatography process. Besides that, the IRR is also one of the key factors in economic feasibility. IRR is the expected return generated by the investment, thus when the IRR is greater than the capital costs of the project the project will gain profit for the company. In this case, both of the processes have high IRR. However, the IRR for aqueous two-phase separation is higher therefore it is a better process comparing the two. Lastly, it is the break–even point where the revenue gain from the process is enough to cover the capital investment on the project. The shorter the break–even point the better because it takes the shortest time to break–even the investment made, and profit will be gain. With aqueous two-phase separation process having only 0.068 years to break–even, it is a better process as it can start to gain profit earlier compared to protein A affinity chromatography process.

11.9

Conclusion

As a conclusion, the process of multiple liquid separation systems is technologies that can improve the economic sustainability compared to the conventional chromatography method and other type of separation such as precipitation. The process of multiple liquid separation by polymer–polymer, polymer–salt, and all the other interactions for different kind of components to separate is very simple and easy as it is a one-step process. This means that if the process is very simple, the operating costs will be much lower due to less labor costs, utility costs, maintenance costs. Also, with the simplified separation process, the less complicated equipment is cheaper to purchase and maintain than the conventional equipment. Moreover, with the ATPS, the process can be run continuously to produce more products. Continuous process is better than batch process in terms of economic sustainability because consistent amount of product is being produced every day to hit the minimum target of the process whereas batch process needs to be monitored frequently with specific schedule. By comparing ATPS with the protein A affinity process, it shows that ATPS is much more advantageous in terms of economic sustainability. Thus, it can be concluded that in the economic point of view, ATPS is a better process and it should be considered for more separation manufacturing plants in the world.

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Hock Chee Lu a, Sze Shin Low a, Shuet Fen Lai b and Kuan Shiong Khoo c a Department of Chemical and Environmental Engineering, Faculty of Engineering, University of Nottingham Malaysia Campus, Semenyih, Selangor Darul Ehsan, Malaysia, b School of Energy and Chemical Engineering, Xiamen University Malaysia, Sepang, Selangor Darul Ehsan, Malaysia, c Department of Chemical Engineering and Materials Science, Yuan Ze University, Taoyuan, Taiwan

12.1

Introduction

Liquid–liquid extraction (LLE), also known as solvent extraction, is a method that separates compounds based on their respective relative solubilities in a mixture that contains two different immiscible liquids, which are usually water, a polar covalent molecule and organic solvent, a nonpolar covalent molecule. The transfer of one or more species from one liquid into another liquid phase driven by chemical potential undergoes a LLE process. Once the transfer process is completed, the overall system of various chemical components consists of solvents and solute are in a more stable and low free energy configuration where the solvent that is enriched in solute is defined as extract and feed solution that is depleted in solute is defined as raffinate. There is only slight difference when it comes to LLE and its counterpart, liquid– liquid separation (LLS): separation principles, equipment, and raw material used. LLS is defined as a system or method to separate two or more immiscible liquids that lie in the form of a mixture. Usually, the physical and chemical properties of these different liquids are well defined before knowing which separation method is suitable to be used such as gravity separation and coalescing separation which will be discussed later on. According to their separation principles, LLS generally takes a longer time for the separation to be done when compared to LLE. However, LLE poses more environmental threat which will be elaborated later. Fig. 12.1 shows the typical illustration of LLS and LLE, respectively. LLE plays an important role when it comes to chemical processing industries. It is widely used in the production of fine organic compounds, perfumes, vegetable oils, biodiesel, and others (Chemat et al., 2019). As new LLE method becomes an efficient and environmentally friendly way to separate a liquid mixture, it was not until decades ago. The modern LLE methods are the successor of conventional extraction method as they are improved in many aspects such as cost, energy saving, efficiency, and environmentally friendly. In a more in-depth manner, LLS also stands as a successor from the conventional extraction method due to its similar improvement Principles of Multiple-Liquid Separation Systems: Interaction, Application and Advancement. DOI: https://doi.org/10.1016/B978-0-323-91728-5.00003-2 c 2023 Elsevier Inc. All rights reserved. Copyright 

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Figure 12.1 Schematic diagram of typical LLS and LLE process.

with LLE. However, unlike LLE, LLS application is usually seen in the oil and gas industries such as wastewater treating where emulsions happen between a low-density liquid and a high-density liquid (Noïk et al., 2013).

12.2 Environmental impact caused by conventional extraction method Conventional extraction method has been in the industries for a long time. There are around six main types of conventional extraction method, namely infusion, decoction, maceration, percolation, reflux extraction, and Soxhlet extraction (Panja, 2018). Other methods are not common or only available in lab scale due to the difficulty to define scale-up parameters. However, these conventional extraction methods result in many disadvantages, leading to environmental impacts. In addition, technology advancements have also driven the LLE boundaries and the new LLE methods have been slowly replacing these conventional extraction methods. In this chapter, we will be discussing the environmental impacts of conventional extraction methods and elaborate on the different kinds of pollution. However, most of them impact the environment in an indirect manner. There are two major drawbacks for conventional extraction and both are interrelated with how it affects the environment negatively. First is its low efficiency when it comes to extracting a desired product. Efficiency is crucial in a processing plant, as the efficiency of a specific production flow decreases, more money, time, and energy are needed to produce a desired number of

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high-quality products. In addition, usually time and energy relate to amount of carbon footprint a process produced, since the longer the time of the process the more energy it requires hence more fossil fuel and coal will be burned resulting in an increase of carbon footprint. For example, the extraction time of phenol is fairly long using conventional method when compared to alternative methods. Because of the long extraction time, the amount of energy needed for the process to achieve the desired amount of product can increase as well, further contributing to global warming. Some of the most common method for the extraction of phenol is through Soxhlet, maceration, infusion, and digestion where long extraction time up to 720 minutes is needed to extract merely 60 mg of phenol content from Vernonia cinerea leaves. Hence, it is known that long extraction time and low yield amount lead to low extraction efficiency. Other than that, Soxhlet and maceration extraction both uses a high solvent to feed ratio of at least 20 for the exhaustive extraction of all compounds from a matrix (OsorioTobón, 2020). The common solvent used for both type of extraction method is ethanol, which is considered as a green solvent due to its nontoxic properties and biodegradable capability. It also means that to extract 10 L of phenolic compound, at least 200 L of ethanol is needed for the extraction process. However, according to various studies (Hoekman et al., 2018; Lu, 2018), ethanol actually poses a major threat to human health and the environment due to its extensive air, water, and soil pollution capabilities (England, 2019). As the demand for ethanol increases, the production of ethanol must be increased too.

12.2.1 Air pollution Many of these solvent chemicals used in conventional approaches are classified as volatile organic compounds which are harmful to the environment such as benzene, formaldehyde, and xylene (Wexler, 2014). These volatile organic compounds may contribute to the formation of ozone in a complex series of reactions that requires the presence of heat, nitrogen oxides which are usually produced from vehicles and sunlight. The ozone formed are called ground-level ozone which creates a smog-filled environment which is not good for human health and the environment (Bredenberg, 2012). When smog fills the air, the sunlight will be partially blocked hence result in low agricultural productivity because of the low photosynthesis efficiency of the affected plants. Large amount of ethanol solvent is used during phenolic compound extraction via conventional extraction method. From here, it is known that a tremendous amount of energy will be used to manufacture this ethanol due to low extraction efficiency and high temperature process of conventional extraction method resulting in more burning of nonrenewable energy such as coal or fossil fuel to keep up with the energy demand. Overall, air pollution caused by conventional extraction method especially in the presence of harmful solvents is significant as they contribute to the destruction of nearby ecosystem and human health. In addition, high carbon footprint from these processes also causes the acceleration of global warming and in the long run, total eradication of many coastal cities.

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12.2.2 Water pollution Since ethanol is a common solvent used in conventional extraction method, and is mainly produced from corn, a major water pollution concern arises as the increased corn production may result in surface runoff and infiltration of groundwater (Hoekman et al., 2018). According to a report by the Environmental Protection Agency, it is noted that corn has the highest fertilizer use per acre when compared to other biofuel feedstock (Integrated Assessment of Hypoxia in the Northern Gulf of Mexico and Hypoxia Assessment Reports, 2019). It has been estimated that 96% of corn field acreage receive nitrogen-based fertilizers at a rate of 155 kg/hectare (National Research, 2011). Other than that, phosphorus is also used as a fertilizer source to optimize ethanol born production in different parts of the world. With all these nutrients supplied for large-scale corn production, a significant amount (roughly 40–60%) of these applied nutrients are not being absorbed into the corn plantation as they are lost due to runoff, tile drainage, or sediment transport (Hoekman et al., 2018). In the presence of these excess nutrients, eutrophication may happen to promote the growth of algae. At a certain point where the algae ecosystem dies and begins to undergo a decay process that consumes oxygen that is dissolved in water. With an unpleasant situation like this, a condition called hypoxia which are typically called dead zones as oxygen concentration falls below 2–3 ppm where the designated environment can no longer support marine animals (Integrated Assessment of Hypoxia in the Northern Gulf of Mexico and Hypoxia Assessment Reports, 2019). These hypoxia zones usually occur in coastal waters may occur due to severe lack of dissolved oxygen in the water. Nutrients related pollutions for water-based sources such as rivers, lakes, reservoirs, and coastal waters also significantly affect drinking water supplies and recreational activities such as fishing and swimming. Nutrients run off leads to excessive growth of algal and other water plant species which in the end giving water supplies weird taste and odor problems due to high levels of nitrogen, phosphorus, and suspended solids. Since conventional extraction method uses large amount of solvent for extraction process, the chance of having solvent spillage is also significant such as improper storage of solvent and poor piping management. Improper storage of these solvents may also cause water pollution as some solvents used in most of the conventional extraction method (maceration, percolation, reflux extraction, and Soxhlet extraction) are considered hazardous and harmful to the environment ecosystem such as methanol (Zhang et al., 2018). Since most solvents are considered toxic and carcinogenic, hence its negative environmental impacts toward flora and fauna are logically known. Other than that, the treating or recovery of large amount of used solvents from conventional extraction method may be expensive, resulting in improper disposal of solvents into nearby rivers and lakes such as the illegal dumping of toxic waste that happened alongside Sungai Kim Kim in Pasir Gudang (Sukaimi, 2019).

12.2.3 Soil pollution Soil pollution may also occur in conjunction of water pollution as rivers may carry harmful solvents that was disposed by factories and ended up dissipating into the

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soil, altering its pH value and fertility rate. With these alterations, crops in agriculture industries and floras around the area may be facing a diminishing of survivability as the change in soil properties does not favor the living conditions. As mentioned for the corn-based ethanol production, soil erosion can occur due to an increase of agricultural activities. The effect of erosion on soil is its diminishing soil quality which will result in lower productivity of natural and agricultural ecosystem as it removes the finest and uppermost soil particles which are usually higher in terms of organic matter, nutrients, and water-holding capacity (Hoekman et al., 2018). Another concern of increased crops production is the reduction of soil organic carbon whereby crops productivity, nutrients, water retention, and soil biodiversity are affected at a significant scale. From the discussions above, it is known that conventional extraction method poses significant environmental damage. While more research and advancement can be done to improve these conventional extraction methods; however, it will be unlikely that the conventional extraction method will be considered a green extraction process.

12.3

Nonconventional extraction method

Alternative extraction methods are created to replace conventional extraction method for the main purpose of better efficiency, lower cost, and more environmentally friendly. Over time, traditional extraction methods are improved to increase their extraction efficiency and reduce extraction time. While on the other hand, modern extraction techniques have been developed for quick and selective extraction that requires lesser amount of solvent. Table 12.1 shows some of the available traditional and advance separation method alongside with their respective industrial applications. It also shows the improvements of modern extraction techniques over various traditional separation methods and their respective industrial applications. In addition, alternative methods can produce extracts that are rich in phenolic compounds using moderate temperature, shorter extraction time, and solvents that are recognized to be safe and eco-friendlier to the environment (Osorio-Tobón, 2020). The advancement of the new extraction methods is developed to replace conventional extraction methods. In addition, the alternative extraction method is the improvement of the conventional extraction method. The respective pre- and post-requisite extraction methods are shown in the following sections.

12.3.1 Microwave-assisted extraction Microwave-assisted extraction (MAE) is considered an excellent separation method due to their high efficiency in extracting many types of chemical compound from natural resources (Osorio-Tobón, 2020). When compared to conventional extraction method, MAE is a more efficient separation technique due to its capability to supply heat to the matrix internally and externally without a thermal gradient (Pena-Pereira and Tobiszewski, 2017). Other than that, higher extraction efficiencies, greater reduction in solvent requirement (around 90%), and short extraction time (a few minutes) are

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Table 12.1 Improvement of various traditional methods (Panja, 2018). Traditional methods Infusion

Advanced methods Pressurized liquid extraction

Decoction

Supercritical fluid extraction

Maceration

Microwave-assisted extraction

Percolation

Ultrasound-assisted extraction

Reflux extraction

Gravity separation

Soxhlet extraction

Coalescing separation

Industrial application – Curcuminoids extraction – Detection of contaminants in food – Extraction of bioactive compounds – Extraction of hop constituents – Decaffeination of tea and coffee – Separation of lecithin from oil – Extraction of active components from medicinal plants – Extraction of phenolic compounds – Textiles application – Extraction of active components from medicinal plants – Food processing application – Extraction of oils from animals – Separation of coffee beans, cocoa beans, rice – Filtration of aviation jet fuel – Removal of liquids from hydrocarbons – Removal of oil from water phase

From Panja, P. (2018). Green extraction methods of food polyphenols from vegetable materials. Curr. Opin. Food Sci., 23, 173-182. https://doi.org/10.1016/j.cofs.2017.11.012.

the advantages of using MAE in extraction processes (Pena-Pereira and Tobiszewski, 2017). These advantages over conventional extraction method are an indirect approach toward greener extraction process because of lower energy and solvent requirement. Hence, the use of MAE in the industries has drastically increased due to its good analytical and eco-friendly performance when compared to conventional extraction method such as Soxhlet extraction. Fig. 12.2 shows MAE environmental impact in terms of lab scale. However, the details shown above are only based on lab-scale experiment results due to insufficient publication data from other sources. Preferable solvent used for MAE are water, enzymatic aqueous extractants, supramolecular solvents, ionic liquids (ILs), and deep eutectic solvent as these mentioned solvents are advantageous in terms of extraction efficiency, selectivity, and environmentally friendliness (Gałuszka et al., 2012). Since MAE has a great reduction in solvent requirement, the amount of pollution caused by solvent being released to the environment is minimal. In addition, the solvents used in MAE are considered green hence further reducing its pollution potential as an alternative method. With all the mentioned advantages of MAE, the scaling of MAE for the purpose of industries is still in its infancy form while this extraction method is largely applied on laboratory scale.

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Figure 12.2 Scheme of the green aspect of microwave-assisted extraction. From Pena-Pereira, F., Tobiszewski, M. (2017). The Application of Green Solvents in Separation Processes. Elsevier Science. https://books.google.com.my/books?id=SZupDQAAQBAJ.

12.3.2 Pressurized liquid extraction In this alternative extraction process, temperature of above solvent boiling point but below the critical point is used to increase the kinetics of extraction with the implementation of applying high pressure to maintain the solvents in their liquid state form (Panja, 2018). Many researchers have also found out that the solubility of chemicals of polyphenols in liquids is drastically increased in pressurized liquid extraction (PLE) (Chaves et al., 2020; Tsao, 2010). A literature revealed that using PLE to extract phenolic compounds from various phenolic sources require a temperature range of 40°C and 275°C, pressure range between 10 and 200 bars (Bursa´c Kovaˇcevi´c et al., 2018). Although some of the extraction process such as the phenolic extraction of pacific oyster requires extreme operating condition of 225°C and 120 bar which raises the energy requirement of the process, while at the same time making conventional extraction method more attractive instead. However, the solvent used is only water which is considered an environmentally friendly solvent. In addition, the extraction time is only a mere 5 minutes which is definitely a more efficient extraction method adding the fact that the yield of total phenolic content extracted using PLE is higher. This is due to a temperature increase which will result in an increase in diffusion of phenolic compounds into solvent as a result, improving the transport efficiency. In addition, using PLE as an extraction method also poses many advantages such as:

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1. Higher amounts of polyphenols are covered at elevated temperature. 2. Process is considered energy saving when compared to conventional extraction method because sensible heat of a liquid is lower than the heat of vaporization hence less heat is required to increase temperature. 3. Solvents used in PLE process are mainly water and other aqueous alcohols where all of them are environmentally friendly and nontoxic.

However, the operating conditions for PLE are still considered extreme because of its high-pressure requirement. As high pressure is required for specific extraction process, its energy requirement may change resulting in a more energy consuming process which increases its overall potential carbon footprint.

12.3.3 Gravity separation Gravity separation or gravity settling can be considered leaning closer toward the principle of LLS as no solvent is used for this separation technique. In short, gravity separation uses the technique where two immiscible liquid phase mixture are separated within a vessel via the difference in density of the two liquids (Bahadori, 2014). With sufficient retention time, the separators allow gravity separation to take place resulting in total extraction of the desired product. If the gravitational force acting on the droplet is greater than the drag force of gas flowing around the droplet, the liquid droplet will settle out of a gas phase. Gravity separation is one of the oldest techniques to separate different liquids. Just as mentioned before it utilizes only gravity for separation process. Hence, this separation method is considered an extremely attractive separation technique due to its low capital and operating cost as no excessive heating or any solvent chemical is needed for the entire process making it one of the most environmentally friendly operations (Falconer, 2003). Although gravity separation is usually more widely used for solid and liquid separation; however, it is still exceptionally going well for LLS applications. Gravity separation is well known in some of the application due to their added advantages compared to other separation methods. Gravity separation has replaced the conventional separation method for oil and water emulsion separation process (Zhao et al., 2020). Due to many researches in the field of finding a more low-cost and eco-friendly process, gravity separation was developed. It was mentioned that gravity separation is an excellent separation method for oil and water emulsion due to their high average separation efficiency of more than 99.5%. In addition, the separation time is extremely low making it an attractive separation choice among other separation methods of only 3 minutes. As its separation time is short, more energy will be saved. Unlike the previous mentioned MAE and PLE which uses green solvent for their extraction process, while most gravity separation system does not require any solvent to be involved making it a low carbon footprint process. In addition, no excessive pressure or heat are used during the process resulting in a low energy consumption when compared to other separation process which also means lower burning of coal, fossil fuels, and other unrenewable energies. Many of the literatures for gravity separation only refer to solid–liquid separation. Hence the yield for LLS in terms of gravity separation is not able to be obtained due to lack of information and literature data. However, its long retention time is bound to be an obvious disadvantage which altered

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its overall efficiency which makes gravity separation an unattractive separation method in some situations, as the process may be more time consuming than the others, which might be less desirable in industrial applications. Gravity separation is mainly used for agricultural application such as removal of impurities, insect damage, admixture, and immature kernels from various type of agricultural harvest. Some of them include wheat, cocoa beans, peanuts, corn, and more (Sapkota, 2017). When it comes to recycling, gravity separation is also being employed to remove any valuable components from a recycling mixture (Bilesan et al., 2021). This eases recycling and further improves the efficiency of the overall recycling process. A research has also been done to show that gravity separation can also be employed in obtaining milk fat or fatty acid from cow milk (Martini et al., 2017).

12.3.4 Coalescing separation Coalescer, a separation tool for a mixture of liquids is commonly mistaken as filters. Although both their equipment structures are fairly similar to those that are defined as filters; however principle-wise, they are very different from each other on the theory spectrum. Filter system uses synthetic membrane and high pressure to separate their desired products, usually in the application field of separating suspended/dissolved solids from a body of liquid such as nanofiltration or reverse osmosis. As for coalescing filters, it separates small particles of one liquid phase from a large quantity of another liquid phase through coalescing effect (Bahadori, 2014). Different types of internal structure of the coalescers must be configured to effectively separate the mixture. This applies to both vertical and horizontal orientation of the coalescers. There are mainly two types of coalescers: mechanical and electrostatic coalescers. Mechanical coalescer is operated by physical alteration or involvement of liquid droplet under the influence of mechanical and physical means. It is more commonly applied in oil and gas industries for the application of removal of water or hydrocarbon condensate and also the purpose of ensuring natural gas quality, protecting downstream equipment. It is also known that their efficiency as a liquid-based coalescers is close to 100%. When compared to conventional separation method, mechanical coalescer does not require a long retention time hence explaining its high efficiency of 99.89%. However, there is a need for regular changing of coalescer cartridges which can lead to exposure and releasing of various chemical to the surrounding, damaging the health of workers, and the environment. On the other hand, no excessive heating is implemented in the separation process which indicates its higher energy saving capabilities and lower carbon footprint. There is also no known literature writing about the presence and usage of external solvents for separation process, eliminating the chance of having unpleasant occurrence of solvent spillage which may cause pollution in terms of air, water, and soil. This further shows that mechanical coalescer separation technique is indeed environmentally friendly. As for electrostatic coalescer, electrical fields produced from electrodes are used to induce droplet coalescence in water from crude oil emulsions to increase its droplet size. The effects on increasing size of water droplet arise from a wide range of different

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dielectric properties of the conductive water droplets that are dispersed in the insulating oil (Mura et al., 2010). As an uncharged droplet is exposed to an AC electric field formed within the electrode, the droplet polarizes and an electric field forms around it to offset the external field (Moukengué Imano and Beroual, 2006). The induced charges would be on the surface of the water droplet since it is considered highly conductive, resulting in no net charge but one positive and one negative side. Hence, when two droplets with induced dipoles get close to each other, a pulling force will be observed resulting droplets getting pulled closer together until they coalesce. In terms of efficiency, electrostatic coalescers are well known to be an effective method to separate water–oil emulsion by using the application of an electrical field to reduce stability of the emulsion which improves the separation of phases due to the polarization effect of the droplets, which favors attraction and thus coalesce. However excessive voltage may destroy the droplets and causes the opposite of improving the emulsion stability (Rossi et al., 2017). Other chemicals, emulsifiers, or additives are added into the process too in order to improve separation between water and oil such as asphaltene, resins which are not considered environmentally friendly due to their nonbiodegradable characteristics (Al-Taq et al., n.d.). However, some alternatives can be done to make electrostatic coalescing a more environmentally sustainable process such as replacing asphaltene and resins with green emulsifiers. Although after changing, the process efficiency may differ. Many researches have examined potential biodemulsifiers to destabilize crude oil emulsion while at the same time being environmentally friendly and its usage does not result in secondary pollution (air, water, and soil) (Saad et al., 2019). As for conventional demulsifier which are higher in efficiency for the demulsification of water–oil mixture, challenges in the separation process still exist such as low compatibility and efficiency. In addition, water extracted by these chemical demulsifiers has to undergo water treatment process before being discharged to the environment to remove any refractory organic polymers which are considered extremely harmful to aquatic organisms.

12.4

Comparison between alternative extraction methods

Using phenolic compound as a natural product, Table 12.2 summarizes the performance of various separation methods, which shows many other separation methods that are not mentioned in this chapter (Zhang et al., 2018). However, due to the lack of literature data for gravity separation and coalescer separation, both of these separation methods are not mentioned in the table. Table 12.2 clearly elaborates the differences between both separation methods mainly in terms of the environmental aspect. Both conventional and modern extraction methods use polar solvents as they play a crucial role in the extraction of phenolic compounds and organics of higher polarity (Osorio-Tobón, 2020). Most of the solvents used are water, aqueous, and nonaqueous solvent.

Method Maceration Percolation Reflux extraction Decoction Pressurized liquid extraction Supercritical fluid extraction (SFE) Microwave-assisted extraction Ultrasonic-assisted extraction Pulsed electric field extraction Enzyme-assisted extraction

Hydro distillation and steam distillation

Solvent Water, aqueous, and nonaqueous solvents Water, aqueous, and nonaqueous solvents Aqueous and nonaqueous solvents Water Water, aqueous, and nonaqueous solvents Supercritical fluid (S-CO2 ), sometimes with modifier Water, aqueous, and nonaqueous solvents Water, aqueous, and nonaqueous solvents Water, aqueous, and nonaqueous solvents Water, aqueous, and nonaqueous solvents Water

Temperature Room temperature

Pressure Atmospheric

Time Long

Volume of organic solvent consumed Large

Room temperature, occasionally under heat Under heat

Atmospheric

Long

Large

Atmospheric

Moderate

Moderate

Under heat Under heat

Atmospheric High

Moderate Short

None Small

Near room temperature

High

Short

None or small

Room temperature

Atmospheric

Short

Moderate

Room temperature or under heat Room temperature or under heat Room temperature, or heated after enzyme treatment Under heat

Atmospheric

Short

Moderate

Atmospheric

Short

Moderate

Atmospheric

Moderate

Moderate

Atmospheric

Long

None

Environmental sustainability of multiphase systems

Table 12.2 Brief summary of various extraction methods for natural products (Zhang et al., 2018).

From Zhang, Q.-W., Lin, L.-G., and Ye, W.-C. (2018). Techniques for extraction and isolation of natural products: a comprehensive review. Chin. Med., 13 (1), 20. https://doi.org/10.1186/s13020018-0177-x.

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As mentioned earlier, water is considered an environmentally friendly solvent as it does not pollute the environment. Most of the modern separation methods have an option of using water for separation process when compared to conventional methods except for supercritical fluid extraction that requires supercritical fluid (S-CO2 ) to act as a solvent. Hence, this puts modern separation method in a more environmentally friendly prospect. In addition, their amount of solvent require is lower hence lowering the chance of having any potential pollution. Among the modern separation methods, hydro distillation and steam distillation are the only separation techniques that require a long separation time while all the conventional separation method requires a moderate to long separation time. This indicates the drastic difference of efficiency between two separation methods. Hence, it is clear that the modern separation method is actually more efficient. In conclusion, it is obvious that modern separation method is a better choice when it comes to separation technique. This is due to their higher efficiency and lower energy demand compared to their conventional method. There are many more alternative methods that have been developed throughout the century for the goal to have a better, more efficient, environmentally friendly process. Other than the mentioned MAE and PLE, supercritical fluid extraction, ultrasonicassisted extraction, and others. Table 12.3 compares the pros and cons between conventional and alternative extraction method. Based on Table 12.3, conventional extraction methods have two major disadvantages when compared to their successors, which is the average usage of higher temperatures and longer extraction time. Since operating conditions such as temperature and extraction time are a key factor that determines a specific extraction process energy consumption, hence it is known that conventional method releases a higher amount of carbon footprint. In addition, the extreme operating condition of conventional extraction method may degrade the extraction compound, lowering the overall yield, and efficiency. For example, in Soxhlet extraction, it is observed that this extraction technique acquires fewer phenolic compounds when comparing MAE and Soxhlet extraction of phenolic compounds from Vernonia amygdalina. MAE produces a greater extraction yield in a much shorter time with a value of 102.04 mg GAE/g d.b. in 10 minutes, while Soxhlet extraction obtained a total phenolic content value of 73.54 mg GAE/g d.w. in 480 minutes. Moreover, Soxhlet uses a higher amount of solvent for extraction process compared to MAE (Alara et al., 2018). Hence it is clear that alternative extraction method like MAE, PLE, and others are more environmentally friendly when compared to its predecessor.

12.5

Environmental sustainability-related industrial applications

To further understand how a change in some parts of a separation process could impact the amount of carbon footprint it produces, some examples will be discussed regarding real industrial processes that adopt environmental sustainability.

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Table 12.3 Comparison between the pros and cons of different extraction methods in terms of environmental sustainability (Panja, 2018). Extraction method Pros Cons Conventional (Soxhlet, – Incomplex separation method – Requires high solvent to maceration, – Occasionally energy feed ratio (above 20) infusion, digestion) requirement may be low resulting in higher potential environmental impact – Low extraction yield (between 48.6 and 71 mg of GAE) resulting in higher carbon footprint – Long extraction time (360–720 minutes) resulting in higher carbon footprint – Nongreen solvent are used – Energy intensive processes – Greatly induce air, water, and soil pollution Alternatives LLE – Shorter extraction time which – May be energy intensive (MAE, PLE, SFE, lowers carbon footprint process occasionally (up to UAE) – Able to accommodate 275°C and 200 bars for different variety of green PLE, 400 bars for SFE, solvents 5500°C and 500 bars for – Higher extraction yield for UAE) many different LLS (132 mg GAE for PLE)

From Panja, P. (2018). Green extraction methods of food polyphenols from vegetable materials. Curr. Opin. Food Sci., 23, 173–182. https://doi.org/10.1016/j.cofs.2017.11.012.

12.5.1 Purification of natural dye carmine It is known that carmine is widely used in the pharmaceutical, food, and dyeing industrial sectors with a total estimated market value of USD$ 75 million per year as the demand for it increases year by year (Mageste et al., 2009). Hence, it has been a continuous interest from many researchers and companies to develop a more environmentally sustainable separation process. To obtain carmine, cochineal is first extracted from a dried female insect, Dactylopius coccus Costa using aqueous alcohol, with the final extraction of carmine from cochineal using aqueous extraction. However, most of the solvents used in this process have caused significant environmental problems due to their carcinogenic, inflammable, and toxic characteristics. With that in mind, there has been a noticeable growing interest in looking for a solvent replacement in industries that employs LLS. Recently, a new separation method has been employed to replace the existing carmine extraction method as it is more environmentally friendly due to its greener solvent usage when compared to the current use of organic solvent. The new separation method is known as aqueous two-phase systems (ATPS) where it is formed by mixtures of polymers, salt, and water.

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ATPS has great potential in extraction, separation, and purification of various biomolecules including carmine while at the same time well known for its high recovery yield and easy scale-up capabilities (Iqbal et al., 2016). However, the partitioning behavior of chromophoric solutes in an ATPS was poorly understood hence more research still needs to be done to make ATPS a greener separation method to replace conventional separation methods. ATPS has proven its separation capabilities in conjunction of its benign characteristics to be a more reliable and attractive alternative toward carmine purification. It is also revealed that carmine molecules partition has a higher preference toward macromolecule-rich phase when it comes to ATPS. In addition, more studies have been done to expand the boundaries of ATPS being a conventional separation method in the future. If such separation method was to be realized, the amount of carbon footprint for separation processes will be greatly reduced and it will be another great leap forward toward a more environmentally sustainable process.

12.5.2 Ionic liquid as green extraction solvent Since the dawn of chemical and energy industries, there has been a clear sign that their environmental pollution capabilities have ultimately change the ecosystem in their respective vicinity and causing global climate change. And for many decades, there has been a social expectation toward scientist and engineers to redesign a more environmentally sustainable process for the goal of generating lesser hazardous materials and pollutants. That being said, a new revolutionized green solvent has been discovered. Ionic solvents are organic salts in liquid form when under room temperature and are new revolutionized green solvent that have been discovered. Due to its nonvolatile properties under 100°C, it is naturally a good substitute toward conventional solvents. In addition, IL as a green solvent is nonflammable, nonvolatile, and recyclable. Due to their outstanding properties, such as outstanding solvating potential, thermal stability, and tunable properties through appropriate cation and anion selection as illustrated in Fig. 12.3, they are widely used (Elsaid et al., 2020). ILs have been under many researchers’ radar for the hope of replacing conventional organic solvents whereby most of them are considered to be harmful to the environment. It is known that one of the main sources of waste from a production plant is solvent which usually ends up in the atmosphere or into rivers and ground waters. Conventional solvents have also been reported to account for around 50% of post-treatment greenhouse gas emission. Hence, ionic solvents have been under many interests to replace conventional solvents. ILs are categorized into four different types based on their cation segment which are alkylammonium, dialkylimidazolium, phosphonium, and N-alkylpyridinium (Minea, 2020). Table 12.4 shows a guide for solvent selection to replace existing conventional solvent that are harmful to the environment. The table not only accounts for ILs only but other suitable alternatives. From Table 12.4, not just heptane or hexane, ILs are also able to replace many other carcinogenic organic solvents such as dichloromethane and ethers (Dietz, 2006).

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Figure 12.3 Advantages of ionic liquids over conventional organic solvents.

Table 12.4 Guide for solvent selection. Preferred Ethanol Ionic liquids Isopropylacetate Acetone 1-Propanol 2-Propanol Water Ethylacetate Supercritical CO2

Usable Isooctane Heptane Xylenes Methyl cyclohexane 2-Methyltetrahydrofuran Acetonitrile Toluene Tetrahydrofuran Cyclohexane

Undesirable Dichloroethane Hexane(s) Dioxane Chloroform Chloroform Dichloroethane Di isopropyl ether Pyridine Pentane

With ILs environmentally friendly properties when compared to those conventional high volatile and toxic organic solvents, ILs is truly a revolutionize replacement. It also mentioned that certain ILs extractant combinations have been shown to have the ability to have greater efficiency yield toward metal ion extraction than molecular organic solvents. However, it also stated that ILs face a few limitations when it comes to solubilization losses and the existing difficulty in recovering extracted metal ions. New discovery is yet to be unrevealed for the greater possibilities of ILs for separation application. As of now, more work and research have to be done to fully understand the toxicity and environmental impact of ILs alongside with their broken-down products/byproducts. It is also mentioned that the long-term stability and recyclability of

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ILs are not fully understood yet especially under certain process relevant operating conditions (Abu-Eishah, 2011).

12.6

Conclusion

The chapter reveals that separation processes are indeed causing significant impact to the environment mainly due to the existence of harmful and hazardous solvents. Other than that, low separation efficiency and high energy consumption of the extraction process also contributes to negative environmental impacts. Hence, new discoveries are made as time passes to lower down the amount of carbon footprint a specific process produce, whether it is optimization, new process creation, or solvent replacement. That is why there are many differences between conventional and modern separation methods. As pointed out in the chapter, it is clearly known that modern separation method is a better separation option when compared to its conventional counterparts due to be more environmental friendliness, higher efficiency, better energy saving, and lower cost. Although in some situation it has minor drawbacks, the use of modern separation method is justifiable. In the near future as technology advances, new and innovative separation method and solvent will be discovered to make sure that the chemical industries get closer to their respective environmental sustainability goals.

References Abu-Eishah, S.I., 2011. Ionic liquids recycling for reuse. Scott T. Handy (Ed.), Ionic LiquidsClasses and Properties 239–272. Alara, O.R., Abdurahman, N.H., Ukaegbu, C.I., Azhari, N.H., 2018. Vernonia cinerea leaves as the source of phenolic compounds, antioxidants, and anti-diabetic activity using microwave-assisted extraction technique. Ind. Crops Prod. 122, 533–544. Al-Taq, A., Alfakher, B., Alrustum, A., Aldarweesh, S., 2019. Alternative environmentally friendly solvents for asphaltenes/paraffins removal from oil producing wells. OnePetro. Bahadori, A., 2014. Natural Gas Processing: Technology and Engineering Design. Elsevier Science, United Kingdom https://books.google.com.my/books?id=1VgXAwAAQBAJ. Bilesan, M.R., Makarova, I., Wickman, B., Repo, E., 2021. Efficient separation of precious metals from computer waste printed circuit boards by hydrocyclone and dilution-gravity methods. J. Clean. Prod. 286, 125505. https://doi.org/10.1016/j.jclepro.2020.125505. Bredenberg, A., 2012. Reducing the Environmental Impact of Industrial Solvent Use. https://www.thomasnet.com/insights/imt/2012/06/11/reducing-the-environmental-impactof-industrial-solvent-use/#:∼:text=Organic%20solvents%20react%20in%20the,building %20materials%2C%20forests%20and%20crops. Accessed date: 30th May 2022. Bursa´c Kovaˇcevi´c, D., Barba, F.J., Granato, D., Galanakis, C.M., Herceg, Z., Dragovi´cUzelac, V., Putnik, P., 2018. Pressurized hot water extraction (PHWE) for the green recovery of bioactive compounds and steviol glycosides from Stevia rebaudiana Bertoni leaves. Food Chem. 254, 150–157. https://doi.org/10.1016/j.foodchem.2018.01.192. Chaves, J.O., de Souza, M.C., da Silva, L.C., Lachos-Perez, D., Torres-Mayanga, P.C., Machado, A.P., da, F., Forster-Carneiro, T., Vázquez-Espinosa, M., González-dePeredo, A.V., Barbero, G.F., Rostagno, M.A., 2020. Extraction of flavonoids from

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natural sources using modern techniques. Front. Chem. 8, 507887. https://www. frontiersin.org/article/10.3389/fchem.2020.507887. Chemat, F., Abert Vian, M., Ravi, H.K., Khadhraoui, B., Hilali, S., Perino, S., Fabiano Tixier, A.-S., 2019. Review of alternative solvents for green extraction of food and natural products: panorama, principles, applications and prospects. Molecules 24 (16), 3007. https://doi.org/10.3390/molecules24163007. Dietz, M.L., 2006. Ionic liquids as extraction solvents: where do we stand? Sep. Sci. Technol. 41 (10), 2047–2063. https://doi.org/10.1080/01496390600743144. Elsaid, K., Kamil, M., Sayed, E.T., Abdelkareem, M.A., Wilberforce, T., Olabi, A., 2020. Environmental impact of desalination technologies: a review. Sci. Total Environ. 748, 141528. https://doi.org/10.1016/j.scitotenv.2020.141528. England, P.H., 2019. https://www.gov.uk/government/organisations/public-health-england. Accessed date: 30th May 2022. Falconer, A., 2003. Gravity separation: old technique/new methods. Physical Separation in Science and Engineering 12, 812865. https://doi.org/10.1080/1478647031000104293. Gałuszka, A., Migaszewski, Z.M., Konieczka, P., Namie´snik, J., 2012. Analytical eco-scale for assessing the greenness of analytical procedures. TrAC Trends Anal. Chem. 37, 61–72. https://doi.org/10.1016/j.trac.2012.03.013. Hoekman, S.K., Broch, A., Xiaowei, L., 2018. Environmental implications of higher ethanol production and use in the U.S.: a literature review. Part I – impacts on water, soil, and air quality. Renew. Sustain. Energy Rev. 81 (P2), 3140–3158. https://EconPapers.repec.org/ RePEc:eee:rensus:v:81:y:2018:i:p2:p:3140-3158. Integrated Assessment of Hypoxia in the Northern Gulf of Mexico and Hypoxia Assessment Reports, 2019. https://www.epa.gov/ms-htf/integrated-assessment-hypoxianorthern-gulf-mexico-and-hypoxia-assessment-reports. Accessed date: 30th May 2022. Iqbal, M., Tao, Y., Xie, S., Zhu, Y., Chen, D., Wang, X., Huang, L., Peng, D., Sattar, A., Shabbir, M.A.B., Hussain, H.I., Ahmed, S., Yuan, Z., 2016. Aqueous two-phase system (ATPS): an overview and advances in its applications. Biol. Proced. Online 18 (1). 18 https://doi.org/10.1186/s12575-016-0048-8. Lu, J., 2018. Ethanol is renewable, but that doesn’t mean it’s good for us. Popular Science https://www.popsci.com/corn-ethanol-smog/. Accessed date: 30 May 2022. Mageste, A.B., de Lemos, L.R., Ferreira, G.M.D., da Silva, M.C.H., da Silva, L.H.M., Bonomo, R.C.F., Minim, L.A., 2009. Aqueous two-phase systems: an efficient, environmentally safe and economically viable method for purification of natural dye carmine. J. Chromatogr. A 1216 (45), 7623–7629. https://doi.org/10.1016/j.chroma.2009.09.048. Martini, M., Altomonte, I., Silva, S., Salari, F., 2017. Fatty acid composition of the bovine milk fat globules obtained by gravity separation. Int. Food Res. J. 24 (1), 148–152. Minea, A.A., 2020. Overview of ionic liquids as candidates for new heat transfer fluids. Int. J. Thermophys. 41 (11), 151. https://doi.org/10.1007/s10765-020-02727-3. Moukengué Imano, A., Beroual, A., 2006. Deformation of water droplets on solid surface in electric field. J. Colloid Interface Sci. 298 (2), 869–879. https://doi.org/10.1016/ j.jcis.2005.12.041. Mura, E., Josset, C., Loubar, K., Huchet, G., Bellettre, J., 2010. Effect of dispersed water droplet size in microexplosion phenomenon for water in oil emulsion. At. Sprays 20, 791–799. https://doi.org/10.1615/AtomizSpr.v20.i9.40. National Research, C., 2011. Renewable Fuel Standard: Potential Economic and Environmental Effects of U.S. Biofuel Policy. The National Academies Press, United States https://doi.org/10.17226/13105.

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Noïk, C., Palermo, T., Dalmazzone, C., 2013. Modeling of liquid/liquid phase separation: application to petroleum emulsions. J. Dispers. Sci. Technol. 34 (8), 1029–1042. https://doi.org/10.1080/01932691.2012.735929. Osorio-Tobón, J.F., 2020. Recent advances and comparisons of conventional and alternative extraction techniques of phenolic compounds. J. Food Sci. Technol. 57 (12), 4299–4315. https://doi.org/10.1007/s13197-020-04433-2. Panja, P., 2018. Green extraction methods of food polyphenols from vegetable materials. Curr. Opin. Food Sci. 23, 173–182. https://doi.org/10.1016/j.cofs.2017.11.012. Pena-Pereira, F., Tobiszewski, M., 2017. The Application of Green Solvents in Separation Processes. Elsevier Science, United States https://books.google.com.my/ books?id=SZupDQAAQBAJ. Rossi, F., Colombo, S., Pierucci, S., Ranzi, E., Manenti, F., 2017. Upstream operations in the oil industry: rigorous modeling of an electrostatic coalescer. Engineering 3 (2), 220–231. https://doi.org/10.1016/J.ENG.2017.02.013. Saad, M.A., Kamil, M., Abdurahman, N.H., Yunus, R.M., Awad, O.I., 2019. An overview of recent advances in state-of-the-art techniques in the demulsification of crude oil emulsions. Processes 7 (7), 470. https://doi.org/10.3390/pr7070470. Sapkota, B., 2017. Seed Desilting Gravity Separator Machine. Tribhuvan University Institute of Engineering Thapathali Campus, Kathmandu https://drive.google.com/ file/d/10FjFcmH2uwzP6awhOZEEubIe7yroLqzH/view. Sukaimi, S.A., 2019. Authorities confirm: latest Pasir Gudang pollution caused by Sg Kim Kim toxic waste. https://www.nst.com.my/news/nation/2019/06/498799/authoritiesconfirm-latest-pasir-gudang-pollution-caused-sg-kim-kim-toxic. Accessed date: 30th May 2022. Tsao, R., 2010. Chemistry and biochemistry of dietary polyphenols. Nutrients 2 (12), 1231– 1246. https://doi.org/10.3390/nu2121231. Wexler, P., 2014. Encyclopedia of Toxicology, third ed. Elsevier Science, United States https://books.google.com.my/books?id=CCiSAgAAQBAJ. Zhang, Q.-W., Lin, L.-G., Ye, W.-C., 2018. Techniques for extraction and isolation of natural products: a comprehensive review. Chin. Med. 13 (1), 20. https://doi.org/ 10.1186/s13020-018-0177-x. Zhao, B., Ren, L., Du, Y., Wang, J., 2020. Eco-friendly separation layers based on waste peanut shell for gravity-driven water-in-oil emulsion separation. J. Clean. Prod. 255, 120184. https://doi.org/10.1016/j.jclepro.2020.120184.

Potential upscaling of multiphase systems

13

Jasmine Tiong Sie Ming a, Chin Kui Cheng b, Shuet Fen Lai c, Kit Wayne Chew c and Kuan Shiong Khoo d a Department of Chemical and Environmental Engineering, University of Nottingham Malaysia Campus, Semenyih, Selangor Darul Ehsan, Malaysia, b Department of Chemical Engineering, College of Engineering, Khalifa University of Science and Technology, Abu Dhabi, United Arab Emirates, c School of Energy and Chemical Engineering, Xiamen University Malaysia, Sepang, Selangor Darul Ehsan, Malaysia, d Department of Chemical Engineering and Materials Science, Yuan Ze University, Taoyuan, Taiwan

13.1

Introduction

Biological molecules are delicate, sensitive to temperature, pH, and shear forces. The specific function of a biological molecule depends on the complex folding within the structure. Under unfavorable surrounding conditions, the three-dimensional structure of the biological molecule will be altered, rendering it ineffective in carrying out its expected function. Thus, the concentration and purification of it is a bottleneck in the industry (Hanke and Ottens, 2014). The need for efficient separation without compromising the biological activity of the molecules is in high demand. With the increasing usage in various industries such as food and beverages, wastewater treatment, cosmetic and pharmaceutical, separation techniques must be improved with its limitation addressed. Separation can be achieved via phase addition, presence of a barrier and inclusion of a solid agent. The commonly employed methods for liquidliquid separation encompass chromatography, membrane, aqueous two-phase system (ATPS) and precipitation, classified as the following: Solid agent – Chromatography Barrier – Membrane separation. Phase addition – ATPS and precipitation. Several factors are involved for the feasibility and practicality of the separation techniques. First, the performance of the method. This includes the yield and throughput. Next, the operating conditions such as feed condition, temperature and pressure. The simplicity of technology is required for the ease of operation, control and maintenance work. Furthermore, the availability and cost of suitable solid agent, barrier and phase forming component is also imperative. Sustainability aspect which is very much emphasized in today’s context must also be taken into consideration during the design and operation stage. The scale-up procedure should also be straightforward for large scale manufacturing process. Moreover, the maturity of the separation technology is also a key in determining the extensive usage. A comprehensive understanding of the technology enables attainment of desired results. Previously, bioseparation technology is mostly in batch mode. To cope with the market demand, a continuous operation mode catering for Principles of Multiple-Liquid Separation Systems: Interaction, Application and Advancement. DOI: https://doi.org/10.1016/B978-0-323-91728-5.00014-7 c 2023 Elsevier Inc. All rights reserved. Copyright 

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both separation and purification is highly desired. This offers benefits such as consistent product quality, higher flexibility and productivity, reducing holding and buffer tanks, smaller equipment volume, and smaller carbon footprint. Hence, it is more economical and environmentally friendly. A versatile and agile manufacturing process is very much sought after to adapt to the ever-changing and growing market demand. The main problem with multiple liquid separation is the cost of final product (Singh and Herzer, 2017). Furthermore, the separation efficiency and the processing time are also crucial. Hence, the objectives are as follows. A brief introduction on the working principle of the technology and its merit and drawbacks will also be discussed. The factors affecting the separation and partitioning of the biological molecule will also be explored to identify the optimum range. Furthermore, the development and advancement of the technology will also be highlighted. Major issue arising from the technology and ways to overcome will also be underlined. Recent advancement such as integration with other technologies will also be included. Despite being in experimental stage, the results are promising and favorable. All these aid in increasing the separation efficiency.

13.2

Chromatography

13.2.1 Introduction to chromatography Chromatography is one of the most employed method for bioseparation as it is a matured technology (Orr et al., 2013). Additionally, its high selectivity nature ensures high separation resolution. The separating agent, conventionally a resin bead, is also insoluble (Agyei et al., 2016). The separation mechanism involves solutes redistribution between two phases, viz., a stationary phase and a mobile phase. As different solutes have different affinities toward the stationary phase, separation could be achieved. Equilibrium is usually established between the stationary phase and the solutes in the liquid phase (Harrison et al., 2015). Two types of chromatographic mode exist, positive chromatography and negative chromatography. Positive chromatography refers to the desired product that has a greater affinity toward the stationary phase and binds toward it, while the undesired molecules will be eluted at a faster pace together with the mobile phase (Lee et al., 2014). A brief working principle of chromatography is shown in Fig. 13.1. Feed containing multiple solutes and the mobile phase will be injected from the top of the column. With the convection action of the mobile phase, the biomolecules will be fractionated into different sections as seen in Fig. 13.1. The transient separation of molecules into different zones will be more evident because of their different partition between the phases (Carta and Jungbauer, 2020). The greater the interaction of solute with the stationary phase, the slower the traveling velocity of the solutes toward another end of the column. Thus, it is retained longer within the column and its elution its delayed (Pfister et al., 2018). The transient concentration profile indicates that the red molecules have weaker interaction and thus elutes at a shorter time. In contrast, the strongest interaction between the green molecules and stationary phase ensures its slowest elution.

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Figure 13.1 Working principle of chromatography, adapted from Carta and Jungbauer (2020). From Carta, G., Jungbauer, A. (2020). Protein Chromatography: Process Development and Scale-Up: Process Development and Scale-Up. Wiley. http://ebookcentral.proquest.com/ lib/nottingham/detail.action?docID=6119414.

13.2.2 Limitation and challenges with chromatography 13.2.2.1

Mode of operation

Commonly, chromatography is operated as a batch-wise process. Several limitations are associated with it. First, resin beads toward the end of the column cannot be fully utilized. The dynamic binding capacity of the column is lowered, reducing the efficiency of separation (Behere and Yoon, 2020). Moreover, the quality of the final product for each batch is subjected to variability. Thus, quality control work is more strenuous. In addition, malfunction of column may result in off-specification product for the entire batch.

13.2.2.2

Transport phenomenon

Solutes are required to diffuse from the bulk solution to the binding sites within the pores of the chromatographic media. Consequently, longer processing time is required as the diffusion is slow. Furthermore, channeling also occurs within the bed. The uneven flow distribution prevents maximum bed utilization. Ultimately, the flow medium unpredictability hinders the scale-up process (Milne, 2017).

13.2.2.3

Scaling up

Process scale-up is one of the most crucial steps in technological development for industrial-scale production. For chromatography, the procedure is simple as it involves linear process parameters scale-up. In the design, column width is increased accordingly while maintaining constant bed height, velocity of fluid, and volume of mobile

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and stationary phases. Notwithstanding the simplicity, this reduces the flexibility in the process as only column width and the number of columns can be varied. From design calculation, the number of columns required to meet production throughput will be obtained. However, due to its rigidness, sometimes an exact number of columns required cannot be obtained. Thus, deliberation between carrying out an arduous optimization process to get an exact number of column or investing in another extra column must be carried out. Both methods are time-consuming and incur extra costs (Staby et al., 2017).

13.2.2.4

Stationary phase—resin

The increase of column diameter reduces the support of resin packing within the column. Additionally, the quality of packing is also affected. This results in dead zone formation. Consequently, flow distribution becomes uneven and larger pressure drop occurs (Milne, 2017). For instance, flow of media can be quicker at the edge of column. Compression of resin can also occur. As a result, this affects the stability of the resin and reduces its operational lifespan (Ingle and Lali, 2017). Resin aging leads to decrease in performance of chromatography separation. The resin is also susceptible to fouling. This can be contributed by improper cleaning procedure and change in feed composition (Rathore et al., 2018). The resin condition is very crucial for the efficient separation of desired and undesired solutes. Thus, it is imperative to ensure that the resins are not subjected to unstable and harsh operating conditions.

13.2.2.5

Environmental concern

Typically, an organic solvent is used for the mobile phase. Furthermore, a large quantity is usually required in the separation process (Li et al., 2020). Improper handling and discharge of the organic solvent is an environmental concern. Its toxicity can pollute water and cause adverse effects on human health. It is carcinogenic and a reproductive hazard. Some of the most common solvents used are methanol, acetonitrile, and tetrahydrofuran (Shen et al., 2015).

13.2.2.6

Other issues

The viscosity of the mixture affects the hydrodynamics pattern. A more viscous mixture possesses greater flow resistance. This may promote adverse issues such as plugging. The velocity of the mobile phase will also be reduced (Agyei et al., 2016). As the solutions are required to flow through the resins, large pressure drop is unavoidable (Rathore et al., 2018). This results in greater energy consumption, where inlet pressure must be increased to compensate for the reduced pressure at the outlet.

13.2.3 Cleaning and regeneration Cleaning and sanitization are vital aspects in the processing of biomolecules. The standard clean-in-procedure (CIP) must be adhered to for thorough cleansing. Failure

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Performance

Pressure drop

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Working range Bed height

Figure 13.2 Working range of a chromatography column based on pressure drop and performance considerations.

to do so results in increases fouling rate of resin (Rathore et al., 2018). Furthermore, contamination of the desired product with microorganisms happens as well. The repeated usage of stationary media is also a concern due to exposure to cleaning agents. Furthermore, the comprehensiveness of CIP and the number of CIP dictate the lifespan of the resin. Other than that, the cost of the media also heavily influences the need for CIP (Milne, 2017).

13.2.4 Development and advancement in chromatography 13.2.4.1

Increasing flexibility in scale-up process

Designing of chromatography column can be made flexible by keeping the residence time constant. Therefore, the height of column is adjustable in process design. This is favorable as height is continuous and can be scaled to the desired volume (Staby et al., 2017). Fig. 13.2 shows the profile with respect to the bed height of chromatography column’s performance and pressure drop along the column length. The performance increases rapidly at the onset and asymptotes as bed height increases. This can be attributed to more surface area of stationary phase for attachment of desired product. Beyond a certain bed height, the performance improve significantly. Conversely, the pressure drop increases with the bed height. Thus, taking both the performance and pressure drop into consideration, a working range could be established.

13.2.4.2

Advancement in resin

This prioritizes in increasing the chemical and physical stability of resin. Thus, the stability and efficiency of the separation process can be improved. This adaptation helps to cope with the ever-changing market demands. First, resin matrices are developed to be rigid but with complementary compressing to form a well-distributed packing in the column. It can be repeatedly used as it can withstand exposure to corrosive

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cleaning agents. Pore structure, pore size, and pore volume are optimized to ensure mass transfer is not limiting the process and enhances the dynamic binding capacity. Additionally, for ligand chemistry, there is success in developing protein A (a 42 kDa surface protein) ligand. This boasts desired characteristics such as higher binding capacity and selectivity (Ingle and Lali, 2017; Rathore et al., 2018).

13.2.4.3

Continuous chromatography

Continuous production is very desired in manufacturing. It aids in ensuring consistent product quality, reduces downtime, increased yield and productivity, and reduces operating cost. Thus, to enable continuous operation of chromatography column, multiple columns are operated in series but out-of-phase with each other. This involves several modes of operation such as periodic countercurrent, simulated moving bed, sequential multicolumn continuous chromatography (Rathore et al., 2018). Typically, three columns are involved with a six-step cycle. Despite that, it is not commonly applied in the industry due to the complexity of controlling the operations in multiple columns. The presence of an additional control loop is required to switch the phase in each column. A precise coordination of loading and elution phase is crucial to ensure the product is within the desired range (Armstrong et al., 2021).

13.2.4.4

Integration with membrane

This technology involves the merging of chromatographic separation and membrane filtration in one unit operation. The superiority of both technologies can be capitalized. It can accomplish better performance in technical, operational, and economical aspects compared to chromatography. The chromatographic media are substituted with a membrane (Orr et al., 2013; Rathore et al., 2018). It has several desired attributes, which makes it a promising technology in the separation of biomolecules. Fouled or exhausted modules can be discarded after a single usage. As a disposable unit, the need for thorough cleaning, regeneration, and sanitization step is no longer required. Revalidation for the equipment can also be eliminated. This saves time as cumbersome and time-consuming procedures are obliterated (Rathore et al., 2018). Single-use and discard of chromatographic media made from membrane are possible as it is simple and inexpensive to manufacture (Liu et al., 2017). The pressure drop is also considerably lower. Compression and channeling are also minimized. This ensures even flow distribution throughout. It is highly desired in processing large flow rate. In addition, the scale-up process is made easier (Orr et al., 2013). Mass transfer resistance is one of the limiting steps in chromatography. There are more steps involved in chromatography compared to membrane chromatography. The absence of the pore diffusion step is beneficial as this shortens the processing time considerably. Thus, membrane chromatography is a favorable technology to overcome the mass transfer resistance issue in chromatography (Ghosh, 2015). The major concern with membrane chromatography is the low binding capacity of solute (Ramos-de-la-Peña et al., 2019). Table 13.1 shows several available commercialized membrane adsorbers.

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Table 13.1 Commercialized membrane adsorbers (Liu et al., 2017). Company Asahi Kasei Medical

Membrane adsorber QYUSPEED

Type Anion exchange chromatography

Pall Corporation

MustangS

Cation exchange chromatography

Sartorius AG

Protein A

Affinity

SartobindPhenyl

Hydrophobic interaction chromatography Multimodal

Natrix Separations, Inc.

Natric HD-Sb

Application Removal of host cell protein, virus, and DNA Removal of positively charged host cell protein Immunoglobulin purification Aggregates removal Monoclonal antibodies capture and aggregates removal

From Liu, Z., Wickramasinghe, S.R., and Qian, X. (2017). Membrane chromatography for protein purifications from ligand design to functionalization. Sep. Sci. Technol., 52 (2), 299–319.

13.2.5 Concluding remarks and future prospects Basis of separation for chromatography depends on the affinity of the solutes for the stationary phase. Despite the various limitations, it is widely applied in bioseparations due to its simplicity and maturity. With the advancement of technology, several improvements were made in scaling up, advancement in stationary phase, and integration with membrane. Furthermore, despite the tedious control strategies with continuous multicolumn chromatography, several advantages can be obtained with continuous operation. Good implementation of control strategies ensures that optimum conditions are maintained and product specification can be met. Real-time monitoring can be achieved. Thus, research and studies can be focused on ensuring a smooth continuous operation by studying column configuration, column control strategies, and good binding capacity of the stationary phase. Addressing the limitations will be beneficial as this helps to improve the applicability of these techniques in multiple liquid separations.

13.3

Membrane

13.3.1 Introduction to membrane Currently, membrane application is gaining interest and momentum in various bioseparation applications. A barrier that allows separation to be achieved by allowing specific molecules to pass through. Several benefits are attributed to it. It allows continuous operation, which is highly desired to produce a consistent quality final product. Furthermore, it has a high throughput. It is also easily scaled for a major production. It can be carried out isothermally and at constant pH (Agyei et al., 2016). This helps in maintaining the function of desired molecules especially proteins and enzymes. As

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Figure 13.3 Fouling mechanism adapted from Arhin et al. (2016). From Arhin, S.G., Banadda, N., Komakech, A.J., Kabenge, I., and Wanyama, J. (2016). Membrane fouling control in low pressure membranes: a review on pretreatment techniques for fouling abatement. Environ. Eng. Res., 21 (2), 109–120.

membrane is specific, it allows only selective molecule to diffuse through, separating it from the bulk mixture. Moreover, the membrane is an insoluble separating agent. Hence, simultaneously, a good separation and purification of molecule can be achieved.

13.3.2 Issues with membrane Despite the advancement and development of membrane, it is still susceptible to fouling. Membrane fouling is the adsorption or deposition of molecules on the surface of membrane or within the pores in the membrane. As a result, the feed pressure must be increased to compensate for the lower flux, incurring higher operational cost. Moreover, frequent chemical cleaning is needed. Consequently, it affects the operational lifespan of the membrane due to the aggressive nature of the cleaning agent (Arhin et al., 2016). Furthermore, membrane selectivity is also altered (Harrison et al., 2015). With increasing membrane resistance, less of the desired molecules diffuse into the retentate reducing the throughput and yield, despite the same operating conditions. Different types of fouling can occur concurrently; this will reduce the permeability of membrane. Membrane fouling is either reversible or irreversible, depending on the nature of the foulant. Some of the foulants are macromolecules, organic colloids, microbes (biofouling), and inorganic molecules (Rudolph et al., 2019). Several fouling mechanisms are shown in Fig. 13.3. Fouling can be categorized into internal and external fouling. Internal fouling is shown in Fig. 13.3(A) and (B). External fouling is deposition or adsorption at the external surface of the membrane as shown in Fig. 13.3(C). Another issue that arises with membrane is the reduced selectivity when there are similar sized molecules as the desired one. Additionally, along the permeate flow, the concentration increases, making the liquid more viscous (Agyei et al., 2016). Membrane fouling is affected by the feed properties, membrane properties, and the operating parameter of the systems. A comprehensive understanding on the effect of

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those parameters is important to reduce the rate of fouling which increases the lifespan of membrane. For feed properties, physiochemical characteristics are involved. The nature of the feed is crucial as this helps to identify possible components, which are foulants as extend of fouling, depend on the chemical nature of the components. For instance, in dairy production, protein is a major contributor to membrane fouling. During fouling, protein can bind to membrane surface electrostatically, hydrophobic interaction, and formation of disulfide bonds. Next, the concentration of feed is also of paramount importance. As concentration increases, permeate flux decreases due to lower mass transfer rate. This also means more contaminant is present. The presence of salt and undesired pH conditions also changes the protein structure and reduces its stability. This increases the deposition rate of foulants. Moreover, the membrane structure is also affected as well (Abdelrasoul et al., 2013; Hu and Dickson, 2015). Membrane properties are affected by its hydrophobicity, surface charge, pore size, and porosity. Two types of membrane are available, namely, hydrophilic and hydrophobic membrane. Hydrophilic membrane can form hydrogen bonds with water. Hydrophobic membrane causes water beading on the surface which promotes fouling. Surface charge is affected by the membrane material as well as the condition of the feed stream. Normally, membrane is negatively charged. With an optimum pH, the selectivity can be increased, providing a higher flux through the membrane. Permeation flux can also be increased with greater pore size. Besides, a lower porosity membrane is more susceptible to fouling. Due to lesser pores present for molecules to travel, the available pores are subjected to higher local concentration polarization which increases fouling rate (Abdelrasoul et al., 2013; Hu and Dickson, 2015). The first operating parameter is the transmembrane pressure. Higher pressure encourages a greater diffusion rate through the membrane. However, beyond the critical pressure, foulants start to aggregate to form a gel or cake layer. Furthermore, the cross-flow velocity should also be monitored. When cross-flow velocity is increased, the permeation rate is increased. With constant removal as well as increased crossflow velocity, this prevents the accumulation of foulant on the membrane surface. As temperature increases, the flux rate increases. This reduces permeate viscosity and increases diffusivity of solute. Despite that, care must be taken to ensure the temperature is not raised until denaturation and deformation of protein (Abdelrasoul et al., 2013; Hu and Dickson, 2015).

13.3.3 Methods to reduce membrane fouling Membrane fouling is inevitable. However, the fouling rate can be controlled and minimized with several methods such as pretreatment of feed, membrane surface modifications, improve operating conditions, and membrane cleaning and sanitization.

13.3.3.1

Pretreatment of feed

Coagulation helps to aggregate smaller suspended and colloidal particles together (Ghernaout et al., 2018). This aids in minimizing reversible fouling. As particles

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are larger, small membrane pores will not be blocked. However, a layer of cake will be formed. It can be easily cleaned by backwashing. Moreover, coagulation process can be coupled together with sedimentation process. This helps heavier particles to be separated out. The supernatant can then be fed into the membrane unit (Abdelrasoul et al., 2013). This is certainly beneficial as the cleaning frequency of membrane can be reduced. Furthermore, exposure to aggressive cleaning chemicals can be minimized. The operational lifespan of the membrane can be lengthened. Furthermore, magnetic ion exchange can be performed as well. Organic acids which are the major contributor to membrane fouling can be removed (Jutaporn et al., 2016). The working principle is where charged species in wastewater will be adsorbed onto the polymer beads. The beads can be easily regenerated using brine solution (Abdelrasoul et al., 2013). Another technique is micellar-enhanced filtration. Surfactants which consist of hydrophobic and hydrophilic region are added to trap foulants. Beyond critical micelle concentration, micelles are formed trapping foulant in it. The size of micelles formed will have similar size to the diameter of membrane pores. Thus, by selecting a suitable membrane, micelles will diffuse through the membrane into the retentate zone (Schwarze, 2017). Additional benefit of surfactant introduction is inhibiting hydrophobic interactions of bacteria with the membrane surfaces. In the presence of surfactant, its cell wall starts to fail, causing bacteria to cease function normally (Abdelrasoul et al., 2013).

13.3.3.2

Membrane surface modification

Membrane can be pretreated to reduce its susceptibility to fouling. Commonly, this is carried out by physical and chemical modification. In physical method, a component with hydrophilic functional group such as hydroxyl group is integrated (Abdelrasoul et al., 2013). Coating is done by physical interaction ensuring that the chemical characteristics of the membrane remain. The selectivity of membrane toward the desired molecule will not be altered. With chemical modification, covalent bonding is involved. First, activation of membrane polymer base chains is done either chemically or by irradiation. Next, membrane surface treatment can be accomplished by coating or grafting. Despite the presence of covalent bonding, the chemical properties of the membrane remain (Singh and Purkait, 2019). As covalent bonding is much stronger, the component integrated for surface modification can be anchored more securely. Desorption issue will not occur with a stronger linkage.

13.3.3.3

Improvement of operating conditions

This approach can be easily carried out and controlled, involving the cross-flow velocity as well as the temperature and pH of the system. Increasing the cross-flow velocity increases mixing preventing the accumulation of particles forming a cake layer. Additionally, turbulence can be induced using vibratory shear-enhanced filtration and rotating disk modules. A much more chaotic flow increases shear rate at the membrane surface (Koh et al., 2014). This is beneficial in reducing aggregation of particle, which subsequently deposit on the membrane pores. Overall, hydrodynamics

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Table 13.2 Chemical agents used for membrane cleaning (Abdelrasoul et al., 2013; Hu and Dickson, 2015). Chemical agents Acid Alkalis Surfactants

Compound removed Calcium salts Metal oxides Silica, Inorganic colloids, Organic foulant Oil and fats Organic foulant

Sequestrants

Metal cations

Enzymes

Proteins Starches Fat and oil Cellulose

Characteristics Solubilizes foulants Solubilizes foulants, Hydrolyze foulant Contain hydrophilic and hydrophobic region which is beneficial Capable of forming chelating complexes Very costly, Effective as enzymes are specific molecules

From Abdelrasoul, A., Doan, H., Lohi, A. (2013). Fouling in membrane filtration and remediation methods. Mass TransferAdvances in Sustainable Energy and Environment Oriented Numerical Modeling, 195. From Hu, K., Dickson, J. 2015. Membrane Processing for Dairy Ingredient Separation. Wiley.

of the system can be improved to reduce fouling. High temperature also leads to increased fouling. For instance, in dairy industries, this enhances the precipitation of calcium phosphate (Tan et al., 2014). Then, its deposition leads to pore narrowing or blockage. With high temperature and high pH conditions, the precipitation of calcium phosphates also accelerates (Koh et al., 2014). Hence, it is crucial to have an optimum condition to effectively control the rate of fouling.

13.3.3.4

Membrane cleaning and sanitization

Generally, membranes can be cleaned by mechanical means and with the addition of chemical agents. Mechanical methods include increasing the flow rate of feed or water, minimizing transmembrane pressure to make fouling negligible, and backflushing (Hu and Dickson, 2015). Higher flow rate increases shear rate at membrane surface. For backflushing, the permeate flow is reversed. This initiates displacement of foulant by loosening its grip toward the membrane surface. However, another unit operation is required to contain the opposite flow. In addition, it is also ineffective for irreversible fouling (Basile and Curcio, 2018). Chemical agents work by competitive adsorption of surface active agent to remove foulant, increase solubility of foulant or chemical modification of foulant (Hu and Dickson, 2015). Typical chemicals used are acid, caustic, surfactants, sequestrants, and enzymes as listed in Table 13.2. The nature of the chemical agents will remove different types of foulant. The downside of chemical cleaning is the shutdown of the membrane filtration unit. This will disrupt the production and reduces yield. Additionally, due to the aggressive nature of the chemical solution, this causes membrane to deteriorate, shortening its lifespan. Consequently, this will incur additional cost.

13.3.4 Advancement in membrane cleaning Advancement in new membrane cleaning method, viz., ultrasonic waves (sonication), enables in situ membrane cleaning, increases efficiency in cleaning, and reduces

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Figure 13.4 Flow diagram for extraction process adapted from Harrison et al. (2015). From Harrison, R.G., Todd, P.W., Rudge, S.R., and Petrides, D.P. (2015). Bioseparations Science and Engineering. Oxford University Press, Incorporated. http://ebookcentral. proquest.com/lib/nottingham/detail.action?docID=1911965.

dependency on chemical agents (Chen et al., 2019). Ultrasound is a sound wave traveling at a frequency beyond the normal human audible range. It has high mechanical power through small mechanical movements (Abdelrasoul, 2020). When the wave travels through the medium in the membrane unit, acoustic cavitation occurs. Bubbles will form and collapse due to fluctuation in pressure. As a result, shear force and turbulence can be generated (Koh et al., 2014). Studies showed that membrane performance improved as a result. The mechanical energy is also able to suspend the particles, preventing them from depositing within the membrane pores.

13.3.5 Concluding remarks and future prospects With high selectivity, effective separation of products could be achieved. As it is easily scaled up, the only issue to be addressed in membrane application is fouling. Fouling hinders the permeability of the desired molecules. This affects the recovery yield. Fouling minimization could be tuned via controlling feed conditions, membrane properties, and operating conditions. Membrane cleaning and regeneration are also a pivotal aspect in operating a membrane. Advancement in membrane properties and cleaning method helps to reduce the fouling rate too. With proper maintenance and cleaning of membrane, operational lifespan of membrane can be prolonged. This reduces cost and increases productivity by avoiding downtime for membrane cleaning.

13.4

Aqueous two-phase system

13.4.1 Introduction to aqueous two-phase system Liquid–liquid extraction involves the partitioning of solute in two immiscible liquids. Fig. 13.4 shows an extraction unit in equilibrium. Raffinate is the spent feed whereas the extract contains the desired product and solvent. The partitioning of solute during equilibrium between the two phases is determined by the partition coefficient. Suitable partition coefficient ensures effective separation with the lowest solvent quantity. Low partition coefficient results in poor extraction. Besides, large partition coefficient involves large quantity of solvent and many more extraction unit in series (Harrison et al., 2015).

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Figure 13.5 Recovery of product based on Aqueous Two Phase System concept.

One of the widely applied liquid–liquid extraction techniques is aqueous twophase system (ATPS) due to enhanced mass transfer between the phases and lower interfacial tension (Pereira et al., 2020). ATPS involves the combination of watersoluble polymers or a polymer and salt, which are immiscible beyond their critical concentration. This results in a biphasic system, an upper and top phase. The two distinct phases show partitioning of solutes in the system between the two immiscible phases. The top phase is usually the polymer while the bottom phase will be polymer or salt. Typically, the targeted compound will be enriched in the top phase whereas the contaminants are enriched in the bottom phase. When equilibrium is reached, solute will migrate to the phase which it has a greater affinity for (Desai et al., 2014; Khan et al., 2019). Fig. 13.5 shows the working principle in partitioning of desired product based on notion of ATPS. Polymer–polymer and polymer–salt are the most common type of ATPS. Several other ATPS types are also available such as alcohol– salt, ionic liquid and micellar (Iqbal et al., 2016). To separate the desired product from the polymer phase, back-extraction process is carried out. This involves process such as ultrafiltration, diafiltration, and crystallization (Khoo et al., 2020). ATPS is an appealing and promising technology in separation and purification of biomolecules. First, water is present in large amount, at a range of 70–80 wt.% (Goja et al., 2013). As the water in a noncorrosive and nontoxic liquid, the biomolecules are not subjected to a harsh environment. This is a favorable condition for target products and polymers to maintain their biological activities (Wu et al., 2014). It is also an environmental-friendly extraction method as water is present and no organic solvents are consumed in the process (Pereira et al., 2020). It is also capable of operating continuously. A high-purity product can also be achieved. Furthermore, it is a simple

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procedure with large processing capacity. Rapid separation can be achieved as well. Ease of scaling up is also another pros. During scale-up, the amount of extraction solvent increases linearly with volume of system (Glyk et al., 2015a). The energy requirement for the process is also low. It can be highly selective which reduces the need for multiple unit operations, thus lowering the costs. The extraction solvent used can also be recycled (Goja et al., 2013; Phong et al., 2018; Wu et al., 2014). Incorporating clarification, concentration, and purification process in one unit operation is feasible as well (Molino et al., 2013). Integration with other liquid separation technologies such as precipitation and ion-exchange chromatography are also showing promising results (Glyk et al., 2015a). ATPS is a promising technology as it is economical and sustainable.

13.4.2 Limitation and challenges There are several obstacles in the usage of ATPS in the industry. First, settling of biomolecules using gravity can take a long time in highly viscous polymer phase. This phenomenon is contributed by the presence of high molecular weight (MW) polymer together with high concentration of polymer. The duration for settling increases with increase in viscosity and phase height. Furthermore, in polymer–salt system, high salt concentration systems lead to corrosion in equipment and pipeline (Soares et al., 2015). This is a major hindrance for wide application of ATPS in the bioseparation industry. Solvent which is used in this technique could be costly. Difficulty in sourcing the suitable solvent is also compounded by the fact that the solvents must be immiscible and will not affect the biological stability of the protein molecules. Moreover, the poor recyclability of the phase-forming component also leads to the increase in operational costs. Proper disposal of solvents after usage also poses an environmental concern (dos Santos et al., 2018). However, the recyclability and sustainable issue have been addressed. For instance, cheaper polymer such as hydroxypropyl starch and crude dextran can be used. Polymer conjugates such as hydrophobic affinity ligand can also be substituted with the conventionally used dextran polymer. Recycling of phaseforming component can also be achieved with back extraction although recovery and purification of the polymer may incur high costs too (Soares et al., 2015). Another hindrance is the lack of knowledge on partitioning behavior of molecule (Goja et al., 2013). Proper depiction must be known for phase diagram to be constructed. Changes in operating parameter will alter the tie line. Thus, optimization of partition process requires rigorous experiments and is time-consuming.

13.4.3 Factor affecting ATPS The partitioning behavior of solute in ATPS is affected by several factors described herein. The effects of each factors should be studied and investigated to ensure optimum condition for enhanced concentration and/or purification of desired target molecules. Selective partition occurs with the recovery of the desired molecule. Separation efficiency can be increased with higher final product purity.

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13.4.3.1

273

Molecular weight and size of polymer

As the MW of polymer increases, the density, refractive index, and viscosity of the phase increases. The concentration of polymer required in phase formation is inversely proportionate to the MW of polymer (Ketnawa et al., 2017). In a viscous mixture, the settling time for solutes is longer due to the mass transfer resistance. Polyethylene glycol (PEG) is one of the most common polymers used. Besides, the increase in PEG concentration results in a lowered partition coefficient (Iqbal et al., 2016). High MW of PEG results in partitioning of desired products to the bottom phase. This is contributed by the increasing chain length of the polymer which results in free volume reduction. Meanwhile, low MW of PEG enables all the biomolecules in the sample to partition to the top phase. Thus, a suitable MW of PEG required is identified by analyzing the partition behavior at different MWs of PEG (Goja et al., 2013). In polymer– polymer system, partition of solute is lower in the phase where MW of polymer is greater. For polymer–salt system, partition of solute to the top phase (polymer) is reduced when concentration of polymer is increased (Iqbal et al., 2016). Hydrophobic interactions play an important role in partitioning of biomolecules in ATPS. A low MW polymer has a shorter chain with hydrophilic groups at the end. Thus, this reduces the hydrophobicity of the molecule. Generally, the extent of hydrophobicity between two phases enhances phase separation. The hydrophobic proteins form at the top layer while hydrophilic proteins will settle at the bottom phase (Teixeira et al., 2018).

13.4.3.2

Impact of temperature

Change in temperature has a significant effect on the composition of top and bottom phases during partition by ATPS. Additionally, the viscosity and density of the system are also changed. For high recovery yield and purity, a tight control on the operating temperature is thus crucial. Generally, the fractionation of molecules is carried out at a range of 20–40°C. For polymer–polymer system, a lower operating temperature with low polymer concentration can achieve good separation. So, in polymer–salt system, the vice versa phenomenon occurs. Another pivotal aspect to monitor is ensure that the operating temperature does not exceed the denaturation temperature of biomolecules (Iqbal et al., 2016; Khoo et al., 2020).

13.4.3.3

Impact of pH

Partitioning of biomolecules such as protein and enzymes to the desired phase, either top or bottom depends on their isoelectric point (pI). For enhanced separation, the pI of the target biomolecule must be lower than the surrounding pH during partitioning. Most of the biomolecules are stable and biologically active at pH 7, enabling optimum condition for partitioning (Goja et al., 2013). At higher pH system, negatively charged molecules will have a greater affinity toward the top phase. The partition coefficient can be increased. Opposite charge attracts improving recovery of target molecule to the top phase (Ketnawa et al., 2013). Nonetheless, proteins are sensitive to changes in pH. Alteration in surrounding pH results in disruption of bonds and interactions within the molecule. Thus, the pH during ATPS must be suitable as changes in charge and

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surface properties will affect the partitioning of the biomolecules to the desired phase (Iqbal et al., 2016). Furthermore, biological stability and activity of the molecule might also be affected. So, pH of the system must be within the range for protein stability and function.

13.4.3.4

Impact of type and concentration of salts

Presence of salt will affect the partitioning behavior of biomolecules in ATPS. Hydrophobic interaction is intensified between biomolecules as addition of salt dehydrates protein, exposing the hydrophobic zones (Khan et al., 2019). Thus, protein– polymer interaction is improved. Commonly used anion salts are sulfate, phosphate, and citrate. Utilization of citrate salt is gaining traction as it has high selectivity and poses no environmental harm due to its biodegradability. Its biocompatibility is also a contributing factor. Cation salts such as ammonium, potassium, sodium, calcium, and magnesium are also used. Despite that, anion salts are much superior in partitioning performance of ATPS. For phosphate salt, the partition efficiency in decreasing order is Na3 PO4 > NaH2 PO4 > Na2 HPO4 . Na3 PO4 is capable in promoting better hydrophobic interaction, increasing the partition coefficient (Goja et al., 2013). The partitioning efficiency of anion salts is SO4 2− > HPO4 2− > Cl− , while for cation salts is NH4 + > K+ > Na+ > Li+ > Mg2+ > Ca2+ (Ketnawa et al., 2017). Salt concentration influences the recovery and partitioning behavior. Generally, the extraction process improves with high salt concentration. This is due to salting out phenomenon. Despite that, beyond the optimum concentration, recovery of desired product decreases (Goja et al., 2013). Furthermore, in a salty environment, the properties of desired solute and pH of the system can be disrupted. This contributes to increased rate of corrosion in equipment (Sankaran et al., 2019).

13.4.3.5

Impact of NaCl addition

Addition of NaCl, a neutral salt promotes recovery of desired protein molecule to the top phase. The purification and recovery of protein can be enhanced. The hydrophobic difference between the phases is increased, so there is stronger interaction between protein and the hydrophobic chain of PEG. This also prompts an electrical potential difference between the two phases, which facilitates movement of protein to the desired phase depending on the protein charge. An extremely high salt concentration also leads to denaturation and precipitation of protein. Thus, a working range of 0–1.0 M is established (Goja et al., 2013; Ketnawa et al., 2017).

13.4.3.6

Tie line length

For ATPS, phase diagram is unique for each temperature and pH. Information about the concentration required to form two phases can be obtained to allow partitioning of biomolecules (Teixeira et al., 2018). Fig. 13.6 can be divided into two region namely monophasic region (below the binodal curve) and biphasic region (above the binodal curve). Tie lines provide the composition of the top and bottom phase. Point 1 indicates the composition of the top phase while Point 3 gives the composition

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Binodal

Polymer X % (w/w)

1

2

Tie lines Critical point

3

Polymer Y / Salt % (w/w)

Figure 13.6 A typical phase diagram for Aqueous Two Phase System. The part below the binodal curve indicates the single-phase area, and the upper part indicates the two-phase area.

of the bottom phase. The critical point is where the composition and volume of the phases are equal. As the length of tie line increases, ATPS become more hydrophobic. This is due to increase in polymer or salt concentration where water content is reduced. This increases the protein partition coefficient. Hence, protein recovery to the top phase is increased. Additionally, in polymer–salt system, salting out effect is enhanced. This facilitates the transfer of desired protein the top phase (Goja et al., 2013).

13.4.4 Development and advancement 13.4.4.1

Strategies for optimization of ATPS for large-scale applications

To obtain the desired recovery yield, the factors affecting partitioning behavior of the molecules must be investigated and optimized. Inevitably, this involves tedious experimental works which is time-consuming and costly. Previously, optimization is done using one-factor/variable-at-a-time. However, this method provides inconclusive results as only one factor is investigated at a time while all other factors are kept constant. Thus, to analyze the interaction between all the factors, a new technique is introduced—“Design of Experiments (DoE).” The steps involved for DoE are described and shown in Fig. 13.7 (Glyk et al., 2015b; Iqbal et al., 2016).

13.4.4.2

Screening of variables

Factors affecting the partitioning behavior will be identified to study the effects. The methods involved are full factorial design (FFD), fractional factorial design (fFD), and

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Screening of variables

• FFD/fFD/PBD

Initial optimization

• Sleepest Ascent/Descent Experiment

Final optimization

• RSM (CCD/BBD)

Analysis of model

Validation of model

Figure 13.7 DoE process adapted from Glyk et al. (2015b). From Glyk, A., Solle, D., Scheper, T., and Beutel, S. (2015). Optimization of PEG–salt aqueous two-phase systems by design of experiments. Chemometrics Intell. Lab. Syst., 149, 12–21. Table 13.3 Explanation for FFD and fFD. Method Number of experiment, N FFD N = 2k

fFD

Eq. (1)

N = 2k − 1 , reduction of N by one half Eq. (2) N = 2k − 2 , reduction of N by one-quarter Eq. (3)

Description – More accurate – More cumbersome as number of experiment increases with the number of factor – Number of experiments can be reduced – Higher fraction can be used

Plackett–Burman design. For FFD and fFD, the factors, k has two magnitude, “+” for high and “−” for low. Table 13.3 provides the explanation for the usage of FFD and fFD. Plackett–Burman design is a linear approach where only the main factors are taken into consideration. The predicted response variable, y can be known using Eq. (4). y = βo +

k  i=1

βi Xi + ε

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where βo/i is the coefficient of regression, Xi is the experiment factor, and ε is the Random error.The significance of the factor for each method is determined using analysis of variance. The magnitude of variation can be known.

13.4.4.3

Initial optimization

The optimum value of the factors involved is identified. For optimum value validation, analysis of the model curvature is carried out by adding several experimental center points to the screening model. A model curvature happens when the difference between the center point experiments and average output is significant. This proves that the output responses are within the optimum experiment value. In the absence of curvature, steepest ascent and descent experiments are carried out, helping to determine the direction of experimental investigation. This works by increasing or decreasing the output response so the value falls near the optimum range. Experiment will be conducted by increasing the output response until there is no change in the value. This provides an indication that the optimal region is within the area. Hence, the points can be used in the next step.

13.4.4.4

Final optimization

Response surface methodology (RSM) is employed in this step. Optimum values for all the factors can be established and interdependence of factor affecting protein recovery yield can be known. Prediction of responses within an optimal range and gathering of experimental data for quadratic equation fitting is aided with the use of RSM. Progress is then made with regression analysis to assess the relationship. RSM can be divided into two, namely, multilevel design central composite design and Box–Behken design. The advantages of central composite design are its rotatability and precision. Thus, it is widely used in polymer-salt system. For Box–Behken design, the number of experiments required is lower.

13.4.4.5

Analysis of model

Next, the quadratic equation obtained has to be solved analytically to obtain the ideal values. Several statistical softwares are available, viz., MATLAB, Design Expert, and Minitab. After analysis is done, the optimum value of the significant factors can be identified.

13.4.4.6

Validation of model

Experiments are executed based on the predicted values for each factor. The final results obtained are compared with those from the estimated values. Validation of model can be substantiated where the difference between the values is not significant. Therefore, prediction of response for combination of multiple variables can be done using the model obtained.

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13.4.4.7

Sustainable and environmental concern

Ionic liquids offer flexibility as its properties are tuneable by permutation and combination of negative and positively charged ions. The properties desired for efficient partitioning are polarity, hydrophobicity, and viscosity (Desai et al., 2014). It also has low vapor pressure, nonflammable, high chemical and thermal stability. Thus, substituting organic solvent which is volatile with, it is a greener approach. Despite that, it is costly and difficult to produce. If recycling of solvent can be achieved, usage of this liquid will be attractive in ATPS. Whence, a new green solvent is being developed to substitute ionic liquids. Being an excellent electrolyte, it poses properties such as high stability, nonvolatile, nontoxic, and high solubilization ability. Also, it is biodegradable, cheap, and easy to synthesize (Wang et al., 2017).

13.4.4.8 13.4.4.8.1

Integration of technologies with ATPS Aqueous two-phase floatation

This technology integrates ATPS and solvent sublation. The working principle is mixing the sample with the lower phase in ATPS followed by the addition of a fresh top phase. Then, gas bubbles are introduced from the bottom. Bubble adsorption occurs where molecules are dragged as air bubbles rise. The top phase consists of organic solvents where the bubbles will accumulate. This technology has shown promising results in the recovery of protein and enzymes such as bromelains and lipase. The benefits of bubble introduction result in a high partition coefficient as recovery of desired product is higher as they are being propelled upward. Thus, the amount of top phase required can also be reduced significantly, making the process more sustainable and environmentally friendly. Design consideration for higher separation efficiency for aqueous two-phase floatation (ATPF) includes physical and chemical characteristics of component present, concentration of component, gas bubble size, aeration rate, and pH of the system. Optimization of these parameters increases the migration of desired product to the top phase (Sankaran et al., 2019; Torres-Acosta et al., 2019). It is a relatively new technology and success has been made in applying these technologies on an industrial scale such as concentration of liquiritin, glycyrrhizic acid, active compound, and antibiotics (Chang et al., 2014). With continuous research and advancement, application can be extended to a wider area such as biotechnology, pharmaceutical, or food and beverage industry.

13.4.4.8.2

Magnetic ATPS

Several strategies were involved in magnetic ATPS involving creation of surface modified magnetic particles (MPs) and phase-forming component as listed herein. 1. 2. 3. 4.

MPs modified with ligands Polymer coated MPs with immobilized protein For micellar system, MPs are coated with silica or sulfonate Usage of magnetic ionic liquid

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Magnetic field can be applied to force phase separation. Hence, desired biomolecules can be recovered and concentrated in a smaller volume. The superiority of introduction of magnetic properties in ATPS is greater partition coefficients, reduces separation times and removal of phase components. As it is less time-consuming, the production cost is lowered. Furthermore, a higher purity and recovery yield can be attained, maximizing the profit. However, the availability of MPs and their cost are major obstacles. One accomplishment of this technology is MPs coated with polymer and gum Arabic then modified with ligand, aminophenyl boronic acid. mAbs can be successfully recovered and partitioning process is quicker (Dhadge et al., 2014; Torres-Acosta et al., 2019). Further research is required for acceptance in large-scale manufacturing.

13.4.4.8.3

Ultrasound-assisted ATPS

Ultrasound-assisted ATPS is an emerging technology in bioseparation of molecules. When the ultrasonic wave is introduced, acoustic cavitation occurs whereby bubbles will form and collapse. High shear force induced by the cavitation will weaken and break down the tough and rigid plant cell wall, exposing the inner content. Thus, proteins can be released for an easier recovery process. Additionally, desired biomolecules will come into contact with extraction solvent more rapidly. Mass transfer resistance within the system can be remarkably reduced. Favorable extraction and recovery yield are obtained from extraction of phenolic compounds and antioxidants. Moreover, incorporation of ultrasonic irradiation in ATPF is also possible. The presence of air bubbles encourages adsorption of desired molecules. Hence, the separation efficiency is higher. The benefits of ultrasound-assisted ATPS are rapid, energy-efficient, inexpensive, and eco-friendly. Notwithstanding the efficiency, collapse of bubbles results in temperature elevation. Biomolecules are labile; therefore exposure to high temperature will result in deformation and lost in biological activity. Temperature within the system must be controlled and maintained at optimum level for partitioning (Ðorđevi´c and Antov, 2017; Khoo et al., 2020; Xu et al., 2017).

13.4.4.8.4

Electricity-assisted ATPS

Cell disruption for extraction of intracellular material can also be achieved with electricity-assisted ATPS. The application of this technology is optimistic for extraction and recovery of desired protein. Pulsed electric field involves the introduction of short electrical pulse through the sample and solvent subjecting disruption to the charges on the cell membrane. The electricity treatment causes pore formation within the membrane, facilitating the movement of proteins toward the external region. This phenomenon is known as electroporation. Furthermore, ATPF can also be electricityassisted. The advantages from both technologies can be exploited to maximize extraction and recovery of protein. Triggering electric pulse through the system reduces the processing time as well as enhanced recovery yield. Furthermore, being a greener technology, promotes sustainability (Khoo et al., 2020).

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Figure 13.8 Precipitation and recovery of protein.

13.4.5 Concluding remarks and future prospects Partitioning of solutes is achieved depending on their affinity toward the top or bottom phase. For smooth operation and performance of ATPS, the factors affecting the partitioning behavior must be known and understood. These factors are also key in establishing the phase diagram. The DoE method for optimization also ensures all base is covered in terms of parameters affecting the performance of ATPS. Additionally, with integration with technologies such as floatation, magnetic, ultrasound, and electric, the future prospect of ATPS is promising. Despite the lack of application in large-scale production, with more research and advancement, this technology can be used in the future. A better separation of desired molecules can be achieved, increasing purity and recovery yield.

13.5

Precipitation

13.5.1 Introduction to precipitation According to Harrison et al. (2015), precipitation is the conversion of solution into an insoluble solid form. It is widely employed in the separation and purification of protein. This phenomenon occurs when the soluble protein becomes insoluble due to changes in surrounding conditions, destabilizing it. Furthermore, the product of interest is usually targeted to precipitate and is recovered via filtration or centrifugation. Precipitation is induced with the introduction of precipitating agents such as salt solution, organic solvents, nonionic polymers, and polyelectrolytes (Martinez et al., 2019). Fig. 13.8 shows the precipitation process and its subsequent recovery process using centrifugation.

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The maturity of the technology contributes to the extensive usage in protein purification. Furthermore, it is a relatively simple and cheap method. Scaling up is also a straightforward process. It has a high throughput and protein separation can also be done in a continuous mode. Through precipitation, protein concentration as well as volume reduction can be achieved (Agyei et al., 2016; Harrison et al., 2015).

13.5.2 Limitation and challenges Unsuitable precipitating agent leads to denaturation of protein by alteration of the three-dimensional structure. Furthermore, selective precipitation of protein cannot be achieved in some cases, where impurities precipitate as well. Hydrophobic proteins have low solubility in aqueous and organic solvent, thus precipitation process will be ineffective. Additionally, a subsequent recovery step is required to separate the precipitate from the precipitating agent (Agyei et al., 2016).

13.5.3 Factors affecting protein solubility Protein solubility is the basis of separation in the precipitation process. Several factors are involved such as temperature, pH value, and precipitating solvent used. Optimum conditions of these factors are required to ensure the precipitation of desired molecules.

13.5.3.1

Temperature

As temperature increases, the formation of hydrophobic interaction dominates and dipolar interaction diminishes (Dos Santos et al., 2017). This favors the precipitation of protein molecules.

13.5.3.2

pH value

The net charge of protein affects its solubility. The higher the net charge difference, the greater the solubility of protein. This is due to the formation of dipole moment on the molecule. To alter the net charge on protein, the pH of medium is change. At isoelectric point, a protein has zero net charge, resulting in minimum solubility (Harrison et al., 2015).

13.5.3.3

Precipitating solvent

Several types of solvent are available such as salts, organic solvents, and polymers. Precipitation is triggered as it affects the ionic strength and hydrophobicity. Salts commonly used are ammonium sulfate and sodium sulfate. Anion salts are more commonly used. The ionic strength of solvent can solubilize (salting in) and precipitate (salting out) protein. Salting out effect is due to the strengthening of the hydrogen bond network, stabilizing. Salting in happens as the hydrogen bonding network is weaken and destabilizing the protein. Hence, the selection of salts is imperative to achieve precipitation of desired molecules. Organic solvents such as methanol, ethanol,

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propanol, and ethanol are used. The hydrophobic part of the solution disrupts the interaction within the structure, causing its solubility to decrease and to precipitate out. However, these alcohol leads to protein denaturation. This can be avoided when precipitation process is carried out at a lower temperature. Typical polymer used is PEG. It can enhance protein–protein interaction. Thus, proteins can aggregate and subsequently precipitate out (Dos Santos et al., 2017; Harrison et al., 2015).

13.5.4 Development and advancement 13.5.4.1

Affinity precipitation

This method provides higher selectivity and recovery as affinity ligand and the solubility-switchable smart polymer have specific interaction for the targeted molecules. Small changes in surrounding condition induce conformational changes to the smart polymer which leads to precipitation. Thus, compared with traditional precipitation process, sample will be subjected to mild operating conditions which is beneficial in maintaining biological activity of protein. It is a simpler process. A smart polymer that is available is poly (N-isopropyl acrylamide), a thermosensitive one. It is capable of becoming more hydrophobic or hydrophilic depending on surrounding temperature (Li et al., 2020; Savina et al., 2019).

13.5.4.2

Scaling up and continuous processing

During precipitation, a perikinetic phase occurs and followed by orthokinetic phase. The former is where protein particles come together while the latter is protein growing to its final size. The orthokinetic phase is crucial in scaling up of equipment. It is affected by the power inputted into the equipment. Thus, a scaling parameter called Camp Number (NCa ) is derived. NCa should be greater than 105 for precipitation to be mechanically stable and also resist the shear forces subjected during mixing (Jungbauer, 2013). Eq. (5) shows the formula for Nca while Eq. (6) defines the shear rate, γ. NCa = γ¯  t where γ is the shear rate and t is the time. 

P/V γ= ρv

 12

where P is the power input of reactor, V is the volume of reactor, ρ is the density of medium, and v is the kinematic viscosity. Precipitation is commonly a batch process. During scaling up, when volume is increased, power input of reactor must be increased. This makes the precipitation process energy intensive which is undesirable. Furthermore, mixing in large reactors also become challenging. Thus, continuous processing is implemented with the

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Figure 13.9 Reactors for continuous processing (Martinez et al., 2019). From Martinez, M., Spitali, M., Norrant, E.L., and Bracewell, D. G. (2019). Precipitation as an enabling technology for the intensification of biopharmaceutical manufacture. Trends Biotechnol., 37 (3), 237–241.

introduction of continuous tubular reactor and coiled flow inversion reactor for largescale manufacturing. Fig. 13.9 shows the reactor configuration. The performance of continuous tubular reactor and coiled flow inversion reactor is comparable to the batch reactor. With continuous processing, higher productivity can be achieved as well (Martinez et al., 2019). Moreover, real-time monitoring of the process can also be achieved (Zelger et al., 2016). The factors affecting the precipitation process such as temperature, flow rate of precipitating agents, pH, and mixing rate can be monitored. Any disturbances to the system can be known and troubleshot. The parameters can also be adjusted back to the desired set point. This ensures a consistent product quality can be achieved.

13.5.5 Concluding remarks and future prospects Precipitation is an established technology in the bioseparations industry. The basis of separation depends on the solubility of the desired protein which is affected by several parameters. A comprehensive understanding on the effect of those parameters is pivotal in achieving a high-efficiency separation process. Scaling up procedure is also aided with the introduction of NCa which ensure precipitation is feasible and mechanically stable. Continuous operation also allows increase in productivity, realtime monitoring, and smaller footprint. To conclude, the advancement and development of this technology help to increase the performance.

13.6

Conclusion

Several options are available for bioseparations. The maturity of each technology after review is as shown in Fig. 13.10. Each method has its own benefits and downside. With recent development, limitations for each technology have been addressed. Each technology is constantly being improvement to achieve the desired separation and at the same time lowering the costs associated with it. For instance, integration with other technologies, converting from batch to continuous manufacturing, invention and

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Figure 13.10 Maturity of technology for separations.

modification of smart biopolymer to induce separation. Moreover, constant research and progress are being achieved with favorable outcomes. So, to ensure the desired separation is achieved, the basis of separation and partitioning for the desired molecule should be known. The affinity of the desired molecules toward the separating agent must be exploited to achieve the desired purification and separation. All in all, the bioseparation industry will be ever to rise and meet the demanding market demand.

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Singh, R., Purkait, M.K., 2019. Microfiltration membranes. Ahmad Fauzi Ismail, Mukhlis A. Rahman, Mohd Hafiz Dzarfan Othman and Takeshi Matsuura (Eds.), Membrane Separation Principles and Applications. Elsevier, UK, pp. 111–146. Soares, R.R., Azevedo, A.M., Van Alstine, J.M., Aires-Barros, M.R., 2015. Partitioning in aqueous two-phase systems: analysis of strengths, weaknesses, opportunities and threats. Biotechnol. J. 10 (8), 1158–1169. Staby, A., Rathore, A.S., Ahuja, S., 2017. Preparative Chromatography for Separation of Proteins. Wiley, US. http://ebookcentral.proquest.com/lib/nottingham/detail.action?docID= 4800257. Tan, T., Wang, D., Moraru, C., 2014. A physicochemical investigation of membrane fouling in cold microfiltration of skim milk. J. Dairy Sci. 97 (8), 4759–4771. Teixeira, A.G., Agarwal, R., Ko, K.R., Grant-Burt, J., Leung, B.M., Frampton, J.P., 2018. Emerging biotechnology applications of aqueous two-phase systems. Adv. Healthc. Mater. 7 (6), 1701036. Torres-Acosta, M.A., Mayolo-Deloisa, K., González-Valdez, J., Rito-Palomares, M., 2019. Aqueous two-phase Systems at large Scale: challenges and opportunities. Biotechnol. J. 14 (1), 1800117. Wang, T., Xu, W.-J., Wang, S.-X., Kou, P., Wang, P., Wang, X.-Q., Fu, Y.-J., 2017. Integrated and sustainable separation of chlorogenic acid from blueberry leaves by deep eutectic solvents coupled with aqueous two-phase system. Food Bioprod. Process. 105, 205–214. Wu, Y., Wang, Y., Zhang, W., Han, J., Liu, Y., Hu, Y., Ni, L., 2014. Extraction and preliminary purification of anthocyanins from grape juice in aqueous two-phase system. Sep. Purif. Technol. 124, 170–178. Xu, Y., Qiu, Y., Ren, H., Ju, D., Jia, H., 2017. Optimization of ultrasound-assisted aqueous twophase system extraction of polyphenolic compounds from Aronia melanocarpa pomace by response surface methodology. Prep. Biochem. Biotechnol. 47 (3), 312–321. Zelger, M., Pan, S., Jungbauer, A., Hahn, R., 2016. Real-time monitoring of protein precipitation in a tubular reactor for continuous bioprocessing. Process Biochem. 51 (10), 1610–1621.

Integrated systems for multiphase development

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Wan You Kho a, Shir Reen Chia b,c and Saifuddin Nomanbhay b a Department of Chemical and Environmental Engineering, Jalan Broga, University of Nottingham Malaysia, Semenyih, Selangor Darul Ehsan, Malaysia, b Institute of Sustainable Energy, Universiti Tenaga Nasional (UNITEN), Jalan IKRAM-UNITEN, Kajang, Selangor Darul Ehsan, Malaysia, c AAIBE Chair of Renewable Energy, Institute of Sustainable Energy, Universiti Tenaga Nasional (UNITEN), Jalan IKRAM-UNITEN, Kajang, Selangor Darul Ehsan, Malaysia

14.1

Introduction

Extraction is an important process to extract valuable ingredients such as bioactive, polysaccharides, and proteins from natural resources and identify and separate them from other impurities. A lot of extraction methods have been developed and the selections of methods are crucial to obtain the maximum extraction yields and the highest quality of products safely and economically. The selection of method is mainly relying on the properties of targeted compounds and raw materials, and financial feasibility (Shirsath et al., 2012). Conventionally, the extraction methods such as maceration, solvent extraction, distillation, and Soxhlet extraction are used. The mass transfer resistance is the major issue in the extraction system as the system usually involves more than one phase of materials. Another important issue affecting the extraction yield is the molecular affinity between solutes and solvents. Besides, some techniques might be energy intensive and time-consuming due to the low diffusion rate of solvents. For the heat-sensitive compounds, they will be degraded thermally during the heating process, resulting in low extraction yields and low quality of products. For solvent or chemical extractions, a large number of solvents or chemicals are required to achieve a higher extraction yield. Furthermore, some solvents used are toxic and harmful to both environment and humans (Danlami et al., 2014; Gligor et al., 2019). The severe environmental issues and human health are more concerned by society nowadays. Hence, the green, clean extraction, and effective technologies are intensively explored to achieve continuous quality improvement or Six Sigma in the research and industries. New technologies such as ultrasound-assisted extraction (UAE), microwave-assisted extraction (MAE), and enzyme-assisted extraction (EAE) are developed and integrated with conventional techniques to overcome these problems. These improved techniques minimize or eliminate the usage of toxic chemicals or solvents while enhancing the extraction efficiency, extraction yields, and quality of the products (Tiwari, 2015). In the work of Xu et al. (2017), the extraction of Principles of Multiple-Liquid Separation Systems: Interaction, Application and Advancement. DOI: https://doi.org/10.1016/B978-0-323-91728-5.00006-8 c 2023 Elsevier Inc. All rights reserved. Copyright 

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Principles of Multiple-Liquid Separation Systems

antioxidants from Limonium sinuatum flowers through Soxhlet extraction and maceration is compared with UAE method. The highest extraction yield was observed in the extracts produced by the UAE method in the shortest time as compared to those conventional methods. This proved that the UAE technique overcome some limitations of conventional methods by accelerating the mass transfer and diffusion processes, resulting in the improvement of extraction efficiency and yields. Yin et al. (2018) compared the extraction yields and antioxidant activities of Lentinus edodes polysaccharides extracted from hot-water extraction, EAE, MAE, and UAE with enzymes–microwave–ultrasound-assisted extraction. As compared to other techniques, the extraction yield of polysaccharides had been improved and the reducing power was the highest using enzymes–microwave–ultrasound-assisted extraction method. In this chapter, the mechanisms, key factors affecting the process, and applications of UAE, MAE, and EAE processes are discussed. Selection of a suitable method is not sufficient to maximize the extraction yields as the efficiency of methods and the nature or quality of products will be influenced by key factors such as temperature and time. Hence, the optimization of factors affecting the extraction process should be conducted to obtain the high quality and quantity of the desired products. For sustainability, environmental friendly, financial feasibility, and high-level bioactivity, the current applications and technologies act as the foundations or fundamentals for finding more sophisticated extraction methods in the near future.

14.2

Ultrasonic-assisted extraction

14.2.1 Mechanism/working principle The frequency of ultrasonic waves is greater than the audible range and lower than the microwave frequency (20 kHz–10 MHz). Ultrasonic effects such as physical effects, chemical effects, and acoustic streaming effects are influenced by the range of frequencies: r Physical effects: 20–100 kHz r Chemical effects: 200–500 kHz r Acoustic streaming effects: >1 MHz

Ultrasound can be divided into two categories, which are low-intensity sonication and high-intensity sonication for different applications. Low-intensity sonication (2.5 μm,