Liquid Chromatography of Synthetic Polymers: Entropy/Enthalpy Compensation and Critical Conditions (Physical Chemistry in Action) 3031348346, 9783031348341

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Physical Chemistry in Action

Muhammad Imran Malik Dusan Berek

Liquid Chromatography of Synthetic Polymers Entropy/Enthalpy Compensation and Critical Conditions

Physical Chemistry in Action

Physical Chemistry in Action presents volumes which outline essential physicochemical principles and techniques needed for areas of interdisciplinary research. The scope and coverage includes all areas of research permeated by physical chemistry: organic and inorganic chemistry; biophysics, biochemistry and the life sciences; the pharmaceutical sciences; crystallography; materials sciences; and many more. This series is aimed at students, researchers and academics who require a fundamental knowledge of physical chemistry for working in their particular research field. The series publishes edited volumes, authored monographs and textbooks, and encourages contributions from field experts working in all of the various disciplines.

Muhammad Imran Malik · Dusan Berek

Liquid Chromatography of Synthetic Polymers Entropy/Enthalpy Compensation and Critical Conditions

Muhammad Imran Malik Third World Center for Science and Technology, H.E.J. Research Institute of Chemistry, International Centre for Chemical and Biological Sciences (ICCBS) University of Karachi Karachi, Pakistan

Dusan Berek Polymer Institute Slovak Academy of Sciences Bratislava, Slovakia

ISSN 2197-4349 ISSN 2197-4357 (electronic) Physical Chemistry in Action ISBN 978-3-031-34834-1 ISBN 978-3-031-34835-8 (eBook) https://doi.org/10.1007/978-3-031-34835-8 © The Editor(s) (if applicable) and The Author(s), under exclusive license to Springer Nature Switzerland AG 2023 This work is subject to copyright. All rights are solely and exclusively licensed by the Publisher, whether the whole or part of the material is concerned, specifically the rights of translation, reprinting, reuse of illustrations, recitation, broadcasting, reproduction on microfilms or in any other physical way, and transmission or information storage and retrieval, electronic adaptation, computer software, or by similar or dissimilar methodology now known or hereafter developed. The use of general descriptive names, registered names, trademarks, service marks, etc. in this publication does not imply, even in the absence of a specific statement, that such names are exempt from the relevant protective laws and regulations and therefore free for general use. The publisher, the authors, and the editors are safe to assume that the advice and information in this book are believed to be true and accurate at the date of publication. Neither the publisher nor the authors or the editors give a warranty, expressed or implied, with respect to the material contained herein or for any errors or omissions that may have been made. The publisher remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. This Springer imprint is published by the registered company Springer Nature Switzerland AG The registered company address is: Gewerbestrasse 11, 6330 Cham, Switzerland

Foreword

I am very pleased to be invited to write the foreword for this exciting new book, Liquid Chromatography of Synthetic Polymers: Entropy/Enthalpy Compensation and Critical Conditions, by Drs. Muhammad Imran Malik and Dusan Berek. Having worked broadly in the field of polymer science, including synthesis, characterization, and applications, for over 45 years, it has become increasingly obvious to me that rigorous characterization of polymer structure is by far the most difficult challenge in the field. This is because all synthetic polymers are mixtures, comprised of chains varying in molar mass, stereochemistry, and end groups even for the simplest linear homopolymers. Introduction of long- and/or short-chain branching, either intentionally or due to side reactions such as chain transfer to polymer, further complicates the task of rigorous macromolecular characterization. Branched synthetic homopolymers are almost invariably mixtures of linear and branched species, with branched components varying in extent of branching and branched chain architectures, as well as possibly containing gel (crosslinked) components. Copolymers, made by incorporating more than one type of monomer unit into the polymer chains, exhibit additional complexity in various copolymer compositions and sequence distributions. Even with the formidable array of polymer molecular characterization techniques available at present, it is not possible to characterize such mixtures to the extent that we can determine the structures of all the individual chains that comprise the material. Instead, we must rely on determining averages of properties, such as molar mass and composition, as well as their distributions. Fortunately, modern liquid chromatography, in particular different modes of interaction chromatography (IC), offers a powerful array of separation techniques that allows polymer scientists to take a “divide and conquer” approach to polymer characterization. While the commonly employed size exclusion chromatography (SEC), an entropy-controlled process, separates polymer mixtures according to their sizes (hydrodynamic volume), this method is severely limited with polymer mixtures because many factors (molar mass, branching, chain conformation, co-monomer content, etc.) affect hydrodynamic volume. Thus, fractions isolated using SEC are themselves invariably complex mixtures. In contrast, as elucidated in Liquid Chromatography of Synthetic Polymers: Entropy/Enthalpy Compensation and Critical v

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Foreword

Conditions, different methods of IC can be chosen to separate polymer mixtures purely, or very nearly so, on the basis of molar mass, composition, architecture, end groups, etc. The authors, top experts in the field, adopt a tutorial style so that little prior knowledge of polymer separations and characterization is required. Thus, this book would be ideal as a text for a course on polymer separations but, due to the thorough referencing and discussion of the latest stationary phases, detectors, and instrumentation, it will also be of great benefit to scientists already working in this field. Furthermore, there are chapters dealing with two-dimensional chromatography and hyphenation of liquid chromatography with a range of spectroscopy techniques. These techniques, as described in Liquid Chromatography of Synthetic Polymers: Entropy/Enthalpy Compensation and Critical Conditions, open the road to achieving new levels of insight into the structural complexity of synthetic polymers, ultimately enabling a critical enhanced understanding of how polymer structure affects polymer properties. Jimmy Mays Professor Emeritus University of Tennessee Knoxville, USA

Preface

After almost a century of the discovery of polymers by Hermann Staudinger, modern technology and life without polymers is impossible to imagine. In past 100 years, numerous novel polymers varying in their molar mass, chemical composition, architecture etc. are synthesized by modern synthesis methods. An obvious requisite after synthesis of such a variety of polymers is the development of reliable characterization tools. Polymers unlike other materials have distribution of properties that makes them an extremely difficult candidate for characterization. A simplest polymer sample (homopolymer) inherently has molecules varying in the number of repeat units that originates the molar mass distribution. Homopolymers are further complicated by end groups, long-chain or short-chain branching, tacticity, mode of monomer insertion (e.g., head-to-tail or head-to-head), chain conformation (flexibility), etc. In the case of copolymers, additional complications are introduced by variations in co-monomer content across the MWD and monomer sequence distributions. These variations in the polymers strongly influence their applications properties. Knowledge of molecular characteristics of polymers is indispensable not only for their proper applications but also for their recycling and remediation. Scientists have realized from very start that complete molecular characterization of all aspects of polymer structure will be near to impossible. Accurate and complete molecular characterization of polymers is still a highly challenging and unachievable task despite momentous developments in the characterization tools. Moreover, thorough characterization of polymers often requires combination of different techniques along with some knowledge on the synthesis method of the polymer under investigation. Most of the characterization tools for polymers especially spectroscopy such as NMR, FTIR, etc. provide average values of the distributed properties which is not sufficient for development of structure-property correlations. Thus, separation techniques are required that reveal distribution of properties instead of only average values. Size exclusion chromatography is the premier and most widely used technique for determination of molar mass distributions of polymers. SEC is an entropy-controlled process that separates polymer molecules with regard to their hydrodynamic volume in dilute solution. However, SEC separation is not strictly according to molar mass and the polymers with similar molar mass vii

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but varying in composition and architecture may have very different hydrodynamic volumes. On the other hand, different modes of interaction chromatography (IC) of polymers can be used to separate them with regard to other distributions such as chemical composition, number of branches, architecture, etc., and also strictly with regard to molar mass. The techniques of IC for polymer analysis can be used independently as complementary techniques and can also be hyphenated with each other and with spectroscopy for deep insight into the polymer composition. The primary focus of this book is to serve as a textbook for a course (academic course or short course) on polymer characterization by HPLC in order to better train the next generation of polymer characterization experts. The book is written in a tutorial style to serve the primary objective. The book contains a comprehensive overview of the basics of chromatographic techniques used in polymer analysis along with an up-to-date account of their recent applications. Thus, it will also be a useful table reference for experienced researcher both in academia and industry. The book provides comprehensive insight on liquid chromatography modes for characterization of complex synthetic polymers. Another mission of the book is to elucidate the peculiar phenomenon of entropy/enthalpy compensation taking place in polymer HPLC. This occurrence plays decisive role in the advanced method of polymer characterization, liquid chromatography at critical conditions, but so far its role in other procedures of polymer HPLC is largely overlooked. The book provides a number of practical advices for users of liquid chromatography methods of synthetic polymers. The book begins with several chapters introducing the complexity of polymers and basic terminology. Chapter 1 introduces the complexity of polymers and highlights the important properties that should be determined. Chapter 2 discusses the behavior of polymers in solution, involved terminology, their solubility, and behavior of polymer solution in contact with the solid particles. Chapter 3 provides a comprehensive discussion on the theory of different modes of liquid chromatography. Chapter 4 describes the instrumentation used for polymer chromatography with an extensive discussion on the peculiarities of different parts of the chromatographic system such as stationary phases, pumps, mobile phases, and detectors. Chapter 5 briefly discusses the non-exclusion methods of polymer liquid chromatography such as hydrodynamic chromatography, field flow fractionation, etc. Chapter 6 focuses on conventional entropy-controlled methods of polymer liquid chromatography mainly size exclusion chromatography. Chapters 5 and 6 are only included to differentiate these techniques from entropy/enthalpy compensation methods for the sake of completeness of the topic. Chapter 7 introduces the role of entropy/enthalpy compensation—critical conditions in liquid chromatography of polymers with historical notes on its developments along with terminology. Chapter 8 focuses on liquid chromatography at critical conditions, LCCC, providing a detailed account of the methods of determination of critical conditions, their sensitivity, and working principle. Finally, applications of LCCC in polymer analysis are comprehensively reviewed followed by associated limitations and opportunities. Chapter 9 discusses the working principle and applications of liquid chromatography under limiting conditions in polymer analysis. Chapter 10 discusses the role of entropy/enthalpy compensation in eluent gradient

Preface

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interaction chromatography, EGIC, along with a comprehensive discussion on its applications for polymer analysis. Chapter 11 focuses on the temperature gradient interaction chromatography, TGIC, which is also based on entropy/enthalpy compensation phenomenon. Recent applications along with associated limitations and opportunities are highlighted. Chapter 12 is focused on coupling of different modes of liquid chromatography for comprehensive analysis of polymers. Approaches and experimental setup are discussed along with detailed account of applications. Chapter 13 describes the possibilities of hyphenation of different modes of LC of polymers with spectroscopic methods such as NMR, FTIR, and MS. Approaches and experimental setup are discussed along with detailed account of applications. Chapter 14 summarizes the achievements and highlights the still unaddressed challenges in polymer analysis. At last, I would like to pay tribute to Prof. Dusan Berek. Prof. Berek and I planned this book couple of years ago and slowly started working on its contents. Unfortunately, Dusan passed away on June 28, 2022 in Ljubljana while he was there for a lecture in a conference. I appreciate his contribution in this write-up especially for first few chapters focusing on the history. Finally, I would like to acknowledge Prof. Bernd Trathnigg, Prof. Harald Pasch, and Prof. Taihyun Chang for their immense contribution in development of my understanding on this particular topic during my stay in their labs and afterwards. I am especially thankful to Prof. Taihyun Chang for comprehensively reviewing the manuscript. Karachi, Pakistan

Muhammad Imran Malik

Contents

1

Polymers and Their Complexity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1 5

2

Polymers in Solution . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1 Basic Terminology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2 Solubility of Polymers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.3 Polymers in Solution in Contact with Porous Solid Particles . . . . 2.3.1 Entropy-Controlled Processes . . . . . . . . . . . . . . . . . . . . . 2.3.2 Enthalpy-Affected Processes . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

7 7 11 14 14 15 19

3

Liquid Chromatography as a Tool for Determination of Distribution of Polymer Molecular Characteristics . . . . . . . . . . . . . 3.1 Separation Mechanisms in Polymer LC . . . . . . . . . . . . . . . . . . . . . . 3.2 Theory of Polymer Chromatography . . . . . . . . . . . . . . . . . . . . . . . . 3.2.1 Size Exclusion Chromatography (SEC) . . . . . . . . . . . . . 3.2.2 Interaction Chromatography (IC) or Liquid Adsorption Chromatography (LAC) . . . . . . . . . . . . . . . . 3.2.3 Liquid Chromatography at Critical Conditions . . . . . . . 3.3 Thermodynamics of Polymer Chromatography . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

4

Instrumentation for Polymer Liquid Chromatography . . . . . . . . . . . . 4.1 Solvent Delivery System and Injector . . . . . . . . . . . . . . . . . . . . . . . 4.2 Columns . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.3 Stationary Phases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.3.1 Physical Structure of Stationary Phases . . . . . . . . . . . . . 4.3.2 Chemical Nature of Stationary Phases . . . . . . . . . . . . . . 4.4 Mobile Phases/Eluents and Sample Solvents . . . . . . . . . . . . . . . . . 4.5 Detectors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.5.1 Concentration Sensitive Detectors . . . . . . . . . . . . . . . . . . 4.5.2 Molar Mass Sensitive Detectors . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

23 24 27 27 28 30 32 33 35 35 36 38 38 39 42 46 47 51 52 xi

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5

6

7

8

Contents

Non-exclusion Methods of Polymer Liquid Chromatography . . . . . . 5.1 Polymer—Mobile Phase Interactions . . . . . . . . . . . . . . . . . . . . . . . . 5.1.1 Precipitation-(Re)dissolution Polymer LC . . . . . . . . . . . 5.1.2 Hydrodynamic Chromatography . . . . . . . . . . . . . . . . . . . 5.1.3 Field-Flow Fractionation . . . . . . . . . . . . . . . . . . . . . . . . . . 5.2 Polymer—Column Packing Interactions . . . . . . . . . . . . . . . . . . . . . 5.2.1 Eluent (Mobile Phase) Gradient Interaction Chromatography (EGIC) . . . . . . . . . . . . . . . . . . . . . . . . . . 5.2.2 Full Adsorption—Desorption Polymer LC . . . . . . . . . . 5.3 Coupled Methods of Polymer Separation . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conventional Entropy-Controlled Methods of Polymer Liquid Chromatography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.1 Size Exclusion Chromatography . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.1.1 Origin and First Stage of Size Exclusion Chromatography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.1.2 Theory of Size Exclusion Chromatography . . . . . . . . . . 6.1.3 Column Packings, Mobile Phases/Eluents for Size Exclusion Chromatography . . . . . . . . . . . . . . . . 6.1.4 Relation Between Elution Volume and Polymer Molar Mass in Size Exclusion Chromatography . . . . . . 6.1.5 Secondary Effects in Size Exclusion Chromatography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.1.6 Applications of Size Exclusion Chromatography . . . . . 6.1.7 Unwanted Enthalpic Effects in Size Exclusion Chromatography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.2 Exclusion—Interaction Combinations . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Entropy/Enthalpy Compensation in Polymer Liquid Chromatography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.1 History of Entropy/Enthalpy Compensation—The Critical Conditions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.2 Terminology of Polymer LC Based on Entropy/Enthalpy Compensation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.3 Molar Mass Versus Elution Volumes in the Process of Entropy/Enthalpy Compensation . . . . . . . . . . . . . . . . . . . . . . . . . 7.4 Rational for Polymer Chromatography Based on Entropy/Enthalpy Compensation . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Liquid Chromatography at Critical Conditions . . . . . . . . . . . . . . . . . . 8.1 Establishment of Critical Conditions . . . . . . . . . . . . . . . . . . . . . . . . 8.2 Sensitivity of Critical Conditions . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.3 Working Principle . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

57 57 57 59 60 60 61 63 64 65 71 72 73 74 76 79 81 84 88 90 90 99 100 101 102 103 104 107 107 109 111

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8.4

Applications of LCCC in Polymer Analysis . . . . . . . . . . . . . . . . . . 8.4.1 LCCC-SEC . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.4.2 LCCC-IC . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.5 Limitations and Opportunities . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.5.1 Sensitivity and Instability of Critical Conditions . . . . . . 8.5.2 Band Broadening . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.5.3 Incomplete Sample Recovery . . . . . . . . . . . . . . . . . . . . . . 8.5.4 Detection Problems . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.5.5 Incomplete Invisibility of the Critical Block . . . . . . . . . 8.5.6 Important Information Accessible Through LCCC . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

112 113 126 136 137 137 138 138 138

Liquid Chromatography Under Limiting Conditions . . . . . . . . . . . . . 9.1 Working Principle . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9.2 Applications of LC-LC in Polymer Analysis . . . . . . . . . . . . . . . . . 9.2.1 Polymer Blends . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9.2.2 Block Copolymers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9.2.3 Statistical Coplymers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9.2.4 Polymers Varying in Tacticity/Microstructure . . . . . . . . 9.3 Limitations and Opportunities . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

153 153 155 156 157 158 160 161 162

10 Eluent Gradient Interaction Chromatography . . . . . . . . . . . . . . . . . . . 10.1 Working Principle . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10.2 Applications of EGIC in Polymer Analysis . . . . . . . . . . . . . . . . . . . 10.2.1 Molar Mass Separation of Homopolymers . . . . . . . . . . . 10.2.2 Oligomer Separation of Homopolymers . . . . . . . . . . . . . 10.2.3 Homopolymer Blends . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10.2.4 End-Functionalized Homopolymers . . . . . . . . . . . . . . . . 10.2.5 Block Copolymers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10.2.6 Graft Copolymers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10.2.7 Polymers Varying in Tacticity/Microstructure . . . . . . . . 10.2.8 Branched Polymers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10.2.9 Statistical Copolymers . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10.2.10 Dendrimers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10.3 Limitations and Opportunities . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

165 166 167 168 169 170 173 175 177 179 182 184 192 193 194

11 Temperature Gradient Interaction Chromatography . . . . . . . . . . . . . 11.1 Working Principle . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11.2 Applications of TGIC in Polymer Analysis . . . . . . . . . . . . . . . . . . . 11.2.1 Molar Mass Separation of Homopolymers . . . . . . . . . . . 11.2.2 Polymer Blends . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11.2.3 End-Functionalized Linear Homopolymers . . . . . . . . . . 11.2.4 Branched Polymers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

205 207 208 208 209 210 211

9

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11.2.5 Star-Shaped Polymers . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11.2.6 Statistical Copolymers . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11.2.7 Polymers Varying in Tacticity/Microstructure . . . . . . . . 11.2.8 Graft Copolymers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11.2.9 Block Copolymers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11.3 Limitations and Opportunities . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

216 217 218 220 221 222 223

12 Two-Dimensional Liquid Chromatography . . . . . . . . . . . . . . . . . . . . . . 12.1 Overlapping Distributed Properties of Polymers . . . . . . . . . . . . . . 12.2 Approaches of Two-Dimensional Analysis . . . . . . . . . . . . . . . . . . . 12.3 Experimental Setup of Orthogonal Chromatography . . . . . . . . . . . 12.4 Applications of Two-Dimensional Liquid Chromatography in Polymer Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12.4.1 SEC as Second Dimension . . . . . . . . . . . . . . . . . . . . . . . . 12.4.2 Interaction Chromatography in Second Dimension . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12.5 Limitations and Opportunities . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

231 231 233 234

13 Hyphenation of Liquid Chromatography with Spectroscopy . . . . . . . 13.1 Hyphenation of Liquid Chromatography with Fourier Transform Infrared Spectroscopy . . . . . . . . . . . . . . . . . . . . . . . . . . . 13.2 Hyphenation of Liquid Chromatography with Mass Spectrometry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13.3 Hyphenation of Liquid Chromatography with Nuclear Magnetic Resonance Spectroscopy . . . . . . . . . . . . . . . . . . . . . . . . . . 13.4 Limitations and Opportunities . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

265

237 237 245 252 253

265 269 273 276 278

14 Challenges and Opportunities . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 287 Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 291

Abbreviations

a ACN AEAm AF4 APCI API aPP BCP BET BHT BPA BPPSTE BR c CAP CCD CCP CDMSS CE CEC CEF CH2 Cl2 CH3 CN CHCl3 CH3 OH CPP CRYSTAF D Ð DAD DCB

Mark-Houwink exponent Acetonitrile N-2-(aminoethyl) acrylamide Asymmetric-flow-field-flow fractionation Atmospheric pressure chemical ionization Atmospheric pressure ionization Atactic polypropylene Block copolymers Brunauer-Emmett-Teller Butylated hydroxytoluene Bisphenol A BiPolar gradient Pulsed STimulated Echo Butyl rubber Concentration Critical adsorption point Chemical composition distribution Chromatographic critical point p-(chlorodimethylsilyl) styrene Capillary electrophoresis Capillary electrokinetic chromatography Crystallization elution fractionation Dichloromethane Acetonitrile Chloroform Methanol Critical point for enthalpic partition Crystallization fractionation Pore diameter Dispersity Diode-array detector 1,2-dichlorobenzene xv

xvi

DCM DEF DEM DHB 2D-LC DMF d-PS DVB EB EEGE 2E1H EGIC EGMBE EH ELSD ENB ENR EO EO EO-PO EP EPDM EPDM-g-PMMA ESI-TOF-MS EVA FAD FFF FTIR G1 G2 G3 GPC HDC HDPE HFIP HT 2D-LC HT-EGIC HT-LCCC h-PS HPLC IC IGG iPP

Abbreviations

Dichloromethane Diethyl formamide Diethyl malonate Dihydroxy benzoic acid Two-dimensional liquid chromatography Dimethyl formamide Deuterated polystyrene Divinyl benzene Ethylene-butylene copolymer Ethoxy ethyl glycidyl ether 2-ethyl-1-hexanone Eluent gradient interaction chromatography Ethylene glycol monobutyl ether Ethene-hexene copolymer Evaporative light scattering detectors Ethylidene norbornene Epoxidized natural rubber Ethene-octene copolymer Ethylene oxide Ethylene oxide-propylene oxide Ethylene-propylene copolymer Ethylene-propylene-diene Ethylene-propylene-diene-graft-poly(methyl methacrylate) Electrospray ionization time-of-flight mass spectrometry Ethylene-vinyl acetate copolymers Full adsorption-desorption Field flow fractionation Fourier Transform Infrared Spectroscopy Gradient-1 Gradient-2 Gradient-3 Gel permeation chromatography Hydrodynamic chromatography High-density polyethylene 1,1,1,3,3,3-hexafluoro-2-propanol High-temperature two-dimensional liquid chromatography High-temperature eluent gradient interaction chromatography High-temperature liquid chromatography at critical conditions Protonated polystyrene High-performance liquid chromatography Interaction chromatography Isopropylidene glycerol glycidyl ether Isotactic polypropylene

Abbreviations

IBA LALLS LCCC LEAC LC-LC LC-LCA LC-LCD LC-LCI LC-LCP LC-LCS LC-LCU LDPE LLDPE LCST LC-PEAT LS M MAA MALDI-TOF MS MALLS MEK MeOH mi Meq mm MMA MMD MP MS Mn Mo Mv Mw Mw /Mn n ni nBA NIPAm

xvii

Isobornyl acrylate Low-angle light scattering Liquid chromatography at critical conditions Liquid exclusion adsorption chromatography Liquid chromatography under limiting conditions Liquid chromatography under limiting conditions of adsorption Liquid chromatography under limiting conditions of desorption Liquid chromatography under limiting conditions of insolubility Liquid chromatography under limiting conditions of partition Liquid chromatography under limiting conditions of solubility Liquid chromatography under limiting conditions of unpartition Low-density polyethylene Linear low-density polyethylene Lower critical solution temperature Liquid chromatography at the point of adsorption-exclusion transition Light scattering Molar mass Methacrylic acid Matrix-assisted laser desorption/ionization time-of-flight Multi-angle laser light scattering Methyl ethyl ketone Methanol Mass of species i Equivalent molar mass Isotactic triad Methyl methacrylate Molar mass distribution Mobile phase Mass spectrometry Number average molar mass Molecular weight of repeat unit Viscosity average molar mass Weight average molar mass Dispersity Degree of polymerization Number of species i n-butyl acrylate N-isopropylacrylamide

xviii

NMR NP NPLC NP-TGIC ODCB ODS P PAMAM PB PB-b-PS-b-PB PCL PDMS PEAT PEMA PE PEG PEMA PEO PEO-b-PPO PGC PI PIB PLA PMA PMMA PMMA-g-PDMS PMMA-grafted-ENR Pn PnBMA PO PP PPG PPO PP-g-MA P(POPA) PRESAT PS PS/DVB PS-b-(PB-g-PBA) PS-b-PB PS-b-PB-b-PS

Abbreviations

Nuclear Magnetic Resonance Normal phase Normal phase liquid chromatography Normal phase temperature gradient interaction chromatography 1,2-dichlorobenzene or Ortho dichlorobenzene Octadecyl silane Degree of polymerization Poly(amidoamine) Polybutadiene Polybutadiene-block-Polystyrene-block-polybutadiene Poly(1-caprolactone) Poly(dimethyl siloxane) Point of adsorption-exclusion transition Poly(ethyl methacrylate) Polyethylene Poly(ethylene glycol) Poly(ethyl methacrylate) Poly(ethylene oxide) Poly(ethylene oxide)-block-poly(propylene oxide) Porous graphitic carbon Polyisoprene Polyisobutylene Poly(L-lactide) Poly(methyl acrylate) Poly(methyl methacrylate) Poly(methyl methacrylate)-graft-poly(dimethyl siloxane) Poly(methyl methacrylate)-grafted-epoxidized natural rubber Number average degree of polymerization Poly(n-butyl methacrylate) Propylene oxide Polypropylene Poly(propylene glycol) Poly(propylene oxide) Polypropylene-grafted-maleic anhydride Poly(propylene phthalate) Transmitter presaturation Polystyrene Polystyrene/divinyl benzene Polystyrene-block-(polybutadiene-graft-poly butyl acrylate) Polystyrene-block-polybutadiene Polystyrene-block-polybutadiene-block-polystyrene

Abbreviations

PS-b-(PB-g-PBA) PS-b-PI PS-b-PI-b-PS PS-b-P2VP PVAc PVC P2VP P2VP-b-PS-b-P2VP Pw RALLS Rg RGD RI rm RP RPLC rr RT SAN SBA SBR SCB SDV SEC SEC-TD SFC S/N SP sPP T TALLS TCB TEA TFE TGIC TGIC-TD THF TLC TMB TMED ToF TREF

xix

Polystyrene-block-[polybutadiene-graft-poly(butyl acrylate)] Polystyrene-block-polyisoprene Polystyrene-block-polyisoprene-block-polystyrene Polystyrene-block-poly(2-vinyl pyridine) Poly(vinyl acetate) Poly(vinyl chloride) Poly(2-vinyl pyridine) Poly(2-vinyl pyridine)-block-polystyrene-block-poly(2-vinyl pyridine) Weight average degree of polymerization Right-angle light scattering detectors Radius of gyration Radius of gyration distribution Refractive index Heterotactic triad Reversed phase Reversed phase liquid chromatography Syndiotactic triad Retention time Styrene-acrylonitrile copolymer Styrene-butyl acrylate copolymer Styrene-butadiene rubber Short chain branching Styrene-divinylbenzene copolymer Size exclusion chromatography Size exclusion chromatography-triple detection Supercritical fluid chromatography Signal-to-noise ratio Stationary phase Syndiotactic polypropylene Temperature Two-angle light scattering detectors 1,2,4-trichlorobenzenec Triethylamine 2,2,2-trifluoro ethanol Temperature gradient interaction chromatography Temperature gradient interaction chromatography-triple detection Tetrahydrofuran Thin layer chromatography 1,3,5-trimethylbenzene Tetramethyl ethylenediamine Time of flight Temperature rising elution fractionation

xx

UCST UP-LCCC UPLC UV VA VE Vi VISC Vp VR Vstat WATERGATE WET Wi

Abbreviations

Upper critical solution temperature Ultrahigh pressure liquid chromatography at critical conditions Ultrahigh pressure liquid chromatography Ultraviolet Vinyl Acetate Elution volume Interstitial volume Viscometer Pore volume Retention volume Volume of the stationary phase WATER suppression by GrAdient-Tailored Excitation Water suppression enhanced through T1 effects Weight fraction

Chapter 1

Polymers and Their Complexity

Synthetic polymers represent an important group of non-metallic materials with enormous wide area of applications in modern technology, healthcare and everyday life. The utility properties of polymeric materials depend on molecular characteristics of the constituent macromolecules and their mutual arrangement, as well as on presence of molecular or particulate additives. The basic molecular characteristics of the non-charged macromolecules are their molar mass, chemical composition along with structure (monomer sequence), and physical architecture. Exact determination of above properties of macromolecules is important for both synthesists, who produce polymers and for technologists, who apply them. This task is very demanding, because molecular characteristics of all synthetic polymers exhibit a distribution unlike monodisperse natural macromolecules such as proteins and nucleic acids. Especially intricated is molecular characterization of the high-performance synthetic polymers, which are composed of two or several distinct monomers. They may either be joined in the copolymer chains or can form blends of different kinds of macromolecules. Accordingly, one speaks about complex polymers and complex polymer systems. In both groups of materials, the distinct distributions of their molecular characteristics often overlap. Synthetic polymers always have several distributed properties irrespective of the polymerization technique employed for their synthesis [1–12]. Molar mass is the primary distribution that is present in all synthetic polymers. A homopolymer is the simplest type of polymer composed of only one type of repeating units and inherently have molar mass distribution. However, even this simple looking macromolecule may be linear, star-shaped, comb-shaped, hyperbranched, and net-work etc. An additional functionality at the ends of chains in the homopolymers introduce a functionality type distribution, Fig. 1.1. Moreover, incorporation of more than one monomer in polymer chains add additional complexities to the polymers. Different repeat units may be arranged differently making block, graft, statistical, gradient copolymers etc. Hence, except for analytical grade non-functional linear homopolymers, an additional distributed property may always be present that is overlapping with the inherent molar mass distribution. An important analytical

© The Author(s), under exclusive license to Springer Nature Switzerland AG 2023 M. I. Malik and D. Berek, Liquid Chromatography of Synthetic Polymers, Physical Chemistry in Action, https://doi.org/10.1007/978-3-031-34835-8_1

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1 Polymers and Their Complexity

Fig. 1.1 Possible molecular structures of synthetic polymers regarding chemical composition, architecture and end-group functionality [7]. Copyright 2005, Elsevier

challenge, still not fully addressed, is the determination of the second type of distribution as a function of, essentially present, molar mass distribution. Commonly occurring distributed properties of complex polymers include molar mass, chemical composition, end-group functionality, molecular topology, sequence length, etc. Changes in the distribution of any of above may lead to completely different physical and performance properties. A summary of different molecular distributions and the physical properties directly affected by them along with the relevant separation/analysis techniques for their determination is given in Table 1.1 [13, 14]. It is important to mention here that these distributions are also present in polyolefins that are simple hydrocarbons. Important distributions in polyolefins are molar mass, molecular topology (branching), chemical composition, and end-groups. Important grades of polyolefins high-density polyethylene (HDPE), low-density polyethylene (LDPE) and linear low-density polyethylene (LLDPE) all have molar mass distribution. HDPE is linear homopolymer while LDPE has branching distribution (long chain branches). Moreover, LLDPE has short chain branching originated from incorporation of other α-olefins comonomers (propene, butene, hexene, octene) and hence also have chemical composition and monomer sequence distribution. Therefore, careful analysis and understanding of these distributed properties as a function of molar mass distribution is imperative. Direct analysis of complex polymers for chemical composition analysis by captivating spectroscopic techniques such as Fourier transform infrared spectroscopy (FTIR), nuclear magnetic resonance spectroscopy (NMR) can only reveal average values for their composition. Furthermore, it is often not possible to differentiate between blend of two homopolymers and a copolymer by spectroscopic techniques. However, distribution of these properties is the target in order to develop structure–property correlations.

1 Polymers and Their Complexity

3

Table 1.1 Polymer distributions, end-use properties, and relevant separation/analysis methods for their determination [13, 14] Polymer distribution

Relevant separation/analysis methods

End-use properties

Molar mass

SEC, FFF, HDC, TGIC, CEC, SFC Tensile strength, adhesion, elongation

Chemical composition

LCCC, LCLC, TGIC, EGIC

Morphology, solubility, toughness, biodegradability

Long-chain branching

SEC–MALLS, SEC-VISCO, TGIC, EGIC

Tack, crystallinity, peel, shear strength

Short-chain branching

SEC-FTIR, SEC-NMR, TREF, CRYSTAF, EGIC, TGIC

Crystallinity, stress-crack resistance, haze

Topology

SEC–MALLS-VISC

Diffusion, viscosity, flow

Tacticity

LCCC, TGIC, SEC-NMR

Solubility, crystallinity, toughness

Copolymer sequence

SEC-spectroscopy, EGIC, TGIC, LCCC, 2D-LC

Haze, flexibility, miscibility

SEC: size exclusion chromatography, FFF: field flow fractionation, HDC: hydrodynamic chromatography, TGIC: temperature gradient interaction chromatography, CEC: capillary electrokinetic chromatography, SFC: supercritical fluid chromatography, HPLC: high performance liquid chromatography, EGIC: eluent gradient interaction chromatography, TGIC: temperature gradient interaction chromatography, LC-LC: liquid chromatography under limiting conditions, LCCC: liquid chromatography at critical conditions, MALLS: multi-angle laser light scattering, VISC: viscometry, TREF: temperature rising elution fractionation, CRYSTAF: crystallization fractionation, 2D-LC: two-dimensional liquid chromatography

In order to determine distribution of their molecular characteristics, as a rule the macromolecules must be separated. The polymer separation methods are dominated by the high-performance liquid chromatography of polymers—polymer HPLC, in which the macromolecules are transported along the collected mass of porous particles, termed as stationary phase (SP), usually arranged in the form of a column. The transporting liquid is called mobile phase (MP) or eluent. In the proceeding text, term eluent is predominantly used for the transporting liquid. Considering determination of polymer molar mass average and distribution, numerous modern approaches employ the methods based on the size separation of dissolved macromolecules usually in the form of statistical coils. At present the field is dominated by the size exclusion (gel permeation) chromatography, SEC [13]. As well-known, SEC is based on the differences in the velocity of movement, elution rate of macromolecular coils with distinct sizes along a column flushed with the eluent. In the “ideal” SEC, the behavior of macromolecules is mainly controlled by their conformational entropy (entropy difference between the two environments, pore/interstitial space). The process is denoted as entropic partition. However, the size of dissolved macromolecules simultaneously depends on their other molecular characteristics too and this brings about important limitations of SEC, especially in case of complex polymers and multicomponent polymers [13, 15, 16]. Multi-detector size exclusion chromatography may help to resolve it to some extent; however, still separation is based only on the differences in the hydrodynamic volumes of different species in the eluent and the

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1 Polymers and Their Complexity

fractions of same hydrodynamic volume may have molecules with different molar mass/branching/chemical composition etc. [13, 15]. To resolve the issue, the entropic partition is supplemented by the enthalpic interactions within the chromatographic system. This enables separation of macromolecules also according to their chemical structure and architecture. The resulting polymer HPLC methods are often collectively called interaction polymer liquid chromatography or simply interaction chromatography (IC) [14, 16, 17]. Interaction chromatography is the term broadly employed for such chromatographic systems throughout this book. They employ interaction of macromolecules either with the eluent or with the stationary phase (SP). Less frequently, the separation methods of polymer HPLC make use of variance in the profile of eluent flow or combine the flow with the external field. Electrical field driven separation is employed in case of charged macromolecules. It is necessary to mention that with exception of oligomers, the entropic partition of macromolecules plays more or less important role in numerous methods of interaction polymer HPLC. Hydrophobic interaction chromatography utilizes entropy driven interaction of solutes with the stationary phase. Similar to SEC, the major drawback of most methods of interaction polymer HPLC originate from simultaneous (concurrent) dependence of elution rate of macromolecules on all their molecular characteristics. Numerous methods of interaction polymer HPLC are affected by temperature changes and rather few of them by pressure variations in the separation system. Very specific polymer separation methods employ remarkable phenomenon of mutual compensation of entropic and enthalpic effects on behavior of macromolecules within chromatographic system, denoted (called) as critical conditions [18]. The entropy/enthalpy compensation process results in the suppression of molar mass effect on the elution of macromolecules from the HPLC column. As a result, other molecular characteristics of synthetic macromolecules can be acquired without molar mass interference. Most important methods of HPLC of the non-charged polymers will be briefly elucidated in this book, but the principal part of the title will be devoted to the above-mentioned phenomenon of the entropy/enthalpy compensation. The critical conditions will be discussed in detail—starting from the history of their discovery, through their theory, the techniques of their practical execution up to their application in the original method of liquid chromatography at critical conditions, LCCC. The discussion of peculiarities, advantages and drawbacks of LCCC will be accomplished with the critical overview of its recent applications. The reason for the detailed treatment of LCCC, lies in the fact that it may render data on complex polymers and complex polymer system, which are not directly available by means of SEC. From this aspect, LCCC can be considered a complementary technique to SEC. Numerous SEC users so far have not yet recognized true potential of LCCC, which can bring about important progress in molecular characterization of complex synthetic polymers to better recognize the true structure–property correlations. The often-overlooked role of the entropy/enthalpy compensation in other advanced polymer HPLC is also outlined in the book. To bridge over above information gaps is the important task of the current book.

References

5

In fact, LCCC is an isocratic liquid chromatography method, which couples’ entropy-controlled exclusion of macromolecules from the porous column packing with their enthalpy-affected retention. After detailed account of critical conditions and its peculiarities, advantages and caveats, other methods of liquid chromatography where entropy/enthalpy compensation is the major driving force are discussed. These methods include liquid chromatography under limiting conditions along with gradient methods of liquid chromatography of polymers such as eluent gradient interaction chromatography (EGIC) and temperature gradient interaction chromatography (TGIC). Finally, hyphenated methods of polymer analysis that include hyphenation of different LC methods and coupling of LC methods with spectroscopic methods is discussed. In these chapters, focus of discussion is role of entropy/enthalpy compensation along with a detailed account of their modern applications.

References 1. Blosch SE, Scannelli SJ, Alaboalirat M, Matson JB (2022) Complex polymer architectures using ring-opening metathesis polymerization: synthesis, applications, and practical considerations. Macromolecules 55(11):4200–4227. https://doi.org/10.1021/acs.macromol. 2c00338 2. Matyjaszewski K (2017) Polymer chemistry: current status and perspective. Chem Int 39 (4):7– 11. https://doi.org/10.1515/ci-2017-0404 3. Gao Y, Zhou D, Lyu J, AS, Xu Q, Newland B, Matyjaszewski K, Tai H, Wang W (2020) Complex polymer architectures through free-radical polymerization of multivinyl monomers. Nat Rev Chem 4(4):194–212. https://doi.org/10.1038/s41570-020-0170-7 4. Polymeropoulos G, Zapsas G, Ntetsikas K, Bilalis P, Gnanou Y, Hadjichristidis N (2017) 50th anniversary perspective: polymers with complex architectures. Macromolecules 50(4):1253– 1290. https://doi.org/10.1021/acs.macromol.6b02569 5. Gregory A, Stenzel MH (2012) Complex polymer architectures via RAFT polymerization: from fundamental process to extending the scope using click chemistry and nature’s building blocks. Prog Polym Sci 37(1):38–105. https://doi.org/10.1016/j.progpolymsci.2011.08.004 6. Guo X, Choi B, Feng A, Thang SH (2018) Polymer synthesis with more than one form of living polymerization method. Macromol Rapid Commun 39(20):1800479. https://doi.org/10. 1002/marc.201800479 7. Matyjaszewski K, Spanswick J (2005) Controlled/living radical polymerization. Mater Today 8(3):26–33. https://doi.org/10.1016/S1369-7021(05)00745-5 8. Stridsberg KM, Ryner M, Albertsson A-C (2002) Controlled ring-opening polymerization: polymers with designed macromolecular architecture. In: Degradable aliphatic polyesters. Springer, Heidelberg, pp 41–65. https://doi.org/10.1007/3-540-45734-8_2 9. Matyjaszewski K, Tsarevsky NV (2009) Nanostructured functional materials prepared by atom transfer radical polymerization. Nat Chem 1(4):276–288 10. Hadjichristidis N, Pitsikalis M, Pispas S, Iatrou H (2001) Polymers with complex architecture by living anionic polymerization. Chem Rev 101(12):3747–3792. https://doi.org/10.1021/cr9 901337 11. Guillaume SM (2013) Recent advances in ring-opening polymerization strategies toward α, ω-hydroxy telechelic polyesters and resulting copolymers. Eur Polym J 49(4):768–779. https:/ /doi.org/10.1016/j.eurpolymj.2012.10.011 12. Xu J, Hadjichristidis N (2023) Heteroatom-containing degradable polymers by ring-opening metathesis polymerization. Prog Polym Sci 139:101656. https://doi.org/10.1016/j.progpolym sci.2023.101656

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13. Striegel AM, Yau WW, Kirkland JJ, Bly DD (2009) Modern size-exclusion liquid chromatography: practice of gel permeation and gel filtration chromatography, vol 2. Wiley Hoboken, New Jersey. https://doi.org/10.1002/9780470442876 14. Pasch H, Trathnigg B (2013) Multidimensional HPLC of polymers. Springer, New York. https:/ /doi.org/10.1007/978-3-642-36080-0 15. Lederer A, Brandt J (2021) Chapter 2—Multidetector size exclusion chromatography of polymers. In: Malik MI, Mays J, Shah MR (eds) Molecular characterization of polymers. Elsevier, pp 61–96. https://doi.org/10.1016/B978-0-12-819768-4.00012-9 16. Malik MI, Pasch H (2014) Novel developments in the multidimensional characterization of segmented copolymers. Prog Polym Sci 39(1):87–123. https://doi.org/10.1016/j.progpolymsci. 2013.10.005 17. Malik MI, Pasch H (2021) Chapter 1—Basic principles of size exclusion and liquid interaction chromatography of polymers. In: Malik MI, Mays J, Shah MR (eds) Molecular characterization of polymers. Elsevier, pp 1–59. https://doi.org/10.1016/B978-0-12-819768-4.00007-5 18. Malik MI (2021) Liquid chromatography at critical conditions in polymer analysis: a perspective. Chromatographia 84(12):1089–1094. https://doi.org/10.1007/s10337-021-04096-x

Chapter 2

Polymers in Solution

This chapter is destined for the readers not involved in the research and applications of macromolecular substances. Its purpose is the brief, simplified elucidation of the selected principles and terms, which may be needed for comprehension of the following chapters of the book, which is devoted to molecular characterization of polymers by advanced methods of liquid chromatography. The methods of polymer synthesis and measurement of their properties in solid state are not discussed. The specific target group are the readers coming from the field of high-performance liquid chromatography, HPLC of low-molecular weight substances.

2.1 Basic Terminology Giant molecules, macromolecules, which are built up from the chains of the basic units, “mers” connected by covalent bonds are designated as polymers [1–3]. Not all macromolecules are polymers but all polymers are considered macromolecules. Some huge molecules, supramolecules are created by arrays of low-molecular weight substances, which are mutually held together by physical interactions. Present book is devoted exclusively to authentic polymeric materials. Most of them are of synthetic nature but numerous natural polymers can also be included into this group of materials. For instance, behavior of certain chemically modified polysaccharides resembles to synthetic polymers in various aspects. The existence of polymeric substances was discovered and described by H. Staudinger in his seminal landmark in first quarter of twentieth century [4]. Subsequently, he with his coworkers extended their work on cellulose, starch, polystyrene and (natural) rubber, and coined the terms “macromolecule” and “polymer”. In 1947 he had initiated the Journal “Die Makromolekulare Chemie”, presently it is “Macromolecular Chemistry and Physics” and five related Journals. Staudinger’s discovery originated extensive research, which led to important scientific and technological breakthroughs in material science [2]. Numerous scientists, contributed to progress © The Author(s), under exclusive license to Springer Nature Switzerland AG 2023 M. I. Malik and D. Berek, Liquid Chromatography of Synthetic Polymers, Physical Chemistry in Action, https://doi.org/10.1007/978-3-031-34835-8_2

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in the area and we will pay tribute to several of them as the share of our historical notes [5]. Synthetic polymers found wide applications in both technology and everyday life. Presently, more than 400 million metric tons of them are produced yearly that is continuously increasing. Following characteristics of polymers influence their utility properties. (i) molecular characteristics of the comprising macromolecules, (ii) mutual arrangement of macromolecules, either amorphous or containing crystallites, (iii) nature and amount of additives, either high or low molar mass substances, including inorganic particles. The primary molecular characteristics of polymers are. (i) molar mass (ii) chemical composition and microstructure (iii) chain architecture. The primary molecular characteristics are often interdependent and mutually affect each other to form secondary molecular characteristics (utility properties). They also influence behavior of macromolecules in contact with their surroundings e. g., their solubility in liquids. Present book deals with determination of molecular characteristics of polymers with the help of liquid chromatography. The molar mass, M is the most important primary molecular characteristic of polymers. It is responsible for many of their valuable physical properties. It refers to the mass of one mole of macromolecular substance, expressed in g/mol or kg/ mol. The IUPAC approved alternative term is the unitless molecular weight. The macromolecular substances with molar mass ranging up to several thousands g/mol are called oligomers while those with molar mass in millions of g/mol are often termed ultra-high molar mass polymers. The term chemical structure of polymers covers both basic chemical composition of monomers and their arrangement in the macromolecules. Macromolecules comprising only one kind of monomeric unit are denoted as homopolymers. Their end-groups often differ from the composition of the main chain. The end-groups may extensively affect the properties of oligomers where their relative role is much more significant than in the macromolecules with high molar mass. Macromolecules built from chemically distinct/different units are called complex polymers. Copolymers, are formed by two and more different monomers, the comonomers. Their sequence in the polymer chains may be different that originate sequential-, statistical-, block-, graft-, etc., polymeric species. The copolymers, in which two distinct monomers are deposited regularly in a row are the alternating copolymers. The short sequences of different length comprising of identical monomeric units render macromolecules exhibiting certain blockiness, termed as statistical copolymers. When the sequences of identical monomeric units in copolymers are long enough to be considered macromolecules of certain nature, they form linear di-, tri-, and multi-block copolymers

2.1 Basic Terminology

9

and graft copolymers. In the graft copolymers, the long side chains with a distinct composition are attached to the main chain, a backbone. Naturally, the chemical structure of macromolecules affects their chemical and physical properties. Polymer blends as a rule exhibit difference in the chemical structure of their constituents. For special purposes, the polymers are blended that differ substantially only in their molar mass. Most technically important commercial polymers are electroneutral. All the above features of chemical structure of polymers can also be found in oligomers but the presence of the functional groups have significant impact on its utility properties compared to high polymers. Molecules of oligomers can also carry polymerizable double bonds and in this case, they are termed as the macromonomers. The physical architecture constitutes the third group of molecular characteristics of the polymers. The architecture, topology of macromolecules may significantly affect properties of otherwise chemically identical polymeric materials. The most common are the linear macromolecules but also the branched, star-shaped, cyclic and dendritic structures were created. If the chemical composition of the polymer main chain, the backbone and the side chains is similar/same, the resulting macromolecule is (long chain) branched. Polymer chains of the same or distinct chemical nature commencing from a common center form the star-shaped polymers or the miktoarm polymers, respectively. Due to the spatial organization, the rotation around the single bond in a polymer backbone is blocked. As a result, the orientation of short side groups on the polymer backbone is fixed. This introduces another type of variation in polymers termed as the stereoregularity or tacticity (isotactic, syndiotactic, heterotactic, and atactic homopolymers). Further important difference in the architecture of macromolecules of the same chemical composition are the cis- and transstructures of the species containing double bonds in their main chain. Generally, the architecture of otherwise chemically identical macromolecules affects their size in solution and also their interactivity in interaction chromatography. Polymer may also exhibit large variability in their secondary molecular characteristics. For example, grafted or branched macromolecules can be composed of distinct statistical copolymers. Evidently, the overall possible number of chemically and physically different kinds of polymers is enormous but practical employment is found for only few of them. The molecular characteristics of synthetic polymers are not uniform in their value. All of them exhibit a distribution, a dispersity (Ð) which can be broad or narrow, unimodal, bi-, and polymodal, continuous or discrete, symmetric or distorted, etc. Theoretically, the shapes of distributions of the molecular characteristics of particular polymeric substance can be described by means of the distribution functions. This is however possible only for the simple polymers. The number of distinct macromolecules in a polymeric material may be enormous. Even a homopolymer with average molar mass of hundred thousand g/mol can contain thousands of species with distinct sizes. Advanced methods of mass spectrometry enable direct determination of real sizes (molar mass) of macromolecules and to calculate their actual molar mass distribution function only up to few ten-thousands g/mol. The individual discrimination of larger macromolecules is so far not possible. The complex

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polymers, such as the binary copolymers represent extremely complicated multicomponent mixtures of different macromolecules with distinct molar mass, overall composition and blockiness. The block copolymers exhibit several overlapping distributions simultaneously. It is evident that the synchronous quantitative description of distribution of all molecular characteristics of complex polymers is hardly possible. This is why the researchers focus only on the determination of average values and distributions of the molecular characteristics of polymers, which are most important from the point of view of their synthesis or application. The relative representation of distinct macromolecules in a particular polymer can be expressed in different ways. The macromolecules of same kind can be either counted or weighed. Correspondingly, the number- or weight fraction of identical macromolecules in the system is obtained and particular average (mean) values of molecular characteristics are calculated. Hence, the terms number- or weight-average of given molecular characteristic are originated. The most frequently measured molecular characteristic of homopolymers is their molar mass. The number average molar mass of a polymer sample is defined as Mn =

N i Mi = Pn Mo Ni

(2.1)

while for the weight average molar mass one can write Mw =

Wi Mi = Wi

N i Mi 2 = P w MO N i Mi

(2.2)

The difference between number and weight average values indicates width of the particular distribution. In the case of polymer molar mass, important and often employed parameter is the ratio of weight average molar mass Mw and number average molar mass Mn of the given polymer, Mw /Mn , which is called the polydispersity (or polymolecularity). Recently IUPAC recommended use of the term dispersity, ÐM for this [6]. Some other averages were proposed to express molar mass of macromolecules, for example the viscosity average, Mv or the z and z + 1 averages Mz and Mz+1 , respectively. The latter two averages are especially important for the polymers with very broad molar mass distribution. Unfortunately, average values of polymer molecular characteristics indicate little about the shape of the distribution function, about the type of the molar mass distribution. For example, the Mw /Mn values may be equal for polymers exhibiting both unimodal and multimodal distributions. The term degree of polymerization is used to define the average number of monomeric units joined together to form a polymer molecule. Pn represent the number average degree of polymerization while Pw represent the weight average degree of polymerization. Mo is the molecular weight of the repeat unit.

2.2 Solubility of Polymers

11

2.2 Solubility of Polymers Most methods of polymer molecular characterization employ measurements of their behavior in solution. Solubility and solution properties of macromolecules substantially differ from those of low molecular weight substances. Therefore, these issues deserve at least a brief and simplified explanation of basic concepts and also terms, which will be employed in this book. We will pay attention mainly to solution of linear macromolecules at atmospheric pressure. The old Latin proverb says: “similia similibis solventur” (similar dissolves similar). The well-known thermodynamic rule states that two substances of different chemical nature are mutually soluble if their mixing brings about a loss in the value of the Gibbs function, G, called also Gibbs free energy or free enthalpy—that is G < 0. The change of Gibbs function is connected with changes of further basic thermodynamic quantities namely enthalpy, H and entropy, S. The mutual dependence of enthalpy and entropy is described by a well-known equation G=

H −T

S

(2.3)

where T is temperature in Kelvin. The gain of the mixing entropy is high in the course of dissolution of the low molecular weight substances, the small molecules in a solvent. Therefore, the lowmolecular weight substances may dissolve even if the enthalpy of mixing is rather unfavorable. The situation is different with the macromolecules, when the contribution of mixing entropy due to dissolution is much lower. Therefore, enthalpic interactions between macromolecules and solvent molecules often decide about polymer (in)solubility. In order to dissolve macromolecules, the attractive interactions between polymer segments and solvent molecules must be at least equal or little larger than the mutual interactions between polymer segments themselves. It illustrate about thermodynamic quality of the solvent for a given polymer. The solvent quality can be poor, weak, medium or (very) good. The terms a (very) strong solvent or highly efficient solvent are sometime used for a thermodynamically good solvent. If the interaction between a polymer segment and liquid molecules is endothermic, it refers to a non-solvent (precipitant), which are also commonly designated weak or inefficient solvents rather than non-solvents. A thermodynamically good solvent exhibits high solvating power, which depends on the all-molecular characteristics of the polymer to be dissolved. To dissolve the crystalline polymers, the crystallites must be disrupted by solvent molecules possessing high solvating power or by their melting at elevated temperature as in case of polyolefins. Dissolved macromolecules can assume various conformations such as the rods, worms, globules or Gaussian statistical coils. The latter conformation is mostly encountered situation in the majority of methods of polymer characterization in solution under theta conditions (not in a good solvent) that is especially valid for IC. In SEC, the most favorable condition is a fully expanded coil in a thermodynamically good and chromatographically strong solvent. In IC, the most favorable condition is a thermodynamically

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good but chromatographically weak solvent. The shape of polymer coils in solution is usually slightly ellipsoidal but it is often approximated by the spherical one. The size of the coils of (linear) macromolecule with given molar mass in solution depends on the thermodynamic quality of the solvent. Polymer coils are expanded in a thermodynamically good solvent while shrunken in a thermodynamically poor solvent. To estimate the solubility of a given polymer and to select the appropriate solvent, the physical properties of the latter should be considered. The data are collected in various books [7–10]. The simplest practice is comparison of the polarity of the macromolecules with that of the solvent, expressed by their solubility parameters. Some other important factors in this context include dispersion force (polarizability), hydrogen bonding, etc. The obvious rule of “similar dissolves similar” is being followed. The quantitative expression of the solvent quality for a polymer is the famous Flory–Huggins polymer–solvent interaction parameter, χ , (also designated α) or the second virial coefficient, from the power series that expresses the concentration dependence of the colligative properties of polymer solutions such as vapor or osmotic pressure, light scattering, etc. Both χ and second virial coefficient values are tabulated for numerous polymer–solvent systems [7–10]. The thermodynamic quality of a solvent for given linear polymer also can be estimated from the Kuhn–Mark–Houwink–Sakurada viscosity law (often called Mark–Houwink equation) [η] = K M α

(2.4)

where [η] is the intrinsic viscosity (limiting viscosity number) of the linear homopolymer with molar mass M in a particular solvent while K and α are constants. The unitless exponent α represents polymer–solvent interaction of linear, flexible, coiled macromolecules. It assumes values between 0.65 and 0.80 while its increase indicates rising solvent quality. The value of 0.5 for α corresponds to a theta solvent. Numerous α values for different polymer–solvent systems are tabulated in various sources [7–10]. It is to be noted that Eq. 2.4 is generally valid only for linear polymers and its validity ceases below molar mass of about 10–15 kg/mol. Intrinsic viscosities of particular macromolecules of the same chemical nature in the same solvent reflect their physical structure such as linearity or branching. As mentioned, the deterioration of the solvent quality, the weakening of attractive interactions between the polymer segments and the solvent molecules brings about the reduction of the polymer coil size—up to the state when the interaction between polymer segments and solvent molecules is the same as the mutual interactions between the polymer segments, the situation is called theta state or theta conditions. Under theta conditions, the Flory– Huggins parameter χ assumes value 0.5, the virial coefficient is zero, and exponent α in the viscosity law is 0.5. Further deterioration of solvent quality brings about the collapse of the macromolecular coils, their merging into associates and eventually to either formation of concentrated phase—to phase separation—or to the formation of solid precipitate. The controlled phase separation of macromolecules can

2.2 Solubility of Polymers

13

be employed for generation of nanoparticles that may be suitable for some specific application. The processes taking place under atmospheric pressure will be considered in the further discussion. Temperature plays important role in polymer solubility as it influences interaction of the segments of macromolecules with solvent molecules [11–13]. Numerous polymer–solvent systems are not miscible over entire concentration scale at any temperature. For a given molar mass, the dependence of the miscible polymer—solvent composition on temperature is called coexistence curve. Usually, polymer solubility increases with rising temperature ( Hmix > 0, , i.e., endothermic) and the coexistence curve has a maximum, a critical point, called upper critical solution temperature, UCST. Over UCST polymer and solvent are miscible in any concentration. However, there do also exist systems, in which polymer solubility decreases with elevating temperature. They exhibit lower critical solution temperature, LCST. The solvent quality can be affected by the addition of further solvent component(s) including a non-solvent, referred as two- or multi-component mixed solvents. In most cases, polymer chains interact preferentially with one of the mixed solvent components, concentration of which increases in the domain of polymer coils, the phenomenon is called preferential solvation. It plays important role in molecular characterization of polymers by advanced methods of liquid chromatography. Surprisingly, sometimes the non-solvent molecules preferentially solvate dissolved macromolecules. This may happen in a certain range of the mixed solvent compositions and it likely results from the repulsive solvent—solvent interactions. It means that not only the extent but, in some cases, also the direction of preferential solvation may depend on the relative concentration of the solvent components [14]. The typical example is the system poly(methyl methacrylate) (PMMA) or poly(dimethyl siloxane) (PDMS)—benzene/methanol, in which macromolecules are preferentially solvated by their non-solvent methanol in the mixtures with a high benzene concentration [15, 16]. Some specific phenomena appear in case of mixed solvents in contact with polymers. A mixture of two non-solvents can become a (good) solvent for a polymer (co-solvency) [15] and vice versa, a mixture of two polymer solvents can behave as a non-solvent (co-non-solvency) [17]. All above occurrences have to be considered when working with polymer solutions. The charged macromolecules represent technologically important materials as the ionizable groups give them some specific properties. In solution of polyelectrolytes, the macromolecular chains carrying charged groups are either expanded or collapsed due to repulsive or attractive interactions between charges of the same or opposite nature, respectively. These phenomena, which are called polyelectrolyte effects, may bring about significant problems while working with solutions of charged polymers. Therefore, it is often attempted to suppress ionization of potentially charged groups in solution by adding the appropriate low-molecular counter-ions. The interactions of the ionizable groups with the counter-ions, bring about similar effects as preferential solvation of macromolecules dissolved in mixed solvents. The low-molecular ions present in polymer solution may also interact with the otherwise uncharged macromolecules in solution e.g., with poly(ethylene

14

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oxide)s (PEO)s. Pseudo-polyelectrolytes are formed, which may exhibit an unexpected behavior. The role of ion nature in the extent of their interaction with the noncharged macromolecules is so far not understood well. Large differences between interaction of distinct ions with natural and synthetic polymers have been observed (Hofmeister series) [18]. Since recently, the interactions are intensively studied between proteins dissolved in water and various kinds of ions. The presence of ions extensively affects the interface water–air and water–macromolecule. Similar phenomena may also take place in case of some synthetic polymers such as poly(Nisopropylacrylamide) dissolved in organic solvents [19–21]. The specific behaviors of ionized macromolecules are mentioned in this book only as a certain sort of caution.

2.3 Polymers in Solution in Contact with Porous Solid Particles In this Section, the behavior of dissolved macromolecules in contact with porous solid particles is outlined. The dynamics of polymer coils is very high. Therefore, the changes of coil conformation are much more rapid than any other process taking place in polymer solutions when in contact with porous solid particles. Hence, polymer coils in their apparent equilibrium (the average value of size) with the solvent molecules are always taken into account. The pores are large enough to accommodate all solvent molecules. On the other hand, only a fraction of macromolecules may fully fit in the pores. Two different volumes namely pore volume and interparticle (interstitial) volume, and two distinct processes are present in the system namely, (i) the entropy controlled selective pore permeation of macromolecules and (ii) the attractive enthalpic interaction of macromolecules with the inner and outer surface of the solid particles.

2.3.1 Entropy-Controlled Processes The pore permeation process, which theoretically does not include any attractive interactions between the dissolved macromolecules, a solute, and the porous particles, is often termed “ideal”, which means H in Eq. (2.3) is zero. The pore permeation and partial pore exclusion of macromolecules is called entropic partition [22–24]. Thermodynamic quality of solvent for macromolecules (Sect. 2.2) determines the polymer coil sizes that directly affects the pore permeation process. The basic force, which pulls dissolved macromolecules into the pore is the tendency to equalize polymer concentration, the chemical potential in the interstitial volume and the pore volume. During the pore permeation, a polymer chain in a pore simply loses some of its conformational degree of freedom, i.e. lower entropy state than in

2.3 Polymers in Solution in Contact with Porous Solid Particles

15

the interstitial space. Their conformational, mixing and possibly also orientational entropy decreases. In case of particular pore and polymer, the penalty of entropy of the macromolecules controls the extent of their pore permeation [25]. The pore permeation process is concluded when the loss of entropy of macromolecules outbalances the pulling force. The extent of pore permeation of macromolecules evidently depends on their size, as well as on the size of pores in the solid particles. As shown in Sect. 2.2, the sizes of synthetic macromolecules in a sample are never uniform, they exhibit a distribution. Similarly, also all pore sizes are not the same and the smallest pores may be inaccessible for any macromolecule. Very large macromolecules, which do not fit even into largest pores, are excluded from the porous particles. However, their long segments may diffuse into the pores. In real effect, the most static systems polymer–solvent-porous particles are far from the thermodynamic equilibrium. On the whole, pore permeation of macromolecules is a very complex process and its quantitative description even for simple, chemically homogeneous homopolymers is hardly possible.

2.3.2 Enthalpy-Affected Processes The enthalpy-affected processes, which accompany contacts of dissolved macromolecules with the surface of the porous solid particles, are often denoted enthalpic interactions. Several parameters are to be considered when analyzing enthalpic interaction of polymers with the solid surface. Besides the pore size and accessibility, the chemical composition of both macromolecules and surface, and also the nature of solvent molecules is important in this context. Consequently, several different kinds of enthalpic interactions can take place in the system. The overall situation may be very complex because “everything interacts with everything”. In order to simplify present qualitative discussion that is oriented to analytical practice rather than to theory, only selected binary interactions will be regarded namely polymer—solid surface, polymer—solvent (molecules), and solvent—solid surface. The polymer—polymer interactions in the sample solutions and the solvent—solvent interactions, which may play specific role in the two- or multi-component solvents will be mentioned only briefly—though in some cases their role may be quite significant. The contemporaneous ternary and quaternary interactions will be neglected. In practice, usually unwanted ionic interactions are suppressed in the practical analytical systems by addition of appropriate low molecular salts, the counterions (Sect. 2.2). The uncontrolled phase separation processes, that can be caused by the insolubility of some sample constituents in given solvent, are undesirable. Herein, only simple systems—solutions of homopolymers will be treated. Evidently, the interaction of macromolecules with inner pore surface is only possible for those polymer coils which enter the pores. Moreover, only the outer part of polymer coils is available for interactions with the walls of the pores. The interaction of macromolecules, which only partially enter the pores, is very important in the analytical procedures discussed in this book. The size of macromolecular coils

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with given molar mass in solution also reflects both the mutual interactions among polymer segments and their interaction with solvent molecules. The intra-molecular interactions depend on the topology of macromolecules and in the complex polymers also on their chemical structure and architecture. Moreover, all molecular characteristics of macromolecules exhibit a distribution. While the “ideal entropic partition” may be operative without any enthalpy contribution, the opposite situation does not exist. The entropic effects are always present in polymer chromatography. The only exception constitutes the short-chain oligomers, which may behave similarly as the low molecular weight substances and can hardly form the coiled structures.

2.3.2.1

Adsorption

According to IUPAC accepted terminology, the process of adsorption is the distribution of molecules of a substance, a sorbate between the volume of a gas or liquid phase and the surface of an (ad)sorbent. The solution of sorbate outside of adsorbent is called supernatant. The adsorption can also take place on the interface between two liquid phases, termed as the interfacial adsorption. Either the chemical bonds or the physical forces keep the sorbate molecules on the sorbent surface. Correspondingly, there can be chemisorption and physisorption. The latter one, which takes place on a solid sorbent from a solution of macromolecules is important for the polymer separation by means of liquid chromatography, treatment of which is the main task of this book. Evidently, the chemical composition of macromolecules, of solvent molecules, and of surface of sorbent jointly influence and determine the extent of adsorption. In the course of the adsorption process, the polymer segments compete for active sites on the sorbent surface with the solvent molecules. Polar sorbents such as bare silica gel are often applied in the analytical practice. Generally, the polar—polar interactions are stronger than the nonpolar London forces. Therefore, the adsorption of polymers of medium- to high-polarity on the polar surfaces is as a rule more intensive than the adsorption of non-polar macromolecules on the non-polar surfaces. However, in terms of H of the HPLC separation, NPLC and RPLC show comparable H. In the former case, the polarity of solvents also called solvent strength is most important. Strong solvents intensively interact with sorbent surface and they may fully prevent adsorption of macromolecules, designated as desorption promoting liquids (desorli). On the contrary, the less polar solvents that do not intensively interact with the sorbent surface, promote adsorption of macromolecules, termed as the weak solvents. If a solvent is very weak, it promotes the full adsorption of macromolecules up to the saturation of sorbent surface, called an adsorli. The thermodynamic quality of solvent for macromolecules usually less intensively affects the extent of adsorption. It is important to reiterate that the adsorption process of macromolecules is always accompanied with a large change of their mixing, conformational and possibly also orientation entropy. The solvent strength strongly influences the adsorption process of macromolecules.

2.3 Polymers in Solution in Contact with Porous Solid Particles

17

The dependence of concentration of sorbate molecules in solution and on the surface (or interface) of adsorbent at constant temperature is called adsorption isotherm. The adsorption isotherm for the low molecular substances usually starts from the origin while the phenomenon of the full adsorption is often observed for macromolecules. In this case, all macromolecules are situated on the sorbent surface and they appear in the supernatant only after sorbent surface is saturated. The full adsorption phenomenon can be employed in the separation of chemically different macromolecules [26]. A specific situation may appear in the system containing a weak solvent along with polar polymer and polar sorbent. Similarly, as in the entropy-controlled pore permeation, if the sorbent pores are small to accommodate entire macromolecules, their segments can be pulled into the pore. This effect is augmented by the attractive forces between the pore surface and the macromolecules. The rest of macromolecules stay outside the pores. A sort of “flower” is formed with a “stem” inside the pore and “crown” outside of it. Gorbunov and Skvortsov were first to describe this phenomenon and called it “flower-like” conformation of macromolecules [27]. The extent of adsorption also depends on temperature. Rising temperature, as a rule, decreases extent of adsorption of low-molecular substances. This is not the general case for macromolecules because their conformational entropy may strongly depend on temperature. At increased temperature, the gain in enthalpy due to adsorption may be outbalanced by the large loss of the conformational entropy. This results in the (not frequently observed) increase of adsorption with increasing temperature. A typical example is the adsorption of poly(methyl methacrylate)s (PMMA) and a poly(ethylene oxide) (PEO) on a bare silica column using toluene or tetrahydrofuran, respectively [26]. Adsorption of macromolecules may be also influenced by pressure [28]. This effect is especially pronounced when polymer is dissolved in the two-component solvents. As a rule, one solvent component from the mixture is preferentially located on the sorbent surface. The extent of preferential solvation of the sorbent surface varies at the low-pressure changes [29, 30]. As result, the pressure variation can affect the extent of sorption of macromolecules. The specific kind of systems represents the additives, which have deliberately or accidentally got into the system. If they exhibit high affinity for the sorbent, they displace original solvent molecules from its surface and affect the adsorption of macromolecules. In analytical practice, small amounts of the high polarity desorlis are often added to polymer solutions in order to suppress unwanted adsorption effects [31, 32]. In most sorbent–polymer–solvent systems, extent of adsorption of macromolecules within accessible pores increases with their molar mass. However, stiff macromolecules with lower molar mass may be preferentially adsorbed from their mixture with the chemically similar flexible polymer chains on the non-porous adsorbents. Adsorption and desorption of macromolecules are very fast processes though they may be strongly decelerated by their diffusion into porous structure of the sorbent. Complete trapping and release of macromolecules onto/from the surface

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of non-porous silica particles was accomplished within fragment of a second under dynamic conditions of the fast solvent flow [33]. Hence, adsorption of macromolecules on the surface of porous particles is a complex process and several aspects must be considered upon its application for the analytical purposes.

2.3.2.2

Enthalpic Partition (Absorption)

The term absorption refers to the distribution of a dissolved substance, a solute between (two) volumes of substances with distinct chemical nature. Surprisingly, the terms adsorption and absorption are often confused in literature though the principal difference of their backgrounds and nature is evident. In order to prevent such confusion, the term enthalpic partition is applied instead of absorption in the numerous papers and in this book. Evidently, large difference in polarity of solvents is necessary to form a twophase system and only few polymers are soluble in both components. Therefore, it is a technical problem to create practically applicable two-phase liquid systems. The slightly cross-linked, insoluble macromolecules swollen in a solvent form a soft gel, a sort of liquid-like phase. When such gel is equilibrated with a polymer solution, the macromolecules can penetrate into it and the enthalpic partition between gel phase and free solution, supernatant takes place. Enthalpic partition of macromolecules can be achieved when certain volume of appropriate liquid is deposited on the surface of solid porous particles. If the deposited liquid is kept on the carrier surface just by the physisorption it is called the stagnant phase. In contact with polymer solution, the stagnant phases are often physically unstable. The specific kinds of stagnant phases are generated by charged molecules, which are kept on the carrier by the ionic interactions [34, 35]. So far, they were not employed in polymer separations. Much more stable are stationary phases, which are formed with the low molecular substances attached to the carrier with chemical bonds. The most commonly employed stationary phases in analytical practice are linear C18 groups bonded to porous silica gel. The molecules of stationary phases are solvated by the surrounding solvent. Difference in solubility of polymer in stationary phase and in surrounding solvent decides about extent of its enthalpic partition. Macromolecules dissolved in a thermodynamically poor solvent may be pushed into the solvated stationary phase, which may be their better “solvent”. Temperature may strongly affect both the extent and course of enthalpic partition as it governs the solubility of the macromolecules in solvent and also in the solvated stationary phase. The situation is rather complex when two-component solvents are applied and the results depend on the preferential solvation of the system constituents. In this case, the indirect effect of pressure on the enthalpic partition of macromolecules may be even more pronounced than in case of adsorption. Enthalpic partition of macromolecules likely proceeds more slowly than the exclusion and adsorption processes.

References

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Thus, the process of enthalpic partition of macromolecules substantially differs from adsorption. Temperature plays more important role in partition compared to adsorption. Molar mass of macromolecules may strongly affect the outcome of enthalpic partition. For given polymer and stationary phase, the extent of the enthalpic partition is controlled primarily by the thermodynamic quality of the solvent toward the system constituents. In any case, the solvent strength must be high enough to suppress adsorption of macromolecules on the free surface of carrier, which supports the stationary phase. Herein, adsorption is an unwanted competitive process. On the whole, the processes of enthalpic partition are complicated and it is difficult to predict their outcome.

2.3.2.3

Interplay of Entropy and Enthalpy

The entropy-controlled permeation of polymers into porous solid particles can theoretically proceed without any participation of enthalpy. In practice, however, slight enthalpic effect can be hardly eliminated. Naturally, the enthalpic interactions can be deliberately appended into procedures, in which entropy continues to dominate. On the other hand, both adsorption and enthalpic partition of polymers are practically always accompanied with important changes of conformational entropy of macromolecular coils. The exceptions of this rule are just low oligomers. A specific situation appears, when the impacts of entropy and enthalpy mutually compensate and G in Eq. 2.3 of the resulting process becomes zero. This occurrence is called critical conditions and it plays important role in several methods of polymer molecular characterization. Major part of present book is devoted to critical conditions, explaining their theoretical backgrounds, practical significance, and practical applications.

References 1. Alger M (2017) Polymer science dictionary, 3rd edn. Springer, Dordrecht. https://doi.org/10. 1007/978-94-024-0893-5 2. AlMaadeed MAA, Carignano MA, Ponnamma D (eds) (2020) Polymer science and innovative applications. https://doi.org/10.1016/C2018-0-00918-4 3. Chanda M (2006) Introduction to polymer science and chemistry: a problem-solving approach, 1st edn. CRC Press. https://doi.org/10.1201/9781420007329 4. Staudinger H (1920) Über Polymerisation. Ber Dtsch Chem Ges 53(6):1073–1085. https://doi. org/10.1002/cber.19200530627 5. Freiburg B-W (1999) The foundation of polymer science By Hermann Staudinger (1881–1965). American Chemical Society 6. Stepto RFT (2009) Dispersity in polymer science (IUPAC Recommendations 2009). Pure Appl Chem 81(2):351–353. https://doi.org/10.1351/PAC-REC-08-05-02 7. Brandrup J, Immergut EH, Grulke EA, Abe A, Bloch DR (1989) Polymer handbook, vol 7. Wiley, New York 8. Mark JE (ed) (2007) Physical properties of polymers handbook. Springer, New York. https:// doi.org/10.1007/978-0-387-69002-5

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9. Wypych G (2012) Handbook of polymers. ChemTec Publishing. https://doi.org/10.1016/ C2011-0-04631-8 10. Brandrup J, Immergut EH, Grulke EA (eds) (2003) Polymer handbook, 4th edn. Wiley, New York 11. Sanchez IC (1978) Chapter 3—Statistical thermodynamics of polymer blends. In: Paul DR, Newman S (eds) Polymer blends. Academic, pp 115–139. https://doi.org/10.1016/B978-0-12546801-5.50009-8 12. Paul DR, Bucknall CB (2000) Polymer blends: formulation and performance, vol 1–2. Wiley, New York 13. Sanchez IC, Lacombe RH (1978) Statistical thermodynamics of polymer solutions. Macromolecules 11(6):1145–1156. https://doi.org/10.1021/ma60066a017 14. Miller-Chou BA, Koenig JL (2003) A review of polymer dissolution. Prog Polym Sci 28(8):1223–1270. https://doi.org/10.1016/S0079-6700(03)00045-5 15. González-Benito J, Koenig JL (2002) FTIR imaging of the dissolution of polymers. 4. Poly(methyl methacrylate) using a cosolvent mixture (Carbon Tetrachloride/Methanol). Macromolecules 35(19):7361–7367. https://doi.org/10.1021/ma020401u 16. Campos A, Borque L, Figueruelo JE (1977) Preferential solvation of poly(dimethylsiloxane) and poly(methyl methacrylate) in benzene-methanol mixtures by gel permeation chromatography. J Chromatogr A 140(3):219–227. https://doi.org/10.1016/S0021-9673(00)935 81-7 17. Zhang X, Zong J, Meng D (2020) A unified understanding of the cononsolvency of polymers in binary solvent mixtures. Soft Matt 16(33):7789–7796. https://doi.org/10.1039/D0SM00811G 18. Kang B, Tang H, Zhao Z, Song S (2020) Hofmeister series: insights of ion specificity from amphiphilic assembly and interface property. ACS Omega 5(12):6229–6239. https://doi.org/ 10.1021/acsomega.0c00237 19. Wang J, Satoh M (2009) Novel PVA-based polymers showing an anti-Hofmeister Series property. Polymer 50(15):3680–3685. https://doi.org/10.1016/j.polymer.2009.05.050 20. Moghaddam SZ, Thormann E (2019) The hofmeister series: specific ion effects in aqueous polymer solutions. J Colloid Interf Sci 555:615–635. https://doi.org/10.1016/j.jcis.2019.07.067 21. Schroffenegger M, Zirbs R, Kurzhals S, Reimhult E (2018) The role of chain molecular weight and hofmeister series ions in thermal aggregation of Poly(2-Isopropyl-2-Oxazoline) grafted nanoparticles. Polymers 10(4):451 22. Pang P, Koska J, Coad BR, Brooks DE, Haynes CA (2005) Entropic interaction chromatography: separating proteins on the basis of size using end-grafted polymer brushes. Biotechnol Bioengin 90(1):1–13. https://doi.org/10.1002/bit.20430 23. Berek D (2003) Adsorption and enthalpic partition retention mechanisms in liquid chromatography of non-charged synthetic polymers. Chromatographia 57(Suppl.):S/45-S/54. https://doi. org/10.1007/bf02492082 24. Berek D (2005) Adsorption and enthalpic partition in liquid chromatography of non-charged synthetic polymers, 3: Interphase adsorption versus enthalpic partition of polystyrene in alkanebonded silica gel. Macromol Chem Phys 206(19):1915–1927. https://doi.org/10.1002/macp. 200500234 25. Berek D (2001) Progress in the liquid chromatography of synthetic non-charged lipophilic macromolecules. Polimery 46(11–12):777–784 26. Simekova M, Berek D (2005) Studies on high-performance size-exclusion chromatography of synthetic polymers: I. Volume of silica gel column packing pores reduced by retained macromolecules. J Chromatogr A 1084(1–2):167–172 27. Gorbunov AA, Skvortsov AM (1995) Statistical properties of confined macromolecules. Adv Colloid Interface Sci 62(1):31–108 28. Ingham KC, Busby TF, Sahlestrom Y, Castino F (1980) Separation of macromolecules by ultrafiltration: influence of protein adsorption, protein-protein interactions, and concentration polarization. In: Cooper AR (ed) Ultrafiltration membranes and applications. Springer US, Boston, MA, pp 141–158. https://doi.org/10.1007/978-1-4613-3162-9_9

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29. Kristl A, Lukšiˇc M, Pompe M, Podgornik A (2020) Effect of pressure increase on macromolecules’ adsorption in ion exchange chromatography. Anal Chem 92(6):4527–4534. https:/ /doi.org/10.1021/acs.analchem.9b05729 30. Zhang J, Wei C, Zhao C, Zhang T, Lu G, Zou M (2021) Effects of nano-pore and macromolecule structure of coal samples on energy parameters variation during methane adsorption under different temperature and pressure. Fuel 289:119804. https://doi.org/10.1016/j.fuel.2020. 119804 31. Malik MI, Mahboob T, Ahmed S (2014) Characterization of poly(2-vinylpyridine)-blockpoly(methyl methacrylate) copolymers and blends of their homopolymers by liquid chromatography at critical conditions. Anal Bioanal Chem 406(25):6311–6317. https://doi.org/10.1007/ s00216-014-8075-2 32. Park S, Chang T (2006) Characterization of Poly(2-vinylpyridine) by temperature gradient interaction chromatography. Macromolecules 39(9):3466–3468. https://doi.org/10.1021/ma0 60326d 33. Li H, Chen X, Shen D, Wu F, Pleixats R, Pan J (2021) Functionalized silica nanoparticles: classification, synthetic approaches and recent advances in adsorption applications. Nanoscale 13(38):15998–16016. https://doi.org/10.1039/D1NR04048K 34. Tallarek U, Vergeldt FJ, As HV (1999) Stagnant mobile phase mass transfer in chromatographic media: intraparticle diffusion and exchange kinetics. J Phys Chem B 103(36):7654–7664. https:/ /doi.org/10.1021/jp990828b 35. Pak AJ, Hwang GS (2016) Charging rate dependence of ion migration and stagnation in ionicliquid-filled carbon nanopores. J Phys Chem C 120(43):24560–24567. https://doi.org/10.1021/ acs.jpcc.6b06637

Chapter 3

Liquid Chromatography as a Tool for Determination of Distribution of Polymer Molecular Characteristics

As stated in the previous chapters, together with mutual arrangement of macromolecules in solid state and presence of additives, molecular characteristics of polymers determine their utility properties. These are molar mass, chemical structure and physical architecture. Naturally, molecular characteristics of polymers deserve considerable attention of polymer synthesists, analysts, and users. Molecular characteristics of polymers can be determined in solid state, in solution, and in few cases also after their pyrolysis within gas phase. In accordance with the focus of this book, we will deal only with polymer solutions. As repeatedly claimed in previous Chapters, synthetic polymers are not uniform substances. Their molecular characteristics exhibit dispersity, which affects the applicability of the materials made of them. Static methods of determination of polymer molecular characteristics mostly provide their average values. In order to assess their distribution, polymers must be separated. This book is devoted to the methods of polymer separation. Along this book, we will pay tribute to scientists, who contributed to progress in this area of research, but the extent of present book does not enable comprehensive excursions into history. We will not parse the non-chromatographic approaches such as separation of macromolecules with the help of Ultracentrifugation, introduced by T. Svedberg in 1923, which is still mainly applied for separation of biopolymers and nanoparticles [1]. Similarly, we will omit the detailed treatment of the countercurrent separation method introduced by Merle and Longtin in 1938 [2], which are also applied for polymer separation by Ito [3]. The dependence of solubility of macromolecules on their molecular characteristics was employed for the separation purposes already in the initial stages of polymer research. The most common is fractionation of polymers applying successive precipitation from their dilute solutions. A non-solvent was gradually added to the solution of polymers. Alternatively temperature of the solution was changed, usually decreased. Phase separation was evoked to form small volumes of concentrated solutions of sample fraction or solid particles of precipitate containing macromolecules with the highest molar mass. Next, the properties, for example molar mass of thus © The Author(s), under exclusive license to Springer Nature Switzerland AG 2023 M. I. Malik and D. Berek, Liquid Chromatography of Synthetic Polymers, Physical Chemistry in Action, https://doi.org/10.1007/978-3-031-34835-8_3

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3 Liquid Chromatography as a Tool for Determination of Distribution …

obtained fractions were determined one-by-one for example by viscometry, osmometry, or light scattering measurements. The procedures were laborious, complicated and slow. Sample consumption was large. Fractional precipitation is older and more crude method which is still employed for preprative fractionation of polymers. The attempts to accelerate and automate the fractionation process lead to the development of the dynamic separation systems. The separation methods were optimized by applying the columns filled with small particles, the column packings (stationary phase). The columns were connected with pumping systems to achieve fast and constant flow of the appropriate liquid, which transport macromolecules along the packing. In this context, the original polymer separation procedure was developed by Baker and Williams [4, 5]. It included the fractional elution of polymer deposited as a thin film on the non-porous (glass) particles by applying temperature gradient. In the course of further development, devices were introduced, which continuously monitored the separated sample fractions in the column effluent by the detectors. Volume of eluate was measured by the siphon systems. Hence, methods of chromatographic separation of macromolecules were born. We will concentrate on the procedures of polymer liquid chromatography, for short polymer LC and related methods. Classical chromatographic polymer fractionation methods were reviewed in numerous review papers and books, for example [6–9]. This chapter is intended to provide basic concept and a brief theoretical background of polymer HPLC.

3.1 Separation Mechanisms in Polymer LC Various phenomena are employed to determine distributions of molecular characteristics of macromolecules. They constitute separation mechanisms of polymer LC. The term retention mechanism is often used in high performance liquid chromatography, HPLC of low-molecular substances. It indicates their separation principles, according to which the elution rates of separated sample components along the column are decelerated, retained to a distinct extent. In polymer LC, especially in case of exclusion-based separations, the elution of macromolecules can be faster or at least equal compared with the velocity of the mobile phase (eluent). Therefore, the term retention is less appropriate for polymer LC. However, it is customary to consider that peak elution time is equivalent to its retention time. Similar to previous Chapters, we will first explain the basic terms to meet the needs of readers not familiar with the overall problematics. The solubility of macromolecules and behavior of their solutions and the peculiarities of polymer solution in contact with porous particles are discussed in Chap. 2. The processes of permeation of macromolecules into pores leading to their entropic partition, as well as to their enthalpic interactions, either adsorption with the pore surface or absorption called enthalpic partition with the deposited stationary phase

3.1 Separation Mechanisms in Polymer LC

25

(SP). Above occurences are the rudiments for various polymer LC separation procedures. As an exception, entropy driven absortion (while endothermic) is possible too—e.g. hydrophobic interaction. The direct interactions of macromolecules with molecules of transporting liquid, mobile phase (MP) (eluent), their precipitation, as well as their response to its flow including its combination with the specific, usually vertical field are further alternatives to achieve polymer separation. The polymer separation procedures, which are solely based on the entropic partition of macromolecules are often called exclusion polymer LC and these, in which also enthalpic interaction of macromolecules plays important role are named interaction polymer LC. One should keep in mind that in most interaction polymer LC the exclusion processes are necessarily, though unintentionally involved. There is also a group of polymer LC methods, in which entropic exclusion is deliberately combined with enthalpic interaction. For this instance, the term coupled polymer LC is used in literature. To the latter groups of methods also belong the advanced polymer separation approaches, which employ the mutual compensation of entropy and enthalpy contribution. As repeatedly stressed, present book is focused just to the entropy/enthalpy compensation phenomenon, the critical conditions, and on the polymer LC methods, in which the critical conditions play a role. We will apply the commonly utilized terms. – – – –

interaction polymer LC exclusion polymer LC coupled polymer LC critical conditions.

For completeness, let us enumerate the most important separation mechanisms employed in polymer LC using constant (isocratic) or changing (gradient) mobile phase composition within a column or a channel. These are isothermal or temperature affected entropic, enthalpic or flow processes, which are caused by, – contact of macromolecules with molecules of mobile phase (eluent) causing their precipitation, (re)dissolution or just changes of the patterns of their transport – contact of macromolecules with the porous particles resulting in their adsorption – contact of macromolecules with the stationary phase situated on the surface of a carrier particles rendering their enthalpic partition – contact of macromolecules with porous particles, which acts only as a barrier controlling equilibration of chemical potential in system and brings about entropic partition of macromolecules, their partial exclusion – combination of the mobile phase flow patterns with the specific field applied vertically on the transported macromolecules – combination of two and more above effects, especially important is already mentioned mutual compensation of entropic and enthalpic separation mechanisms, which gives rise to cancellation of their manifestation. To stress again, the detailed description of this phenomenon and its practical utilizations is the essential task of the present book.

26

3 Liquid Chromatography as a Tool for Determination of Distribution …

In any chromatographic system, the selective distribution of analyte molecules between MP and SP makes the basis of separation, as described by Ve = Vi + V p K d

(3.1)

where Ve represents the elution (retention) volume of the analyte, Vi the interstitial volume of the column, Vp the pore volume of the packing, and Kd the distribution coefficient. The ratio of the analyte concentration in the MP and the SP is given by the distribution coefficient. A relation of Kd to the Gibbs free energy G is given below, value depends upon the partitioning of the analyte between interstitial and pore volume [8]. G◦ =

H◦ − T

S ◦ = −RT ln kd

(3.2)

The van’t Hoff plot (logarithmic plot) of the distribution coefficient render the determination of the contributions the entropic ( S) and enthalpic ( H) changes in the distribution coefficient: ln K d =

S H − R RT

(3.3)

Limited dimensions of macromolecules in the pores of the SP render a decrease in their conformational entropy while adsorptive interactions of macromolecules with the SP bring about changes in the enthalpy. However, it is pertinent to mention that adsorption is never a two-component interaction since SP and macromolecules are saturated with solvent molecules. If mixed solvent system is used, preferential sorption may be there too. Adsorption of macromolecules replaces the already-adsorbed solvent molecules (that may be in different composition from the bulk solvent) and the released solvent should mix with the bulk solvent. These will bring about enthalpy and entropy changes also. The changes in both entropy and enthalpy coactively contribute in the overall changes in the Gibbs free energy. As mentioned earlier, separation in SEC is based on hydrodynamic volume of the polymer species in their dilute solution. The characteristic pore size distribution of a SEC SP render variations in the access of the polymers to the pores depending on their hydrodynamic sizes. The polymer molecules having large size cannot enter the pores and elute at the interstitial volume (Vi ) while polymer molecule of the size of the MP molecules have full access to the pores and elute at the void volume. Void volume is the sum of interstitial and pore volume (Vo = Vi + Vp ). Hence, the value of distribution coefficient in case of SEC is in the range of 0–1, i.e., 0 < KSEC < 1. As already mentioned, ideal SEC behavior is only achieved when the value of distribution coefficient is totally dependent upon the changes in the entropy without any involvement of enthalpic interactions, which is however not possible (or at least very difficult to achieve) in the real world. On the same lines, the value of distribution coefficient in polymer IC also depends upon co-active entropy and enthalpy effects. Hence, the separation modes in polymer LC depends upon the dominant factor of

3.2 Theory of Polymer Chromatography

27

either entropy or enthalpy. The dominance of entropic interactions ( G > 0 since S < 0) renders size exclusion of polymeric species that corresponds to the negative G. On the contrary, enthalpic interactions dominate in case of IC ( G < 0 since H > 0) that corresponds to positive G value. The compensation of enthalpic and entropic effects ( H = T S) corresponds to value of G equal to zero. The mode of polymer LC operative at zero value of G is termed as liquid chromatography at critical conditions (LCCC) and this specific point is termed as chromatographic critical point (CCP). At the CCP of a polymer in a certain MP composition on a SP at a given temperature, G and Kd assumes a values of zero and one, respectively. This is effectively a narrow range of MP composition and temperature where polymers elute independent of their molar mass near the void volume of the column. Slight changes in MP composition and/or temperature shift the elution to either SEC or IC regime. The dependence of separation modes of polymers on the MP is noticed for the first time by Belenkii et al. [10] and Tennikov et al. [11]. In SEC mode of polymer separation, high molar mass polymers elute earlier than the low molar mass polymers before the void volume of the column. The elution order in IC is reversed compared to SEC i.e., low molar mass polymers elute early compared to high molar mass polymers. Moreover, the elution of polymers take place after the void volume of the column. At the transition point of SEC and IC, polymers elute independent of their molar mass near the void volume of the column.

3.2 Theory of Polymer Chromatography Classical concept of distribution coefficient is valid for the description of retention of an analyte on the SP in HPLC. K =

Ve − Vi Vp

(3.4)

The value of distribution coefficient is the determining factor of the operative mode of LC of polymers. Distribution coefficient may assume different values in different modes of LC of polymers. A more convenient term the interaction parameter, c is introduced by de Gennes in this context [12, 13]. Similar to distribution coefficient, interaction parameter also depends on the composition of MP and temperature for any given polymer-SP combination, having unit of inverse length (nm−1 ).

3.2.1 Size Exclusion Chromatography (SEC) The distribution coefficient ‘Kd ’ assumes value between zero and one (0 < K < 1) while interaction parameters ‘c’ assumes negative value in SEC mode of polymer LC.

28

3 Liquid Chromatography as a Tool for Determination of Distribution …

Movement of differently sized polymers through chromatographic column induce entropy changes owing to their different access to the pores of SP. In an ideal case, elution volume in a SEC separation is given by [14] 4 R VS EC = Vi + V p 1 − √ π D

(3.5)

where D represent the pore diameter of the SP while R is the radius of gyration of the analyte. The radius of gyration is generally expressed in terms of length and number of the repeat units R=a

n 6

(3.6)

where n represents the number of the repeat units while a is length. In principle, SEC separations are independent of composition and functionality while depending on molecular dimensions (hydrodynamic size in dilute solution) of the polymers.

3.2.2 Interaction Chromatography (IC) or Liquid Adsorption Chromatography (LAC) As mentioned earlier, the strength of the interaction of the analyte with the SP is the major driving force of separation in IC. In this case, Kd assumes values more than one (k > 1) while ‘c’ has positive values. Martin’s rule is often employed to describe the retention in IC [15, 16]. ln k = A + Bn

(3.7)

The dimensionless factor ‘k’ in the above equation is based on solute elution volume and hold-up volume of the column. The hold-up volume is treated different from the widely used term “void volume” [17]. The theory explaining the interaction of flexible homopolymer with SP is developed by Gorbunov and Skvortsov [18, 19]. Accordingly, the elution of a polymer chain without any functional groups follows following equation, 4 R Ve = Vi + V p 1 − √ π D

−

2V p 2V p + exp(c R)2 [1 + er f (c R)] cD cD

(3.8)

wherein D, R, c, and erf(cR) represent the pore diameter of the SP, the radius of gyration of the macromolecule, the interaction parameter, and the error function,

3.2 Theory of Polymer Chromatography

29

respectively. Herein, the value of c is independent of D, Vp , Vi or R (i.e., the degree of polymerization n). Moreover, at sufficiently strong interaction the value of erf(cR) approaches unity. By taking it into account, Eq. (3.8) can be rewritten as 4 R Ve = Vi + V p 1 − √ π D

−

2V p 2V p + exp(c R)2 cD cD

(3.9)

Or Ve = Vo∗ +

4V p c2 a 2 exp η cD 6

(3.10)

wherein the accessible volume for the polymer chain is represented by V0∗ . Vo∗ = Vi + V p 1 −

4R √ D π

−

2V p 2V p = VS E C − cD cD

(3.11)

The accessible volume is an important term here, which is represented by the intercept of the plot of elution volume of oligomers (Vn ) versus difference in the elution volume of consecutive oligomers ( Vn = Vn − Vn−1 ). The accessible volume obviously has smaller value than the void volume. Vn = Vo∗ + γ Vn

(3.12)

The slope of the plot γ = e B / e B − 1 can be used to calculate the interaction parameter c, wherein the slope in the Martin’s rule is B = c2 a 2 /6 (see Eq. 3.7) c=

γ 1 6 ln a γ −1

(3.13)

At a sufficiently strong interaction, determination of the accessible volume and the interaction parameter does not require the individual peak number (n). In this context, a software is developed by Gorbunov et al. for determination of interaction parameters in all modes of polymer LC [20]. A modified form of Eq. 3.10 in terms of the retention factor k* is k∗

4V p Ve − Vo∗ c2 a 2 = exp n Vo∗ cDVo∗ 6

(3.14)

The Martin’s rule corresponds to the logarithmic form of the equation. ln k ∗ = ln

4V p cDVo∗

+n

c2 a 2 6

(3.15)

30

3 Liquid Chromatography as a Tool for Determination of Distribution …

A modified form of the Martin’s rule for non-functional chains is given as ln k ∗ = ln

2S p (ca)2 + n cVo∗ 6

(3.16)

However, for determination of the pore surface from the Martin’s plot peak identification is required. For mono-functional chains, an additional parameter ‘q’ is required [21] which corresponds to the difference in the free energy of adsorption of the end-group and the repeat unit [22]. Determination method of ‘q’ is given in ref. [23]. The retention of mono-functional chains carrying an adsorbing end-group in IC regime of polymer LC is given by ln km∗ = ln

c2 a 2 4 Vp + n + qc) (1 ∗ cD Vo,m 6

(3.17)

∗ where V0,m represents the accessible volume. The accessible volume for monofunctional chains is smaller than the non-functional chains. ∗ Vo,m = Vo∗ −

Vp 2 q √ D π Rc

(3.18)

Increase in number of repeat units render an exponential increase in the value of ‘k’. The slope of plots remains same for the plot of lnk vs n, however, intercepts are different. The separation of oligomers of non-functional and mono-functional polymers is possible by IC. However, larger difference in the interaction strength of the end-group from that of repeat unit render poor resolution of individual oligomers.

3.2.3 Liquid Chromatography at Critical Conditions As iterated earlier, SEC is an entropy-dominated process while separation in IC is dominated by enthalpic effect. At the intersection of these two situations, exists a point where enthalpic effect and entropic effect compensate and cancel each other resulting in zero and one values for the interaction parameter and the distribution coefficient, in the same order. This so-called transition point is termed as chromatographic critical point (CCP). At this very point of a polymer, a combination of polymerSP-MP-temperature, molar mass independent elution of non-functional polymer is obtained near the void volume of the column. In case of functional polymer, however, interaction strength of the end-group has to be taken into account. Molar mass independent elution of mono-functional polymers is still possible at the CCP of the repeat

3.2 Theory of Polymer Chromatography

31

unit with an increased elution volume depending upon the interaction strength of the end-group [22, 24]. Va ≈ Vi + V p (1 − qa ) ≈ Vo + qa V p

(3.19)

However, the elution behavior is not same for di-functional chains [22, 25]. If the end-groups at both ends of the polymer chains of a di-functional polymer are same then, q2 D Vaa ≈ Vi + V p 1 + 2qa + √a π R

(3.20)

The influence of the radius of gyration ‘R’ of the critical polymer chain is indicated. The same relation holds for the asymmetrical di-functional polymer chains (polymer chains with different end-groups at both ends), qaqb D Vab ≈ Vi + V p 1 + qa + qb + √ π R

(3.21)

where ‘a’ and ‘b’ are end-groups The elution volume of di-functional chains at the CCP of polymer chain depends on the contribution of the end-groups similar to mono-functional polymers. It also contains an additional contribution from the ratio of the pore diameter of the SP and radius of gyration R of the polymer molecules [22, 25]. Consequently, order of elution follows SEC for di-functional polymer chains having adsorbing end-groups at the CCP of the polymer chain. Another peculiar situation is when polymer chain elutes in SEC order while endgroup have stronger enthalpic interaction with the SP. The term liquid exclusion adsorption chromatography (LEAC) is coined for such situation [26]. Obviously, the positive interaction parameter ‘cB ’ of the end-group and negative ‘c’ for the repeat unit act oppositely in context of elution behavior. The combined effect results in elution of mono-functional polymers in SEC order but beyond the void volume of the column. The combination renders oligomeric separation of mono-functional polymers in decreasing order of number of repeat units (SEC order) but after the void volume of the column [26–28]. The elution volume VAB of short mono-functional polymer chains under LEAC conditions is given by √ V AB ≈ VB 1 −

π CB RA 2

√ = VB 1 − C n A

where ‘A’ is the repeat unit while ‘B’ is the end group

(3.22)

32

3 Liquid Chromatography as a Tool for Determination of Distribution …

LEAC has proved to be an excellent technique for oligomer separation of monofunctional polymer chains containing 10–15 repeat units. The isocratic separation in LEAC allows employment of a RI detector for accurate quantification [27].

3.3 Thermodynamics of Polymer Chromatography Gibbs free energy changes in a chromatographic process are the determining factor for the value of the distribution coefficient. The well-known Eqs. (3.2) and (3.3) show that Gibbs free energy is the function of oppositely occurring entropy and enthalpy effects. Entropy changes dominate in SEC while enthalpic change dominates in IC regime. As repeatedly stressed, the complete avoidance of any of them in polymer LC is not possible. The compensation of both entropic and enthalpic effects render critical mode of polymer LC, G = 0, as H = T S. Absence of any enthalpic interaction in “ideal SEC” render insignificant temperature dependence of the elution volume. Opposite is the case for IC, enthalpic interactions are the major driving force of the separation. Hence, retention in polymer IC depends on both entropy and enthalpy changes during the chromatographic process. A balance of entropy and enthalpy changes render critical conditions. Therefore, retention in IC depends on MP composition and temperature, though MP composition have more pronounced effect compared to the changes in temperature. The van’t Hoff plot allows determination of changes in entropy and enthalpy, ln K vs 1/T. While the thermodynamic parameters can be determined by various approaches that primarily differ in the calculation of the distribution coefficient [29, 30]. The distribution coefficient K is directly proportional to the retention factor k = (Ve − V0 )/V0 Hence, ln k = −

S◦ H◦ + + ln ϕ RT R

(3.23)

wherein generally unknown MP and SP ratio is represented by term ϕ, principally indicating the pore volume and interstitial volume. Hence, the thermodynamic paramS∗ R = S ◦ R + ln ϕ represent the slope and intercept eters H ◦ R and in a plot of ln K vs 1/T. Nonetheless, direct correlation between the distribution coefficient K and the retention factor k is not clear from the Eqs. 3.24 and 3.25. Ve − Vo Vo

(3.24)

V e − Vi Ve − Vi = Vp Vo − Vi

(3.25)

k= K =

References

33

Incorporation of the above equations render following relationship between the distribution coefficient K and the retention factor k k = (K − 1)

Vp Vo

(3.26)

Hence, no direct proportionality is found between K and k. However, for a K 1 the relation can be approximated as K = k · φ. The determination of characteristic volumes Vi , Vp and V0 are also required. Moreover, void volume determination is not a trivial task. The definitions of the void volume, the dead volume, and the hold-up volume may be situation dependent [17, 31–33], and their determination methods are elaborated in several articles [17, 34]. Gravimetric determination of the total amount of solvent in a column is usually taken as the void volume while elution volume is considered as the hold-up volume. The elution volume of a completely excluded polymer from the pores of SP is taken as the interstitial volume, determined by inverse SEC. It is pertinent to mention here that dissimilar values for the same column may be obtained in different MP (eluents).

References 1. Walter M, Lars B (2006) Analytical ultracentrifugation of polymers and nanoparticles. Springer, Heidelberg. https://doi.org/10.1007/b137083 2. Randall M, Longtin B (1938) Separation processes general method of analysis. Ind Eng Chem 30(9):1063–1067. https://doi.org/10.1021/ie50345a028 3. Ito Y (2020) Two-phase motion in hydrodynamic counter-current chromatography. Curr Chromatogr 7(2):76–81. https://doi.org/10.2174/2213240606666190912161221 4. Williams RJP (1952) Gradient elution analysis. Analyst 77(921):905–914. https://doi.org/10. 1039/AN9527700905 5. Baker CA, Williams RJP (1956) 456. A new chromatographic procedure and its application to high polymers. J Chem Soc 2352–2362. https://doi.org/10.1039/JR9560002352 6. Glöckner G (1982) Polymercharakterisierung durch Flüssigkeitschromatographie. Hüthig 7. Francuskiewicz F (1994) Polymer Fractionation. Springer, Heidelberg. https://doi.org/10.1007/ 978-3-642-78704-1 8. Glöckner G (1991) Gradient HPLC of copolymers and chromatographic cross-fractionation. Springer, New York 9. Glöckner G (2012) Gradient HPLC of copolymers and chromatographic cross-fractionation. Springer Science & Business Media, New York 10. Belenky BG, Gankina ES, Tennikov MB, Vilenchik LZ (1978) Fundamental aspects of adsorption chromatography of polymers and their experimental verification by thin-layer chromatography. J Chromatogr A 147:99–110. https://doi.org/10.1016/S0021-9673(00)851 21-3 11. Tennikov MB, Nefedov PP, Lazareva MA, Frenkel SJ (1977) Vysokomolekulyarnye Soedineniya. Seriya A 19:657 12. de Gennes PG (1969) Some conformation problems for long macromolecules. Rep Prog Phys 32(1):187–205 13. Gorbunov AA, Skvortsov AM (1995) Statistical properties of confined macromolecules. Adv Colloid Interface Sci 62(1):31–108

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14. Casassa EF, Tagami Y (1969) An equilibrium theory for exclusion chromatography of branched and linear polymer chains. Macromolecules 2(1):14–26 15. Tchapla A, Colin H, Guiochon G (1984) Linearity of homologous series retention plots in reversed-phase liquid chromatography. Anal Chem 56:621–625 16. Tchapla A, Heron S, Lesellier E, Colin H (1993) General view of molecular interaction mechanisms in reversed-phase liquid chromatography. J Chromatogr A 656(1–2):81–112 17. Rimmer CA, Simmons CR, Dorsey JG (2002) The measurement and meaning of void volumes in reversed-phase liquid chromatography. J Chromatogr A 965(1–2):219–232 18. Gorbunov AA, Skvortsov AM (1986) Theory of behavior of flexible polymers in limited volumes. Vysokomol Soedin, Ser A 28(10):2170–2176 19. Skvortsov AM, Gorbunov AA (1986) Adsorption effects in the chromatography of polymers. J Chromatogr 358(1):77–83 20. Gorbunov AA, Vakhrushev AV, Trathnigg B (2009) Calibration of chromatographic systems for quantitative prediction of chromatography of homopolymers. J Chromatogr A 1216(51):8883– 8890. https://doi.org/10.1016/j.chroma.2009.10.038 21. Gorbunov AA, Vakhrushev AV (2004) Theory of chromatography of linear and cyclic polymers with functional groups. Polymer 45(21):7303–7315 22. Gorbunov A, Trathnigg B (2002) Theory of liquid chromatography of mono- and difunctional macromolecules—I. Studies in the critical interaction mode. J Chromatogr A 955(1):9–17 23. Nguyen VC, Trathnigg B (2010) Determination of thermodynamic parameters in reversed phase chromatography for polyethylene glycols and their methyl ethers in different mobile phases. J Sep Sci 33(4–5):464–474. https://doi.org/10.1002/jssc.200900638 24. Gorbunov AA, Skvortsov AM, Trathnigg B, Kollroser M, Parth M (1998) Reversed-phase highperformance liquid-chromatography of polyethers—comparison with a theory for flexiblechain macromolecules. J Chromatogr A 798(1–2):187–201 25. Rappel C, Trathnigg B, Gorbunov A (2003) Liquid chromatography of polyethylene glycol mono- and diesters: functional macromolecules or block copolymers. J Chromatogr A 984:29– 43 26. Trathnigg B, Gorbunov AA (2001) Liquid exclusion-adsorption chromatography: new technique for isocratic separation of nonionic surfactants I. Retention behaviour of fatty alcohol ethoxylates. J Chromatogr A 910 (2):207–216 27. Trathnigg B (2001) Liquid exclusion-adsorption chromatography, a new technique for isocratic separation of nonionic surfactants II. Quantitation in the analysis of fatty alcohol ethoxylates. J Chromatogr A 915(1–2):155–166 28. Trathnigg B, Kollroser M, Rappel C (2001) Liquid exclusion adsorption chromatography, a new technique for isocratic separation of nonionic surfactants; III. Two-dimensional separation of fatty alcohol ethoxylates. J Chromatogr A 922(1–2):193–205 29. Trathnigg B, Nguyen Viet C, Ahmed H (2009) A data base for polymer chromatography: Temperature dependence of interaction parameters in liquid adsorption chromatography. J Sep Sci 32(17):2857–2863 30. Kim Y, Ahn S, Chang T (2009) Martin’s rule for high-performance liquid chromatography retention of polystyrene oligomers. Anal Chem 81(14):5902–5909 31. Gritti F, Kazakevich Y, Guiochon G (2007) Measurement of hold-up volumes in reverse-phase liquid chromatography: Definition and comparison between static and dynamic methods. J ChromatogrA 1161(1–2):157–169 32. Wang M, Mallette J, Parcher JF (2008) Strategies for the determination of the volume and composition of the stationary phase in reversed-phase liquid chromatography. J Chromatogr A 1190(1–2):1–7 33. Oumada FZ, Roses M, Bosch E (2000) Inorganic salts as hold-up time markers in C-18 columns. Talanta 53(3):667–677 34. Trathnigg B, Veronik M, Gorbunov A (2006) Looking inside the pores of a chromatographic column. J ChromatogrA 1104(1–2):238–244

Chapter 4

Instrumentation for Polymer Liquid Chromatography

Due to dissimilar nature of separation for different modes of polymer LC, several additional challenges are posed compared to HPLC of small molecules. These challenges can be addressed by modern state-of-the-art HPLC instrumentation. Polymer HPLC require a reliable pump (isocratic or gradient), stable temperature conditions through an oven, and a reliable set of detectors. Any HPLC instrument has a solvent delivery system, a sample injector, a detection system, and a data acquisition system along with the columns which contain the SP of different nature that makes the basis of any separation. Generally, the polymer LC instruments are used as delivered and their choice depends on the separation problem and the budget. Their list can be found in the yearly edition of LC.GC Magazine. Therefore, we will only briefly overview the LC hardware and attention will be paid to the components, which are chosen by the operator. These are column packings, mobile phases/eluents, and detectors. In case of polyolefins, all the system has to be at high temperature (>140 °C). While any advanced HPLC system can be used for ambient temperature applications in polymer analysis, applications of HPLC to high temperature have just been reported recently. HT-SEC has been used for molar mass of polyolefins for about half century [1, 2]. The applications of HT-IC to analysis of polylolefins is a recent development pioneered by Macko and Pasch [3, 4]. The first commercial HT-HPLC instrument was introduced by Polymer Laboratories, Ltd. (England) (now a part of Agilent Inc.) [5] in collaboration with Pasch and Macko [6].

4.1 Solvent Delivery System and Injector A reliable, reproducible, and constant flow of eluent is the most important requirement of any HPLC system. In the process of development, several types of pumps have been used whose working principles are different. For instance, syringe pump functions like a syringe and produces pulseless flow. The reciprocating pumps are © The Author(s), under exclusive license to Springer Nature Switzerland AG 2023 M. I. Malik and D. Berek, Liquid Chromatography of Synthetic Polymers, Physical Chemistry in Action, https://doi.org/10.1007/978-3-031-34835-8_4

35

36

4 Instrumentation for Polymer Liquid Chromatography

now widely used that are available in various modifications such as single piston, and dual piston (parallel or in series). In principle, pump for HPLC should have precise flow rate (0.2%), a pressure output of 6,000 psi, less than 1% pressure pulsation at typical flow rate (1 mL/min), flow rate range of 0.01–10 mL/min, small hold-up volume, and should be made of chemically resistant materials. Modern instruments are equipped with in-line degassing system. Syringe pumps are suitable for SEC or LCCC owing to their stable flow and minimized evaporation of the MP. The preferred choice for gradient elution is reciprocating pumps that allow efficient low-pressure mixing. One of the avoidable reasons of band broadening in HPLC is a broad band of injection. Sample injection valves are typically composed of two-piston and 6-ports that may be operated manually or automatically. A completely filled sample loop ensures the precise measurement. The injection volume and in-turn the size of the injection loop is decided based on dimensions of the column, detector sensitivity, and separation mode. Dilute sample solutions in larger loops (50–100 µL) are preferred over concentrated sample solutions in smaller loops for SEC. Conversely, IC and LCCC preferably require higher concentrations in smaller loops (10–50 µL) compared to dilute concentrations in larger loops. Modern HPLC equipment have autosamplers that can inject any volume in a range of 0–2000 µL with a precision of ±0.5%, without any intervention of operator. The state-of-the-art autosamplers have built-in sample filtration, variable speed and temperature mixing, and needle wash etc. Especially important is the autosamplers for HT-SEC and HT-HPLC that require stirring at high temperatures of 150–180 °C for 1–6 h [7–9]. To avoid thermo-oxidative degradation of polyolefins at high temperatures 0.2–1.5 mg/mL of phenolic antioxidants such as butylated hydroxytoluene (BHT) is added [9].

4.2 Columns The separations of dissolved polymers usually take place in the straight cylindrical tubes called columns. Sometimes also rectangular channels are employed. The attempts to apply twisted and conical columns were not successful. The size of columns and channels depends on the particular separation method under use and may have a diameter of one to tens of millimeter, between four and ten mm is most cases. Their length can also be different, usually in the range from 10 to 1,000 mm. The columns are filled with the column packing, usually with the porous particles of preferably spherical shape, termed as stationary phase (SP). The nature and properties of SP will be accounted in more detail in Sect. 4.3. The separation channels are usually rectangular devices with length of few mm and diameter of few µm. The macromolecules are transported along the column or channel with a liquid called mobile phase (MP) or eluent. While the role of eluent is to solely provide transport of sample along the column or channel, mobile phase is often directly involved in the separation process. It interacts either with separated macromolecules or with the column packing. Therefore, the term “eluent” is in a way subordinated to the term

4.2 Columns

37

“mobile phase”. However, in this book the terms “eluent” and “mobile phase” are used interchangeably. The typical characteristics of MP/ eluent will be systematically specified in Sect. 4.4. The mode of chromatographic operation is the determining factor of the type and dimensions of the columns. For SEC, the separation is based on the access of macromolecules to the porous non-interactive SP. The pore volume of SEC columns typically makes about 30–40% of the void volume of the column. Fairly longer columns (25–60 cm) containing larger volume of SP are generally required for efficient separation. Multiple-column sets are typically used in SEC while performing separations in a single solvent. SEC is not the focus of this book and is only discussed up to certain level required for discussion of SEC phenomena in interactive columns and also as a part of two-dimensional LC. Hence, it is only briefly discussed and interested readers are referred for more in-depth discussion to refs. [1, 10–14]. There is a clear trend of using smaller columns with high efficiency in HPLC of micro-molecules. This is also true for polymer HPLC, however, since exclusion also plays a part in separation of polymers the column lengths and pore sizes must be appropriate to provide sufficient exclusion effect. In principle, retention in HPLC is based on relative interaction of the analyte with MP and SP. The strength of eluent/ MP is the determining factor of the separation. Smaller particles provide more surface area and higher efficiency but are associated with higher back pressures. Moreover, the diameter and length of connecting capillaries must be kept minimum to avoid band broadening. Some interactions, especially enthalpic partition of macromolecules, are strongly affected by temperature. Therefore, it is useful to keep at constant temperature not only the separation column but also the mobile phase. Majority of common polymer LC separations are performed near ambient temperature. The column temperature is raised in order to keep dissolved the low solubility samples such as polyolefins, polyamides or polyesters [7–9], in order to suppress the unwanted enthalpic interactions, to decrease viscosity of mobile phase, to accelerate the separation process, or just in the course of the column cleaning. On the other hand, temperature of a column may be gradually changed during separation process to assume temperature gradient. This is for example the case of temperature gradient interaction chromatography; radial temperature gradient should be avoided. Appropriate HPLC column thermostats are well available. To secure exact temperature control, the liquid medium is preferred over air for the heat transmission. Due to friction of MP with the packing particles, temperature within the LC column may gradually increase. This is especially acute at high elution rates and in the columns packed with small particles (less than 5 µm). Both longitudinal and transverse temperature gradients are created. Also unexpected temperature fluctuations may appear within column, which result in both baseline and elution volume instabilities.

38

4 Instrumentation for Polymer Liquid Chromatography

4.3 Stationary Phases Stationary Phase (SP) is the heart of any liquid chromatographic system. In contrast to other liquid chromatography instrumentation, its selection is in the hands of the operator, who wants to solve a particular separation task. Evidently, proper SP is the important requirement for obtaining accurate separation results. Thus, it is necessary to pay close attention to its properties. Therefore, some conclusions introduced in the previous sections will be stressed again. The choice of SP depends on the applied separation method, on nature of the studied macromolecules and on their anticipated molecular characteristics. The speed of separation and available instrumental facilities are also important considerations.

4.3.1 Physical Structure of Stationary Phases Most polymer LC measurements are performed with the particulate SP. The monolithic columns, which found applications in HPLC of low-molecular substances [15, 16], have not yet found broader use in polymer LC. The outer size of, preferably spherical, SP particles ranges from 1.5 to 20 µm. Particle size in a range of 5– 10 µm are applied mostly because of high back-pressure produced by transport of MP through smaller particles. Larger column particles render peak broadening; however, the shearing degradation of very large macromolecules is diminished. Cross-linked organic polymer particles are employed in numerous polymers LC methods, especially in size exclusion chromatography. The high-speed polymer LC, which applies pressures up to 100 MPa requires mechanically stable inorganic (silica gel) column packings. In physical sciences, the effective pore diameters (termed also pore size) of the porous particles are designated “micro”—below 20 nm, “meso”—up to about 50 nm, “macro”—up to about 100 nm, and “giga”—over 100 nm. The common pore sizes employed in polymer LC ranges from 6 and 400 nm, most frequently between 6 and 100 nm. The 3 nm silica gel is no more commercially available. The pore sizes over 100 nm are used mainly for polymer exclusion. The information on SP pore size is usually provided by its producer, who may also disclose data on its pore volume and specific surface area. Pore size distribution of the SP particles is carefully controlled to avoid presence of micropores smaller than 2 nm. The diffusion rate within small pores is too low and it results in the adverse effect called chromatographic peak broadening. The pore volume of common LC SPs varies between 0.5 and 1.2 mL g−1 , the most abundant being 0.8 mL g−1 . The mechanical stability of most SPs decreases not only with increasing pore volume but also with their effective pore diameter. For example, silica gels with 400 nm pores are rather pressure sensitive even if they possess only medium pore volume. Little information is available about the actual SP pore geometry, exact pore structure is not known. Their shape is approximated by the cones or cylinders with irregular

4.3 Stationary Phases

39

surface. General pore characteristics can be more or less precisely assessed from the BET argon or nitrogen absorption isotherms and bulk density measurements in dry state. The pore size distribution of mechanically stable column packings within the medium range of pore sizes can also be determined with help of mercury porosimetry. Majority of researchers, however rely on the value of average pore size given by the column supplier. A specific issue is the dependence of column packing interactivity on the pore size. Wang et al. concluded from their simulation study that the net interactivity of silica gels should increase with rising effective pore diameter [17]. Evidently, this conclusion is valid only for effective pore diameters which match the size of the macromolecules under study.

4.3.2 Chemical Nature of Stationary Phases Starch and homogenously cross-linked soft dextran particles were applied in the advent of polymer LC separations [18]. Later, dextran was chemically modified by hydroxypropylation to be swelled in organic solvents. It was subsequently replaced with homogeneously cross-linked synthetic polymers, especially polystyrene. Depending on their size, oligomers selectively permeated polymer network. The difference in the polarity of the modified polymer network due to its swelling with distinct solvents was employed for enhancing separation selectivity of oligomers [19–21]. Homogeneously cross-linked soft gels are hardly applicable in the highperformance polymer LC systems, which work at increased flow rate and require application of pressurized MP. Therefore, the pores of pressure resistant materials such as silica gel were filled with the framework of homogeneously cross-linked polymers, such as polystyrene [22]. In order to separate large macromolecules, soft column packings were substituted by hard, meso- and macro-porous materials. Typically, these are either silica gels or heterogeneously cross-linked polymers. Commercially available HPLC columns for the separation of small molecules are equally applicable for IC of polymers. In contrary to SEC, both adsorption/interaction and exclusion effects play their role in separation of macromolecules, and thus pore size is very important. Silica gel column packings are often used in interaction polymer LC. The attempts to employ porous alumina and aluminosilicates were not successful. Bare silica gel is especially suitable for application of adsorption separation mechanism. The general problem is irregular chemical structure of its surface. The interactive properties of silica gel with otherwise identical pore sizes may substantially differ in dependence on the concentration and topology of the free silanols situated on its surface. The overall concentration of silanols depends on the method of silica gel synthesis and it is usually not disclosed by the producer. There are present isolated, geminal and vicinal silanols, which exhibit distinct adsorptivity [23, 24]. Due to hydrolysis of Si–O–Si bonds, both topology and concentration of silanols may change in the course of silica

40

4 Instrumentation for Polymer Liquid Chromatography

gel application. The hydrolysis can take place not only in aqueous mobile phases but also with humid organic solvents. For the high-precision measurements, it is useful to check the actual interactivity of column under use with injection of suitable polar marker, for example an amine such as triethylamine (TEA), and tetramethyl ethylenediamine (TMED) and comparison of its elution volume [25, 26]. To mitigate the problems with free silanols, the silica gel surface is often modified with various small polar groups, for example glyceryl, amino, nitro and nitril units, which are attached to silica gel via a n-propyl spacer. The adsorption of macromolecules takes place on these polar groups. It is rather better controllable and fast. However, the polar adsorption may be accompanied with the non-polar interaction with the groups of the n-propyl spacer. Silica gel with surface bonded short alkyl or phenyl group exhibit reduced polarity. Polymers can also be deposited on the silica gel surface. Poly(ethylene oxide), PEO coated silica gel is commercially available. It seems that the PEO chains lay flat on the surface of the silica gel carrier instead of forming grafts which would protrude over it. This means that the adsorption of macromolecules in polymer LC with this material is operative, in contrast with expected enthalpic partition. Silica gels prepared by hydrolysis of water glass are thermally unstable. On the other hand, the pore geometry of material produced from silica sol is not changed by heating up to 550 °C while the silanols are removed. They are rapidly restored when in contact with the traces of humidity. Fully dehydroxylated silica gel was found to be much less adsorptive than the material containing the intermediate concentration of isolated silanols [23, 24]. On the other hand, the silica gel which was fully re-hydroxylated in boiling water, exhibited decreased adsorptivity compared to the common silica gel. It is anticipated that the hydrogen bonds between the adjacent silanols have reduced their adsorptivity. The unequal accessibility of different kinds of silanols for macromolecules with distinct sizes may be responsible for often observed irregularities of elution volumes in polymer LC. The heterogeneously cross-linked macro-porous polymers for example polystyrene/divinylbenzene, alone divinylbenzene and poly(hydroxyethyl methacrylate)s dominate the exclusion polymer LC. Porous styrene-based materials, which were originally developed as the ion exchange resins, are prepared with various pore sizes, up to giga-pores. Both their synthesis and chromatographic properties in the exclusion polymer LC are subject of numerous papers and books [1, 10–14]. Free polymer chains usually protrude over surface of heterogeneously cross-linked macro-porous gels. The concentration of protruded polymer chains is too low for their application of enthalpic partition-based separation mechanism. Porous graphite SP developed by Knox et al. in 1980s, has been employed in HPLC of low molecular weight substances [27], the main separation mechanism was found to be adsorption. Its surface homogeneity and thermal stability (even long term at 160 °C) are high. Recently it was applied in polymer LC at high temperature [28, 29]. As explained earlier, polymer LC that applies enthalpic partition-based separation mechanism needs a volume of SP distinct from the MP. The macromolecules under study are discriminated due to their uneven distribution between both the volumes.

4.3 Stationary Phases

41

The suitable materials are prepared by chemical attachment of selected long groups or polymer chains to solid, pressure resistant carrier, which is usually silica gel, Fig. 4.1. Similar to short groups attached to silica gel surface, the free silanols are employed for bonding reactions. This type of SPs is termed as chemically bonded phases. Linear C18 alkyl groups, octadecyl, are mostly employed in HPLC of lowmolecular substances and they also found rather broad application in the interaction polymer LC. Silica gels with alkyl bonded groups are often called reverse phase (RP). They mainly apply aqueous MPs in HPLC of low-molecular substances. Several other alkyl groups, from C4 to C30 are also bonded to silica gel to create stationary phase with various volume. It was shown that in some cases, C8 bonded groups provide better service because they render larger free pore volume compared to silica gel bonded with C18 groups [30, 31]. It seems that macromolecules do not enter entire volume of the bonded phase. The alkyl bonded silica gels are prepared by reaction of chloro- or ethoxy-silanes with free silanols present on the silica gel surface. It is necessary to stress that due to the spatial hindrances, all silanols cannot be reacted with long alkyl groups and the resulting material contains rather high concentration of unreacted polar silanols. Surprisingly, polar macromolecules can “snake around” the alkyl groups to be adsorbed on the free silanols [25, 26]. The process is called “U-turn adsorption”. As a result, two distinct separation mechanisms namely adsorption and enthalpic partition can be abreast operative in the alkyl bonded silica column packing. The unreacted silanols can be partially blocked with small methyl groups using the procedure called end-capping. However, the “end-capped” methyl groups are rather unstable and easily hydrolyze in humid eluents. Moreover, despite of application of the sophisticated, strictly confidential capping reactions, about 50% of initially present silanols still remain non-reacted. Hence, it is reasonable to block the free silanols on the surface of alkyl bonded silica gels with the addition of appropriate low molecular additives to MP such as triethylamine (TEA), and tetramethyl ethylenediamine (TMED) [25, 26]. The selection of a SP is based on the separation problem. Classically, chromatographic separations are divided into reverse phase (RP) and normal phase (NP). The polarity of SP is more compared to MP for NP chromatography while polarity of MP is more compared to SP for RP chromatography. Typical NP and RP SPs are listed in Fig. 4.1. Modified silica-based SPs are obtained by reacting silica with silanes. The cleaning of the used columns in order to restore their separation properties is an often-neglected procedure. The column properties may quite dramatically change due to retained low molecular impurities from eluent and also by macromolecular remnants of the previous measurements. The high polarity solvents such as dimethylformamide (DMF) usually remove quite reliably the adsorbed macromolecules from the polar SP such as silica gel and graphite. The thermodynamically good solvents are suitable for removal of the residuals of previously eluted non-polar polymers, for example tetrahydrofuran for alkyl bonded SPs. The SPs should be carefully reequilibrated with the MP under use, which often requires quite a bit of patience. The re-equilibration can be accelerated by increased temperature. Generally, it is easier to restore interactive properties of polar column packing when passing from a weak to a

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4 Instrumentation for Polymer Liquid Chromatography

Fig. 4.1 Typical polar (normal phase) and non-polar (reversed phase) stationary phases for liquid chromatography [11]. Copyright 2021, Elsevier

strong solvent. Similarly, it is useful to clean the alkyl bonded silica gel by applying a better solvating liquid. The danger of the full retention of separated macromolecules within polymer LC column rapidly rises with decreasing effective packing pore size. It is likely a result of the “flower-like” conformation of adsorbed macromolecules [32]. Evidently, the poor sample recovery is the reason of limited use of SPs with narrow pore diameters, without explanation, in numerous polymer LC applications [11, 13, 14]. The absence of narrow packing pores may cause insufficient separation of the noninteracting sample constituents with lower molar mass. On the contrary, the wide pore column packings have low surface area and limited interaction capability. Hence, a compromise is to be sought.

4.4 Mobile Phases/Eluents and Sample Solvents As stated above, the macromolecular analytes are transported along the column or channel with the liquid called mobile phase or eluent. According to the separation task, it should have specific properties. Generally, it must dissolve the analyte and not have high viscosity at temperature of separation. Some liquids employed in polymer LC actively participate on the separation processes. They will be designated mobile phase. The term “eluent” is reserved for liquids, which only carry macromolecules

4.4 Mobile Phases/Eluents and Sample Solvents

43

along the polymer LC system without direct involvement in the separation process. In this book, both terms mobile phase (MP) and eluent are used interchangeably. In SEC, single solvents are typically used that must be thermodynamically good solvents for the polymer under investigation. Low toxicity, an appropriate refractive index, low viscosity, and UV- transparency are some of other required attributes. The nature of the polymer and SP are the determining factors of the MP. Tetrahydrofuran, toluene, esters, chloroform, ketones, and dimethylformamide, are the most widely used solvents for SEC analysis. As mentioned, several times in the previous text, addition of low-molecular weight electrolytes is sometimes necessary to overcome unwanted enthalpic interactions with the SP. Polyolefins are only soluble at elevated temperatures in high-boiling solvents. 1,2,3-trichlorobenzene is the most widely used solvent for SEC analysis of polyolefins. To avoid thermos-oxidative degradation, small amounts of (0.2–1.5 mg/mL) of phenolic antioxidants such as butylated hydroxytoluene (BHT) are added [9]. The basic information about behavior of dissolved macromolecules and about particularities of their contact with solid porous particles are described in Chap. 2. Once the appropriate separation mechanism and column packing were chosen, the suitable mobile phase is to be selected. Its choice may be decisive for a success of the practical polymer LC separation. In most conventional methods especially in SEC, single solvents are used as mobile phases. however, in the numerous advanced polymer LC methods, two- or multi-component mixed mobile phases are applied. Their composition is usually given in volume percent (vol. %). However, weight percent data are more suitable as they are not susceptible to temperature variations. Dissolved polar macromolecules in contact with polar solid surface are often subject of adsorption. They “compete” with solvent molecules for free space. The extent of interaction of a liquid with solid surface is called solvent strength. Concerning the polar surface, strong are polar solvents, while the weak are the nonpolar solvents. The strong solvent, which prevents adsorption of macromolecules on the solid surface is called a desorli. The weak solvent, which promotes adsorption of macromolecules on given surface is called an adsorli. The solvent strength concept was introduced by Snyder in liquid chromatography of low-molecular substances eluted from alumina column packings [33–36]. It was later applied also to other column packings, including bare silica gel [37]. Solvent strength to a large extent reflects polarity of the particular liquid. It is a unitless constant designated as εo . In liquid chromatography of low-molecular substances, as well as in polymer LC, the appropriate solvent strength of mobile phase is usually attained with mixing of strong and weak solvents. The solvent strength of a mixture of liquids is in the first approximation considered additive. The εo data of most common liquids applied in HPLC of low molecular substances are tabulated [38], however, some values for important mobile phases for polymer LC are missing. It is also necessary to note that the solvent strength values represent only estimates of actual potency of the particular solvent toward particular macromolecules concerning their interaction with the given SP. For example, a solvent with a higher tabulated εo value may be a less efficient desorli for given polymer than a solvent possessing lower εo value. Moreover, the actual desorli-adsorli action

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4 Instrumentation for Polymer Liquid Chromatography

of a given solvent on the same bare silica gel may differ between two distinct polymers. For example, a solvent may act as an adsorli for a less polar polymer while act as a desorli for a more polar polymer. This may be caused by differences in polymer– solvent interactions for both solvents and by the effect of the conformational entropy of polymer coils. Moreover, adsorption activity of the most common polar column packing, silica gel strongly depends on the concentration and topology of surface silanols. In practice, the trial-and-error approach is recommended in the interaction polymer LC to adjust the appropriate solvent strength of a mixed eluent. To conclude, the literature data can only be employed for the first approximation and the actual experiments are usually decisive. In case of absorption (enthalpic partition), two volumes compete for macromolecules. Extent of enthalpic partition of non-polar or low polarity macromolecules between the non-polar, alkyl bonded SP and MP is mainly controlled by the thermodynamic quality of both phases toward polymer under study. The SP is solvated with MP and the difference of attractiveness for distinct macromolecules between two phase volumes may be rather small. In many cases, the MP needed for attaining suitable extent of enthalpic partition of a polymer is also its poor solvent. It should “push” macromolecules into SP. The deterioration of MP thermodynamic quality slightly below theta conditions may lead to the association or aggregation of macromolecules. It is invisible to operator and therefore it may cause misleading results. Often a small change in the MP composition and temperature variation may even bring about the phase separation. The micro-droplets are created and their travel along column is usually uncontrolled. Most sensitive to precipitation are usually the largest macromolecules present in the sample. They may undergo selective phase separation. In any case, elution volumes may become irreproducible. Thus, the application of thermodynamically poor MPs should be avoided in polymer LC. In order to adjust the adsorptive strength or thermodynamic quality of MP, two or more liquids are mixed. Separation mechanism is often controlled by the MP composition. Generally, two-component MP are preferred over multi-component systems whenever possible. An exception is the addition of small amount of substances, which block the free silanols present in alkyl bonded silica gels [25, 26]. Due to problems associated with extreme adsorptivity or low solubility of some polymers, multi-component MPs are hardly avoidable. Typical examples are poly(amide)s and aromatic poly(ester)s that are soluble at ambient temperature in very expensive 1,1,1,3,3,3-hexafluoro-2-propanol, HFIP or in 2,2,2, trifluoro ethanol, TFE, and in their mixtures with some other solvents, for example with chloroform, CHCl3 . Sometimes, it is possible to avoid high-cost MPs by using the small amount of HFIP or TFE just for polymer dissolution and then add a cheaper solvent like chloroform, while containing low concentration of expensive fluorinated solvents in MP [39]. In some cases, two different mobile phases are applied in two distinct but mutually related separation steps to achieve separation of complex polymers or complex polymer systems. Corresponding two- or multi-dimensional approaches

4.4 Mobile Phases/Eluents and Sample Solvents

45

are discussed in Chap. 12. When the MP contains two and more components, preferential solvation of macromolecules present in the sample is often inevitable. Peculiarities and solving problems connected with preferential solvation, especially with its impact on sample detection are discussed in Sect. 4.5. In common practice, the exponents α of Kuhn-Mark-Houwink-Sakurada viscosity law are considered for solvent quality. Thermodynamic quality for a given polymer in a solvent can also be estimated by comparing solubility parameters, δ for solvent and the polymer. Similarity of their δ values roughly indicates polymer solubility. However, both the parameters can serve only for orientation and preliminary experiments are often unavoidable. For the basic orientation, few examples of appropriate adsorli solvents are, – low polarity polymers such as polystyrene or polydienes: tetrachloro methane (suspect carcinogen!), hexane, heptane, dichloromethane, dichloroethane, cyclohexane, 1-chlorobutan etc. – medium polarity polymers as poly(methyl methacrylate): 1-chlorobutan, toluene, dichloromethane, dichloethane, chloroform, acetone etc. – high polarity polymers such as poly(2-vinyl pyridine) or poly(ethylene oxide): tetrahydrofuran. A prerequisite for HT-HPLC is the solvents having boiling points above 150 °C. The polar solvents having such a high boiling point include 1-ethyl1-hexanol, n-decanol, cyclohexyl acetate, hexyl acetate, cyclohexanone, ethylene glycol monobutyl ether while non-polar solvents in this context could be n-decane, TCB, DCB etc. [4, 40]. As suitable desorli solvents for low and medium polarity polymers can be tetrahydrofuran and methylethylketone, while dimethylformamide is an efficient desorli for high polarity polymers. Usually, MP is also employed as sample solvent. However, in some cases, especially if interaction of macromolecules with MP is used as a means for achieving polymer separation, sample solvent may differ from the MP. Most MPs appropriate for control of polymer adsorption are mixtures of strong and weak solvents. Concerning management of enthalpic partition it is necessary to mix a thermodynamically good solvent with a poor solvent or a non-solvent for polymer under study. Good polymer LC results are often obtained with the “symmetrical mixed MPs”, in which the relative concentrations of strong and weak, or good and poor solvents are similar. Sensitivity of sample elution volume toward both eluent composition and temperature variation of such systems is less pronounced and repeatability of results is usually improved. The actual suitable mobile phases and sample solvents will be parsed in connection with particular polymer LC methods in the corresponding chapters. It is recommended that the caps of the MP containers under use must be provided by a desiccant to prevent absorption of humidity. Follow the general information about solvent safety, such as health hazards, flammability and explosivity, for example due to forming peroxides in the course of oxidation especially for tetrahydrofuran (THF). The (possibly) present peroxides must be decomposed before its

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4 Instrumentation for Polymer Liquid Chromatography

distillation by adding appropriate chemicals, like sodium benzophenone along with distillation under nitrogen. However, THF should never be distilled into dryness. The solvents susceptible to oxidation should be well-stabilized and keep in dark, preferably under nitrogen. Both the polymer solubility and their interaction strength with the column packing often depend on the solvent purity. Two different batches of solvent of the same grade from the same producer may behave in a different way. Typical examples are the “HPLC grade solvents”, which became popular in HPLC of low molecular substances. From them, the traces of the UV absorbing impurities were removed. However, in some cases, less attention is paid to the water content in hydrophilic solvents, such as the very important, above-mentioned polymer LC solvent, THF. Evidently, the differences in water content play little role in HPLC of low molecular substances, where water is often the main mobile phase constituent. However, in the interaction polymer LC, presence of small water content may strongly affect the solvent strength and adsorption of polymer on polar surface and similarly, the thermodynamic quality of solvent for the given macromolecules. If the control of water presence—for example by K. Fischer titration or by a more sophisticated method is not possible, it is advised to make stocks of the stabilized and from both light and humidity protected solvents for the entire set of experiments. As iterated above, solubility of sample and comparative interactions between sample, the solvent and the SP need to be considered. Mixture of solvents are often used that can be classified with regard to their chemical nature and polarity. The classification of solvents with regard to polarity is usually presented in terms of “eluotropic series”. The polarity index and UV-cutoff of important HPLC solvents are listed in Table 4.1. Different values of polarity index are reported in literature; however, the order remain more or less similar. Moreover, spectroscopic behavior of the solvent must be considered while using spectrophotometric detectors such as UV, FTIR, or NMR.

4.5 Detectors In the initial studies on separation of macromolecules according to their molecular characteristics, the amount and some properties of obtained fractions were measured one-by-one by different methods. The breakthrough in polymer LC as to its speed and precision rendered devices, which continuously monitor the amount of the separated macromolecules in the column effluent. These flow-thru devices are called detectors. Their principles, sensitivity and applications, as well as general advices for users are described in numerous studies, reviews and books [10–13]. We will briefly mention several kinds of detectors applied in polymer LC but we will focus only to the devices, which can be employed with multi-component mobile phases. The latter are almost exclusively availed in the methods of entropy/enthalpy compensation, to which this book is primarily devoted. A broad classification of detectors employed for HPLC of polymers may be based on the nature of their sensitivity. These can be concentration detectors and molar mass detectors. The attempts to create devices, which would

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Table 4.1 Typical solvents used in liquid chromatography of polymers [13]. Copyright 2013, Springer Nature Class Alkanes Aromatics

Ether

Alkyl halides

Esters Ketones Alcohols

Solvent

Polarity index

UV-cutoff

Hexane, Heptane

0.0

200

Cyclohexane

0.2

200

Benzene

2.7

280

Toluene

2.4

285

Xylene

2.5

290

Diisopropyl ether

2.2

220

Methyl-tert.-butyl ether

2.5

210

Tetrahydrofuran

4.0

215

Dioxane

4.8

215

Tetrachloromethane

1.6

263

Dichloromethane

2.5

235

Dichloroethane

4.0

225

Trichloromethane

4.8

245

Butyl acetate

4.0

254

Ethyl acetate

4.4

260

Methyl ethyl ketone

4.7

329

Acetone

5.1

330

n-Butanol

3.9

215

i-Propanol

3.9

210

n-Propanol

4.0

210 205

Methanol

5.1

Nitriles

Acetonitrile

5.8

190

Amides

Dimethylformamide

6.4

268

Carboxylic acids

Acetic acid

Water

6.2

230

9.0

200

continuously measure osmotic pressure either in its vapor pressure or membrane arrangement were so far not successful. Detectors used in HPLC of polymers can be broadly classified into two major categories, concentration detector and molar mass detectors.

4.5.1 Concentration Sensitive Detectors The detectors included in this category have concentration-dependent signal of the analyte. Further sub-classification of these detectors could be selective detector that measure property of solute, and universal detector that measure the bulk property

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of the MP/eluent. Multiple detection is often required for simultaneous analysis of various overlapping distributions of complex polymers.

4.5.1.1

Selective Detectors

It is important of mention here that not all the available selective detectors for HPLC are compatible with polymer HPLC. For instance, fluorescence and electrochemical detectors are not applicable to polymers. The most widely employed selective detector for polymer analysis is UV detector. Detection in the IR region is limited to eluents that don’t absorb radiations in the relevant detection wavelength. The applications of IR detection in polymer HPLC are mostly conducted through a evaporative interface in an quasi on-line mode [41, 42]. The spray of eluate on a rotated Germanium disc is followed by analysis of the disk by FTIR, rotating at same speed in both instruments to correlate chemical composition as a function of elution time. An additional concentration detector is however required for accuracy of quantification. UV Detector The first flow-thru polymer LC detectors were photometers equipped with mercury discharge tubes and optical filters. They produced nearly monochromatic light 254 nm and also 280 nm from an additionally supplemented fluorescent source. The photometric detectors were gradually improved concerning their sensitivity and size of the measuring cell. The fixed wavelength devices were supplemented with fluorescence detectors, with variable wavelength detectors, and with the diode array detectors, which can monitor macromolecules simultaneously at different wavelengths. They are most important detectors in HPLC of low molecular substances but their applicability to polymers is restricted due to low UV absorption of most of the polymers. Noel and Monerie have even observed differences between the ultraviolet light absorption of certain binary styrene statistical copolymers and the corresponding mixtures of the homopolymers [43]. These effects are likely a function of the position of the phenyl ring along the copolymer chain, as well as of the overall chain conformation. The latter may favor or hinder electronic interactions between neighboring chromophore groups. Gallo and Russo also observed marked hypochromism of statistical styrene-methyl methacrylate copolymers, which has affected the signal of UV detector in chloroform, dichloroethane, dioxane, tetrachloroethane and tetrahydrofuran [44]. Such effects may complicate the application of UV detectors even for copolymers containing UV-absorbing repeat units. The unexpected results were also observed when UV detectors were applied to monitor system peaks caused by preferential solvation [45]. Mori proposed the UV detection at wavelength near 237 nm for monitoring of polystyrene, poly(alkyl methacrylate)s and their copolymers [46]. These polymers exhibit practically identical absorbance at this wavelength and thus can be jointly detected as homopolymers. The online attached UV photometer working at 254 nm detected only polystyrene. Nonetheless, the UV detector is the most widely employed solute property detector. The basic principle is absorbance of a light from UV region (180–350 nm)

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49

by a chromophore-containing repeat unit. Another associated caveat of UV detector is the UV absorbance of the eluent, must have low UV-cutoff. UV-cutoff of commonly used HPLC solvents is given in Table 4.1. Commercially three types of UV detectors are available namely fixed wavelength detector, variable wavelength detector, and diode-array detector (DAD). A single wavelength lamp emitting light at 254 nm is used for fixed wavelength detector while variable wavelength detector allows selection of appropriate wavelength. Finally, DAD measures simultaneous absorbance of the whole UV spectrum for the eluate. IR Detector The detectors working at visible wavelengths provide valuable services in monitoring of some polymers. Important applications are found for the photometric detectors working in the infrared range of electromagnetic radiation, fixed or scanning wavelength, and Fourier-transform devices. They are mainly employed in the high temperature separations of polyolefins [1, 2]. The overview of infrared detection can be found in refs. [47, 48]. Coupling of FTIR to HPLC can be realized via a flow cell in an on-line mode [49, 50] or through solvent elimination interface in an off-line mode [51–53]. Flow cell interface cannot be used with eluent gradients and is less sensitive but provide reliable quantitative information. On the other hand, solventelimination interface can be used for gradient separations, is highly sensitive, and provide only qualitative information. Most of the solvents employed in HPLC have strong absorption in mid-IR region that hinder the application of flow cell devices. In context of solvent-elimination interface, essential progress brought application of rotating germanium disk as the sample carrier. It enables quantitative sample capture and storage. In the necessary cases, sample can be accumulated by repeated separations. It follows independent sample analysis, for example by an independent infrared instrument. Several other kinds of detectors developed for HPLC of low-molecular substances can be used for monitoring polymers but their applicability in polymer LC is rather limited. One can consider the mass spectrometers based on molecular fragmentation by electrical field such as electrical conductivity detectors, charged aerosol detector, electrochemical detectors, chiral detectors and pulsed amperometric detectors. Moreover, a specific group represent the devices with sample transfer. Column effluent is continually deposited on appropriate moving carrier from which eluent is removed usually by increased temperature and the non-volatile sample is analyzed. Originally, samples were pyrolyzed and the products of pyrolysis were subject to measuring of the amount of the products of pyrolysis with help of gas chromatographic detectors. The aim was to determine concentration of polymer in column effluent. The transport devices were round wires, tapes or chains. The important problem comprised removal of remains of the pyrolyzed sample. Pyrolysis of discrete polymer fractions by independent gas chromatograph may provide additional information about composition and structure of sample. Important limitation of this approach is the compositional dependence of products of pyrolysis on the structure of statistical copolymers.

50

4.5.1.2

4 Instrumentation for Polymer Liquid Chromatography

Universal Detectors

The change in a bulk property of the eluent is measured by universal detectors such as refractive index, density, conductivity and evaporative detectors. Obviously, the sensitivity of universal detectors is limited compared to specific detectors. RI detectors have been employed extensively in polymer HPLC whereas applications of conductivity detector are not common. The detection of change in the density based on mechanical oscillator principle is an effective detector in polymer HPLC. Detection of non-volatile polymers by scattering of the light beam after evaporation of eluent is the principle of evaporative light scattering detector. RI Detector The breakthrough in polymer LC detection brought the construction of the deflection type differential refractometer at the beginning of sixties by J. Waters. It was used in initial exclusion-based separations of synthetic polymers. The refractometric detectors underwent significant developments. Christiansen effect, reflexion and interference-based instruments were constructed with increased sensitivity, smaller measuring cells and reduced susceptibility to outer temperature fluctuations. Their general drawback is the limited applicability with multi-component mobile phases because they respond to preferential solvation of macromolecules with one of eluent constituents. Commercially three types of RI detectors are available that include deflection refractometers, Fresnel refractometers, and interferometric refractometers. Among them, deflection refractometer has large cell and fairly wide linear range, therefore, most commonly employed. On the other hand, interferometric refractometers are more sensitive compared to its other counterparts. However, response of RI detector is not independent of molar mass and chemical composition which ensue the requirement of an additional concertation detector for reliable analysis of copolymers and blends of homopolymers [54]. Furthermore, detector signal may also be affected by preferential solvation of the polymer in one component of the eluent [12]. In case of copolymers, the preferential solvation may be multi-dimensional due to multiple components of both polymers and eluent. Density Detector Density detectors monitor the changes of specific weight of column effluent due to presence of macromolecules. The working principle of density detector is based on mechanical oscillator [55, 56]. It consists of a U-shaped oscillating capillary whose periods are dependent upon the density of the flowing eluent. However, their sensitivity is pretty limited. They provide good service in the liquid chromatography procedures, which enable work with higher sample concentrations, especially oligomers. Moreover, it has to be used in combination of UV or RI detectors. Evaporative Light Scattering Detector (ELSD) A decisive advantage in monitoring fractions acquired by polymer LC, which employs mixed mobile phases is presented by evaporative light scattering detectors, ELSD. Any non-volatile component of the eluate can be detected by ELSD,

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hence it is regarded as a universal detector [57, 58]. In principle, nebulization of the column effluent is followed by evaporation of the solvents from the droplet. Each droplet forms particles comprised of the non-volatile components of the column effluent. The incident light beam is scattered by these particles which is detected. However, the signal of ELSD depends on number of factors that include number, size, and RI of the particles along with the eluent composition [59, 60] and nature of the polymer [60, 61]. Moreover, the operating conditions such as flow rate of the carrier gas and eluent, oven temperature, viscosity and surface tension of the eluent directly influence the detector signal and performance [62, 63]. Hence, the quantification by ELSD is tricky due to its non-linear response and other above-discussed factors [64, 65]. Furthermore, ELSD does not reveal any information on the polymer itself.

4.5.2 Molar Mass Sensitive Detectors A breakthrough in detection of exclusion-based polymer LC constituted viscometry and light scattering measurements. Their basic features are presented in the several books and review articles [10, 12, 14, 66]. Unfortunately, their application to polymer LC, which employ multi-component mobile phases is very limited due to unavoidable phenomenon of preferential solvation. Hence, only brief discussion of molar mass sensitive detectors is presented here. Molar mass sensitive detectors are now widely accepted for SEC of polymers in order to obtain information on branching and absolute molar mass. These detectors have to be used in conjunction with concentration sensitive detector in multi-detector system since signal in molar mass sensitive detectors depends both on molar mass and the concentration of the analyte. There are basically two types of molar mass sensitive detectors namely viscometric and light scattering detectors [12, 67]. There are several types of light scattering detectors such as low-angle light scattering detectors (LALLS), two-angle light scattering detectors (TALLS), right-angle light scattering detectors (RALLS), and multiangle light scattering detectors (MALLS). Among which MALLS is by far the most widely employed detector in polymer analysis. Moreover, combination of light scattering and viscosity detector is of synergistic nature that provide important information that is not obtained otherwise [68]. Absolute molar mass and radii of gyration can be obtained by light scattering detection while distribution of intrinsic viscosity as a function of SEC profile is accessible by viscometeric detector. A triple-detector system containing a concentration detector along with light scattering and viscosity detector provide reliable results and reveal information on branching distributions [69–72]. In principle, all the component of ambient temperature HPLC of polymers and high temperature HPLC instrument are same. The only major difference is the temperature of the whole system.

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References 1. Pasch H, Malik MI (2014) Advanced separation techniques for polyolefins. Springer labortary series. Springer, London. https://doi.org/10.1007/978-3-319-08632-3 2. Pasch H, Malik MI, Macko T (2013) Recent advances in high-temperature fractionation of polyolefins. Adv Polym Sci 251:77–140. https://doi.org/10.1007/12_2012_167 3. Macko T, Pasch H, Kazakevich YV, Fadeev AY (2003) Elution behavior of polyethylene in polar mobile phases on a non-polar sorbent. J Chromatogr A 988(1):69–76 4. Macko T, Pasch H, Denayer JF (2003) Adsorption of polyethylene standards from decalin on liquid chromatography column packings. J Chromaogr A 1002(1–2):55–62 5. https://polymer-laboratories.com/. 6. Heinz L, Macko T, Williams A, O’Donohue S, Pasch H (2006). The column (electronic J) 7. Parth M, Aust N, Lederer K (2003) Molecular characterization of ultrahigh molar mass and soluble fractions of partially cross-linked polyethylenes. Int J Polym Anal Charact 8:175–186 8. Mes EPC, de Jonge H, Klein T, Welz RR, Gillespie DT (2007) Characterization of high molecular weight polyethylenes using high temperature asymmetrical flow field-flow fractionation with on-line infrared, light scattering, and viscometry detection. J Chromaogr A 1154(1–2):319–330. https://doi.org/10.1016/j.chroma.2007.03.116 9. Pasti L, Melucci D, Contado C, Dondi F, Mingozzi I (2002) Calibration in thermal field flow fractionation with polydisperse standards: application to polyolefin characterization. J Sep Sci 25(10–11):691–702 10. Lederer A, Brandt J (2021) Chapter 2 - Multidetector size exclusion chromatography of polymers. In: Malik MI, Mays J, Shah MR (eds) Molecular characterization of polymers. Elsevier, pp 61–96. https://doi.org/10.1016/B978-0-12-819768-4.00012-9 11. Malik MI, Pasch H (2021) Chapter 1—Basic principles of size exclusion and liquid interaction chromatography of polymers. In: Malik MI, Mays J, Shah MR (eds) Molecular characterization of polymers. Elsevier, pp 1–59. https://doi.org/10.1016/B978-0-12-819768-4.00007-5 12. Striegel AM, Yau WW, Kirkland JJ, Bly DD (2009) Modern size-exclusion liquid chromatography: practice of gel permeation and gel filtration chromatography, vol 2. Wiley, Hoboken, New Jersey. https://doi.org/10.1002/9780470442876 13. Pasch H, Trathnigg B (2013) Multidimensional HPLC of polymers. Springer, New York. https:/ /doi.org/10.1007/978-3-642-36080-0 14. Malik MI, Pasch H (2014) Novel developments in the multidimensional characterization of segmented copolymers. Prog Polym Sci 39(1):87–123. https://doi.org/10.1016/j.progpolymsci. 2013.10.005 15. Rashed NS, Zayed S, Abdelazeem A, Fouad F (2020) Development and validation of a green HPLC method for the analysis of clorsulon, albendazole, triclabendazole and ivermectin using monolithic column: Assessment of the greenness of the proposed method. Microchem J 157:105069. https://doi.org/10.1016/j.microc.2020.105069 16. Asmari M, Wang X, Casado N, Piponski M, Kovalenko S, Logoyda L, Hanafi RS, El Deeb S (2021) Chiral monolithic silica-based HPLC columns for enantiomeric separation and determination: functionalization of chiral selector and recognition of selector-selectand interaction. Molecules 26(17):5241 17. Zhu Y, Ziebarth JD, Wang Y (2011) Dependence of critical condition in liquid chromatography on the pore size of column substrates. Polymer 52(14):3219–3225. https://doi.org/10.1016/j. polymer.2011.04.059 18. Berek D, Bakoš D (1974) Effect of solvent on the separation of oligomer polyethers on sephadex LH-20. J Chromatogr A 91:237–245. https://doi.org/10.1016/S0021-9673(01)97903-8 19. Trathnigg B (2001) Liquid exclusion-adsorption chromatography, a new technique for isocratic separation of nonionic surfactants II. Quantitation in the analysis of fatty alcohol ethoxylates. J Chromatogr A 915(1–2):155–166 20. Trathnigg B, Gorbunov AA (2001) Liquid exclusion-adsorption chromatography: new technique for isocratic separation of nonionic surfactants I. Retention behaviour of fatty alcohol ethoxylates. J Chromatogr A 910(2):207–216

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41. Provder T, Kuo CY, Whited M, Huddleston D (1994) GPC FTIR characterization of copolymer compositional heterogeneity. Abstr Pap Am Chem Soc 208 (AUG):186-PMSE 42. Cheung PC, Balke ST, Schunk TC (1995) Size-exclusion chromatography fourier-transform ir spectrometry using a solvent-evaporative interface. Adv Chem Ser 247:265–279 43. Noël C, Monnerie L (1976) UV absorption of styrene statistical copolymers in solution. Polym J 8(4):319–323. https://doi.org/10.1295/polymj.8.319 44. Gallo BM, Russo S (1974) Ultraviolet absorption spectra of styrene copolymers. I. Solvent effects on the hypochromism of Poly(styrene-co-methyl Methacrylate) at 2695 Å. J Macromol Sci: Part A Chem 8(3):521–531. https://doi.org/10.1080/00222337408065847 45. Buszewski B, Bleha T, Berek D (1985) UV detection of solvent peaks in liquid chromatography with mixed eluents. J High Resol Chromatogr 8(12):860–862. https://doi.org/10.1002/jhrc.124 0081214 46. Mori S (1991) Separation and Detection of Styrene-Alkyl Methacrylate and Ethyl Methacrylate-Butyl Methacrylate Copolymers by Liquid Adsorption Chromatography Using a Dichloroethane Mobile Phase and a UV Detector. J Chromatogr A 541(1–2):375–382 47. Patterson BM, Danielson ND, Sommer AJ (2003) Attenuated total internal reflectance infrared microspectroscopy as a detection technique for high-performance liquid chromatography. Anal Chem 75(6):1418–1424. https://doi.org/10.1021/ac0205917 48. Rogalski A (2002) Infrared detectors: an overview. Infrrared Phys Technol 43(3):187–210. https://doi.org/10.1016/S1350-4495(02)00140-8 49. Hellgeth JW, Taylor LT (1987) Optimization of a flow cell interface for reversed-phase liquid chromatography/Fourier transform infrared spectrometry. Anal Chem 59(2):295–300. https:// doi.org/10.1021/ac00129a016 50. Wang CP, Sparks DT, Williams SS, Isenhour TL (1984) Comparison of methods for reconstructing chromatographic data from liquid chromatography Fourier transform infrared spectrometry. Anal Chem 56(8):1268–1272. https://doi.org/10.1021/ac00272a017 51. Housaki T, Satoh K, Nishikida K, Morimoto M (1988) Study on molecular weight dependence of short-chain branching of linear low-density polyethylene using the gel permeation chromatography/Fourier transform infrared method. Macromol Rapid Commun 9(8):525–528. https://doi.org/10.1002/marc.1988.030090803 52. Nishikida K, Housaki T, Morimoto M, Kinoshita T (1990) Gel permeation chromatography— Fourier transform infrared study of some synthetic polymers: II. Instrumentation for the characterization of polyethylene. J Chromatogr A 517:209–217. https://doi.org/10.1016/S0021-967 3(01)95722-X 53. DesLauriers PJ, Rohlfing DC, Hsieh ET (2002) Quantifying short chain branching microstructures in ethylene 1-olefin copolymers using size exclusion chromatography and Fourier transform infrared spectroscopy (SEC–FTIR). Polymer 43(1):159–170. https://doi.org/10.1016/ S0032-3861(01)00574-2 54. Philipsen HJA (2004) Determination of chemical composition distributions in synthetic polymers. J Chromatogr A 1037(1–2):329–350 55. Trathnigg B (1990) Determination of chemical composition of polymers by SEC with coupled density and RI detection. I. Polyethylene glycol and polypropylene glycol. J Liq Chromatogr 13(9):1731–1743 56. Malik MI, Trathnigg B, Kappe CO (2007) Selectivity of PEO-block-PPO diblock copolymers in the microwave-accelerated, anionic ring-opening polymerization of propylene oxide with PEG as initiator. Macromol Chem Phys 208(23):2510–2524. https://doi.org/10.1002/macp.200 700320 57. Rissler K, Fuchslueger U, Grether HJ (1994) Separation of polyethylene-glycol oligomers on normal-phase and reversed-phase materials by gradient high-performance liquidchromatography and detection by evaporative light-scattering—a comparative-study. J Liq Chromatogr 17(14–15):3109–3132 58. Lafosse M, Elfakir C, Morin-Allory L, Dreux M (1992) The advantages of evaporative lightscattering detection in pharmaceutical analysis by high-performance liquid-chromatography and supercritical fluid chromatography. HRC J High Resolut Chromatogr 15(5):312–318

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59. Trathnigg B, Kollroser M, Berek D, Nguyen SH, Hunkeler D (eds) (1999) Quantitation in the analysis of oligomers by HPLC with evaporative light scattering detector, vol 731. ACS Symposium Series, Chromatography of Polymers 60. Kawai T, Teramachi S, Tanaka S, Maeda S (2000) Comparison of chemical composition distributions of poly(methyl methacrylate)-graft-polydimethylsiloxane by high-performance liquid chromatography and demixing-solvent fractionation. Int J Polym Anal Charac 5(4–6):381–399. https://doi.org/10.1080/10236660008034634 61. Braun D, Kramer I, Pasch H, Mori S (1999) Phase-separation in random copolymers from high conversion free-radical polymerization, 2—chemical heterogeneity analysis of poly(StyreneCo-Ethyl Acrylate) by gradient HPLC. Macromol Chem Phys 200(5):949–954 62. Trathnigg B, Kollroser M, Gorbunov AA, Skvortsov AM (1997) Liquid adsorption chromatography of polyethers—theory and experiment. J Chromatogr A 761(1–2):21–34 63. Hopia AI, Ollilainen VM (1993) Comparison of the Evaporative Light-Scattering Detector (Elsd) and Refractive-Index Detector (Rid) in lipid analysis. J Liq Chromatogr 16(12):2469– 2482 64. Van der Meeren P, Van der Deelen J, Baert L (1992) Simulation of the mass response of the evaporative light-scattering detector. Anal Chem 64(9):1056–1062 65. Michael FJ (2002) Product review: are ELSDs coming out of the shadows? Anal Chem 74(23):631 A–634 A. https://doi.org/10.1021/ac022171n 66. Podzimek S (2011) Light scattering, size exclusion chromatography and asymmetric flow field flow fractionation: powerful tools for the characterization of polymers, proteins and nanoparticles. Wiley, Hoboken, New Jersey 67. Podzimek S (1994) The use of GPC coupled with a multiangle laser-light scattering photometer for the characterization of polymers—on the determination of molecular-weight, size, and branching. J Appl Polym Sci 54(1):91–103 68. Jackson C, Yau WW (1993) Computer-simulation study of size-exclusion chromatography with simultaneous viscometry and light-scattering measurements. J Chromatogr 645(2):209–217 69. Alexeev EM, Catanzaro A, Skrypka OV, Nayak PK, Ahn S, Pak S, Lee J, Sohn JI, Novoselov KS, Shin HSJNl (2017) Imaging of interlayer coupling in van der Waals heterostructures using a bright-field optical microscope. 17(9):5342–5349 70. Lee S, Chang T (2017) Branching analysis of comb-shaped polystyrene with long chain branches. Macromol Chem Phys 218(12):1700087. https://doi.org/10.1002/macp.201700087 71. Lee S, Lee H, Chang T, Hirao A (2017) Synthesis and characterization of an exact polystyrenegraft-polyisoprene: a failure of size exclusion chromatography analysis. Macromolecules 50(7):2768–2776. https://doi.org/10.1021/acs.macromol.6b02811 72. Lee H, Yang J, Chang T (2017) Branching analysis of star-shaped polybutadienes by temperature gradient interaction chromatography-triple detection. Polymer 112:71–75. https://doi.org/ 10.1016/j.polymer.2017.01.070

Chapter 5

Non-exclusion Methods of Polymer Liquid Chromatography

In this chapter, we will briefly elucidate the most important polymer LC procedures, which employ the enthalpy-based separation mechanism, generally designated as enthalpic interactions. As mentioned previously, such methods are often called “interaction polymer liquid chromatography” and we will also adopt this term. It must be stressed again, that exclusion separation mechanism is present in many interaction polymer LC methods but it does not render the basic part of the separation process.

5.1 Polymer—Mobile Phase Interactions 5.1.1 Precipitation-(Re)dissolution Polymer LC Processes of dissolution and precipitation of polymers, as well as properties of their solutions are outlined in Chap. 2. The differences in the solubility of distinct macromolecules were employed for their separation already in the initial stages of polymer research. Large volumes of polymer solution are applied and macromolecules are gradually precipitated by adding non-solvent or by changing temperature. Phase separation is evoked to form small volume of concentrated solutions of sample fraction or solid particles of precipitate containing similar macromolecules. The concentrated solution of macromolecules is often called the gel phase. The properties such as molar mass of obtained fractions are determined one-by-one by viscometry, osmometry or light scattering measurements. The procedures are tedious, laborious, and complicated. However, fractional precipitation is still a very useful tool for a preparative fractionation (g scale) in the laboratory. For analytical purposes, effort to accelerate and automate the separation procedures led to development of the dynamic systems. One of the pioneers of separation science of macromolecules, Jerker Porath proposed protein discrimination by their selective precipitation-redissolution and termed the process “zone precipitation” [1]. Mixtures of proteins are introduced into a column flushed with a water-based MP. Its dissolution power is controlled by © The Author(s), under exclusive license to Springer Nature Switzerland AG 2023 M. I. Malik and D. Berek, Liquid Chromatography of Synthetic Polymers, Physical Chemistry in Action, https://doi.org/10.1007/978-3-031-34835-8_5

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concentration of added salts and it is gradually changed to achieve separate elution of distinct proteins. The history of numerous important achievements of Porath in the separation sciences are summarized in the editorial of Journal of Chromatography [2]. Other important initial progress in polymer separations was by work of Baker and Williams [3]. Polymer LC was modernized step-by-step. New separation mechanisms are developed and instrumentation is improved (Chap. 4). Pumps were developed to achieve fast, reliable, and constant flow of MP. The detectors were introduced, which continuously monitored polymer concentration in the column effluent. Volume of the column effluent was measured by siphon systems. Glöckner studied solubility of synthetic polymers by turbidimetric titrations, proposed efficient polymer separation method, which employed precipitation of macromolecules followed by their continuous dissolution [4]. It is called precipitation-(re)dissolution polymer liquid chromatography. The sample solution, usually a polymer blend or a statistical copolymer is injected into the column flushed with a non-solvent. Sample is precipitated near the column inlet. The non-solvent should ensure complete polymer precipitation. The column is then washed with an eluent with gradually increasing concentration of a (good) solvent, non-solvent to solvent gradient. Depending on their solubility, fractions of macromolecules are sequentially eluted from the column [5–7]. Glöckner has studied various peculiarities of method in detail and successfully separated various statistical copolymers. The results of chromatographic separations are compared with outcomes of turbidimetric titrations [8]. The surface properties of porous column packing play secondary role, unless strong sample adsorption is not present [9, 10]. Monoliths were employed for precipitation-(re)dissolution separations of synthetic polymers [11]. Staal proposed the name “gradient polymer elution chromatography” for precipitation(re)dissolution procedure claiming to develop a new separation method [12]. However, this term is not appropriate, because there are several other polymer LC methods, which make use of an eluent gradient. The selectivity of precipitation-(re)dissolution polymer LC method is very high but the procedure suffers from several drawbacks. One of the major problems is the sudden pressure rise after sample injection due to its precipitation that necessitate the low injection concentration. To ensure successful separation, solvent and nonsolvent properties, as well as the profile of their gradient must be carefully adjusted that usually requires several preliminary tests. Polymer re-dissolution is a relatively slow process compared to diffusion and pore permeation of their solutions. The identical macromolecules hardly dissolve at exactly same time and they must catch up with each other. Therefore, the elution rate must be slow. On the other hand, the dissolved macromolecules cannot outrun the MP due to their pore exclusion and the peaks of the well-optimized experimental conditions are usually narrow. Polymer solubility not only depends on its molar mass but also on other molecular characteristics for instance chemical structure, and physical architecture. Therefore, quantitative interpretation of separation results is hardly possible in case of complex polymers, for instance for statistical copolymers unless a detector can be employed,

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which monitors the composition of the copolymer fractions. The general drawback of precipitation-(re)dissolution polymer LC method is reduced sample recovery. This is an issue with all polymer LC methods, in which sample is temporarily fully-retained near the inlet of the column packed with the porous particles. It may result from the “flower like conformation” of macromolecules, which partially permeate the packing pores [13]. A blank precipitation–(re)dissolution experiments with repeated eluent gradient without any new sample injection have shown the identical elugrams as the original ones. Peaks were small but their positions remained the same. The possible solution of this problem could be application of small “precolumn” packed by non-porous particles.

5.1.2 Hydrodynamic Chromatography Pedersen has drawn attention to the important process that affects the movement of dissolved macromolecules carried by the laminar flow of liquid along a capillary [14]. It is known that due to its friction, the liquid flowing along a capillary moves faster in its center than near its walls and a parabolic flow pattern is formed. If macromolecules travel within such system, the profile of their elution is affected. The faster, central part of capillary is accessible for macromolecules of all sizes, while the large polymer coils cannot enter the slow current near the walls of the capillary. As a result, the larger macromolecules situated in the center of capillary move quicker than the smaller ones on average. This effect can be employed for separation of macromolecules according to their size. The column filled by small non-porous particles can be considered an array of capillaries and therefore the hydrodynamic phenomenon plays a role in all polymer LC methods, which apply packings of small particles. DiMarzio and Guttman [15–17] as well as Verhoff and Sylvester [18] studied features of flow of macromolecules within capillaries. Small et al. recognized potential of this phenomenon for solving the separation problems, which could not be executed by exclusion mechanism [19–21]. Brownian motion cannot help escaping the pores of column packing for species larger than 40 nm. Small coined the name “hydrodynamic chromatography” to the process [20]. Peculiarities of hydrodynamic chromatography and its applications were studied by numerous authors and the results can be found in several review papers [22–28]. Despite its relatively low separation selectivity, hydrodynamic chromatography can provide valuable service in the separation and characterization of giant macromolecules, capsules, micelles, cells, latices as well as inorganic particles. The important advantage of hydrodynamic chromatography is the possibility to apply single eluent, which fairly simplifies effluent detection. Separation of very large flexible macromolecules by hydrodynamic chromatography can be interfered by the phenomenon called “slalom chromatography”. Several authors observed inconsistencies in the exclusion separation of polymers using column packed with small particles [29–37]. Very large macromolecules elute later than the smaller ones. This behavior was explained by the de-coiling of giant, flexible polymer species caused

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by shearing forces in the laminar eluent flow and their “snaking” around the particles of the SP. As a result, the large particles are decelerated and their elution rate is decreased. The process is called slalom chromatography and it is employed in separation of DNA and RNA [31]. The occurrence of this phenomenon can affect results of various polymer LC methods.

5.1.3 Field-Flow Fractionation Excellent theoretician and experimentalist J. Calvin Giddings proposed original concept of separation of macromolecules and particles through combination of flow and external field [38]. It is based on the phenomenon that appears in the flowing rivers. Caused by the combination of flowing water and gravitation, the particles of different sizes and weights, from the microscopic clays up to large stones gradually sediment on the river bottom. Giddings termed his method as field-flow-fractionation, FFF. The mechanism of analytical separation of dissolved macromolecules and particles includes a combined action of laminar flow of transporting liquid, eluent, along a channel and a perpendicular field. Various kinds of fields are applied by Giddings and his coworkers, as well as by his numerous followers, that includes gravitation, centrifugation, magnetism, electricity, or temperature [39–43]. Several review articles and book chapters comprehensively elaborate the basic concept and peculiarities of flow-based separation techniques [44–53]. Important progress into FFF brought the application of transversal eluent flow as a complementary “field” to sustain separation. In a simple arrangement, one wall of separation channel is substituted by a semipermeable membrane which allows the oust of the eluent but does not allow penetration of separated macromolecules or particles. Typical semipermeable membranes are made from cellulose or ceramic materials. The process is designated as asymmetric flow-field-flow fractionation (AF4). AF4 has been applied for high temperature separations too [54–57]. The important area of application of FFF and especially AF4 are large linear and branched macromolecules as well as various biological and synthetic particles. The advantages of FFF methods includes the absence of both shear degradation of macromolecules and unwanted enthalpic interactions among the system constituents, as well as the application of single component eluents, which simplifies detection of the effluent. Recently, application of thermal-field is also getting considerable attention in polymer analytics [48, 58–62].

5.2 Polymer—Column Packing Interactions The polymer LC methods in which the separation mechanism is based on enthalpic interactions of macromolecules with the SP are often called interaction chromatography (IC). The extent of interactions is profusely affected by the MP composition

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or temperature. In the case of oligomers up to molar mass of several hundred g mol−1 , the composition of MP can be constant and it may even contain just one liquid component. On the other hand, interactions between macromolecules and SP are very sensitive to the polymer coil size, shape, and composition. In order to safeguard its subtly control, very small changes of external conditions are required. They are usually achieved with gradual mixing of two and more different solvents under precise adjustment of their proper composition. This is termed as gradient of MP or eluent. In this book, this mechanism is termed as eluent gradient interaction chromatography, EGIC. As repeatedly stressed, the exclusion processes accompany most polymer LC methods based on enthalpic interactions, as far as porous particles of column packing are employed. The role of entropy in such separation processes may be only subsidiary but it should not be fully ignored. It may cause secondary dependence of polymer elution on its molar mass. A specific situation is attained, when the effects of entropy and enthalpy are analogously large so that they mutually compensate. This phenomenon is discussed in detail in Chaps. 7 and 8. In present Chapter, we will parse only the polymer LC methods dominated by enthalpy. Similar to the precipitation–(re)dissolution polymer LC, the other methods which employ the interaction between macromolecules and SP, sample is temporarily fully retained on the inlet of LC column and then gradually eluted. The danger of reduced sample recovery, which may be caused by such mechanisms is described in Sect. 5.1.1.

5.2.1 Eluent (Mobile Phase) Gradient Interaction Chromatography (EGIC) As repeatedly stated, the liquid employed in the polymer LC methods, which actively participates on the separation process is called mobile phase (MP). Eluent is the liquid, which only transports separated macromolecules along the chromatographic system. In this book, we used both terms interchangeably. Present Section deals with the discrimination of macromolecules based on the differences of their interactions with particulate material filled in a cylindrical tubing making the chromatographic column. The target of interaction of macromolecules with the porous column packing is its outer and inner surface, termed as the stationary phase. Column can also be filled with non-porous particles or monoliths can be employed. Alternatively, macromolecules can interact with the volume of a substance, which is deposited and usually chemically attached to the surface of the otherwise inert carrier particles or monolith. Accordingly, the separation mechanisms of polymer LC are called adsorption or enthalpic partition (absorption) (Chap. 2). The materials, on which the separation takes place are termed as adsorbent and absorbent accordingly.

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As stated, the control of separation of high molar mass polymers, for example binary polymer blends or statistical copolymers, usually requires at least twocomponent MP/eluent with continuously or stepwise changing composition in the form of a gradient. Consequently, we will employ the term eluent gradient interaction chromatography, EGIC. However, strictly, this method should be termed as mobile phase gradient interaction chromatography while eluent gradient should be solely for transport of macromolecules, for instance in hydrodynamic liquid chromatography and field-flow fractionation. However, in order to avoid any confusion, we will employ eluent gradient interaction chromatography, EGIC, to the methods in which composition of solvent mixture is changed (either continuously or step-wise) during the chromatographic run. For the sake of understanding, consider separation of two distinct kinds of macromolecules. As stated above, sample is injected into MP or eluent, which promotes its full retention near the column inlet. It is to be stressed again that the composition of sample solvent must ensure its full retention near the column inlet and prevent its breakthrough [63–66]. Subsequently, MP/eluent with increasing eluting power for one sample component is applied into column. It is a desorli in case of adsorption separation mechanism or a thermodynamically good solvent when enthalpic partition is applied. Consequently, one sample component starts eluting when in contact with eluent with composition, which enables its release. Another sample component is liberated later when eluent with increased elution power arrives. As a result, the sample constituents leave the column at different elution volumes and get separated. The first known attempts to discriminate proteins with help of enthalpic interactions were conducted by Porath [67]. Synthetic copolymers according to their composition were separated by Inagaki and coworkers [68–71] and Belenki and coworkers [72]. Inagaki’s and Belenkii’s groups used thin layer (planar, TLC) chromatographic layout. Later, Teramachi attempted to transfer TLC procedure into polymer LC column without success [73]. It took five more years till Teramachi and coworkers succeeded to separate copolymers with eluent gradient in the column arrangement [74]. They extended their study to several other copolymer systems [75, 76]. Thereafter, more researchers applied MP/eluent gradient polymer LC, which was called liquid adsorption chromatography even if the separation mechanism was enthalpic partition [77–83]. Theory and practical applications of separation of homopolymers and copolymers with mixtures of solvents is elaborated by research groups of Armstrong and Boehm [84–86], as well as Mourey and Snyders [87–90]. They considered effects of both polymer solubility and adsorption. In most copolymer separations, which employ the enthalpic interactions of the polymers with SP, size and composition of macromolecules mutually interfere. Evidently, above-mentioned polymer exclusion may disturb the separation as per composition. Therefore, numerous authors suggested application of small-pore column packings. Van Hulst and Schoenmakers compared the influence of pore size on separation of polymers by interaction chromatography [91]. They applied packing particles with different pore sizes in a range of 5–30 nm, as well as of non-porous silica particles, all of them bare and C18 bonded, and the monolithic column also. They showed dependence of the elution of statistical copolymers on the pore size of

5.2 Polymer—Column Packing Interactions

63

column packing. Non-porous, large pore materials, and monoliths showed insignificant molar mass elution dependence, separation is overall governed by copolymer composition. Numerous copolymers are successfully separated applying optimized experimental conditions such as column packing, separation mechanism, mobile phase composition and gradient shape. In these systems, the competitive effect of sample molar mass on separation according to copolymer composition was suppressed. The molar mass independent elution of copolymers was tentatively explained by the “barrier effect” of the front of mobile phase/eluent gradient [92, 93]. Knowledge about entropy/enthalpy compensation (Chap. 7) helped to understand particularities of polymer EGIC. Like precipitation—redissolution polymer LC, EGIC may also suffer from the incomplete sample recovery. Part of the injected sample, which was fully retained near the column inlet could not be fully released by MP with increased elution power. Unexpectedly, it is eluted by repeating eluent gradient without sample injection [94, 95]. Identically positioned chromatograms are produced as the original ones, however, with small sized peaks. It can be result of the “flower like conformation” of retained macromolecules, which are released from the column packing pores during second or third gradient run. This phenomenon may affect not only the actual but also the succeeding analyses. Surprisingly, this phenomenon has not attracted the attention of researchers so far. It may be solved or at least mitigated by applying a small “precolumn” packed by non-porous particles on which the injected sample is fully retained before it is eluted by the MP. Alternative, though more demanding, would be the employment of thin layer of non-porous particles of appropriate sorbent or SP at the column entrance. A detailed account of recent applications of EGIC is given in Chap. 10.

5.2.2 Full Adsorption—Desorption Polymer LC As shown by numerous authors, the continuous change of mobile phase/eluent composition, the gradient, applied to a packed LC column enables separation of complex polymers such as statistical and gradient copolymers according to their composition, and discrimination of polymers with distinct architecture. Similarly, the constituents of complex polymer systems, such as polymer blends can be separated with the help of gradual changes of the MP/eluent composition. The polymer sample can also be fully retained from a suitable liquid on a small device, a microcolumn filled by the appropriate material. One part of sample is selectively released by a narrow pulse of an appropriate liquid. A sudden change of eluent, a sort of a step gradient can also be applied for release of one component of the sample. The released sample fraction can be further analyzed by an independent on-line or off-line method. This approach is successfully applied to a series of polymer blends and copolymers [96–98]. The retention mechanism is adsorption

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and the technique is termed full adsorption–desorption, FAD [97–99]. The second characterization method was size exclusion chromatography, SEC and the entire procedure was designated FAD-SEC [99]. In agreement with the observations on precipitation—dissolution polymer LC and EGIC of polymers, a part of sample is retained within FAD column when porous silica gel is applied as its packing. The full sample recovery is achieved with the non-porous silica particles. The microcolumns with volumes from 0.9 to 5 mL are packed by 6 µm non-porous silica particles. The adsorption capacity of the particles was large enough to accommodate enough polymer sample for subsequent SEC analysis. Using appropriate adsorli, the adsorption process was fast enough to retain complex polymer sample in the microcolumn within less than a second. On the other hand, a short pulse of suitable desorli completely released the adsorbed macromolecules and transported them to the attached SEC column, which was flushed with tetrahydrofuran. FAD in comparison with other polymer characterization methods has high potential, especially in characterization of complex polymer systems such as polymer blends and block copolymers, which contain parent homopolymers. Instead of an FAD microcolumn, a narrow layer of adsorptive particles can be situated at the inlet of the separation column to trap the sample component [100, 101]. Evidently, the capacity of such protective material should be checked. The full retention procedure can help to solve numerous problems associated with analysis of contaminated industrial samples. The polymer LC employing enthalpic interactions between macromolecules and column packing is reviewed by several authors [102–110].

5.3 Coupled Methods of Polymer Separation The selectivity of SEC separation can be enhanced by controlled addition of enthalpic interactions and vice versa. In this case, both exclusion and interaction processes are coactive while enthalpic interactions still prevail resulting in elution of the polymers after the void volume of the column. The situation is practical for oligomers having a strongly adsorbing monodisperse block or end-group, a typical example is fatty alcohol ethoxylates. Strong interaction of end-group results in elution of oligomers after the void volume of the column in “interaction region” but the elution follows SEC order. The basic form of the dependence of elution volume on molar mass (log M vs. VE ) remains same but the elution volumes increase with decreasing molar mass [111]. Trathnigg termed this technique as “liquid exclusion-adsorption chromatography” [112–118]. Enthalpy and entropy are coactive and not fully compensated in this essentially an isocratic mode of liquid chromatography of polymers.

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99. Nguyen SH, Berek D (1997) Full adsorption-desorption/SEC coupling in characterization of complex polymers. Abstr Pap Am Chem Soc 214(SEP):93-PMSE 100. Berek D, Nguyen SH, Hild G (2000) Molecular characterization of block copolymers by means of liquid chromatography: I. Potential and limitations of full adsorption–desorption procedure in separation of block copolymers. Eur Polym J 36(6):1101–1111. https://doi.org/ 10.1016/S0014-3057(99)00178-0 101. Nguyen SH, Berek D, Chiantore O (1998) Reconcentration of diluted polymer solutions by full adsorption/desorption procedure—1. Eluent switching approach studied by size exclusion chromatography. Polymer 39(21):5127–5132. https://doi.org/10.1016/S0032-386 1(97)10192-6 102. Belenkii BG (2012) Modern liquid chromatography of macromolecules. Elsevier 103. Trathnigg B (1995) Determination of MWD and chemical-composition of polymers by chromatographic techniques. Prog Polym Sci 20(4):615–650 104. Kilz P, Pasch H (2006) Coupled liquid chromatographic techniques in molecular characterization. In: Encyclopedia of analytical chemistry. Wiley, New York. https://doi.org/10.1002/ 9780470027318.a2018 105. Berek D (2000) Coupled liquid chromatographic techniques for the separation of complex polymers. Prog Polym Sci 25(7):873–908 106. Pasch H, Trathnigg B (2013) Multidimensional HPLC of polymers. Springer, New York. https://doi.org/10.1007/978-3-642-36080-0 107. Malik MI, Pasch H (2014) Novel developments in the multidimensional characterization of segmented copolymers. Prog Polym Sci 39(1):87–123. https://doi.org/10.1016/j.progpolym sci.2013.10.005 108. Chang T (2003) Recent advances in liquid chromatography analysis of synthetic polymers. Adv Polym Sci 163:1–60 109. Malik MI, Pasch H (2021) Chapter 1—Basic principles of size exclusion and liquid interaction chromatography of polymers. In: Malik MI, Mays J, Shah MR (eds) Molecular characterization of polymers. Elsevier, pp 1–59. https://doi.org/10.1016/B978-0-12-819768-4.000 07-5 110. Chang T (2021) Chapter 3—Temperature gradient interaction chromatography of polymers. In: Malik MI, Mays J, Shah MR (eds) Molecular characterization of polymers. Elsevier, pp 97–128. https://doi.org/10.1016/B978-0-12-819768-4.00009-9 111. Berek D, Bakoš D (1974) Effect of solvent on the separation of oligomer polyethers on sephadex LH-20. J Chromatogr A 91:237–245. https://doi.org/10.1016/S0021-9673(01)979 03-8 112. Trathnigg B, Rappel C, Raml R, Gorbunov AA (2002) Liquid exclusion-adsorption chromatography: a new technique for isocratic separation of non-ionic surfactants; V. Twodimensional separation of fatty acid polyglycol ethers. J Chromatogr A 953(1–2):89–99 113. Trathnigg B, Rappel C (2002) Liquid exclusion-adsorption chromatography, a new technique for isocratic separation of nonionic surfactants - IV. Two- dimensional separation of fatty alcohol ethoxylates with focusing of fractions. J Chromatogr A 952(1–2):149–163 114. Trathnigg B, Kollroser M, Rappel C (2001) Liquid exclusion adsorption chromatography, a new technique for isocratic separation of nonionic surfactants; III. Two-dimensional separation of fatty alcohol ethoxylates. J Chromatogr A 922(1–2):193–205 115. Trathnigg B, Gorbunov AA (2001) Liquid exclusion-adsorption chromatography: new technique for isocratic separation of nonionic surfactants I. Retention behaviour of fatty alcohol ethoxylates. J Chromatogr A 910(2):207–216 116. Trathnigg B (2001) Liquid exclusion-adsorption chromatography, a new technique for isocratic separation of nonionic surfactants II. Quantitation in the analysis of fatty alcohol ethoxylates. J Chromatogr A 915(1–2):155–166 117. Berek D (2001) Liquid chromatography of macromolecules at the point of exclusionadsorption transition. Mater Res Innovations 4(5–6):365–374 118. Baran K, Laugier S, Cramail H (2000) Liquid chromatography of polymers at the exclusionadsorption transition point: physicochemical interpretation. Int J Polym Anal Charact 6(1– 2):123–145

Chapter 6

Conventional Entropy-Controlled Methods of Polymer Liquid Chromatography

This chapter is devoted to the polymer LC methods, in which the processes of permeation and exclusion of macromolecules into/from the porous column packing particles play a decisive role. For obvious reasons, we will focus on the size exclusion chromatography (SEC) also called gel permeation chromatography (GPC). Size exclusion chromatography is not the major focus of the current book. However, due to its importance otherwise and its applications in multi-dimensional chromatographic setup a brief discussion is presented up to necessary details. In case of polymer LC methods governed by enthalpic interactions of macromolecules, entropy is often present as an unavoidable partner in the “separation process”. On the contrary, in most polymer LC methods, which are based on the entropy-controlled permeation/exclusion separation mechanism, enthalpic processes are not spontaneously present. This is the case of “ideal size exclusion chromatography”. However, enthalpic interactions may accidentally or purposefully take part in SEC measurements to constitute “real size exclusion chromatography”. These undesired enthalpic effects should be prevented that may otherwise considerably affect precision of the results. The following Chapter is divided into two sections. The former one is devoted to methods, in which entropy-controlled processes fully dominate and presence of enthalpic effects is considered as drawback. The second section briefly overviews the procedures, in which enthalpy is intentionally added to the permeation/exclusion separation mechanism, which still preponderates. The third option, the deliberate controlled combination of entropy and enthalpy enables their mutual compensation. Such approach represents an important, modern tool to accomplish specific polymer separations.

© The Author(s), under exclusive license to Springer Nature Switzerland AG 2023 M. I. Malik and D. Berek, Liquid Chromatography of Synthetic Polymers, Physical Chemistry in Action, https://doi.org/10.1007/978-3-031-34835-8_6

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6.1 Size Exclusion Chromatography Without any doubt, size exclusion chromatography is at present, and will be, the most widely employed method for molecular characterization of synthetic polymers, especially considering determination of their molar mass average and distribution. It is hardly possible to find an academic or industrial laboratory devoted to polymer synthesis, modification or application, which does not employ this method. The enormous importance of SEC for polymer technology is also evident. Most polymeric materials leaving production are analyzed by means of SEC. However, as outlined above, SEC deserves considerable attention not only due to both its high importance and wide applications but also because its separation mechanism plays a role in several advanced polymer LC methods, to which this book is primarily devoted. In discussion about SEC, it has to be repeatedly stressed that decisive separation parameter is the sizes of polymer coils or nanoparticles. Numerous research papers, books and review articles dedicated to SEC are available [1–9]. Most of them contain great number of references of articles devoted to theory, instrumentation, processing of results and applications of SEC. Recently, comprehensive discussion on modern approaches of multi-detector size exclusion chromatography is presented in an interesting monograph by Striegel et al. [3]. In a critical review article, both positive and negative sides of SEC are presented [10]. It shows that this outstanding, extremely useful, simple, robust, and repeatable method may produce erroneous results if appropriate attention is not paid to the conditions of measurement and to the data processing. Only elementary features of SEC are presented in this chapter along with a brief description of its theory and mechanism. On the other hand, attention is paid to practical aspects of the method, which are important for its users. Unconventional, less-known and seldom SEC applications are presented. The instrumentation, most experimental procedures, and basic data acquisition of SEC are identical with other polymer LC methods in principle. Herein, these details will be discussed mainly regarding specificity to SEC. Different is the SEC data processing, which may affect precision and accuracy of the final results. The SEC method applications are also assessed from the viewpoint of their employment in the new, advanced polymer LC especially in two-dimensional setup. The most important SEC outcome is the polymer molar mass average and distribution, majority of the method applications are devoted to their determination. Further important results of SEC are related to polymer molecular characteristics, for example information about shape of macromolecules such as branched or cyclic structures. Other SEC outcomes include knowledge on size of nanoparticles present in the system, supplementary often only semi-quantitative data about copolymers, as well as comprehension of some complex processes taking place in polymer systems. As stated, the fundamental separation mechanism of the SEC method is permeation and exclusion of macromolecules into/from a set of porous particles. Therefore, some authors denoted the method “network limited separation” [11]. The basic

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events are accompanied by several processes often reflecting operational parameters, which are called secondary effects. The latter may affect the obtained data and thus also precision of the results. We will briefly elucidate backgrounds of both fundamental and collateral SEC events. They should be considered in selection and control of applied materials, experimental conditions, as well as in data processing and evaluation. This is needful when the aim of measurements is focused on obtaining highly precise and absolute data. However, the purpose of many practical SEC measurements is just generation of elementary, qualitative information about the given polymer. In such a case, the meticulous adherence to the strict measurement protocol may be superfluous and the user should decide about details of measurements.

6.1.1 Origin and First Stage of Size Exclusion Chromatography The first attempts to separate molecules according to their dimension are dated back to fifties of twentieth century. Deuel et al. [12], Gregory and Craig [13], and Wheaton and Baumann [14] published their observations about size-based separation of various low-molecular substances on ion-exchanging resins and on polysaccharide particles. Lathe and Ruthven described size-based separations of different proteins on the starch particles [15–17]. Separation of proteins according to their size on cross-linked dextran have been demonstrated by Porath and Flodin [18–20]. They termed the method as “gel filtration” and later the term “chromatography” was added. Porath and Flodin are considered as the inventors of the method based on permeation and exclusion of macromolecules into/from porous bodies. Later, Vaughan reported fractionation of polystyrenes (PS) based on the “filtration” principle [21], Brewer used vulcanized natural latex to separate organic polymers dissolved in low paraffins [22, 23], and Determann applied cross-linked PMMA for separation of PS [24–26]. The breakthrough in the wide application of exclusion-based method for separation of synthetic polymers was experiments of Moore [27]. He noticed that linear polystyrenes of various molar mass eluted in different volume of aromatics or chlorinated liquids from the column packed by porous polystyrene particles. The latter were intended as raw materials for production of ion-exchangers. Evidently, macromolecules of distinct size were mutually separated. Moore comprehended the significance of his observation and proposed the term “gel permeation chromatography” for this separation procedure. His results attracted considerable attention and opened the way to rapid acceptation of the method. Numerous meetings of academic and industrial researchers devoted to dissemination of technical information helped fast spread of the procedure. Important contribution to the method acceptance brought also invention of the flow-through differential refractometer, which enabled monitoring the non-UV absorbing polymers in the column effluent.

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The method successively obtained various names. Apart from the original terms gel filtration (chromatography) and gel permeation chromatography, there were suggestions like “gel chromatography”, “steric exclusion chromatography”, “molecular sieve chromatography”, and “exclusion liquid chromatography”. The group of Bly proposed and via ASTM enforced to replace the designation of Moore “gel permeation chromatography” and its short version “gel chromatography” with the term “size exclusion chromatography” [28–30], which was eventually accepted by IUPAC and now widely used. At present, however, the name gel permeation chromatography is also employed often.

6.1.2 Theory of Size Exclusion Chromatography Numerous researchers attempted to quantitatively describe processes taking part in the SEC column. The aim was to develop a universal theory of pore permeation and exclusion-based polymer separation. The general representation of relation between size of the polymer coils and the volume of liquid needed for their release from the SEC column, their elution volume was successful but creation of comprehensive relations, which could enable a priori calculation of their mutual dependence has not yet been successful. Several different processes take place when solution of macromolecules is in contact with porous particles (see Chap. 2). In case of polymer LC, we usually consider a column packed with porous particles, along which a narrow zone of dissolved macromolecules of distinct size, structure and composition is transported with an appropriate liquid called eluent or mobile phase. The polymer sample solution undergoes mixing and diffusion in the inter-particle volume, in the column endpieces, and in the connecting capillaries. These parasitic processes impair separation and should be suppressed as much as possible. Macromolecules present in sample solution selectively permeate the pores and collide with the packing walls. As subsequently shown, the process renders polymer separation based on size. Pore walls may attract macromolecules or they can only form an indifferent barrier preventing intrusion of polymer species. The pore permeation, which theoretically does not include any attractive interactions between macromolecules and column packing, is often termed as “ideal”. This is the target situation in size exclusion chromatography and is termed as ideal SEC, the value of term H in Eq. 3.2 is zero. The differences of pore permeation and exclusion of distinct macromolecules are responsible for their separation. Exclusion of macromolecules from the outer surface of packing particles is less important, and its influence is usually neglected. Due to their pore permeation, the progression of macromolecules along the column is selectively decelerated. Very large macromolecules, which do not fit into any pore may be fully excluded from the inner volume of the column packing and elute freely in the interstitial (dead) volume of column. On the contrary, the smallest sample constituents may permeate all pores and “accompany” the eluent molecules. As shown in numerous experiments, a sizeable segment of large, pore excluded macromolecules may diffuse into

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the pores to be decelerated and even teared due to strain of the eluent flow. Very big macromolecules, which are completely excluded from the pores, may de-coil and flow around packing particles in the form of a “slalom” that results in an increase in their elution volume [31]. Evidently, eluent molecules permeate all the pores and their transport is the slowest. The extent of permeation, which depends on both size of the pores of the column packing and the size of the macromolecules in solution, affects volume of the eluent or mobile phase needed for the elution of sample molecules from the column. It is their elution volume, VE . Thermodynamic quality of solvent for macromolecules determines the polymer coil sizes that subsequently affect their pore permeation process. To stress again, the elution volumes of macromolecules in the ideal SEC depend exclusively on their effective size in the mobile phase. Enthalpic interactions between polymer and column packing are absent. However, several secondary effects and especially enthalpic phenomena may be active in the real SEC. Most surveys of the SEC process consider structure of macromolecules in the solution in form of statistical coils, which exhibit certain conformational and orientational entropy. The size of polymer coils reflects both their molar mass and interaction with the solvent molecules. Interactions among polymer segments inside its coils also plays important role, which depend on their chemical composition. The situation is rather complex in case of copolymers because the dimensions of the segments of the same and distinct compositions play a role. Evidently, the situation is different for the alternating copolymers, for the block copolymers, and for the statistical copolymers with different blockiness (length of their micro-blocks). Differences in the topology of macromolecules for instance, linear versus cyclic versus branched versus stereo-regular structure render another obstacle in the quantitative expression of the overall effective size of the polymer coils in the solution. The situation is further complicated due the presence of distributions of all molecular characteristics of synthetic macromolecules. For these reasons, the quantitative description of permeation/exclusion processes on the molecular basis and prediction of elution volumes of macromolecules is hardly possible. Concerning the SEC separation mechanism, the idea of Casassa is widely accepted [32–36]. The basic force, which pulls dissolved macromolecules from the plug of sample solution into a pore is the tendency to equalize polymer concentration, the chemical potential of the system in the interstitial volume and the pore volume. During the pore permeation, polymer coils are compressed. Their conformational mixing and possibly also orientational entropy decreases. The loss of entropy of the macromolecules controls the extent of their pore permeation. Hence, entropy controls pore permeation and partial exclusion of macromolecules that is the basic separation mechanism of SEC [35–38]. The pore permeation process of macromolecules of a given size is concluded when the loss of their entropy outbalances the pulling force. Larger macromolecules cannot enter pores available for smaller species because the loss of entropy would be too large. Next, the zone of sample solution continues its transport along the column and the pulling of macromolecules is replaced by their withdrawal. Macromolecules sequentially return to eluent depending upon their size.

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Large macromolecules, which entered smallest volume of pores, leave the packing as first. As a result, polymer species of different size will elute in different eluent volumes and get separated. It can be concluded that size exclusion of macromolecules is the entropy controlled (NOT entropy driven) process. The macromolecules are distributed between two volumes of the eluent. One of them is situated in the interstices among packing particles and another one exists within the pores of the particles. Consequently, one can speak about entropic partition. Due to their exclusion, transport of polymer coils is accelerated in comparison with molecules of both their original solvent and eluent/ mobile phase. In spite of that, exclusion process of macromolecules is sometimes generally denoted “retention”. Besides permeation and exclusion processes, the hydrodynamic phenomena may affect the elution of macromolecules from the column packed with small particles. The corresponding phenomena is evaluated in detail initially by Small [39, 40], and Di Marzio and Guttman [41–43], while more recent work is done by Striegel and Brewer [44–50].

6.1.3 Column Packings, Mobile Phases/Eluents for Size Exclusion Chromatography The peculiarities of the basic parts of high-performance liquid chromatography equipment such as mobile phase containers, thermostats, degassers, pumps, and sample injectors are discussed in Chap. 4. A detailed discussion is devoted to the components generally applied in polymer LC, column packings, eluents (mobile phases), and detectors. In this section, the major focus of discussion will be the column packings and eluents (mobile phases), which are specially employed in SEC. Soon after inception of size-based separation of macromolecules, intensive research has been launched of materials, theory, experimental procedures, and applications of the method. The prime attention was devoted to the column packings. Formally, they can be divided into three groups: – Soft, homogeneously crosslinked natural or synthetic polymers – Semi-rigid heterogeneously crosslinked synthetic polymers – Rigid inorganic materials, especially silica gels, alumina, porous glass, amorphous carbon and graphite. The porous structure is of utmost importance for the column packings suitable for exclusion separations [9]. The size of pores in homogeneously crosslinked polymers depends on the average length of polymer chains between the crosslinking points and on extent of their solvation with mobile phase molecules. They behave similarly as concentrated polymer solutions. Separated macromolecules are distributed between transported solution and a quasi-liquid gel phase. Such systems present

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interesting object for theoretical studies of the behavior of macromolecules in twophase systems. Homogeneously crosslinked soft gels have been applied for separation of proteins and oligomers. However, their mechanical stability is too low for most practical polymer LC applications. There were also attempts to fill the pores of mechanically strong particles with soft polymer network as carrier. The pores of semirigid, heterogeneously crosslinked polymers are formed with voids between primary spherical nano-sized globules. Generally, larger globules render larger pores. The structure often contains irregularities, voids, which decrease their mechanical stability. Basic concept of synthesis of mechanically stable porous polymer particles was created in the course of development of ion exchangers. They are prepared by copolymerization of a vinyl monomer with the crosslinking agent, usually a comonomer containing double vinyl bond. Alternatively, single monomers containing two double bonds are employed. Polymerization is performed in presence of diluents, the liquids which cause phase separation of formed copolymer, that results in the creation of globules. In this class, porous polystyrenes crosslinked with divinylbenzene (PS/DVB) and sole crosslinked divinylbenzene (DVB) gels are widely applied in SEC of different polymers. It is assumed that the PS/DVB and DVB based SEC column packing are highly noninteractive concerning polar macromolecules. Surprisingly, some commercial PS/ DVB and DVB column packings exhibit rather high enthalpic interactivity toward medium polar poly(methyl methacrylate)s (PMMA) in low polarity eluent such as toluene, and polar poly(2-vinyl pyridine) is adsorbed from THF. Polyacrylate based porous particles found applications in separation of highly polar polymers and also water-soluble macromolecules [51]. The pores of most hard porous materials are formed by voids among irregularly arranged amorphous nano phases (silica gel) or crystals (alumina-corundum). Silica gels employed in polymer LC are formed by heterogeneous polycondensation of silicic acid often in the presence of porogen or by agglutination of silica sol particles. Their high mechanical stability proved favorable for use of very small particles even below 2 μm at ultra-high pressures. Silica gel exhibits rather strong enthalpic interactivity with polar polymers. It can be suppressed by surface modification with appropriate organic groups or with application of polar eluents such as dimethylformamide (DMF). Suitable additives such as triethylamine (TEA), and Tetramethyl ethylenediamine (TMED) are also reported to be used to avoid unwanted enthalpic interactions [52, 53]. A favorable structure of porous materials for polymer LC should exhibit large pore volume and good mechanical stability. It can be created by the incomplete phase separation of two highly viscous immiscible liquids, for example borosilicate glass at high temperature. After cooling, B2 O3 phase is dissolved by a base, while porous SiO2 phase remains untouched. Similar structure can also be formed by cautious leaching of silica gel by alkalis or HF. Unfortunately, porous glass particles cannot be formed in spherical shape and leached silica gel exhibits low mechanical stability. Effective pore volume of column packings for polymer LC is rather small. Their pore volume lies in the range of 50–80% and large part of packing occupies the interstitial column volume, usually about 40%. From the viewpoint of exclusion

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separation, the latter represents a sort of “waste space”, also called “dead volume”. As result, the separation selectivity of SEC is rather low. Schure and Moran have compared SEC performance of totally porous and superficially porous particles [54]. They claim that the lower elution range (smaller effective pore volume) of superficially porous particles can be compensated by their higher efficiency and specific resolution. The selection of SP for SEC depends upon the polymer to be analyzed. Styrene– divinylbenzene (SDV) is the preferred choice for organic SEC while modified silica or cross-linked hydrophilic polymers are used for aqueous SEC. SEC columns filled with different column packings are available which further vary in their pore sizes. Initially single pore-sized columns were available that covers only a limited range of the molar mass, two decades of molar masses (e.g., 103 –105 g/mol). Recently, SEC columns are available that contain particles having different pore sizes. These columns are termed as linear or mixed SEC columns and offer analysis of a wide molar mass range compared to single pore columns. Several reviews and books on SEC column packings have been published [9, 55, 56]. They contain important relevant information that may be very helpful in appropriate column selection. On the other hand, the researchers rarely produce their own column packings anymore and the synthesis of commercially available, efficient SEC column packings is a strictly confidential issue. Concerning elution of sample along the SEC column, the transporting liquids only seldom actively participate on the separation process itself so that we mostly suffice with term eluent and reserve the designation mobile phase for specific cases only. However, both terms eluent and mobile phase are used interchangeably in this book. The basic information about liquids employed as polymer LC eluents and mobile phases are given in Sect. 4.3. Therefore, only some basic data are repeated here. Tetrahydrofuran, THF was introduced already in the initial stage of SEC development and it still dominates in most standard applications. Both the outstanding properties and also shortcomings of THF are described in detail in Sect. 4.3. Some liquids can help elution of polymers which are insoluble or poorly soluble in THF [57]. Expensive solvents like 1,1,1,3,3,3-hexafluoro-2-propanol, HFIP and 2,2,2, trifluoro ethanol, TFE, which can be mixed with some other solvents, for example with chloroform, CHCl3 , can also be used [58]. Solvents with different polarity are applied in SEC with added enthalpic interactions. The mobile phase additives may properly adjust interaction of macromolecules with the column packing. This is especially important in case of advanced interaction polymer LC, which will be discussed in Chap. 7 and onwards. Dimethyl formamide (DMF) and diethyl formamide (DEF) are applied as eluent for high polarity polymers [59, 60]. In this case, specially packed PS/DVB or DVB columns should be employed. The low polarity polymers like PS and poly (meth)acrylates are retained on PS/DVB or DVB column in DMF and DEF. It is useful to prefer polar SEC column packings for polar macromolecules. Solvents with high boiling points such as 1,2 dichlorobenzene, decalin, 1,2,4 trichlorobenzene, as well as cyclohexanol found application in high-temperature SEC [61–63].

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6.1.4 Relation Between Elution Volume and Polymer Molar Mass in Size Exclusion Chromatography As repeatedly stressed, separation mechanism of SEC is based on selective column packing pore permeation and exclusion of polymers. It depends on dimensions of the polymer coils in the solution. Therefore, the primary product of SEC, elution volumes of macromolecules, reflect their size. This means that SEC alone is not absolute method, which directly provide polymer molar mass averages and distributions. SEC elution volumes must be converted into molar mass data by a suitable calibration. Alternatively, a detector can be attached to the separation column, which continuously monitors molar mass of the macromolecules in the effluent. Unfortunately, such detectors necessitate single-component eluents. Therefore, they are only exceptionally applicable in the advanced methods of interaction polymer LC, to which this book is mainly devoted. The SEC calibration is a dependence of polymer elution volume VE on its molar mass M. For practical reasons, the SEC calibrations are plotted with logarithm of M on the abscissa. To acquire the calibration dependence, a series of narrow molar mass distributed “standards” are successively injected into SEC column and their chromatograms (elugrams) are recorded, dependences of sample molar mass on elution volume are determined. Maximum VE values on the peak-shaped dependences are considered. They are called peak elution volumes. Only few suitable linear calibration standards are available such as polystyrenes, poly(methyl methacrylate)s and polyethylene oxides (for organic solvents), as well as pullulans and dextran (for aqueous eluents). Polyethylene glycols are often employed for VE calibration of oligomers. Narrow polyethylene fractions prepared by large scale SEC are no more available. In the same column and eluent, as well as under otherwise identical experimental conditions the elution volumes of linear polymers depend on their chemical nature. It means that the log M versus VE calibration dependence obtained with one kind of calibrant cannot be directly applied for another polymer to calculate its absolute molar mass. Therefore, constructions of calibration dependences using chromatograms of macromolecular substances with broad, known molar mass distribution were proposed in the first stages of SEC development for organic [64] and also for water-soluble polymers [65]. Courses of calibration dependences are also influenced by the nature of the eluent. Several researchers looked for parameters, which would universally describe dependence of polymer elution volume on its molar mass irrespective of calibrant and eluent. Such parameter would permit employment of SEC results obtained with one kind of polymer and mobile phase for another polymer–solvent system. For instance, Moore and Hendrickson proposed dependence of ratio of polymer molar mass and length of fully extended polymer chain [66], and Mayerhoff proposed product of M1/2 with effective hydrodynamic radius [67], which can be determined from limiting viscosity number or by means of dynamic light scattering measurement. Eventually, Benoit et al. managed to solve the problem [68, 69]. Their universal calibration parameter called also hydrodynamic volume of macromolecules is

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a product of polymer molar mass M and its limiting viscosity number, [η]. Benoit’Asisclears concept proved wide applicability as “universal calibration parameter” and it is used in most softwares for polymer molar mass determination without applying detectors, which monitor molar mass of the polymers in the column effluent. The shifts between the courses of [η] M versus VR dependences for different polymer-eluent-column packing systems were even employed for assessments of interaction of macromolecules with the SEC column packing, as well as for estimation of mutual interactions, and incompatibility of jointly injected distinct polymers [69]. When the absolute values of polymer molar mass are not needed, available calibration standards and their [η] M data can be directly used for molar mass calculation and the resulting relative data are, according to IUPAC, called “polystyrene (or poly(methyl methacrylate, etc.) equivalent molar mass”. As evident from preceding discussion, the permeation/exclusion-based separation of macromolecules almost exclusively takes place in the pores of the column packing. The hydrodynamic processes and surface exclusion of macromolecules only slightly assist the SEC separation outcome. Both the average and the distribution of pore sizes of the column packing determine magnitude of dimensions of macromolecules, which undergo separation. It identifies the selectivity of process. In other words, there is direct relation between pore size distribution of the column packing and the range of molar mass of the macromolecules, which can be separated. Mutual connection between particular volumes is evident from Eq. 6.1. VE = Vo + K sec · V p

(6.1)

where VE is elution volume of separated macromolecules, V0 is the interstitial (dead) column volume, Ksec is the distribution coefficient, and Vp is the pore volume. As stated above, V0 lies in the range of 40–50% of column volume, Ksec acquires values between 0 and 1, and pore volume assumes from 50 to (rarely) 80% of particles constituting column packing. It is clear that if the packing pore size distribution within a SEC column is narrow, its molar mass separation range is limited. To extend it, several columns with a distinctly different pore sizes can be connected. On the other hand, to increase effective pore volume and augment SEC separation selectivity a tandem of identical columns can be applied. In both cases, time of experiment and volume of consumed eluent increase substantially. Instead of applying several identical columns, sample can be repeatedly eluted along the same column. The procedure is called recycling. Several authors studied peculiarities of recycling procedures, especially in the first stages of the SEC development [70]. In most recycling procedures, effluent from the first column was returned through the pumping system and therefore its zone got significantly broadened. An interesting recycling procedure was proposed Biesenberger et al. [71]. Two columns are mutually connected with a recycling valve, which alternatively directs column effluent between them without passing the pumping system. The recycling procedure can be repeated unless the first sample fraction

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Fig. 6.1 SEC calibration curve of PS on SDV columns in THF of different pores sizes (A) single pore size columns, (B) Linear or mixed columns; Data provided by Polymer Standards Service [73]

would reach the last one. With the development of high-performance SEC columns, recycling procedure is rarely employed. As is clear from the above discussion, the SEC separation directly depends on the distribution of column packing pores. Linear size-separation range of macromolecules should necessitate continuous pore size distribution. Yau et al. have shown that the combination of bimodal, mutually not overlapping pore size packings enables surprising extension of linear log M versus VR calibration dependence [72]. These bimodal pore sized SP are available commercially. Dependance of elution volume on molar mass of narrow PS standards on SDV column in THF as eluent on various pore sizes in demonstrated in Fig. 6.1. Evidently, the dependance of elution volume on molar mass is not strictly linear in case of single pore sized columns Fig. 6.1A. Moreover, only two decades of molar mass (e.g., 103 –105 g/mol) can be separated. Presently the “universal” SEC columns became popular. They contain particles with pores of different sizes so adjusted that they provide linear or nearly linear calibration dependence of log M versus VE . It covers broad molar mass range, usually from oligomer up to several millions g mol−1 , Fig. 6.1B. However, the selectivity of separation is considerably sacrificed while using columns of multiple pore size distributed packing. In conclusion, general performance of SEC is rather limited. It is often necessary to choose what to prefer—separation selectivity or range of molar mass. The operator must decide, whether just the indicative or the high precision data are needed. This also applies to the processing of the results.

6.1.5 Secondary Effects in Size Exclusion Chromatography Both position (elution volume) and width of SEC peaks are mainly employed for determination of molar mass average and distribution of polymer sample. However, apart from size of the polymer coils, both the peak positions and their widths can

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also be affected by several specific phenomena. Important are undesired enthalpic interactions between system constituents, discussed in detail in the Sect. 6.1.7. SEC results can also be biased by specific impacts called experimental or operational variables or “secondary effects”. They were studied in detail in the first stages of the method development. The main effort was focused not only on understanding, evaluation and removal of secondary effects but also on their employment. Several procedures were evolved to mitigate or fully overcome the impact of secondary effects by improvement of equipment and by appropriate data correction. Progress in SEC instrumentation, mainly concerning the column efficiency, significantly reduced the chances of negative consequences of secondary effects. Although, secondary effects are mostly overlooked now, they can still affect results of routine SEC measurements. Especially susceptible are the procedures, in which SEC is combined with other polymer LC methods to create advanced interaction polymer LC. Therefore, a brief overview of elementary secondary effects of SEC with selected literature references will be presented in this section. Injected sample size, both its volume vm and concentration ci , can affect its elution volume VE . SEC sample volume is usually predetermined by volume of the injector loop. It is increased by the operator when the peak size is insufficient for its unambiguous detection, as well as in preparative separations. Net effect of vm on VE in routine SEC measurements is usually small and it is neglected. Still, there is a limit in vm , which should not be exceeded [10, 74]. Changes of VE with ci result from different viscosity of the injected sample and the eluent [75], from mutual interaction of sample macromolecules in injected solution [76], from osmotic effects in the system [77–79], from ionic interactions in case of charged macromolecules [80, 81], and from phenomenon of “secondary exclusion” [82, 83]. As a rule, SEC elution volumes linearly increase with rising sample concentration, VE –ci dependences are usually linear. Their slopes Î decrease with both dropping molar mass and declining thermodynamic quality of the eluent toward the separated polymer. As result, ci value may affect the determined molar mass of the polymer. SEC peaks become distorted when column is overloaded. Even the peak splitting, called viscous fingering may appear [84]. Several authors attempted to quantitatively describe backgrounds of SEC concentration effects and to express connection between eluent nature, injected polymer concentration, and peak elution volume [85]. The corrections of the impact of sample concentration on elution volumes were also developed [86]. To remove influence of sample concentration in some further calculations, the VE values extrapolated to ci = 0 were applied. In most common systems, temperature, T near the ambient one, only slightly affects SEC elution volumes. In general, rising temperature augments diffusion rate of the separated macromolecules and increases the separation efficiency. However, except for polymers, which undergo conformational transition due to temperature change, the coil sizes respond relatively little to varying T [87]. The value of intrinsic viscosity and thus polymer hydrodynamic volume is rather stable at temperatures, where most practical SEC separations are performed. On the other hand, in some

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polymer LC systems, temperature may strongly impact interaction between macromolecules, solvent molecules, and column packing. In any case, it is useful to thermostat eluent, injector, column, and detector for high precision SEC measurements. At least effect of temperature changes on eluent flow patterns is reduced. Hightemperature SEC (HT-SEC) measurements are performed at well-controlled temperature conditions in order to prevent local precipitation of polymers like polyolefins or polyamides [62, 63]. Similar to temperature, moderate change of linear eluent velocity has little though non-zero effect on polymer elution volumes [88]. Due to diffusion role in the separation process, SEC peaks become broader with growing elution rate [89]. Even medium size macromolecules can be degraded by friction at high elution rates. This is especially important for large macromolecules with molar mass over one million g mol−1 [90]. Large column packing particles and low eluent flow rates are to be applied. The optimization of experimental conditions was systematically studied by Aust et al. with the aim to minimize degradation of polystyrenes with molar mass up to 17,000 kg mol−1 [91]. They employed 20 μm column packing particles at different eluent flow rates, as well as at various injected sample volumes and concentration. The popular measure of efficacy of liquid chromatographic systems is its number of theoretical plates. It is determined by injection of appropriate low molecular probe and evaluation of the width of the resulting peak. The concept of theoretical plate was introduced into chromatography at the advent of this group of separation methods [92]. It is a hypothetical stage, in which two phases establish an equilibrium with each other. The number of theoretical plates of SEC column does not provide information about its separation selectivity. However, it offers knowledge about column packing conditions, help to detect possible loss of its homogeneity. Its sudden change may also indicate instrumental problems such as pumping failure or leaking eluent. The important problem of classical SEC separations was significant extra-column band broadening in the column end-pieces, detectors, and connecting capillaries. These effects are markedly diminished by using narrow connecting capillaries and improving design of column end-pieces and detectors. The intra-column SEC peak broadening is result of both diffusion and mixing processes, which accompany sample permeation/exclusion into/out of the packing pores. It also takes place in the interparticle column volume. Their evaluation is complicated because it includes the molar mass distribution of samples employed for evaluation since strictly uniform synthetic polymers do not exist. Several scientists tried to solve the problem by neglection or approximative expression of polydispersity of samples, which were employed for peak broadening evaluation. Theoretical approaches for peak broadening correction were proposed [93]. The solution brought the famous method of reversed flow by Tung [94]. Polymer sample is eluted to the mid of the column and then the flow direction is reversed. It is assumed that this reversal of flow direction eliminates the effect of polydispersity of the test sample. Within the last period of time, the SEC column technology is substantially improved. Important improvements present the small-sized, strictly spherical column packing particles with optimized pore structure, which are regularly deposited in the columns. As result, the SEC peak broadening phenomenon is substantially reduced.

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Due to increased sensitivity of detectors, both injected sample volume and concentration could also be markedly decreased. On the whole, impact of most secondary effects in SEC is strongly diminished. Moreover the “absolute” detectors, multiangle light scattering photometers and viscometers contributed to the increased precision of results especially those for homopolymers [3, 4]. The SEC data processing is almost exclusively done by computers and the advanced softwares inherently include basic SEC data corrections. Consequently, SEC became a routine method in many laboratories and less attention is paid to the good laboratory practice of measurements. According to recommendations, SEC columns should be frequently recalibrated in order to check their conditions. A broad test polymer sample with precisely known molar mass average and distribution is to be repeatedly injected into column followed by evaluation of invariability of results. Interacting and polar polymers such as poly(methyl methacrylate)s in THF provide appropriate service for this purpose. They unveil possible changes in the column packing geometry, pore structure and overall column interactivity due to retained macromolecules and eluent impurities. Some high precision SEC systems especially in industrial laboratories work uninterruptedly. Pumps are not stopped and eluent is continuously distilled under nitrogen and circulated. Once every few months the instrument is completely overhauled.

6.1.6 Applications of Size Exclusion Chromatography Separation of macromolecules according to their size is the basic task of SEC. The most important molecular characteristics of polymers, which can be directly determined with the help of SEC is the molar mass average and distribution. Hundreds of research papers and reviews, as well as numerous monographs, which describe corresponding applications are published on oligomers and polymers of different molar mass [1, 3, 4, 95]. The application area of SEC is very wide. The method provides invaluable service for all those who synthesize, modify and apply synthetic macromolecular substances. It became a sort of blessing of science and technology of synthetic polymers. It is however necessary to also mention the less bright sides of SEC. As explained earlier, SEC suffers from the limited separation selectivity, detector sensitivity, and sample capacity. Several secondary effects may bias the results. These features should not be overlooked when application potential of SEC is evaluated and the obtained results are processed and judged. Precision of SEC measurements, its intra-laboratory repeatability is generally very high. The scatter of molar mass values obtained with repeated measurements is usually negligible. This may evoke a false notion of high accuracy of method. Unfortunately, SEC exhibits high sensitivity toward both careful execution of measurements and appropriate data processing. Especially proper base-line and peak limits setting may be problematic [10, 96]. In practice, SEC became a widely used ordinary method. The experiments are frequently performed in an inappropriate way by not well-trained operators. The inherent pitfalls of method are ignored, and the results

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are incorrectly processed and interpreted. The risks of such situation are often overlooked. Consequently, the inter-laboratory reproducibility of SEC results often lies far from optimum and the obtained data may lead to erroneous conclusions. The accuracy of SEC results may be surprisingly low. Some SEC applications require particular experimental conditions due to limited sample solubility. Important group present high temperature SEC measurements of polymers, which are insoluble at ambient temperature [62, 63]. Apart from polymer molar mass average and distribution, some other valuable information can be obtained from the primary SEC separation data, especially with the help of suitable detectors and special software. These are longchain branching [97–99] cis–trans conformation of polyisoprenes, and tacticity of poly(alkyl metacrylates) [100]. The SEC data processing is straight-forward in case of homopolymers. It is almost exclusively done by computers. However, for the complex polymers such as statistical copolymers the data processing may be complicated. Usually, multi-detector systems are employed [3, 4, 101]. Considering complex (multicomponent) polymer systems such as polymer blends and block copolymers, which often contain parent homopolymers, SEC can only seldom directly provide complete data. The independent discrimination of sample constituents is usually required, which is followed by their individual characterization by means of two- and multi-dimensional polymer LC. Mutual separation of distinct kinds of macromolecules often requires advanced methods of polymer LC for complete characterization. As a rule, conventional SEC presents an important part of such multi-stage processes. A detailed account of SEC is beyond the scope of this book and interested readers can refer to numerous books, monographs, and reviews [1–9]. Herein, discussion on SEC is only limited to appropriate length for the sake of completeness of the topic and its applications in multi-dimensional LC system. As iterated repeatedly, SEC separation is based on differences of the hydrodynamic volume of different polymer chains in dilute solution. An appropriate calibration curve is then employed for information on the molar mass and molar mass distribution of the unknown sample. However, a substantial variation in the hydrodynamic volumes of different polymers of same molar mass in a solvent lead to significant errors [3, 8, 53, 59, 102, 103]. Hence, the calibration curve must be build using polymers of same composition and architecture for reliable results, which are not available mostly. In case of complex polymers, a SEC slice may contain numerous chains differing in their length, composition, architecture but having same hydrodynamic volume in that particular solvent. A dual detection approach may be employed, that work on principle of different response factors of different repeat units, to obtain chemical composition as a function of molar mass distribution. The combination of RI detector with UV [8, 102, 104, 105] and density detector [106–110] can provide useful information. It is important to mention here that dual detection approach is viable only for single solvent systems, as typical for SEC. An additional third concentration detector may be required for

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mixed solvent systems used in other LC methods. Mathematical treatment of the dual-detector approach is outlined by Pasch and Trathnigg [102, 111]. The direct correlation of elution volume with molar mass using only concentration detectors may be erroneous. Employment of molar mass sensitive detectors such a light scattering or intrinsic viscosity to monitor the SEC effluent along with concentration sensitive detector provides an accurate molar mass of any eluting fraction [3, 112–114]. Moreover, some less-common applications of SEC require attention. Exclusion separation mechanism is employed for polymer purification, by removal of lowmolecular or particulate impurities. Preparative SEC is applied for production of polymer samples intended for special purposes. The attempts to employ SEC for production of narrow molar mass distributed polyethylene standards proved economically unsustainable. Already in the first stage of SEC boom, numerous unconventional applications of method were proposed. Some of them are still occasionally employed, other are not in service any more. As a reminiscence, some of them are outlined below. Hummel and Dreyer proposed SEC for assessment of interactions between molecules of proteins and drugs [115]. They employed solution of protein P as mobile phase in which drug D was dissolved and injected. Two peaks appeared on the UV detected elugrams, large associates P-D eluted first. The second, negative peak, a “throw” belonged to the rest of the drug, which was partially consumed for association with protein. Its amount could be calculated from the size of the “throw”, called system peak. The method was applied by numerous authors for study of association of various biopolymers, either mutually or with low molecular ligands, as well as for assessment of complexation of synthetic polyelectrolytes bearing opposite charges [116, 117]. The extent of preferential solvation of a polymer dissolved in the two-component mobile phase can be determined with help of SEC [118–121]. Narrow pore column packings and differential refractometers are applied. Solvated polymer molecules elute at low VE . The peak of solvent, which is in excess consumed by preferential solvation of macromolecules forms the system peak, a vacancy with VE near Vi . The coefficient of preferential solvation can be calculated from its size. Surprisingly, the deficiency of preferentially solvated solvent elutes as a narrow, well-defined peak, not in form of a broad zone. Similarly, chemical reactions and physical interactions of biopolymers or synthetic polymers with low molecular reactants R or ligands L can be assessed. Sébille et al. used ternary systems polymer plus R/L plus solvent as a series of mobile phases [122]. Mobile phase could either play active role to support interactions or just serve as an eluent for transport purposes. Similarly, column packing does not participate on polymer—R/L interaction and its role was only to separate the interacting system constituents. Solutions of R/L with different concentrations without polymer were repeatedly injected into the column. Two peaks were observed on elugrams. One of them belonged to a deficiency of polymer and the second to excess of R/L. At a certain R/L concentration, its peak disappeared. This was just the amount of R/L, which formed complexes with polymer in equilibrium. Sébille

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termed this approach “saturation method”. Alternatively, column was flushed with mobile phase, which contained constant amount of both polymer and R/L [123]. Small amounts of R/L were gradually added to the injected solution. The original negative system peak belonging to deficient R/L, which was consumed by polymer, turned smaller and finally it became positive. The amount of added R/L when the system peak has just disappeared was the sought amount of R/L. The method was called “bulk solvent adjustment”. Finally, mobile phases containing both polymer and R/V were employed, whereas the concentration of dissolved polymer was changed [123]. The system peak disappeared at the desired value of polymer concentration. The approach was denominated as “eluent adjustment method”. The slope Î of the concentration dependence of VE depends on the thermodynamic quality of mobile phase toward polymer. Therefore, Î value indicate extent of polymer solvent interaction. The value of Î assumes zero if mobile phase is a theta solvent for polymer and this fact can be employed for determination of theta conditions [124, 125]. Three concentration dependences of VE of a polymer under study are measured in distinct solvent-nonsolvent mobile phases and the Î values are extrapolated to zero. Thus, SEC can substitute more exacting viscosity, light scattering, or osmotic pressure determination of theta conditions. If the mobile phase is a solution of polymer and a distinct macromolecular substance is dissolved and eluted, polymer–polymer interactions can be estimated from the slope of concentration dependence of VE [126]. The dependence between the slope Î of the VR –ci relation and product of second virial coefficient polymer–solvent, A2 with polymer molar mass M, A2 M is practically identical for different polymer–solvent systems. It enables estimation of unknown A2 value for a polymer from its SEC concentration dependence. Guixian et al. proposed method for determination of radius of gyration of macromolecules from the concentration dependences of their SEC elution volume [127]. Yoshikawa et al. estimated number of THF molecules solvating macromolecules of phenol–formaldehyde condensation products through SEC [128]. Extent of polymer aggregation does not depend on the polymer concentration. Using SEC, Lyngaae-Jorgensen determined extent of PVC aggregation in THF and thermal stability of aggregates [129]. Abdel-Alim and Hamielec found a correlation between extent of PVC aggregation and its tacticity [130]. Assessment of polymer association using SEC is much more complex. Extent of association of macromolecules in solution depends on polymer concentration. Associates decompose during their travel along the column due to sample dilution. Various procedures were proposed to follow this process and Brumbaugh and Ackers even introduced direct optical scanning of glass columns during elution of associated macromolecules [131]. There are several proposals to determine limiting viscosity numbers, intrinsic viscosity data and also constants of Kuhn-Mark-Houwing-Sakurada dependence with the help of conventional SEC. Naturally, procedures are instrumentally less demanding if the conventional viscometers are applied. However, the determination is laborious and requires careful filtration of sample solution. Evidently, the application of SEC instrument comprising viscometric detector is the best solution.

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As already mentioned, SEC is not the major focus of this book and discussion is limited to only necessary details for the sake of completeness of the topic and required for multi-dimensional approaches of LC of polymers. Numerous applications of multi-detector SEC are discussed in great detail in refs. [1–9, 95].

6.1.7 Unwanted Enthalpic Effects in Size Exclusion Chromatography As repeatedly stated, SEC is based on permeation/exclusion of macromolecules within porous particulate materials evenly deposited in columns. Outer and inner walls of particles should act only as impermeable barrier inert from the point of view of enthalpic interaction with the macromolecules. Such favorable conditions are called “ideal”. However, in many real SEC systems, undesirable enthalpic interactions are present between the column packing and the macromolecules. They can be either attractive or repulsive and as a result they increase or decrease polymer elution volumes. Molecules of MP may affect their impact. The enthalpic interactions in common SEC systems are often just minute and can be ignored. Still, omission of greater interactions may significantly affect data calculated with help of VE − logM and VE − logM[η] calibrations. Attractive enthalpic interactions decrease sample recovery. Especially the latter phenomenon is often overlooked though the retained macromolecules may gradually change column packing pore geometry [76]. The repulsive enthalpic interactions are caused by mutual incompatibility of polymer and column packing. Their extent usually depends on the nature of the MP and can be estimated from the shifts of courses of universal calibration [132]. Enthalpic interaction represent a specific problem if separated macromolecules carry very low, barely detectable concentration of charged groups. They still can affect VE values due to ion inclusion, exchange or exclusion effects. Moreover, the attractive interactions in SEC are manifested not only with increased sample elution volume but also with peak broadening, especially with their tailing. Usually, interactions increase with rising polymer molar mass because number of interacting polymer segments increases. Simultaneously surface of column packing available for interaction decreases. These two effects act in the opposite way and mutually diminish each other. Adsorption of macromolecules within column packing as a rule increases SEC elution volumes and decreases apparent sample molar mass. The situation is more complex in case of enthalpic partition, which can be present if composition of mixed mobile phase differs inside and outside of the packing particles and when separated macromolecules are substantially incompatible with the column packing. The basic relation for SEC elution volume VE (Eq. 6.1), can be substituted with dependence VE = Vo + Ksec · Ka · Kp · Vp

(6.2)

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where Ka and Kp are the distribution coefficients describing adsorption and partition effects, respectively [132]. Both Ka and Kp may depend on polymer molar mass and the practical application of the Eq. 6.2 including VE corrections. The role of adsorption on the outer surface of column packing particles is analyzed by Pefferkorn et al. [133]. They considered procedure called surface area exclusion chromatography. Their theoretical considerations were accomplished with experiments using non-porous glass beads and highly adsorptive poly(4-vinyl pyridine) fractions. In order to obtain precise molar mass data based on VE − logM and VE − logM[η] calibrations, the enthalpic interaction in SEC systems are to be suppressed. The optimum way is application of symmetrical system polymer-eluent-column packing in which interactions between separated polymer, column packing, and eluent are similar. Kilz proposed “magic triangle” to manifest this situation, Fig. 6.2 [134]. In the optimum case, three SEC system components namely macromolecules, mobile phase, and column packing would be situated symmetrically within a triangle. However, if the magic triangle is asymmetrical, undesired enthalpic interactions are present that lead to changes in the retention volume. Unfortunately, the choice of column packings and partially also of mobile phases is limited. Numerous authors just suppress the strong polymer–column packing adsorptive interactions by adding small amount of substance such as triethylamine (TEA), and tetramethyl ethylenediamine (TMED) to SEC mobile phase, which is assumed to block active sites on the column packing [52, 53]. Fig. 6.2 The magic triangle concept in SEC [134]. Copyright 1999, Academic Press

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6.2 Exclusion—Interaction Combinations The SEC separation selectivity can be sometimes enhanced by controlled addition of enthalpic interactions. The basic permeation/exclusion separation mechanism is preserved so that entropy/enthalpy compensation is not attained. The unwanted additive can be deleted by applying column packing, which selectively retains it. The approaches called “full adsorption–desorption” enable efficient separation of multicomponent polymer blend [135–137]. Its components are temporarily retained in an microcolumn and then gradually released by appropriate desorbing liquid and transported to SEC column for characterization. In case of oligomers, the interaction of their end-groups with column packing can be utilized. The basic form of log M versus VE dependence remains same but the elution volumes increase with decreasing molar mass leading to an improvement in the separation selectivity [138]. The technique is later termed as “liquid exclusionadsorption chromatography” by Trathnigg [139–145]. The selectivity of SEC separation of some high polymers can also be improved by controlled addition of enthalpic partition or adsorption to permeation/exclusion separation mechanism near entropy/enthalpy compensation, the critical conditions. At critical conditions, elution volumes of macromolecules of low and medium molar mass polymers are close to total volume of liquid within the column, vi and elution is independent of their molar mass. However, for over excluded polymer molar mass, the elution volumes may start rapidly decrease with their size and enable selective separation. This surprising phenomenon was observed for polystyrenes and poly(nbutyl acrylate)s using ODS column packing [146]. Critical mobile phase contained a mixture of DMF or diethyl malonate (DEM) with THF, while DMF and DEM promoted and THF prevented enthalpic partition of macromolecules. The behavior was denoted as “enthalpic partition assisted SEC”. In the same SP-MP system, second polymer with similar molar mass, which is not partitioned, eluted in the SEC mode and could be easily discriminated. The extent of enthalpic partition strongly depends on temperature, which can be employed for the control of the process. Similar effect was also observed with bare silica gel using adsorption-based separation mechanism with PS and polyisobutylene polymers where THF act as desorli and cyclohexane as adsorli, termed as “adsorption assisted SEC” [147].

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Chapter 7

Entropy/Enthalpy Compensation in Polymer Liquid Chromatography

As iterated repeatedly, the hydrodynamic size of macromolecules in their dilute solutions may be larger than the size of the pores of the SP. This limits their access to the pores of the SP that corresponds to the differences in the hydrodynamic volume of the polymer chains. This is the major phenomenon that operates in the most widely used techniques for polymer analysis i.e., size exclusion chromatography. In ideal situation, SEC columns are non-interactive and there is no other coactive force that render separation of macromolecules exclusively as per their hydrodynamic sizes that is determined by the extent of their access to the pores of the SP. However, in the case of interactive SPs entropy-controlled exclusion is supplemented by an opposite force generated by enthalpic interactions of the macromolecules with the SP. The phenomenon is termed as entropy/enthalpy compensation. These two phenomena, that result in opposite outcome in terms of elution order, can be adjusted by changing MP/eluent composition as well as temperature of the system. Several approaches in the chromatography of polymers are based on entropy/enthalpy compensation phenomenon. The isocratic methods based on entropy/enthalpy compensation are liquid chromatography at critical conditions (LCCC), and liquid chromatography at limiting conditions (LCLC). Temperature gradient interaction chromatography (TGIC) is also an isocratic method in which change is temperature during the chromatographic run is employed to maneuver the elution behavior. In TGIC, the initial dominance of enthalpic effect is transformed into dominance of entropic effect at the end of the analysis while going through the so-called entropy/enthalpy compensation point during the process. The eluent composition is varied during the chromatographic run to maneuver the elution behavior in eluent gradient interaction chromatography (EGIC). Similar to TGIC, initial dominance of entropic effect is reduced during the chromatographic run to achieve dominance of entropic effect at the end of the run while going through the point of so-called entropy/enthalpy compensation. In this chapter, history of the development of the methods based on entropy/enthalpy compensation will be followed by rational of using these compensation methods for comprehensive characterization of polymers.

© The Author(s), under exclusive license to Springer Nature Switzerland AG 2023 M. I. Malik and D. Berek, Liquid Chromatography of Synthetic Polymers, Physical Chemistry in Action, https://doi.org/10.1007/978-3-031-34835-8_7

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7.1 History of Entropy/Enthalpy Compensation—The Critical Conditions During the Summer school on chromatography near Uzhgorod, Ukraine (that time the Soviet Union) in August 1968, B. G. Belenkii and E. Gankina of Institute of macromolecular compounds, Academy of Sciences from Skt. Petersburg (that time Leningrad) presented a series of unexpected results on elution of polystyrenes on bare silica gel TLC plates [1]. The positions of TLC spots, which marked elution volumes of narrow molar mass polystyrene standards, have changed their order by raising the polarity of the mobile phase. The original elution order, which increased with the polymer molar mass altered to that, which decreased with the size of the macromolecules. The typical adsorption interaction elution behavior is transformed into size exclusion governed phenomenon. Authors also identified the mobile phase composition, at which polystyrenes eluted irrespective of their molar mass as a single TLC spot. They designated this situation “critical eluent composition”. This surprising behavior remained unexplained for next couple of years till the Belenkii’s group transferred corresponding measurements from TLC plates to LC columns and published several papers that explained the entropy/enthalpy compensation as a ground for the molar mass independent elution of macromolecules [2, 3]. The procedure is called liquid chromatography at critical conditions, LCCC. Gorbunov and Skvortsov, also of Skt. Petersburg, added theoretical explanation of the entropy/enthalpy compensation phenomenon, which takes place at “the critical conditions” [4, 5]. They proposed several practical applications of the new method [6–10]. They introduced the term “chromatographic invisibility” for macromolecules eluting under critical conditions to stress the fact that the effect of molar mass could be suppressed or even fully eliminated [11]. According to the proposal of Skvortsov and Gorbunov, one block in a block copolymer can be eluted under critical conditions irrespective of its molar mass that allows the determination of molar mass of the SEC/GPC eluted block [4, 7, 9, 10, 12, 13]. Zimina et al. applied the idea of the chromatographic invisibility (a more appropriate term would probably be “the column invisibility”) to characterization of various block copolymers [14–16]. The concept of critical conditions was, probably independently, applied in Moscow to separation of functional oligomers by Evreinov et al. [17–21]. As a rule, the main chain of the oligomer under study eluted under critical conditions irrespective of its molar mass while the observed retention volumes reflect both the number and topology of functional groups including functionality distribution. The phenomenon and applications of critical conditions in polymer LC was studied in detail by German and Austrian researchers, mainly by Schultz et al. [22–25]. Especially, Pasch published a series of papers on LCCC separation of polymer blends and block copolymers, while Trathnigg applied LCCC to separation of oligomers. Afterwards, Blagodatskikh [26, 27] and Baran [28–31] separated linear from cyclic macromolecules of identical molar mass with help of LCCC. They

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also applied LCCC for discrimination of macromolecules according to their stereoregularity. In the meantime, Chang applied critical conditions for analysis of cyclic polymers [32] and block copolymers [33–35]. By the end of nineties of twentieth century, LCCC has already found rather broad applications in separation and molecular characterization of various complex synthetic polymers and complex systems of synthetic macromolecules [22–25]. Most recent and significant development is the applications of LCCC to high temperature HPLC for analysis of polyolefins [36–38]. An overview and the details of selected representative applications of LCCC are collected in Chap. 8.

7.2 Terminology of Polymer LC Based on Entropy/Enthalpy Compensation In the early stage of polymer LC, which was dominated by the size exclusion/ gel permeation chromatography, the enthalpic effects were fully neglected. Their undesired presence was admitted only cautiously- and their uncontrolled deleterious effects often remained ignored. The intentional addition of enthalpy—interaction of separated macromolecules with SP to the entropy, exclusion-controlled separation processes appeared later while applying eluent gradient arrangements. At the initial stages of polymer LC development, it has been incorrectly assumed that the enthalpic (interaction) and entropic (exclusion) processes take place in the polymer HPLC column fully independently. Later only, it has become evident that any enthalpybased process taking place in the chromatographic column is inevitably accompanied with the entropy change. Subsequently, the co-operative effect of entropy and enthalpy in numerous polymer LC is employed and, eventually the idea of their mutual compensation was born. Depending on the site and kind of interactions applied in the compensation process, the method was denoted: liquid chromatography at the critical adsorption point (LC CAP) or LC at critical point of adsorption (LC CPA)—considering the adsorption retention mechanism, and liquid chromatography at the critical point of enthalpic partition, (LC CPP) for enthalpic partition (absorption) retention mechanism [39]. The term liquid chromatography at point of exclusion-adsorption transition (LC PEAT) considers only adsorption retention mechanism but includes also liquid chromatography under limiting conditions of enthalpic interactions [40]. The critical adsorption point and critical partition point are often poorly defined, the alternative term was proposed, namely liquid chromatography in the critical (interaction) range [41], where the macromolecules elute nearly independent of their molar mass. In any case, it is evident that the conformational entropy of macromolecules in the chromatographic system directly responds to the enthalpic interactions of polymer segments with both the solvent molecules and the stationary phase. Therefore, the general term liquid chromatography of polymers under critical conditions, or even colloquial, concise but not fully appropriate term “critical polymer chromatography”

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can be found in literature. The term liquid chromatography at critical conditions and abbreviation LCCC will be used in this book. Similarly, different terms like critical adsorption point (CAP), critical point for enthalpic partition (CPP), point of exclusion-adsorption transition (PEAT) for that particular point have been used based on the compensation process. In this book, the term chromatographic critical point (CCP) will be used for this particular point that corresponds to the outcome of coelution of a molar mass range of the polymers, irrespective of either adsorption or partition compensation process.

7.3 Molar Mass Versus Elution Volumes in the Process of Entropy/Enthalpy Compensation Hence, there are three modes of LC of polymers with regard to elution pattern namely entropy-controlled exclusion, enthalpy-driven interaction, and at the transition point of both having fully compensated enthalpic and entropic effects called liquid chromatography at critical conditions, Fig. 7.1. On an interactive porous column, these modes can be switched from one to the other by changing mobile phase composition, and/or temperature. Retention of polymers increases with decrease in the molar mass in entropy-controlled exclusion which is the most widely applied mode of LC of polymers on ideally non-interactive columns. In this case, the polymer molecules are more attracted towards eluent molecules and have no enthalpic interaction with the SP. Higher molar mass polymers elute early than lower molar mass polymers before the void volume of the column. SEC situation can also be achieved on porous interactive columns in a chromatographically strong solvent that does not allow any enthalpic interactions of the analyte with the SP. Changing MP composition to chromatographically weak MP composition render enthalpic interactions of polymer molecules that reverses the elution order from low to higher molar mass after the void volume of the column. In between these two situations, there is a point where enthalpy and entropy fully compensate each other that render molar mass independent elution of that particular polymer. The situation is termed as liquid chromatography at critical conditions (LCCC) and this particular point is termed as chromatographic critical point (CCP). It is important to mention here that temperature changes can only be used when MP has near critical behavior especially for fine-tuning of the method.

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Fig. 7.1 Modes of liquid chromatography of polymers

7.4 Rational for Polymer Chromatography Based on Entropy/Enthalpy Compensation The chromatographic critical conditions correspond to the concept of “chromatographic invisibility” of one particular (so-called critical) component of the complex polymers. The conditions refer to the inability of the separation system to differentiate among different molar masses of that particular polymer. The loss of separation ability by chromatography, basically a separation technique, seems to be an inappropriate idea. However, the concept opens new avenues for comprehensive characterization of complex polymers which were not possible otherwise. The suppression of molar mass discrimination of one segment leads to magnification of separation with regard to some other variable such as number and type of end-groups, molar mass of the non-critical block, architecture and topology of the polymer chains etc. It is important to mention here that the bulk analysis for the chemical composition or other characteristics by spectroscopic techniques can only provide average values in comparison to distributions of that particular property as obtained by chromatographic analysis in general and by LCCC in particular. Obviously, LCCC is the method in which CCP of any particular polymers is established beforehand in an isocratic mode through entropy/enthalpy compensation established after multiple chromatographic runs in different MP compositions for a range of molar mass of that particular polymer. There are other methods such as eluent gradient interaction chromatography (EGIC), temperature gradient interaction chromatography (TGIC), liquid chromatography under limiting condition (LCLC) etc. in which entropy/ enthalpy compensation phenomenon plays a decisive role. The peculiarities, principles and the applications of these are discussed in detail in the proceeding chapters namely LCCC (Chap. 8), LCLC (Chap. 9), EGIC (Chap. 10), and TGIC (Chap. 11). Moreover, the hyphenations of chromatographic separations with another chromatographic approach in a two-dimensional set-up reveal inter-dependence of two

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distributed properties as a function of each other (Chap. 12). Similarly, hyphenation of LC separation with spectroscopic methods also reveals distribution of any particular property as a function of the separation property by LC (Chap. 13).

References 1. Belenkii BG, Gankina ES (1970) Thin-layer chromatography of polymers introductory lecture. J Chromatogr A 53(1):3–25. https://doi.org/10.1016/S0021-9673(00)86699-6 2. Belenky BG, Gankina ES, Tennikov MB, Vilenchik LZ (1978) Fundamental aspects of adsorption chromatography of polymers and their experimental verification by thin-layer chromatography. J Chromatogr A 147:99–110. https://doi.org/10.1016/S0021-9673(00)851 21-3 3. Belenkii BG, Gankina ES, Zgonnik VN, Malachova II, Melenevskaya EU (1992) Critical thinlayer chromatography as a novel method for the determination of the molecular-weight and compositional inhomogeneity of block copolymers. J Chromatogr 609(1–2):355–362 4. Skvortsov AM, Gorbunov AA (1986) Adsorption effects in the chromatography of polymers. J Chromatogr 358(1):77–83 5. Gorbunov AA, Lukyanov AY, Pasechnik VA, Soloveva LY (1986) Adsorption-exclusion behavior of polyethylene-glycol macromolecules during chromatography. Vysokomol Soedin Ser A SSSR 28(9):1859–1864 6. Skvortsov AM, Gorbunov AA (1980) Influence of adsorption effects on chromatographic fractionation of oligomers according to functionality. Vysokomol Soedin, Ser A 22(12):2641–2648 7. Gorbunov AA, Skvortsov AM (1988) Theory of chromatographic-separation of 2-block copolymers according to composition. Vysokomol Soedin, Ser A 30(2):453–458 8. Skvortsov AM, Zhulina EB, Gorbunov AA (1980) Theory of chromatographic fractionation of polymers according to functionality of end groups. Vysokomol Soedin, Ser A 22(4):820–829 9. Skvortsov AM, Gorbunov AA, Berek D, Trathnigg B (1998) Liquid chromatography of macromolecules at the critical adsorption point: behaviour of a polymer chain inside pores. Polymer 39(2):423–429 10. Skvortsov AM, Gorbunov AA (1990) Achievements and uses of critical conditions in the chromatography of polymers. J Chromatogr 507(MAY):487–496 11. Gorbunov AA, Skvortsov AM (1988) Invisibles method in chromatography of polymers and boundaries of its applicability. Vysokomol Soedin, Ser A 30(4):895–899 12. Gorbunov AA, Skvortsov AM (1988) Adsorption and universal laws of chromatography of polymers. Vysokomol Soedin, Ser A 30(1):3–8 13. Gorbunov AA, Skvortsov AM (1988) Methods of investigation theory of chromatographic separation of two-block copolymers according to composition. Polym Sci USSR 30(2):439– 445. https://doi.org/10.1016/0032-3950(88)90144-X 14. Zimina TM, Kever JJ, Melenevskaya EY, Fell AF (1992) Analysis of block copolymers by highperformance liquid chromatography under critical conditions. J Chromatogr A 593(1):233–241. https://doi.org/10.1016/0021-9673(92)80291-2 15. Zimina TM, Kever YY, Melenevskaya YY, Zgonnik VN, Belen’kii BG (1991) Experimental verification of the concept of chromatographic “invisibility” in critical chromatography of block copolymers. Polym Sci USSR 33(6):1250–1254. https://doi.org/10.1016/0032-3950(91)902 35-I 16. Zimina TM, Kever YY, Melenevskaya YY, Zgonnik VN, Belen’kii BG (1991) On the experimental checking of the chromatographic “invisibility” concept in critical chromatography of block copolymers. Vysokomolekularnye Soedineniya Seriya A 6:1349–1353 17. Evreinov VV, Filatova NN, Gorshkov AV, Entelis SG (1995) Chromatographic study of functionality-type distribution in branched polyesters containing hydroxy groups. Vysokomol Soedin, Ser A Ser B 37(12):2076–2080

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18. Evreinov VV, Gorshkov AV, Prudskova TN, Guryanova VV, Pavlov AV, Malkin AY, Entelis SG (1985) Applications of the critical phenomena concept in liquid-chromatography for functionally type separation of macromolecules. Polym Bull 14(2):131–136 19. Filatova NN, Gorshkov AV, Yevreinov VV, Entelis SG (1988) Separation according to functionality types of hydroxyl-containing oligoethers and oligoesters by liquid-chromatography in critical conditions. Vysokomol Soedin, Ser A 30(5):953–957 20. Gorshkov AV, Evreinov VV, Entelis SG (1983) Critical conditions and adsorption effects in the chromatography of functional oligomers. Zh Fiz Khim 57:2665–2673 21. Gorshkov AV, Prudskova TN, Gur’yanova VV, Evreinov VV (1986) Functional type separation of oligocarbonates by liquid chromatography under critical conditions. Effects of pore size. Polym Bull 15(5):465–468 22. Baumgaertel A, Altunta¸s E, Schubert US (2012) Recent developments in the detailed characterization of polymers by multidimensional chromatography. J Chromatogr A 1240:1–20 23. Macko T, Hunkeler D (2003) Liquid chromatography under critical and limiting conditions: a survey of experimental systems for synthetic polymers. Adv Polym Sci 163:61–136 24. Malik MI, Pasch H (2014) Novel developments in the multidimensional characterization of segmented copolymers. Prog Polym Sci 39(1):87–123. https://doi.org/10.1016/j.progpolymsci. 2013.10.005 25. Malik MI, Pasch H (2021) Chapter 1—Basic principles of size exclusion and liquid interaction chromatography of polymers. In: Malik MI, Mays J, Shah MR (eds) Molecular characterization of polymers. Elsevier, pp 1–59. https://doi.org/10.1016/B978-0-12-819768-4.00007-5 26. Blagodatskikh IV, Gorshkov AV (1997) A study of the adsorption properties of ring-shaped macromolecules in the critical region. Polym Sci Ser A Polym Phys 39(10):1135–1141 27. Blagodatskikh IV, Gorshkov AV (1997) Adsorption properties of ring-shaped macromolecules in the critical region. Vysokomol Soedin, Ser A Ser B 39(10):1681–1689 28. Baran K, Laugier S, Cramail H (1999) Investigation of molecular dimensions of polystyrene as a function of the solvent composition: application to liquid chromatography at the exclusionadsorption transition point. Macromol Chem Phys 200(9):2074–2079 29. Baran K, Laugier S, Cramail H (2000) Liquid chromatography of polymers at the exclusionadsorption transition point: physicochemical interpretation. Int J Polym Anal Charact 6(1– 2):123–145 30. Lepoittevin B, Dourges M-A, Masure M, Hemery P, Baran K, Cramail H (2000) Synthesis and characterization of ring-shaped polystyrenes. Macromolecules 33(22):8218–8224. https://doi. org/10.1021/ma000059q 31. Baran K, Laugier S, Cramail H (2001) Fractionation of functional polystyrenes, poly(ethylene oxide)s and poly(styrene)–b–poly(ethylene oxide) by liquid chromatography at the exclusion– adsorption transition point. J Chromatogr B 753(1):139–149 32. Lee HC, Lee H, Lee W, Chang TH, Roovers J (2000) Fractionation of cyclic polystyrene from linear precursor by HPLC at the chromatographic critical condition. Macromolecules 33(22):8119–8121 33. Lee H, Chang T, Lee D, Shim MS, Ji H, Nonidez WK, Mays JW (2001) Characterization of Poly(l-lactide)-block-Poly- (ethylene oxide)-block-Poly(l-lactide) Triblock copolymer by liquid chromatography at the critical condition and by MALDI-TOF mass spectrometry. Anal Chem 73(8):1726–1732. https://doi.org/10.1021/ac001219z 34. Lee W, Park S, Chang T (2001) Liquid chromatography at the critical condition for polyisoprene using a single solvent. Anal Chem 73(16):3884–3889 35. Lee S, Lee H, Thieu L, Jeong Y, Chang T, Fu C, Zhu Y, Wang Y (2013) HPLC characterization of hydrogenous polystyrene-block-deuterated polystyrene utilizing the isotope effect. Macromolecules 46(22):9114–9121. https://doi.org/10.1021/ma4018247 36. Malik MI, Pasch H (2021) Chapter 5—Characterization of polyolefins. In: Malik MI, Mays J, Shah MR (eds) Molecular characterization of polymers. Elsevier, pp 173–222. https://doi.org/ 10.1016/B978-0-12-819768-4.00016-6 37. Pasch H, Malik MI, Macko T (2013) Recent advances in high-temperature fractionation of polyolefins. Adv Polym Sci 251:77–140. https://doi.org/10.1007/12_2012_167

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38. Pasch H, Malik MI (2014) Advanced separation techniques for polyolefins. Springer, London. https://doi.org/10.1007/978-3-319-08632-3 39. Pasch H (1996) Liquid chromatography at the critical point of adsorption—a new technique for polymer characterization. Macromol Symp 110(1):107–120. https://doi.org/10.1002/masy. 19961100110 40. Berek D (2010) Two-dimensional liquid chromatography of synthetic polymers. Anal Bioanal Chem 396(1):421–441. https://doi.org/10.1007/s00216-009-3172-3 41. Trathnigg B, Malik MI, Pircher N, Hayden S (2010) Liquid chromatography at critical conditions in ternary mobile phases: gradient elution along the critical line. J Sep Sci 33(14):2052–2059

Chapter 8

Liquid Chromatography at Critical Conditions

In liquid chromatography of polymers, exclusion of polymer molecules from the pores of column packing is always present owing to larger hydrodynamic size of polymer molecules that restricts their access to the whole pore volume. The phenomenon results in an entropy-controlled elution of polymers in order of decreasing molar mass before the void volume of the column. On the other hand, enthalpic interactions of polymer molecules with the SP render an enthalpy-driven retention of polymers. In this case, enthalpy dominates over entropy that results in elution of polymers in order of increasing molar mass after the void volume of the column. Hence, entropy and enthalpy are coactive in any chromatographic separation of polymers and the elution behavior depends upon the dominance of one over the other. In most of the entropy/enthalpy compensation methods, the dominance of enthalpy is slowly transformed to dominance of entropy during the chromatographic run. Liquid chromatography at critical conditions is the only method in which the compensation of entropy and enthalpy (H = TS) is adjusted prior to analysis that must strictly remain on the same conditions throughout the analysis.

8.1 Establishment of Critical Conditions As mentioned in preceding chapters, the elution of polymers through a column is a function of nature of the polymer to be analyzed, the stationary phase (SP), the mobile phase (MP)—eluent, and the temperature. For any targeted system, generally the polymer and the stationary phase are preselected. The next important question is the selection of an appropriate solvent system that can be maneuvered with regard to polarity in a reasonable range as per nature of the SP and the polymer to be analyzed. Initially, temperature needs to be kept constant which can however be employed for fine-tuning of the system. In principle, for a two-component solvent system, one component has to be thermodynamically good as well as chromatographically strong solvent for the polymer that should evade any interaction of the polymer with the © The Author(s), under exclusive license to Springer Nature Switzerland AG 2023 M. I. Malik and D. Berek, Liquid Chromatography of Synthetic Polymers, Physical Chemistry in Action, https://doi.org/10.1007/978-3-031-34835-8_8

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SP. Use of the pure solvent in this case results in dominance of entropic effect that render elution of polymer sample in size exclusion regime in order of decreasing molar mass earlier than the void volume of the column. On the contrary, the second solvent should be chromatographically weak solvent (that may also be thermodynamically poor) and should allow preferred thermodynamic interactions of the polymer chains with the SP, elution of polymer in order of increasing molar mass after the void volume of the column. The combination of the above-mentioned solvents renders any of the above situation depending upon the dominance of one effect over the other. Hence, both of the situations can be realized for any polymer on a given SP by changing MP composition of an appropriate solvent combination. At the intersection point of the two opposite elution modes, there is a MP composition that renders elution of the polymers at the same elution volume independent of their molar mass at a particular temperature. This specific point with regard to MP composition and temperature corresponds to the balance of entropic and enthalpic effects operative in the process. The entropy/enthalpy compensation leads to molar mass independent elution of the polymer and referred as chromatographic critical point (CCP) [1–5]. Traditionally, different molar mass standards of the target homopolymer are systematically analyzed in different MP compositions for evaluation of their elution pattern. Polystyrene (PS) is one of the most widely used polymer standard that is commercially available in a broad range of molar masses. For the sake of demonstration, elution behavior of PS standards of different molar masses on a RP ODS column in different compositions of the MP composed of dichloromethane (DCM) and acetonitrile (ACN) is shown in Fig. 8.1A [6]. Triethylamine (TEA) is added just to overcome unwanted polar interactions of the polymers with the free silanol groups of the SP, especially required for nitrogen containing polymers such as poly(2-vinylpyridine) (P2VP). In this case, the addition of TEA is just to avoid the unwanted polar interactions of the non-critical component of the polymer to be analyzed (P2VP). For PS and most of other polymers, binary solvent systems are generally employed. For instance, in this particular case CCP of PS should be in DCM/ACN: 60/40 v/ v. DCM is a strong eluent on RP column compared to ACN. A higher content of DCM (70 v%) render elution of PS standards in order of decreasing molar mass, a typical SEC regime. Conversely, reduction of DCM content to 50 v% switched the elution order to IC regime, higher molar masses eluted later than the lower molar masses. At the intersection of the above-mentioned modes of LC of polymers, the point where entropic effect compensates enthalpic effect, the whole range of molar mass of PS eluted at the same elution volume at its so-called chromatographic critical point (CCP). This particular situation is realized in a MP composition consisting of 60 v% DCM and 40 v% ACN with an additional 1% of TEA (not necessary in general). The chromatographic conditions render inability of the system to discriminate among different molar masses of that particular polymer (PS in this case). The first impression of the situation seems to be pointless as it suppresses the separation ability of the system as per molar mass which is arguably the most important characterization parameter for any polymer. However, this seemingly useless situation provides an excellent opportunity for analysis of other parameters of critical polymer (PS in this case) containing copolymers such as length of the other block

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or functional end-groups or architecture of the critical (PS) homopolymer [1–4]. As can be seen, at the CCP of PS, P2VP eluted in SEC regime which provides an ample opportunity for analysis of P2VP block length of PS-P2VP block copolymers (will be discussed in more detail in later sections). The traditional procedure for establishment of establishment of CCP of any polymer is rather tedious. An alternative method based on linear eluent gradient chromatography for the quick estimation of MP composition at the CCP of any polymer is also developed [7–13]. The eluent strength of the MP is increased during the course of the chromatographic run. The applicability of the approach is demonstrated for PS standards on both NP and RP SPs. Figure 8.1B depicts the elugrams of different molar mass PS standards on a NP silica column using n-hexane and THF linear gradient from zero to 100% THF in 10 min. In the initial MP composition (0 v% THF), PS strongly interacted with the SP and even low molar mass PS could not elute. As the content of stronger eluent (THF) is increased the adsorbed polymers start eluting in order of increasing molar mass. Initially, the discrimination of different molar masses was quite obvious. The difference of elution volume start decreasing for successive molar masses during the course of the gradient. At the latter stages of the gradient, high molar mass standards (>66,000 g/mol) eluted at the same elution volume and discrimination among them was not possible. The composition of the MP at the elution of the high molar mass standards is near to the critical composition (THF/hexane: 40/60 v/v), Fig. 8.1C. The method renders considerable reduction in the number of experiments for establishment of CCP. However, this is only an estimation of the MP composition at the CCP that needs to be validated and fine-tuned by traditional isocratic runs requiring significantly less experiments.

8.2 Sensitivity of Critical Conditions The chromatographic critical point (CCP) of any polymer is a compensation of entropic effect and enthalpic forces operating concurrently during the process. This so-called compensation point CCP is sensitive to both MP composition and temperature. A MP having higher eluotropic strength results in the dominance of entropycontrol over enthalpy-driven process rendering elution in SEC regime while a MP with low eluotropic strength favors enthalpic interactions that subsequently leads to dominance of enthalpic effect and render elution in the IC regime. The selectivity and sensitivity of MP composition and temperature for the elution pattern of PS on a ODS column is excellently demonstrated by Chang and coworkers [14]. A MP composed of DCM-ACN having less than 57 v% DCM content favors enthalpic interactions over entropic effect that render elution of PS in IC regime, Fig. 8.2A. Furthermore, the MP containing more than 57 v% DCM enhanced entropic control that dominate over enthalpic forces rendering elution of PS in SEC order. In a MP DCM/ACN: 57/43 v/v, molar mass discrimination of the PS standards is lost that indicates the compensation of entropic and enthalpic effects resulting in elution of whole molar mass range at the same elution volume as a single sharp peak, termed as CCP of PS

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Fig. 8.1 LCCC method development (A) Classical method: Elution behavior of PS in different mobile phase compositions on Jupitor RP octadecyl column, pore size 300 Å, 250 × 4.6 mm, column oven temperature: 30 °C, detection: ELSD, Mobile phase: DCM/ACN/TEA, (a) 70:30:1 (v/v) (Exclusion); (b) 50:50:1 (v/v) (Interaction); (c) 60:40:1 (v/v) (Chromatographic critical point of PS); (d) Elution behavior of P2VP at CCP of PS [6], Copyright 2019, American Chemical Society; (B, C) LCCC method development by gradient chromatography on Nova-Pak® Silica column, 300 × 3.9 mm, 60 Å column using a gradient of n-hexane → THF (B) Chromatograms of different molar mass PS standards while using gradient elution as shown in the graph, (C) Plot of molar mass vs mobile phase composition at the elution point [10], Copyright 2010, John Wiley and Sons

using that particular MP-SP-temperature. It must be mentioned here that temperature was kept constant at 30.5 °C during above-mentioned MP variations. The selectivity of temperature for the elution pattern of PS while using the above-mentioned critical MP (DCM/ACN: 57/43 v/v) is demonstrated in Fig. 8.2B. Reduction of the temperature resulted in stronger enthalpic interaction (dominance of enthalpic effect) of the PS with the SP rendering elution in the IC regime. However, an increase in the column temperature resulted in weakness of the enthalpic interactions rendering elution of PS in the SEC regime. It is important of mention that temperature variations are generally applicable for fine-tuning of the conditions which require binary mixture of solvents of suitable polarity [15, 16]. In another study, the effect of pore size on the CCP is theoretically predicted and experimentally verified [17]. It has been shown that at a fixed critical MP composition, temperature of column has to be higher for larger pore size column compared to smaller pore size column. Binary mixtures of suitable solvents are traditionally used for LCCC analysis which often suffer from preferential solvation. This limitation can be overcome by using a single solvent provided LCCC situation can be realized by changing the column temperature [18–20]. This approach has very limited scope since such a solvent-polymer combination is rather scarce [1–4]. As a special case, existence of two close CCP for PEO in methanol/water and acetonitrile/water lead to a fairly wide MP composition showing near critical behavior that can be exploited for extending the analyzable

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Fig. 8.2 Retention of PS standards on a ODS column, molar mass in kg/mol (1) 2.5, (2) 12, (3) 29, (4) 165, (5) 502, (6) 1800 (A) effect of MP composition (DCM/ACN) at 30.5 °C, (B) effect of temperature, MP DCM/ACN: 57/43 (v/v) [14]. Copyright 1999, John Wiley and Sons

molar mass range of the interacting block provided it has appropriate interaction strength with the SP [21, 22].

8.3 Working Principle The most widely used entropy/enthalpy compensation chromatographic method for analysis of polymer is based on pre-established critical conditions by changing MP composition through the above-mentioned methods. These established conditions are the function of MP, SP, critical polymer, and temperature. The established entropy/ enthalpy compensation point can be subsequently employed for the analysis of the non-critical segment of the complex polymers. The conditions must strictly adhere during the whole process of analysis of the target polymers. Complex polymers refer to the polymeric systems that have additional property distributions beyond typical molar mass distribution of simple homopolymers. These distributions of properties may be due to additional monomers making copolymer or different architectures and functional end-groups of the homopolymers etc. Chromatographic critical point is generally established by using linear non-functional homopolymers of a relevant molar mass range. These conditions are then insensitive to the length of the linear critical homopolymer. Any additional variation to

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the critical homopolymer such as inclusion of different repeat units, architectural variations or a functional end-groups may induce changes in the critical elution behavior. In any case, the system is theoretically blind to the length of the linear homopolymer and deviation from the critical point is the function of the alien segment in that homopolymer. The chromatographically visible non-critical segment may either elute in SEC regime owing to dominance of entropic effect [2, 6, 23–25] or in IC regime due to predominant enthalpic effect [21, 23, 26–28]. The elution behavior of the non-critical segment decides the elution order of the complex polymer that is subsequently employed for meticulous analysis of its non-critical segment. Hence, the analysis at the CCP will follow either SEC or IC regime while separating the complex polymer from the unwanted linear critical homopolymers. The relative polarity of different segments of a copolymer or a functional homopolymer is the determining factor for the selection of the nature of the SP and MP for analysis at CCP. There can be different situations in this context. A principle of correlation of polarity of the SP, the MP, and the polymer in context of analysis at CCP is demonstrated in Fig. 8.3. Polarity of MP and SP is the major factor in this selection along with several other comparatively less-influencing factors. A high relative polarity of the MP is appropriate for the establishment of the CCP of polar polymers which will lead opposite behavior of the non-critical segment of relatively non-polar segments on NP and RP columns. On RP column, the non-polar segment will have stronger enthalpic interactions with the SP at the CCP of the polar segment in contrary to dominance of entropic effect resulting in exclusion of the same on the NP column at the CCP of the polar segment. Similarly, establishment of CCP of comparatively non-polar segment is possible on both RP and NP columns while using low polarity MP/eluent, with opposing elution behavior of the polar segment. In this case, entropic effect will dominate on RP while enthalpic effect will dominate on NP column [1, 2, 29, 30]. All the depicted situations have their peculiarities, specific potential and applications. As can be imagined, when the polymeric segments of different nature are connected to each other, they cannot elute separately but only together. Nonetheless, the molar mass of the critical polymer would not affect the elution behavior of the non-critical polymer to a large extent either in exclusion or in interaction regime. Hence, the elution of segmented copolymers in this case would follow the elution behavior of the non-critical segment.

8.4 Applications of LCCC in Polymer Analysis The possibilities of LCCC with regard to applications for analysis of complex polymers are demonstrated in the previous section. Herein, we make two major divisions of the LCCC applications namely LCCC-SEC and LCCC-IC that will be further sub-divided into numerous sub-sections as per reported literature. This terminology is basically based on the elution behavior of the non-critical segment, the first part of term LCCC refers to the critical segment (invisible) while the second part SEC

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Fig. 8.3 Schematic representation of different chromatographic situations in LCCC of complex copolymers related to the polarity of polymer segments, mobile phase and stationary phase [1]. Copyright 2021, Elsevier

or IC refers to the elution behavior of the non-critical segment (visible). Both situations have their peculiarities and implications that subsequently make either of them suitable for any specific application.

8.4.1 LCCC-SEC The most widely employed and straight forward LCCC approach is adjusting the elution of non-critical segment under dominance of entropic effect, size exclusion regime before the critical segment, termed as LCCC-SEC [1–4, 30, 31]. This approach is applicable to high molar mass polymers having fairly high individual block lengths. The important aspects that need to be considered prior to any LCCCSEC application is appropriate pore diameter as per lengths of the individual segments and the dependence of molar mass of the non-critical segment on its hydrodynamic volume in the critical MP [23]. For higher lengths of the individual segments, large pore size SP are preferred in order to cover appropriate range of the molar mass of non-critical segment at CCP of the critical segment. On the other hand, small pore size SP is preferred for low molar mass polymers to cover the appropriate molar mass range of the non-critical excluded segment. The analyzable molar mass range of non-critical block can be extended by using a combination of multiple columns of same nature but different pore sizes [32].

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Polymer Blends

Polymers having complementary properties are often blended to achieve a synergistic combination of their properties [33, 34]. The molar mass of the parent polymers and the relative content of the blend are important criteria in order to achieve the target properties. LCCC provides an excellent platform for separation of the constituents of polymer blends provided one of them is made chromatographically invisible while other elute under either predominant enthalpic or entropic effect. If the non-critical component of the polymer blend elutes under predominant entropic effect, it will elute as per its molar mass earlier than the critical polymer. This provides an opportunity to analyze the relative content of both polymeric components along with molar mass of the non-critical component [1, 2, 6]. Obviously, the analysis at critical conditions of the second component is required for confirmation of relative content and the molar mass of the first component (critical in the first case). As a typical example, blends of different molar mass PS and P2VP are analyzed at both LCCC-PS and LCCC-P2VP [6]. In the first case, CCP of PS was established on a RP column that render elution of P2VP in SEC regime in order of decreasing molar mass, Fig. 8.4A. The same blend was then analyzed at CCP of P2VP on a NP phase column where P2VP lost its resolution of molar mass at its CCP while PS eluted earlier in order of decreasing molar mass, Fig. 8.4B. An important caveat of LCCC is the reduced recovery of the critical polymer with increase in the molar mass which has considerable selectivity for the polymer-MP-SP combination [17, 35–37]. Hence, determination of relative content and molar mass of both components of the blend require analysis at both CCPs. The sensitivity of LCCC to chemical composition, functionality, molecular topology has been established by number of examples [1–4, 30, 31]. However, degree of deuteration is another aspect that also deserves attention. Although deuterated polymers possess similar physical and chemical properties compared to their

Fig. 8.4 Analysis of blends of PS and P2VP of similar molar masses by LCCC-SEC (A) LCCCP2VP on a RP ODS column in DCM/ACN/TEA: 60/40/1 (v/v), (B) LCCC-PS on a NP silica column in THF/DMF: 84/16 v/v [6]. Copyright 2019, American Chemical Society

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protonated counterparts, they find applications in specialized fields for instance in neutron scattering by utilizing very different coherent scattering cross sections for neutrons of H and D [38]. Sensitivity of chromatographic critical conditions to deuteration has been shown by taking model protonated polystyrene (h-PS) and deuterated polystyrene (d-PS) having otherwise similar molar mass, chemical composition, and functionality [39]. CCP of both d-PS and h-PS were established so that other constituent of the blend gets excluded from the pores of the SP. Deuterated-PSs are polar nature in nature compared to h-PS. Hence at CCP of d-PS on NP, h-PS are expected to elute in SEC regime. On the contrary at the CCP of h-PS on RP, d-PS should have no enthalpic interactions with the SP rendering its elution in SEC regime. The difference of polarity of h-PS and d-PS is not large and temperature can be used for switching of elution behavior of both h-PS and d-PS in an appropriate eluent composition. Figure 8.5 demonstrates separation of blends of h-PS (Mp: 72,450 g/mol) and d-PS (Mp: 112,000 g/mol) on a RP ODS column in an eluent composed of THF/ACN: 47/53 v/v. A baseline separation of both constituents of blend is achieved on all three temperatures, however, the elution behaviors of h-PS and d-PS are different. This is an excellent demonstration of entropy/enthalpy compensation by varying temperature. At 41 °C d-PS eluted at its CCP while hPS show enthalpic interactions with the SP and eluted late, Fig. 8.5A. Figure 8.5B depicts the elution at 45 °C, d-PS is excluded while h-PS is still eluted in interaction regime. However, further increase in the column temperature to 54 °C rendered CCP of h-PS where it lost molar mass discrimination while d-PS still get excluded from the pores of SP (Fig. 8.5C). The study demonstrates the switching of elution behavior from SEC to IC through CCP by changing temperature of the column. Evidently, more than half of the commercial plastics are Polyolefins. Polyolefins are the simplest of polymers that are composed of only carbon and hydrogen. New grades are continuously being introduced having enormous potential to grow further [40]. Introduction of new non-olefinic co-monomers and new possibilities of tailoring of so-called simplest of the polymers by modern catalysts [41, 42] have paved the way for the novel applications and properties of polyolefins. Presence of multiple distributions in polyolefins such as molar mass distribution (MMD), chemical composition distribution (CCD), branching (molecular topology), and tacticity have enormous influence on their application properties. Bulk analysis techniques such as NMR,

Fig. 8.5 Separation of blends of d-PS (112,000 g/mol) and h-PS (72,450 g/mol) on RP ODS column in THF/ACN: 47/53 v/v (A) 41 °C, (B) 45 °C, (C) 54 °C [39]. Copyright 2012, Elsevier

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FTIR etc. can only provide average information on these properties. The current state of the art of analysis of polyolefins by HT-HPLC has been comprehensively reviewed in refs. [43–46]. The major caveat in analysis of polyolefins by solution-based techniques such as HPLC is their insolubility in common solvents. High boiling solvents are generally required for their dispersion above their melting points which are obviously limited in number. In this context, 1,2,4-trichlorobenzene (TCB) is the most widely employed solvent for SEC/GPC of polyolefins [44, 45, 47–49]. Other solvents that can dissolve polyolefins at high temperature are decalin, 1-decanol, n-decane, orthodichlorobenzene (ODCB) etc. It is pertinent of mention here that polyolefins are insoluble even in these solvents at ambient temperature and require heating them above their melting point (typically 130–160 °C) for dissolution rather dispersion [47]. While SEC/GPC has been used for molar mass analyses of polyolefins, LC methods based on entropy/enthalpy compensation are developed only in twentyfirst century. If a polyolefin chain is a part of any polymer system, the analyses in solution have to be performed at elevated temperature in high boiling solvents, HT-LCCC [44, 45, 47–49]. Blends of olefinic and non-olefinic polymers are being used for numerous applications [50–52]. The composition of the blend as well as the other characteristics of the blend components are important for the application properties. In this context, CCP of non-olefinic component would render characterization of the olefinic component. However, CCP must be established at high temperature in a chromatographic system. Critical conditions of PS [53] and PMMA [54] have been reported at the appropriate chromatographic conditions for polyolefins and are employed for analysis of their blends with PE. CCP of PMMA is established on a NP silica column in a eluent composed of TCB and cyclohexanone in a ratio of 34.5/ 65.5 v/v [54]. At the chromatographic critical conditions of PMMA at 140 ºC, PEs eluted in SEC regime before the CCP of PMMA in order of decreasing molar mass, Fig. 8.6. Fig. 8.6 LCCC-SEC analysis of blends of PE and PMMA at the CCP of PMMA on a NP Si column in TCB/cyclohexane: 34.5/ 65.5 (v/v) [54]. Copyright 2006, De Gruyter

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On the same lines, HT-LCCC of olefinic part is important in context of analysis of olefin copolymers and polyolefins having tacticity distribution. The first study of LCCC of polyolefins at high temperature (HT-LCCC) has been reported on a Hypercarb (porous graphite) column using several combinations of weak and strong solvents for instance 1-decanol/1,2,4-trichlorobenzene (TCB), n-decane/ODCB, and n-decane/TCB, and 1-decanol/ortho-dichlorobenzene (ODCB) [13]. CCPs of linear PE are realized in 48.5/51.5, 75/25, 53/47, and 90/10 v/v of TCB/n-decane, ODCB/ndecane, TCB/1-decanol, and ODCB/1-decanol, respectively. Under these conditions poly(1-octene) eluted in SEC regime, hence, the conditions can be applied for analysis of polyethylene blends with poly(1-alkene)s. Polypropylene (PP) can have different arrangements of the pendant methyl groups on the chain that leads to different tacticity such as isotactic, syndiotactic, and atactic. This small microstructure difference is enough for their different interaction behavior with the SP in a chromatographic run. CCPs of iPP and sPP were realized on a porous graphite column using an eluent composed of TCB/ODCB and 2-ethyl-1-hexanol/2-octanol [55]. At CCP of sPP, both aPP and iPP get excluded from the pores of the SP and eluted earlier in order of decreasing molar mass.

8.4.1.2

Block Copolymers

Block copolymers represent a special class of copolymers in which fairly long polymeric segments are attached to each other. The block copolymers not only allow to achieve the individual properties of the polymeric segments but also some special properties which are not possible to attain by blending of two homopolymers such as micellization and self-assembly [56–59]. The analysis of block copolymer is often a challenge since several aspects have to be addressed for complete characterization that include total molar mass, molar mass of individual blocks, chemical composition and so on. The major application of LCCC-SEC is the analysis of block copolymers where enthalpic/entropic compensation for one of the blocks is established while other block is eluting in size exclusion mode under predominant entropic effect. The situation is possible for a comparatively polar block of the block copolymer in a high polarity MP on a NP column. On the same lines, the CCP of comparatively less polar block on a RP column in a MP of low polarity will render exclusion of the polar block. The analysis of block copolymers by LCCC-SEC reveals individual block length of the non-critical block along with the relative content of critical homopolymers in the block copolymer sample. The individual block length of the visible block at CCP of the invisible block is determined through the calibration curve made by homopolymers of the non-critical block under the same conditions. The applicability of the above-mentioned approach to analysis of block copolymers has been demonstrated by number of authors [2–4, 18, 24, 26, 29, 35, 60–96]. LCCC-SEC is effectively the only approach that can reveal the individual block length and their distribution in block copolymers along with the presence and relative content of unwanted homopolymers in the block copolymer samples. The theory of separation of diblock copolymers through LCCC was presented by Gorbunov and Skvortsov [97].

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As a typical example, independent analyses of PS-P2VP block copolymer at CCP of both PS and P2VP revealed complementary information [6]. Entropy/enthalpy compensation of P2VP and PS on columns of different nature allowed predominant entropy-controlled elution of the non-critical block (either PS or P2VP). At the CCP of PS on a RP column, PS-b-P2VP get excluded having elution in order of decreasing length of the P2VP block, Fig. 8.7. The analysis not only separated PS homopolymer from the rest of the sample but also revealed the individual length of the P2VP block. Similarly, CCP of P2VP on a NP column allowed determination of individual block length of PS while separating PS homopolymers from the sample. In case of HTHPLC for polyolefins, analysis of block copolymers of PE with PS [53] and PMMA [54] at their corresponding CCPs at temperatures above 140 ºC have been reported for the analysis of PE block length. The most widely applied LCCC approach for analysis of high polymers is the elution of non-critical block under predominant entropic effect that has a broad scope in context of the individual and total molar mass of the polymers. Important considerations for analysis of block copolymers at CCP through LCCC-SEC approach are the pore diameter of the SP and hydrodynamic volume of the noncritical segment in the critical MP [23]. Large pore SP are required for high polymers while small pore SP are preferred for comparatively low molar mass polymers in order to achieve appropriate selectivity of the hydrodynamic volume of the noncritical segment in a narrow SEC window. For this reason, one may have to select SP of the same nature but different pore size for polymers only differing in their total molar mass and individual block lengths. As mentioned earlier, the analyzable molar mass range of non-critical block can be extended by using a combination of multiple

Fig. 8.7 LCCC-SEC analysis of PS-b-P2VP block copolymers at CCP of PS (NP silica column in THF/DMF: 84/16 v/v); and P2VP (RP ODS column in DCM/ACN/TEA: 60/40/1 v/v) [6]. Copyright 2019, American Chemical Society

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columns of same nature but different pore sizes [32]. The analyzable range of noncritical block length of a block copolymer is also influenced by selectivity of the MP for the non-critical block. Another limitation in analysis by LCCC-SEC is related to reduced recovery of the critical homopolymers that is especially important for higher molar mass polymers. The molar mass dependence of recovery is a function of the polymer-MP-SP-pore diameter combination [17, 35–37]. Another important aspect to consider is effect of the critical block length on the elution behavior of the block copolymer. The influence of critical block length on the elution behavior of the block copolymer is systematically demonstrated by Chang and coworkers [18, 98, 99]. The authors concluded that the accuracy of the determined block length decreases with increase in the molar mass of the critical segment. On the other hand, Falkenhagen showed little effect of the length of the critical block on the elution of the block copolymers [100]. In this case, however, the lengths of both blocks were kept same compared to more exhaustive analysis by Chang and coworkers where they systematically varied the length of the critical block while keeping the noncritical block length constant. Hence, the effect of the critical block length on the elution behavior of the block copolymers may lead to under- or over-estimation of the non-critical block length. As per results of Chang, a difference of factor of two in the molar mass of critical and non-critical block leads to an error of about 10% of the non-critical block length. Theoretically, it has been shown through the selfavoiding walk chain model that excluded volume of the block copolymer should not exactly follow the calibration curve made by homopolymers owing to incomplete invisibility of the critical block at the CCP [99, 101–105]. The statistical theory of polymer chromatography described the elution behavior of various structures and block copolymers [103, 106, 107]. Deviations of experimental results from theory can be attributed to over-simplification of the system by neglecting excluded volume effect [103]. The intensity of this effect however varies from case to case that should be carefully evaluated before concluding about the individual block length of the non-critical block [18, 98, 99]. A prudently developed method of LCCC-SEC can reveal a fair estimation of the individual block length of the block copolymer despite associated challenges and peculiarities [1–5]. A possible additional variation in the block copolymers is the chain architecture. Two blocks may be attached to each other by a single junction point making an ‘AB’ diblock copolymer. On the other hand, two blocks of ‘A’ may be attached to one block of ‘B’ making ‘ABA’ type triblock copolymer or two blocks of ‘B’ attached to one block ‘A’ making ‘BAB’ triblock copolymer. Theoretically, elution of triblock copolymer having critical block on the sides ‘ABA’ will follow the typical ‘AB’ type behavior in chromatography and the length of the critical block should not effect the elution behavior at the CCP of ‘A’ [103, 106, 107]. However, the length of the critical block in the center should influence the retention of a ‘BAB’ that increases with the length and dispersity of the critical block ‘A’ which limits the potential of the technique for determination of molar mass of two ‘B’ blocks attached to the critical block in the center. A prerequisite of obtaining critical behavior of a segmented copolymer requires free chain-end of the critical segment which is not available in ‘BAB’ triblock copolymer at CCP of ‘A’ [107]. Wang and coworkers

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Fig. 8.8 LCCC-SEC separation of styrene-butadiene block copolymers varying in architecture namely SB, SBS, and BSB on a RP ODS column in 1,4-dioxane [26]. Copyright 2003, American Chemical Society

predicated through Monte Carlo simulations that the length of visible block will be underestimated for ‘AB’ and ‘ABA’ copolymer while it will be over-estimated for BAB triblock at the CCP of A [101]. To verify the theoretical predictions, Chang and coworkers demonstrated a LCCC-SEC separation of three block copolymers varying in the position of the critical block [26]. They established CCP of polybutadiene (PB) using 1,4-dioxane as a MP on a three-column set of ODS columns varying in the pore size, polystyrene (PS) was the non-critical block. They used three different products having equal PS and PB block lengths but varying in architecture as SB, SBS, and BSB. According to theory, the SB and BSB should have similar retention while SBS should elute earlier due the architecture effect. As expected, SB and BSB eluted at the same elution volume compared to SBS which eluted much earlier, Fig. 8.8.

8.4.1.3

Star-Shaped Copolymers

Star-shaped polymeric architectures have been employed for myriad of peculiar applications in diverse fields [108–112]. Star copolymers is the term applied broadly for branched polymers in which all the branches root from a single center point [113]. There can be further subdivisions such as star homopolymers, miktoarm star copolymers, star block copolymers, statistical copolymer star etc. Monte Carlo simulations predict the exclusion of the star or branched polymer from the pores of the SP at the CCP of linear polymer in the absence of any strongly interacting end group [114–116]. LCCC-SEC approach is applicable to star copolymers where one of the polymer segment of the star copolymer has preferential exclusion from the pore of the SP at the CCP of the other segment that results in an early elution of the star copolymer [117–121]. An excellent separation of star block copolymers composed of PCL-PEO blocks employing LCCC-SEC is demonstrated by Malik et al. [121]. The CCP of star PCL is established on a RP ODS column in a MP composed of

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Fig. 8.9 LCCC-SEC separation of four arm star-shaped (PEO-PCL)4 at CCP of PCL on RP ODS column in ACN/THF/TEA: 85/15/1 v/v [121]. Copyright 2018, Royal Society of Chemistry

ACN/THF/TEA: 85/15/1 (v/v). At the CCP of star PCL, entropic effect dominates for PEO leading to its decreased retention with increase in the molar mass before the critical star PCL, Fig. 8.9. The synthesis of four arm star (PCL-PEO)4 is done by coupling of four MeO-PEO5K with four arm-star PCL10K . The star block copolymer containing four MeO-PEO should be further excluded due to increase in the PEO molar mass of the final product. The effect is pronounced for MeO-PEO5k whose mass in the star block copolymer will ideally reach to 20,000 g/mol. The analysis successfully separated parent materials from the star block copolymer. However, it must be mentioned that the analysis of stars will not reveal accurate length of all PEO blocks due to reasons cited earlier. Nonetheless, a single step separation of all three components is excellently demonstrated.

8.4.1.4

Graft Copolymers

Graft copolymers is a special class of copolymers that consists of a fairly long polymer backbone attached to numerous shorter branches/grafts of usually different chemical nature [122, 123]. The major area of applications of graft copolymers is membrane science among others. The length of the backbone, grafting density and graft lengths have direct correlation with their application properties. Hence, the analysis of the above-mentioned parameters is very much pertinent for the development of structure–property correlations. LCCC-SEC analysis at critical conditions of backbone and grafts may reveal different information. LCCC of backbone indicate the presence of un-grafted backbones in the sample that elutes at its CCP after the early eluting grafted copolymer. In principle, the elution of graft copolymer at the CCP of backbone will depend upon the number and length of the grafts. On the other hand, at the LCCC of grafts, the grafted copolymer will be separated according to the length of the backbone while allowing detection of free grafted polymer

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segments in the sample. The applications of LCCC for analysis of graft copolymers are rather scarce [61, 73, 124–127]. Grafting of PMMA onto ethylene-propylenediene rubber (EPDM) yields a EPDM-g-PMMA [126]. Such polymeric systems are often a complex mixture of the targeted product along with homopolymers of both types. In the above-mentioned case, CCP of PMMA on a Nucleosil CN column in THF/cyclohexane: 63/37 v/v render molar mass independent elution of PMMA while exclusion of EPDM. The method allowed separation of PMMA homopolymers from the rest of the sample at its CCP. However, the LCCC method was unable to separate EPDM from EPDM-g-PMMA. Same method CCP of PMMA is also employed for analysis of PMMA grafted on epoxidized natural rubber (ENR 50), PMMAgrafted-ENR 50 [124]. PMMA homopolymers elute at an elution volume of 4.6 mL as a distinct peak while comparatively non-polar segment ENR is excluded from the pores of the SP, Fig. 8.10. Slight variation in the elution volume of the grafted product, PMMA-grafted-ENR 50, may be attributed to different length and density of grafts for three products, as has been experimentally shown for block copolymers [18, 98, 99] and predicted by theory [99, 101, 103, 104, 128]. Moreover, the presence un-grafted backbone in the sample may also contribute in broadening of the peak. However, EGIC has proved to be a better choice for separation of backbone, grafted product, and grafts from each other in a single run.

Fig. 8.10 LCCC-SEC analysis of PMMA-grafted-ENR 50 at CCP of PMMA on a Nucleosil CN column in THF/cyclohexane: 63/37 v/v [124]. Copyright 2003, John Wiley & Sons

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8.4.1.5

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Polymers Varying in Tacticity/Microstructure

Relative stereochemistry of adjacent chiral centers in the polymer chain is termed as tacticity. The polymer chains having otherwise similar characteristic but varying in tacticity type distributions may have different physical properties. Two adjacent repeat units of a polymer chain is termed as diad. Diads can be further classified into meso diads and racemic diads. Meso diads refer to a pair of repeat units that are oriented on the same side whereas racemic diads refer to two adjacent repeat units that are oppositely oriented. For macromolecules, the most precise way of presenting the tacticity is based on triads. Two adjacent meso diads make an iso triad (mm) whereas two adjacent racemic diads make a syndio triad (rr). On the same line, adjacent meso and racemic diads make a hetero triad (rm). Isotactic polymers have substituents located on one side of the chain in contrary to syndiotactic polymers where substituents are located alternatively along the chain. Atactic polymers have no defined arrangement of the substituents along the polymer chain. Average value of tacticity can be obtained by direct NMR analysis. Online hyphenation of SEC-NMR is an excellent technique for determination of tacticity distribution if tacticity varies with molar mass in a defined way [129, 130]. However, presence of polymer chains with same molar mass but varying in tacticity will lead to erroneous results. The potential of LCCC for separation of polymers according to tacticity is demonstrated by Berek by using poly(methyl methacrylate) (PMMA) and poly(ethyl methacrylate) (PEMA) as model polymers [131–133]. CCP of it-PMMA was established on a bare silica column in a MP composed of CHCl3 /THF: 87.7/2.3 w/w [131]. At these chromatographic conditions, st-PMMAs eluted in SEC regime, Fig. 8.11A. The study provides one of the first proofs of sensitivity of CCP to stereochemistry of the macromolecules. Moreover, an increase in the broadness of the peak at CCP (~8 mL) with increase in the molar mass clearly demonstrates one of the limitations of the LCCC that may also be supplemented by the distribution in the stereoregularity. Same authors extended their study to online hyphenation of LCCC-NMR for separation according to tacticity using poly(ethyl methacrylate) as model polymer [133]. As expected, SEC-NMR analysis of four polymers having similar molar mass but varying in the triad content (mm, rr, mr) was unable to separate according to tacticity ratio. Chromatographic critical point of syndiotactic PEMA (rr) was established on amino-functionalized silica column in a MP composed of 61% cyclohexane in acetone. Under established chromatographic conditions, presence of other triads (mm and mr) resulted in exclusion of the polymer chains, the extent of exclusion had direct correlation with the amount of the non-critical triads in the polymer chains. Four PEMA samples having similar molar mass but varying in tacticity ratios were mixed and subjected to analysis at the CCP of syndiotactic PEMA (rr). The earliest eluted fraction has 92.4% mm triad that is followed by the fraction with 53% mr and 11.9% mm triad. The third eluting peak has 27.3% mr and 4.8% mm triad along with 67.9% rr triad while PEMA fraction having 86.6% rr triad eluted close to the CCP of the syndiotactic PEMA, Fig. 8.11B. Moreover, separation according to microstructure of polyisoprene (1,2-, 1,4-, and 3,4-isoprenes) by LCCC-NMR is shown by Hiller et al. [129]. Chromatographic

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Fig. 8.11 LCCC-SEC separation of polymers according to tacticity (A) CCP of it-PMMA on a bare silica column in CHCl3 /THF: 87.7/12.3 w/w [131]. Copyright 1997, Springer Nature; (B) Elution of PEMA with different tacticity distributions at the CCP of syndiotactic PEMA (rr) on amino functionalized silica column in a MP composed of cyclohexane/acetone: 61/39 v/v [133]. Copyright 2000, American Chemical Society

critical point of 1,4-PI was established on a set of three ODS columns varying in their pore size (100, 300, 1000 A) in MP composed of butanone/cyclohexane (92/8 v/v). At the established CCP of 1,4-PI, PI having predominant 3,4-PI microstructure eluted in SEC regime. On-line coupling with NMR further endorsed the composition of separated fractions. In the context of polyolefins, tacticity type distribution has significant effect on the properties of polypropylene (PP). On a Hypercarb column in an eluent composed of a combination of TCB/DCB (desorption promoting solvents) and 2-ethyl-1-hexanol/ 2-octanol (adsorption promoting solvents) allow for establishment of CCP of iPP and sPP in a temperature range of 140–180 ºC [55]. CCP of PP with a particular tacticity render its molar mass independent elution. Any other type of tacticity distribution should have elution either in order of increasing (typical IC) or decreasing molar mass (SEC). At CCP of sPP, elution order of iPP and aPP follow typical SEC regime. Using the established CCP of iPP and sPP, authors separated PP according to their tacticity. Moreover, high impact PP that constitutes a semicrystalline component (e.g., iPP) and rubbery phase (e.g., EP copolymers) are separated accordingly.

8.4.1.6

Statistical Copolymers

The analysis of non-critical segment at CCP of the other segment is more applicable to segmented copolymers where the non-critical segment has fairly long lengths that allows unhindered phenomena of either exclusion or interaction. In case of statistical copolymers, the same repeat units are not continuously arranged that results in deviation in the elution volume compared to CCP of one segment. However,

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Fig. 8.12 LCCC-SEC analysis of ethylene-octene statistical copolymers varying in content of 1-octene on a Hypercarb column in TCB/n-decane (48.5/51.5 v/v) [13]. Copyright 2013, Elsevier

the correlation of elution volume with the composition may not be conclusive. As mentioned earlier, linear PE has its CCP in different combinations of eluent namely TCB/n-decane (48.5/51.5 v/v), ODCB/n-decane (75/25 v/v), TCB/1-decanol (53/47 v/v), and ODCB/1-decanol (90/10 v/v) on a Hypercarb (porous graphite) column. Poly(1-octene) was excluded from the pores of SP in the above-mentioned eluent composition [13]. The analysis was done at temperatures above the melting point of polyolefins and is termed as HT-LCCC. Separation of ethylene/1-octene statistical copolymer at LCCC of PE is possible provided total molar mass of the copolymers is similar and the samples differ only in the content of the non-critical 1-octene. Ethylene/1-octene statistical copolymers eluted in order of decreasing 1-octene content before the critical PE, Fig. 8.12. However, for dissimilar total molar mass separation did not follow this trend. In another study, a polymer synthesized by copolymerization of methacrylic acid (PMA) and poly(ethylene glycol) (PEG) macromonomer is analyzed at LCCC of PEG where PMA is excluded from the pores of the SP [72]. Moreover, statistical copolymers may have similar triads throughout polymer chain depending upon its overall composition. Statistical polymers behave like a homopolymer in chromatography, equivalent to homopolymer of some effective segment (such as a triad) that is repeated throughout the chain [9, 10, 12, 134– 136]. This typical behavior of statistical copolymers has a theoretical background as well as practical examples. It is important to mention here that CCP of statistical copolymer depend upon the chemical composition of the copolymer. Statistical copolymers differing in their monomer composition may show critical behavior in different eluent compositions. The analysis of EO-stat-PO copolymers using concept of CCP is demonstrated by Falkenhagen et al. [136]. The authors show the shift of CCP eluent composition with the monomer composition of the polymer. On these basis, successful separation with regard to end-groups is achieved for statistical EO-stat-PO copolymers.

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8.4.2 LCCC-IC The second possibility for analysis at the entropy/enthalpy compensation point of the so-called critical segment is preferential enthalpic interaction of the non-critical segment with the SP [1–4, 30, 31]. The analysis under such conditions allows separation of functional homopolymers with regard to the number and interaction strength of the non-critical segment for instance, end-group/s. LCCC-IC approach have rather limited applications for copolymers.

8.4.2.1

Polymer Blends

The applications of LCCC-IC to polymer blends are generally limited to the oligomer range of constituents [22, 98, 137–139]. The approach requires a workable combination of CCP of one component and fairly weak interaction of the repeating unit of the other polymer. This peculiar combination does not often exist for many combinations of polymer blends [1–4, 30]. However, there are some special cases where this type of combination is available such as PEO & PLA [98, 140], PS & PI [137, 141], and PEG & PPG [21–23, 70, 76, 77, 142–145]. A baseline separation of oligomers of PEG at the CCP of PPG is shown in Fig. 8.13A. CCP of PPG was found on a Nucleosil Si column in an eluent composition of acetonitrile/water: 80/20 v/v. Under these conditions PEG have an enthalpic interaction with the SP that allows its oligomeric separation. A blend of different molar masses of PPGs elutes as a sharp peak irrespective of their molar mass at its critical point while PEGs get separated into individual oligomers under these conditions. The situation can be reversed on a RP octadecyl column where at CCP of PPG, PEGs get separated into individual oligomers in the interaction regime.

Fig. 8.13 LCCC-IC separation of blends of (A) PPG and PEG on a NP Si column in acetone/water: 80/20 v/v; (B) PMMA and P2VP on a RP ODS column in acetonitrile/THF/TEA: 94.5/5.5/1.0 v/v [27]. Copyright 2014, Springer Nature

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The applications of LCCC-IC approach to polymer blends of high molar mass are rather limited. Various molar masses of PMMA eluted as a sharp peak at its critical point on a RP C18 column in an eluent composed of acetonitrile/THF: 94.5/ 5.5 v/v with 1% of TEA as an additive to overcome unwanted polar interactions of polymers with the free silanol groups of the SP [27]. Under these conditions, P2VP have enthalpic interactions with the SP and elute in the interaction regime as a broad peak after PMMA in order of increasing molar mass. The molar mass range is stretched to some extent compared to previously discussed case, however, it is still limited to only 11,000 g/mol.

8.4.2.2

End-Functionalized Linear Homopolymers

Critical conditions are established by using non-functional linear homopolymers that render a chromatographic system that is blind to the number of repeat units of that particular polymer. However, the presence of functional groups at the chain-end, that may have stronger interaction compared to the repeat units of the polymer, will result in the late elution of end-functionalized homopolymers [22, 24, 29, 77, 146– 148]. The elution of the end-functionalized critical homopolymers will be delayed compared to the non-functional critical homopolymer as per interaction strength of the end-group under the experimental conditions. However, the end-functionalized critical polymers still elute irrespective of their molar mass [149]. The examples of such separations include functionalized PS [80], poly(propylene oxide) [22, 146, 150], PMMA [102, 151], poly(butyl terephthalate) [152], telechelic polyisobutylene [153], polycarbonates [154] etc. Generally, end-groups are polar compared to the repeat units and NP SPs are more appropriate for such separations. However, the separation according to end-group functionality is also possible on RP SP provided the end-group is less polar than the repeat unit. A comprehensive study in this context is reported in which poly(propylene glycols) (PPG) of different molar masses were synthesized using n-alcohols having different lengths of the alkyl chains (from nbutanol to n-octadecanol) as initiators [146]. The CCP of the PPG was realized on a RP column in aqueous MP containing 80% THF. In this MP composition, PPGs (having two hydroxyl end-groups) elute independent of molar mass at elution volume of 2.6 mL. Introduction of an allylic group at one side of the chain render increased interaction with the SP and allylic-PPGs eluted at 2.75 mL. On the same lines, introduction of an alkyl end group at one end of the PPG chain resulted in late elution as per interaction strength of the alkyl group with the RP. As can be seen in Fig. 8.14A, n-decyl-terminated PPG eluted at 3.10 mL. In fact, the elution volume increased with increase in the length of the alkyl group. It is pertinent to mention here that even better separation between PPG and allylic-PPO was achieved on a NP column under critical conditions of PPO, however, order of elution was reversed [22]. An excellent demonstration of the nature of the end-group selectivity for separation of linear homopolymers at critical condition of the repeat unit, isobutylene in this case, is presented on a RP column in THF/methanol: 80.5/19.5 w/w MP

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Fig. 8.14 LCCC-IC separation of end-functionalized linear homopolymers (A) PPGs with different end-groups on a RP ODS column THF/water: 80/20 v/v [146]. Copyright 2007, Elsevier; (B) Polyisobutylene with different end-groups on RP ODS column in THF/methanol: 80.5/19.5 w/w [153]. Copyright 2015, Elsevier

[153]. LCCC chromatograms of polyisobutylene (PIB) having different functional end-groups but similar molar mass (2000 g/mol) are demonstrated in Fig. 8.14B. As expected, PIB with most polar functionality (hydroxyl) at both ends eluted first compared hydroxyl functionality at one end (PIB-monool) on a RP column. Moreover, PIBs with non-polar functional end-groups (chloride, diallyl and olefin) eluted after PIB-monool.

8.4.2.3

Block Copolymers

In case of block copolymers, the applications of LCCC-IC face multiplex complications and are not universally applicable to every block polymer samples [22, 98, 137–139]. The approach requires a workable combination of CCP of one block and fairly weak interaction of the repeat unit of the other block. This peculiar combination does not exist for all the block copolymers [1–4, 30]. However, there are some special cases where this type of combination is available which includes block copolymers of PEO-PLA [98, 140], PS-PI [137, 141, 155], PEO-PS [82], and industrially important PEO-PPO, also called poloxamers [21–23, 70, 76, 77, 142–145]. LCCC-IC situation is fairly flexible and pragmatic for poloxamers owing to several reasons namely existence of CCP of both blocks with workable interaction strength of the non-critical block, presence of two close critical points of PEO on RP SP, and industrial importance of the oligomeric products. In aqueous MP containing methanol or acetonitrile as organic component, the molar mass independent elution of PEO is reported for a fairly wide range of eluent composition on RP columns. This behavior actually refers to existence of two close critical points for PEO while elution behavior does not deviate much from critical in between these two critical points [21, 22, 156]. Interestingly, in this critical MP range the elution behavior of non-critical PPO block has strong selectivity. This allows applying shallow MP gradients within

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limits of two CCPs in order to extend the molar mass range of PPO oligomers [70, 76, 77]. The analyzable PPO molar mass range can be further stretched by employing gradient of two different critical MPs for PEO such as aqueous-acetone and aqueous-methanol, termed as critical chromatography in ternary solvent [21, 145, 157]. Excellent baseline oligomeric separations with regard to both EO and PO has been demonstrated at the CCP of the other block by Malik et al. [21–23, 29, 76]. Critical behavior of PEO was found in acetone/water: 37/63 v/v (solvent A) and methanol/water: 87/13 v/v (solvent B) on a RP column. However, the interaction strength of PPO repeat units greatly varies in the two critical MPs. Only first few oligomers can be resolved in acetone/water which indicate fairly strong interaction of PO repeat unit with the SP. On the other hand, the interaction strength of PO in methanol/water is comparatively less that allows a baseline resolution of fairly high oligomers. Interestingly, any combination of these two MPs did not have any pronounced effect on the retention time of PEO in the molar mass range of 200–5000 g/mol. Hence, dispersity in the PEO block length should not contribute in the PO oligomer separation of EO-PO block copolymers using any combination of above-mentioned critical MPs. A gradient of the critical MPs compositions can be designed as per length of the PPO block of EO-PO block copolymer. Different lengths of the PPO block will require a peculiar slope and duration of gradient for better resolution [21]. As an example, PO oligomer separation of PO11 –EO27 –PO11 employing gradient of critical MPs is shown in Fig. 8.15A [21]. Sharpness of peaks clearly indicate that dispersity of EO block length is not significantly contributing in the retention time. Individual peaks in the chromatogram represent an EO-PO block copolymer with a monodisperse PO block that is attached to polydisperse EO block. The number on peaks correspond to PO oligomer number which is deduced from MALDI-TOF MS analysis of the individually fractionated peaks. Similarly, CCP of PPO was found in acetonitrile/water: 80/20 v/v on a NP column. Under these conditions, PEO have fairly weak adsorptive interaction with the SP that allows its oligomeric separation. These conditions should be able to resolve the EO-PO block copolymers according to oligomers of PEO. Figure 8.15B demonstrate analysis of PO11 –EO27 –PO11 at CCP of PPO. PPO homopolymers get separated from rest of the sample at elution volume of 3 mL. The separation according to EO oligomers of the block copolymer starts from retention time of 5 mL. An excellent separation of EO-PO block copolymer according to EO oligomers is possible using these conditions. The number on each peak correspond to the EO repeat units as obtained by MALDI-TO MS analysis of the fractionated individual fractions. Distinct peaks in the chromatogram represent an EO-PO block copolymer with monodisperse EO block that is connected to a polydisperse PO block. There are several critical experimental parameters that can be maneuvered skillfully for improvement of resolution of oligomers of interacting block in LCCC-IC. These include pore diameter of the SP, temperature of the column, critical MP composition, slope and duration of gradient within range of critical MP composition, slope and duration of gradient between two critical MP compositions varying in selectivity for the interacting block [23]. Hence, a well-pondered selection of pore diameter, initial MP composition, slope, and duration of solvent gradient is imperative for better oligomeric resolution of

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Fig. 8.15 LCCC-IC separation of PO11 –EO27 –PO11 (A) PPO at CCP-PEO by gradients of two critical mobile phases (solvent A: acetone/water: 37/63 v/v; solvent B: methanol/water: 87/13 v/ v) on a RP ODS column; (B) PEO block at CCP of PPO (acetonitrile/water: 80/20 v/v) on a NP silica column. The numbers on individual peaks as obtained by MALDI-TOF-MS analysis of the collected fractions and corresponds to oligomer number of non-critical block [21]. Copyright 2016, Elsevier

the non-critical block. The analysis at LCCC-IC is comparatively more sensitive to topology compared LCCC-SEC. The position of critical block in case of triblock copolymers has more influence on retention behavior compared to LCCC-SEC [26, 101, 107]. Separation of di-block copolymers according to oligomers of one block at CCP of the other block can be conducted either way [22]. However, the position of the critical block, either in the center or on the sides, of a triblock copolymer influence the retention behavior significantly. The elution pattern of B oligomers of AB di-block copolymer should be similar to ABA triblock copolymer, where A is at CCP [144, 158, 159]. However, the dispersity of the critical block A will affect the retention behavior of oligomers of B in case of BAB triblock copolymer compared to AB di-block or ABA triblock copolymer. The applications of LCCC-IC approach to block copolymers having high molar mass are rather limited [27].

8.4.2.4

Star Polymers

Star polymers show various peculiarities and applications which are not possible to attain by their linear counterparts [113]. Separation and characterization of branched and star homopolymers with regard to number of arms is a challenging task. Similar value for distribution coefficient for linear and star polymers at the CCP of the repeating unit is predicted by statistical theory of polymer chromatography [106, 115, 160]. Moreover, it has been predicted theoretically and shown experimentally that stars with short arms elute in the interaction regime while stars with long arms elute in exclusion regime before the LCCC retention time of their linear counterpart [161, 162]. Linking of anionically polymerized star-shaped polystyryl anions with divinylbenzene yielded star-shaped PS having different number of branches of the

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same length [161]. The first number of the sample code represent the length of the arm while second number corresponds to the total molar mass of the star, Fig. 8.16A. At CCP of linear PS on a RP column of smaller pore size (100 Å), star with smaller branches (BS2.5-8.8) eluted in interaction regime after CCP of linear PS. On the contrary, star with longer branches and higher total molar mass (BS10-148 and BS26-174) eluted at both sides of the CCP. The elution behavior of star-shaped PS was comparatively less complicated on a 500 Å column. All the star-shaped PS eluted in the interaction regime after the linear PS at its CCP. The retention of star-shaped PS has different elution behavior from their linear counterparts, the extent of which depends upon the pore size of the SP. This peculiar behavior of star-shaped PS at the CCP of linear PS was theoretically verified by taking into account the weak interaction of the chain-end and excluded volume for Monto Carlo simulations. It is concluded that initially retention time increased with the increase in the branch number, followed by decrease in retention time with further increase in the branch number. The turn-over point for star-shaped PS is at high branch number for low molar mass arms compared to the stars with higher molar mass arms. Subsequently, the elution volume crossed the CCP elution volume and star-shaped PS with very high molar mass eluted in SEC regime. End-group may play a decisive role in the separation of star polymers. LCCC provides an ample opportunity for separation of star polymers with regard to number of arms based on the higher interaction strength of the end-groups with the SP at CCP of the repeating unit. Herein, we discuss the separation with regard to number of interacting end-groups of the same nature that are paralleled with the number of branches. The chromatographic elution behavior of star polymers is theoretically predicted [114–116]. The separation of star-shaped polymers with regard to number of branches exploiting the entropy/enthalpy compensation of the repeat unit has been reported by several authors [36, 116, 118, 147, 149, 161, 164–167]. The separation is based on the number of interacting end-groups which are present at the end of each branch. As a typical example, a MP composition of 1,4-dioxane / n-hexane (56.25/ 43.75 v/v) on a set of two NP silica columns render enthalpy/entropy compensation of PLA which lead to molar mass independent elution behavior of non-functional linear PLA. Under these conditions, referred as CCP of PLA, hydroxyl end-groups will have stronger enthalpic interaction with the SP [165]. The increase in the elution volume corresponds to the number of hydroxyl groups which are equal to number of branches, Fig. 8.16B. In this particular case the molar mass of all the PLAs, varying in number of hydroxyl groups and in turn number of branches, was kept similar for fair comparison which was further supplemented by typical molar mass independent elution behavior of PLA under these conditions. The results of separation of starshaped PLA according to number of arms were also theoretically endorsed [115]. In the studies mentioned in this section, the difference in the elution behavior of linear and star polymers at CCP of linear polymer is based on the strong adsorptive interaction of the end-group with the SP.

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Fig. 8.16 (A) Elution behavior of star-shaped PS of different arm length and total molar mass of star at the CCP of linear PS on RP ODS columns of different pore sizes (100 Å & 500 Å) using CH2 Cl2 /CH3 CN (57/43, v/v) [161]. Copyright 2008, American Chemical Society; (B) LCCC-IC separation of PLA stars with different number of arms on a NP column in 1,4-dioxane/n-hexane: 56.25 / 43.75 v/v [165]. Copyright 2003, Elsevier

8.4.2.5

Branched Polymers

Branching in polymer chains offer improved rheological and mechanical properties compared to their linear analogs [169]. The separation of polymers with regard to degree of branching is a challenging task. Generally, multidetector size exclusion chromatography specifically SEC–MALLS is used for branching analysis of the polymers. However, SEC-MALS analysis is mainly based on the detector response to linear and branched polymers and to small extent on the elution behavior [15, 170–173]. SEC separates with regard to hydrodynamic volume of the analyte in the dilute solution which may lead to coelution heterogenous fractions with regard to branching and molar mass. LCCC can provide an opportunity to separate branched polymer with regard to degree of branching based on topology differences. Initially, it was theoretically predicted that the retention of branched polymers at CCP of linear polymer would be independent of topology which means linear and breached polymer should elute at the same elution volume [106, 160]. However, Wang et al. have shown the effect of excluded volume using Monte Carlo simulations [162, 174, 175]. The separation of branched polymers with regard to degree of branching, based on topology, have been demonstrated for several polymers by employing LCCC [164,

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166]. As per theory, the star and branched polymers may elute on either side of the critical point depending upon the branch number and ratio of molecular size to pore size. In this context detailed analysis of linear, branched, and hyperbranched polyester using interaction chromatography techniques was presented by Samman et al. [168]. The authors employed eluent gradient interaction chromatography (EGIC) and liquid chromatography at critical conditions (LCCC) for the analysis. For fair analysis of the dependence of degree of branching on the elution behavior at CCP of linear polymer, fractions of the polymers were collected by EGIC followed by their molar mass analysis by SEC–MALLS. Finally, these fractions were analyzed at the CCP of linear polyester. In a MP composed of acetone/THF: 94/6 v/v, linear polyester eluted independent of their molar mass at an elution volume of 2.7 mL. Any change in the MP composition in either direction resulted in a deviation from the critical behavior. Under these conditions, the branched polymer fractions of similar molar mass eluted later in interaction regime showing dependence of interaction strength on the degree of branching. The interaction strength increased with increase in the number of branches. Hence, hyperbranched polyester eluted later than similar molar mass of branched polyesters having fewer branches. Figure 8.17A demonstrates the real chromatograms of fractions of linear, branched and hyperbranched polyester having similar molar mass. Figure 8.17B depicts a plot of log M w versus peak elution volume of polyesters having different degree of branching. Other examples of LCCCIC separation with regard to degree of branching can be found in refs. [26, 147, 149, 161, 164, 166]. Hence, the LCCC-IC provides an excellent platform for separation with regard to topology of the branched polymers. However, traditional method of branching analysis by SEC based on chain contraction would remain the major approach for this analysis owing to its consistent results and easy operation.

Fig. 8.17 LCCC-IC separation of polyesters having similar molar mass but varying in degree of branching on a Nucleosil ODS column in acetone/THF: 94/6 v/v (A) elugrams, (B) Plot of log M w versus peak elution volume [168]. Copyright 2010, American Chemical Society

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Cyclic Polymers

Cyclic polymers are emerging as an important class of polymers for applications in diverse fields such as vehicles for drugs and genes, gels, self-healing, and shapememory systems [176]. Cyclization of polymeric chains based on reaction of two carbanion end-groups intramolecularly in very dilute conditions is the widely used method for preparation of cyclic polymers. However, it is near to impossible to get pure cyclic products due to intermolecular reactions leading to formation of dimers, trimers, and so on. Hence, fractionation of cyclic polymer from linear polymers is often required that is usually conducted through fractional precipitation or preparative SEC. Both of the above-mentioned methods have their associated limitations such as low resolution and limited choice of scale-up. Theoretically, cyclic polymers should elute after the elution volume of their critical linear precursor in LCCC [177, 178]. The theoretical behavior of cyclic polymers at critical conditions of linear polymers is verified by several authors [36, 105, 162, 163, 178–186]. At the CCP of linear polymer, cyclic polymer eluted in interaction regime after the retention volume of the critical linear precursor. In the pioneering study, Pasch and coworkers showed the critical behavior of cyclic polymers at the critical conditions of linear polymer by using relatively low molecular weight polymers in range of 1800–25,000 g/mol [180]. In this context, a more comprehensive study by using linear precursors of a wide molar mass range (5000–200,000 g/mol) is conducted by Chang and his team [179]. The whole molar mass range of linear PSs eluted at 5.4 mL on a RP ODS column in DCM/ACN: 57/43 v/v irrespective of their molar mass. On the other hand, the cyclic polymers prepared by using the same precursor eluted later than the critical point, Fig. 8.18. It is worth mentioning that the retention time of cyclic PS increased with increase of the molar mass which is consistent with theory of polymer chromatography.

8.4.2.7

Gradient Copolymers/Influence of Microstructure

As iterated several times, critical conditions are generally established by using linear non-functional homopolymers. Any variation in the architecture or composition may result in deviation from the critical behavior. The selectivity of microstructure on the elution behavior of copolymers has been demonstrated for EGIC by Brun et al. [9, 10, 12]. LCCC is generally not employed for analysis of statistical copolymers. Very recently, a comprehensive study in this context is conducted by Zargar and coworkers [187]. Authors demonstrated elution behavior of gradient copolymers by using isocratic mode of entropy/enthalpy compensation—LCCC. The copolymers used in the study consists of poly(propylene oxide) (PPO) and poly(propylene phthalate) [P(POPA)] with comparable molar masses and average chemical compositions. A gradient copolymer refers to a copolymer in which chemical composition varies along the chain. Gradient copolymers show gradual transition from predominant repeat unit ‘A’ to predominant repeat unit ‘B’ along the polymer chains [188]. The strength of the gradient in the gradient copolymer indicates the speed of transition.

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Fig. 8.18 LCCC-IC chromatograms of cyclic PSs and their linear precursors [179]. Copyright 2000, American Chemical Society

Copolymer without any gradients are classical alternative copolymers. Introduction of a weak gradient starts transition from dominance of one repeat unit in the start of the chain to the dominance of the second repeat unit at the other end of the chain, however, both comonomers are present in sufficient amount at both ends of the chain. On the other hand, in a strong gradient the transition from one repeat unit to the other repeat unit is rapid and the copolymer has a blocky structure. The properties of gradient copolymers with strong gradient are similar to their block copolymer analogs. Gradient copolymers have gained significant attention recently in stabilization of emulsions, compatibilization of polymeric blends, and as thermoplastic elastomers etc. [188, 189]. Three different gradient copolymers and a block copolymer were synthesized having similar chemical composition and comparable molar masses [187]. Three gradient copolymers differ from each other in strength of the gradient, gradient-1 is weak followed by gradient-2 and gradient-3 is the strongest. G1 is the copolymer with slow transition of any dominance of comonomers and resulting copolymer is more like a statistical copolymer. G2 has comparatively strong gradient resulting in blocky structures having dominance of blocky structure of A in start to the blocky structures of B at the other end of the chain. G3 has the strongest gradient with even longer segments of both types of repeat units at different ends of the chain,

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making it more like a block copolymer. The focus of this study was to evaluate the influence of microstructure on the retention behavior of polymers in different modes of interaction chromatography. CCP of the P(POPA) was established on a PS-DVB RP column in an eluent composed of THF/ACN: 15.6/84.4 v/v. Under established chromatographic conditions, P(POPA) having molar masses 28.5 and 51.9 kg/mol eluted as a sharp peak at void volume of the column while PPO had stronger enthalpic interaction with the SP and eluted late in order of increasing molar mass, Fig. 8.19. In fact, only PPO-1 (17.1 kg/mol) could be eluted under isocratic LCCC conditions for P(POPA). A high molar mass PPO-2 (33.5 kg/mol) interacted strongly and was retained completely in the critical MP composition for P(POPA). The molar mass of PPO component of all three gradient copolymers and block copolymers was similar to PPO-1. The retention of the copolymers under study should not only depend on the molar mass of PPO constituent but also on the average segment length of PPO i.e., the microstructure. As expected, gradient strength of gradient copolymers has significant impact on the elution order. The first eluting polymer after P(POPA) homopolymer was the gradient copolymer with lowest gradient strength, G1. Other two gradient copolymers G2 and G3 follow in the order of increasing gradient strength. All these gradient copolymers have similar molar mass and ratio of PO and POPA repeat units. The only difference is the segment length. Longer PPO segments allowed more interaction of the PPO repeat unit with the SP that resulted in an increased retention. Moreover, the gradient copolymer with strongest gradient, G3, eluted slightly after block copolymer, B. However, this increased retention is attributed to somewhat higher PPO content of G3 compared to B. In principle, G3 should elute slightly earlier or at the most similar to B if the PPO content is exactly same. As expected, both samples of P(POPA) have sharp peaks and peak broadness and retention increased with the gradient strength of copolymer while PPO homopolymer has the broadest peak among all. The pioneering and comprehensive study reveal the influence of microstructure on the retention behavior of copolymers in interaction modes of LC of polymers. Most recently, selectivity of CCP for chemical composition of EP copolymers was shown by using a binary mixture of 1,2,4-TCB and 1,3,5-trimethylbenzene (TMB), and also in 2-cholortoluene as a single eluent [190]. Different combinations of former (TCB/TMB) are required for CCP of polymers with different EP compositions while different column temperatures serve the purpose for the latter (2-cholortoluene). The developed CCP was subsequently used for analysis of ethylene propylene diene (EPDM) that eluted in interaction regime as per ethylidene norbornene (ENB) content.

8.5 Limitations and Opportunities The entropy/enthalpy compensation in a chromatographic separation is an excellent idea and can provide important information that is not possible otherwise. Nonetheless, there are several caveats that must be considered before drawing any conclusion

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Fig. 8.19 Elution behavior of gradient copolymers varying in gradient strength, block copolymer, and homopolymers of PPO at the CCP of the P(POPA) on a PS-DVB RP column in THF/ ACN: 15.6/84.4 v/v [187]. Copyright 2022, American Chemical Society

about the outcome of any LCCC experiments. These limitations are especially associated with the method based on pre-established critical conditions, LCCC, in which strict compliance with the LCCC behavior is imperative throughout the experiment.

8.5.1 Sensitivity and Instability of Critical Conditions Establishment of CCP is a fine balance of entropy and enthalpy compensation which is very much dependent upon the SP-MP-polymer-temperature combination. As have been shown in Sect. 8.2, slight changes in MP composition and temperature results in deviation from critical behavior [14]. The stability and sensitivity of CCP varies from case to case [1–4, 191]. In most of cases, CCP is a sharp point with regard to MP composition and temperature and slight variation (triad) that allowed for establishment of its CCP. Hence, molar mass independent elution at CCP is observed for the copolymers with the same composition. The polymeric chains having different chemical composition will elute at the eluent composition that corresponds to the CCP of that particular combination of monomers [2, 7]. Moreover, changes in the

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microstructures at different moments in the statistical process have no significant contribution to the retention [4, 5, 118]. Thus, a statistical copolymer is mostly a blend of different copolymers that vary in chemical composition. Different compositions of polymer chains lead to different CCP and in turn different retention that makes the basis for separation of statistical copolymers by EGIC. Brun was the first to report the shifting of elution behavior of statistical polymers as a function of eluent composition from IC to SEC through CCP [7]. Later, Bashir and Radke have demonstrated the applicability of typical equations developed for homopolymer for the retention of styrene-ethyl acrylate copolymers [2]. LCCC is generally not suitable for analysis of statistical copolymers especially for composition analysis since it is an isocratic mode. A LCCC run in an eluent composition corresponding to a specific composition of copolymer, will render elution of other copolymer compositions either in SEC or IC regime. The approach is further associated with incomplete recovery of polymer chains eluting in the IC regime. Hence, isocratic mode based on complete entropy/enthalpy compensation (LCCC) is not appropriate technique for analysis of statistical copolymers. The methods based on continuous change in the entropy/enthalpy balance have proved to be more favorable for chemical composition analysis of statistical copolymers. Numerous literature have appeared for chemical composition analysis of a variety of statistical copolymers using EGIC such as, styrene-butyl acrylate copolymers [119], cellulose acetates by degree of substitution [120], poly(styrene-co-ethyl acrylate) [39, 121, 122], poly(styrene-co-methyl acrylate) [123], gradient copolymers of n-butyl acrylate (nBA) and isobornyl acrylate (IBA) [124], partially hydrolyzed poly(vinyl alcohol) according to degree of blockiness of hydrolyzed and unhydrolyzed segments [125, 126], and so on. For the sake of demonstration, the EGIC elugram of blend of poly(styrene-co-ethyl acrylate) copolymer varying in their composition is shown in Fig. 10.8 [122]. The authors synthesized number of statistical poly(styrene-co-ethyl acrylate) copolymers having different compositions along with polystyrene and poly(ethyl acrylate) homopolymers by free radical mechanism. The copolymers have comparable overall molar mass while having different chemical composition. Sample-1 is poly(ethyl acrylate) and sample-6 is polystyrene while samples 2,3,4, and 5 are poly(styrene-co-ethyl acrylate) (SEA) copolymers having 40, 50, 60, and 80% styrene units, respectively. The starting eluent of the EGIC method was 100% ACN on a RP ODS column. In ACN, poly(ethyl acrylate) had no enthalpic interactions with the RP SP and separation is dominated by entropy changes rendering elution of poly(ethyl acrylate) in SEC regime. However, the other component of the statistical copolymer (styrene) has strong enthalpic interactions with the SP in the initial eluent. THF is used as the stronger component of the eluent that promotes desorption of the styrene units from the SP. Different composition of the copolymer render a predominant triad arrangement (or even longer) of the repeat units throughout polymer chain and this extended segment behaves like pseudo repeat unit. An increase in the THF content of eluent during the chromatographic run brings the eluent composition corresponding to the CCP of any particular polymer composition that render desorption and subsequent elution of polymer chains having that specific composition. Sample-2 is a SEA copolymer having 40% styrene content that

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eluted behind the poly(ethyl acrylate). Other SEA copolymer samples eluted later in order of increasing styrene content. The eluent composition at the elution of any composition of SEA corresponds to the CCP of that particular composition. Finally, polystyrene elute in the eluent composition corresponding to the CCP of PS under applied experimental conditions. Recently, Peltier et al. demonstrated the effect of monomer distribution on retention behavior of a library of acrylamide copolymer systems using EGIC [127]. The authors synthesized copolymer systems having similar degree of polymerization (DP) and relative content of repeat units but varying in segmentation such as statistical, multi-block, and di-block copolymers. The examples shown in Fig. 10.9 are copolymers of N-2-(aminoethyl) acrylamide (AEAm) and N-isopropylacrylamide (NIPAm). All three sets of polymers have DP of 100 while varying in relative content of both repeat units from 70–30 (A), 50–50 (B), and 30–70 (C). The copolymers also vary in segmentation such as statistical, multi-block, and di-blocks. EGIC profiles were optimized using water and ACN for homopolymers of AEAm and NIPAm. The analysis of the synthesized polymers, varying in their segmentation but similar in molar mass and chemical composition, revealed the selectivity of the chromatographic behavior for the segmentation of repeat units. Similar elution trend is followed in all three cases, homopolymer AEAm eluted first followed by statistical, multiblock, di-block, and finally homopolymer NIPAm. It is pertinent to note that the elution volume of copolymers increased with an increase in the NIPAm content for three different segmentations, however, the elution trend remains the same.

Fig. 10.8 EGIC elugrams of styrene-ethyl acrylate copolymers (SEA) on a RP ODS column using acetonitrile → THF gradient [122]. Copyright 2009, De Gruyter

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Fig. 10.9 EGIC elugrams of AEAm/NIPAm (DP = 100) varying in segmentation (A) 70/30, (B) 50/50, C 30/70 using water → ACN gradient on a RP ODS column [127]. Copyright 2018, Royal Society of Chemistry

The selectivity of microstructure on the elution behavior of copolymers using EGIC have been demonstrated by several authors [2–7]. Very recently, a comprehensive study in this context is conducted by Zargar and coworkers [128]. The copolymers used in the study consists of poly(propylene oxide) (PPO) and poly(propylene phthalate) [P(POPA)] with comparable molar mass and average chemical composition. A gradient copolymer refers to a copolymer in which chemical composition

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varies along the chain. Gradient copolymers have a gradual transition from predominantly repeat unit A to predominantly repeat unit B along the polymer chains [129]. The strength (steepness) of the gradient in a gradient copolymer determines the speed of transition from dominance of one repeat unit to the dominance of the other repeat unit. Copolymer without composition gradients will be alternative copolymers. Introduction of a weak composition gradient starts transition from dominance of one repeat unit in the start of the chain to the dominance of the other at the other end of the chain, however, both comonomers are present in sufficient amount at both ends of the chain. Conversely, the transition from one repeat unit (A) to the other (B) is rapid and the copolymer has a blocky structure for a copolymer with a strong composition gradient. The properties of gradient copolymers with strong gradient are close to their block copolymer analogs. Gradient copolymers have gained significant attention recently in stabilization of emulsions, compatibilization of polymeric blends, and as thermoplastic elastomers etc. [129]. Three different statistical copolymers and a block copolymer were synthesized having similar chemical composition and comparable molar mass [128]. Three statistical copolymers differ from each other in strength of composition gradient, gradient-1 (G1) is weak followed by gradient-2 (G2) and gradient-3 (G3) is the strongest. G1 is the copolymer with slow transition of any dominance of comonomers and resulting polymer is more like a random copolymer. G2 has comparatively strong gradient resulting in blocky structures having dominance of blocky structure of A in start to blocky structures of B at the other end of the chain. G3 has the strongest gradient with even longer segments of both types of repeat units at different end of the chain, making it more like a block copolymer. The focus of this study was to evaluate the influence of microstructure on the retention behavior of polymers in different modes of interaction chromatography. For this purpose, two RP columns PS-DVB and ODS were used for the analysis of abovementioned products by EGIC. EGIC method comprised of a linear gradient of 0 → 35% THF in ACN and 0 → 65% THF in ACN for PS-DVB and ODS columns, respectively (Fig. 10.10). In both systems, PPO interacted strongly with the SP and eluted later compared to P(POPA). The influence of molar mass on retention was much pronounced on PS-DVB column compared to ODS column. Statistical copolymers having same average chemical composition but different gradient strength eluted in the order of increasing gradient strength. Although, the molar mass resolution of both PPO and P(POPA) homopolymers was better on PS-DVB column, the difference in resolution of gradient polymers remained similar. In the context of polyolefins, EGIC is the most widely employed interaction based chromatographic method that operates at elevated temperature (>150 °C). Separation of polyolefins by EGIC is based on continuous change in the polarity of the eluent during the run. The choice of solvents in this context is rather limited since many of common chromatography solvents have boiling point below 100 °C. The polar solvents having high boiling point include 2-ethyl-1-hexanol, n-decanol, cyclohexyl acetate, hexyl acetate, cyclohexanone, ethylene glycol monobutyl ether while nonpolar solvents in this context could be n-decane, TCB, DCB etc. The eluent gradient of any combination of above-mentioned polar and non-polar solvent render elution of adsorbing constituent of the polymer sample. HT-EGIC has been applied for chemical

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Fig. 10.10 EGIC elugrams of P(POPA) and PPO homopolymers, block copolymer, and gradient copolymers with different gradient strengths (A) PS-DVB column using 0 → 35% THF in ACN, (B) ODS column using 0 → 65% THF in ACN [128]. Copyright 2022, American Chemical Society

composition analysis of copolymers of ethylene with non-olefinic comonomer [130]. Typical examples in literature are ethylene-norbornene copolymers [104], carboxylic acid-functionalized polyethylene [131], ethylene–vinyl acetate copolymers (EVA) [23, 132–134], and ethylene-acrylate copolymers [92, 135, 136], ethylene-propylenediene terpolymers (EPDM) [137, 138], oxidized polyolefins (waxes) [78]. EVA copolymers are industrially important polymers whose applications are very much dependent upon their chemical composition (VA content) for instance, EVA having VA content 2–15, 20–30, >50% are employed for their application in films, foams, and hot melt adhesives, respectively. On a plain silica column using an eluent gradient of decalin to cyclohexanone allows exclusion of PE in 100% decalin (nonpolar component of eluent) whereas poly vinyl acetate (PVAc) is strongly retained on the column in initial eluent composition (100% decalin), Fig. 10.11A [134]. EVA copolymers of similar molar masses but different chemical composition (VA content) eluted in order of increasing VA content. The method provides a window of 12 mL for separation of EVA copolymers as per VA content of the copolymer. On the contrary,

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separation of EVA copolymer is recently demonstrated on a porous graphite Hypercarb column using linear eluent gradient from cyclohexanone/2-ethyl-1-hexanone to TCB [23]. The developed method renders separation of EVA copolymer in whole composition range of EVA copolymer (0–100% VA content). In this case, PE is strongly adsorbed on the SP in the initial eluent composition. It is important to mention here that PVAc also retained in the initial eluent composition [100% cyclohexanone or 2-ethyl-1-hexanone (2E1H)] and elute only after start of the gradient. However, the order of elution reversed as compared to previous report on NP silica column, Fig. 10.11B. Higher content of VA render early elution of EVA compared to the samples with lower content of VA. Above-mentioned separations address functionalized olefin polymers containing non-olefinic constituents. Obviously, the more relevant question is the development of separation systems for olefinic copolymers, where all commoners belong to the class of olefins such as ethene, propene, butene, hexene, octene etc., according to their chemical composition. As repeatedly iterated, EGIC method may be based on combined effects of precipitation-redissolution or adsorption–desorption phenomenon. The approach based predominantly on precipitation-redissolution phenomenon has been reported for separation of ethylene-rich and propylene-rich fractions of EPC [139, 140], and for chemical composition distribution of LLDPE [141]. In this approach, the SP is not very important as no enthalpic interactions are involved and the transport of the analyte in the column is based on its redissolution after precipitation in the initial eluent composition. The major advancement in HT-HPLC of polyolefins is the development of adsorption–desorption phenomenon. It is well-known that PE and PP can have irreversible adsorption on zeolites [61, 62]. However, any chromatographic system requires adsorption followed by systematic desorption of the analytes. The introduction of carbon based SP (Hypercarb) in polymer chromatography opens new possibilities for interaction-based separations of polyolefins [54, 55, 57]. This development overcomes the classical limitation in characterization of polyolefins which is access

Fig. 10.11 HT-EGIC elugrams of ethylene–vinyl acetate copolymers (A) on a NP Si column using decalin → cyclohexanone gradient, sample solvent: decalin for EVA and TCB for PVAc standards [134]. → Copyright 2007, American Chemical Society; (B) on a Hypercarb column using 2E1H TCB gradient [23]. Copyright 2019, Springer Nature

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to only crystallizable part through crystallization-based techniques like CRYSTAF, TREF, and CEF. Polyolefins can be adsorbed and subsequently desorbed from porous graphite SP selectively based on the eluent composition [142]. Statistical polyolefin copolymers are one of the major industrial polymers that are employed for a variety of applications. The chemical composition of these important polymers is the major criterion for their potential applications. HT-EGIC has been employed for separation of these copolymers according to chemical composition, for instance ethene/propene (EP) [55, 56, 143–148], Ethene/1-butene (EB) [60, 149], ethene/1-hexene (EH) [54, 150], ethene/1-heptene [151], ethene/1-octene (EO) [151–156], LLDPE [157, 158], ethene/1-alkene [110, 111, 142], and propene/1-alkene [110, 111]. As mentioned earlier LLDPE is basically a linear copolymer composed of ethene and n-alkene repeat units. HDPE, a linear long alkane, have strong enthalpic interactions with the flat surface of Porous graphitic carbon (PGC). In case of LLDPE, short alkyl pendant group (C1-C6) on the repeat unit of n-alkene hinders the adsorption of the linear alkane on the flat surface of PGC. The homopolymer of poly(nalkene) is excluded from the pores of the PGC SP in the initial eluent composition (100% 2-ethyl hexanol or decanol). On the contrary, HDPE strongly interact with the SP and require a desorption promoting solvent such as TCB for its elution. A linear gradient starting from adsorption promoting solvent (2-ethyl hexanol or decanol) to TCB render elution of HDPE. The elution volume of PE remains fairly independent of its molar mass above 20 kg/mol [55]. Statistical polyolefins in this case, elute between the elution volume of two homopolymers in order of decreasing content of n-alkene [111]. The separation in this case is very much dependent on the column temperature and the adsorption promoting solvent. A column temperature of 140 °C [159] allowed separation of ethene/1-octene copolymer in whole composition range compared to only 0–60% at 175 °C [160] using otherwise similar system (1decanol/TCB/Hypercarb). On the same lines, separation of whole composition range of ethene-/1-octene copolymer is possible using 2-octanol as adsorption promoting solvent at 160 °C [137]. There is a strong inverse dependence of retention volume on the n-alkene (1-propene, 1-butene, 1-hexene, 1-octene, and 1-decene) content in polyolefin copolymers [111]. Herein, we demonstrate by taking examples of EP [148], EB [60], EH [54], and EO [155] on a Hypercarb SP using linear gradient starting from a weak chromatographic solvent 2-ethyl-1-hexanol, 1-decanol, EGMBE, and n-decane to TCB. Difference in the elution volume/retention times is attributed to different column dimensions used in different studies. Figure 10.12A demonstrates separation of EP as per propene content [148]. Isotactic PP eluted earliest while EP copolymers eluted in order of decreasing P and increasing E content. Same trends follows for EB copolymers using 1-decanol → TCB Fig. 10.12B [60], EH using 2-ethyl-1-hexanol → TCB Fig. 10.12C [54], and EO using EGMBE or 1-decanol → TCB Fig. 10.12D [155] linear gradient on Hypercarb SP. Hence, judiciously selected eluent system (components and slope of gradient) render separation of LLDPE samples as per their chemical composition covering whole composition range of 0–100%. It is pertinent to mention here that all these separations are based on preferential interactions of the repeat units with the PGC SP irrespective of their crystallization behavior. Hence,

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Fig. 10.12 HT-EGIC elugrams of polyolefin copolymers on Hyprcarb columns using 2-ethyl-1hexanol/1-decanol/ EGMBE/ n-decane → TCB gradient (A) ethene-propene (EP) [148]. Copyright 2014, American Chemical Society; (B) ethene-butene (EB) [60]. Copyright 2015, Elsevier; (C) ethene-hexene (EH) [54]. Copyright 2010, Elsevier; (D) ethene-octene (EO) [155]. Copyright 2010, American Chemical Society

HT-HPLC methods opens inroads into the analysis of amorphous part of polyolefins which was not accessible earlier.

10.2.10 Dendrimers Dendrimers are the macromolecules consisting of tree-like arms or branches having two different environments, surface chemistry based on terminal functional groups and a shielded interior. Dendrimers have gained significant attention for biomedical applications such as drug and gene delivery, and as diagnostic reagent [161–163]. An important link between synthesis and applications is the comprehensive characterization of dendrimers in context of number of generations to build a structure– property correlations. Several techniques have been employed for characterization of dendrimers such as SEC, FTIR, NMR, Raman, UV–Visible, fluorescence, X-ray, circular dichroism, diffraction, mass spectrometry, intrinsic viscosity, SANS, SAXS, Laser Light Scattering, microscopy, DSC, EPR, electrochemistry, electrophoresis, and dielectric spectroscopy [164–166]. The major limitation of the above-mentioned techniques is decline in their performance with the increase in the size of the

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193

Fig. 10.13 EGIC chromatograms of amine-terminated dendrimers (G1NH2-G9NH2), gradient 0 → 40% ACN in 30 min (balance water) on a C5 silica-based RP-HPLC column [168]. Copyright 2005, American Chemical Society

dendrimers. HPLC is complementary technique to comprehensively characterize dendrimers overcoming the limitations of the previously employed techniques [167– 170]. In this context, the pioneering example of employing EGIC for separation of poly(amidoamine) (PAMAM) dendrimers according to different generations is demonstrated by Islam et al. [168]. Water → acetonitrile gradient in a range of 0–40% in 30 min was used for successful separation of nine generations of amineterminated-PAMAM, see Fig. 10.13. The construction of dendrimers is accomplished through repetitive alkylation and amidation steps. The controlled synthesis yielded next generation having well-defined molar mass. The separation in HPLC is based on interaction of the surface amine groups with the SP. In context of HT-EGIC, separation of linear, branched and dendric polyethylene is shown on a Hypercarb column using two different gradient system namely decane → ODCB and 1-decanol → TCB [113].

10.3 Limitations and Opportunities EGIC is a versatile and flexible technique that provides a wide range of possibilities owing to numerous solvent combinations along with different gradients shapes. EGIC has been effectively applied for separation of complex polymers composed of distinct segments varying in their interaction strength with the SP. The applications of EGIC in polymer analysis includes homopolymers, end-functionalized homopolymers, block copolymers, graft copolymers, branched copolymers, statistical copolymers, dendrimers, and polymers varying in microstructure etc. EGIC requires a reliable gradient pump that must be able to mix the solvents in exact proportions and deliver it to the column at a constant flow rate. Hence, repeatability of the performance of gradient pump for exact mixing and flow rate is often compromised. Moreover, continuous change in the eluent composition during

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EGIC restricts the use of bulk property detectors such as RI. Use of UV detector is also limited since not many polymers have a chromophore in their repeat units. The choice of detectors is limited to evaporative light scattering detector which has a non-linear response that makes quantification tricky [35, 36]. Moreover, response of ELSD may be influenced by several parameters such as eluent composition [37, 38], and nature of the polymer [38, 39]. ELSD only reveal the elution of the nonvolatile component without any additional information on the polymer such as dn/ dc (RI detector) or absorptivity (UV detector). For an analysis of polymers with a constant composition (including homopolymer), it can provide useful information (after appropriate correction of non-linearity). However, no additional information on the nature of the polymer can be obtained. The back-pressure generated by chromatographic system on the pump may increase after injection due to precipitation of the sample in the initial eluent composition [21]. The precipitation results in the breakthrough peaks [171]. The breakthrough peaks can be avoided or at least minimized by using the weakest possible injection solvent, injecting small volumes carrying high concentrations of the sample, and weakest possible initial eluent [33]. Best scenario is to use the solvent that is the same as the initial eluent if possible. Strangely, EGIC also suffers from incomplete sample recovery [172, 173].

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Chapter 11

Temperature Gradient Interaction Chromatography

Different sizes of macromolecules in solution render their separation based on their limited access to the pores of the SP. This hindered access results in pathways of varying lengths that depend upon hydrodynamic sizes of the analyte molecules. The larger analyte molecules have limited access to the porous SP and elute early compared to smaller molecules that have more access to the porous SP, the elution order in this process is from larger to smaller hydrodynamic size in a so-called exclusion regime. Exclusion is an entropy-controlled process that is widely used in polymer chromatography on non-interactive SPs. On interactive porous SPs (RP & NP), sizebased separations of polymers are possible using strong eluent that don’t render any enthalpic interactions of the polymers with the SP. Polymers elute before the void volume of column in order of decreasing molar mass, similar to traditional SEC on non-interactive SPs. However, a weak eluent will render enthalpic interactions of the analyte with the SP that results in elution of polymers in order of increasing molar mass after the void volume of the column. Similar to interaction chromatography of small molecules, separation is based on the interactive strengths of the polymer molecules with the SP. The two opposing elution orders of polymers are majorly the function of polarity of the analyte, eluent, SP, and temperature. Composition of the eluent and temperature are the most facile variables for switching the elution order of any polymer analyte. Using strong eluent composition render elution in SEC regime in contrary to a weak eluent that render elution in the interaction regime. Entropy/enthalpy compensation in a chromatographic system can also be realized by changing the temperature of the column since each adsorptive event during a chromatographic run is either exothermic or endothermic [1, 2]. The elution behavior based on enthalpic interactions can be maneuvered by continuous change in the temperature of the separation system, termed as temperature gradient on the similar principles as for gas chromatography. As mentioned earlier, entropic effect is always present for polymeric analytes while using a porous chromatographic column owing to variable access of different sized polymer molecules to the pores. A temperature gradient may lead to reduction in the interaction strength that may subsequently render dominance of exclusion phenomenon at some point. However, no switching © The Author(s), under exclusive license to Springer Nature Switzerland AG 2023 M. I. Malik and D. Berek, Liquid Chromatography of Synthetic Polymers, Physical Chemistry in Action, https://doi.org/10.1007/978-3-031-34835-8_11

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of elution behavior to SEC is observed in EGIC or TGIC since all the polymer molecules elute when reach the critical MP composition or critical temperature, respectively. Temperature gradient interaction chromatography (TGIC) is a chromatographic technique in which separation of analytes is realized by varying temperature of the separation system (column) during the run while using isocratic eluent. Similar to other interaction-based chromatographic techniques for polymers, TGIC opens new possibilities for characterization of complex polymers with regard to functional groups, microstructure, chemical composition, or chain architecture. In any IC methods, separation of polymer molecules is based on their interaction strength with the SP that needs to be controlled during the elution process, depends both on chemical composition and molar mass in contrary to SEC where elution is controlled by hydrodynamic size (also function of molar mass and chemical composition) of the polymer molecules. Polymers are heterogeneous materials whose constituents may have very different interaction strength with the SP. For practical purposes, interaction strength of polymers during a chromatography run needs to be weakened. EGIC is the classical method used for this purpose where composition of eluent is changed as a function of time (see Chap. 10). In this case, the content of desorption promoting solvent of the eluent is increased in the course of chromatographic run. However, change in composition of eluent during the chromatographic run render a background drift in the detector signal of many commonly used detectors for chromatography such as differential refractometry, light scattering, or viscometry. Moreover, EGIC separations are often not exactly reproducible due to sensitivity of elution to the eluent composition and small differences in eluent composition at any particular run-time may lead to a different elution profile. TGIC allows use of the traditional HPLC detectors since eluent composition remains same throughout the process. Although, temperature gradient only provides a limited control over retention behavior compared to EGIC, it is more reproducible. In TGIC, the retention of the analytes is reduced by increasing temperature during the run. The temperature gradient allows shift of retention behavior from interaction to exclusion through CCP. It is pertinent to mention here that reversal of elution order is never observed in any TGIC or EGIC run because all the analyte molecules elute before reaching these conditions. In context of TGIC, a suitable eluent composition (in interaction regime near CCP) is the first prerequisite. Typically, increase in temperature results in desorption and early elution of polymers, if the sorption of the polymer to the SP is a typical exothermic process [1, 2]. In rare cases, however, inverse behavior is also observed, an endothermic sorption process, increase in retention with increase in temperature [3–7]. In a multi-component polymeric system, separation of different components with regard to functional group, chemical composition, and topology is possible by TGIC.

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11.1 Working Principle Reduction in the interaction strength of the strongly retained analyte with the SP as a function of run-time is the basic principle of any gradient run. EGIC is the most widely used and versatile gradient method to achieve this target by changing the eluent composition. Change in temperature is the other option in this context since most of the sorptive interactions are enthalpic in nature. Chromatographs equipped with gradient pumps are commercially available for EGIC which is not the case for TGIC. However, modification of a typical chromatograph to TGIC is rather trivial. Main requirement of any TGIC chromatograph is provision of changing the temperature of SP and eluent in a programmed manner during the run. A circulating fluid in the column jacket from a programmable circulator allows systematic variations in the temperature of the system, Fig. 11.1. An isocratic eluent having composition close to the CCP allows only weak interaction of the analyte with the SP. According to molecular statistical model of TGIC, elution order of polymers may follow either increasing or decreasing order of molar mass [8]. Although, elution in order of increasing molar mass is the major behavior based on exothermic interactions, there are cases where elution order is reversed due to endothermic interactions [3–7]. Selection of a suitable eluent is the prerequisite for any TGIC separation since it is essentially an interaction-based process where chemical nature of the analyte, polarity of the eluent and SP are of crucial importance. In EGIC, eluent composition is changed from IC to SEC through CCP during the run. On the same lines, column temperature is changed from IC to SEC through CCP in any TGIC during the run using an isocratic eluent which is typically close to CCP of the analyte. However, polymer samples don’t experience SEC regime during either EGIC or TGIC since all the polymer molecules get eluted while reaching at critical MP composition or critical temperature, respectively. If sorption is an exothermic process, switching from IC to SEC can be realized by increasing temperature, the typical case. On the other hand, if sorption of polymer on the SP is an endothermic process, an inverse temperature gradient is required for separation [3–7, 9]. An IC separation in this context is based on positive entropy change that overcomes the enthalpy penalty [7, 9].

Fig. 11.1 Schematic diagram of the TGIC apparatus [1]. Copyright 2021, Elsevier

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After selection of the appropriate eluent for the analyte of interest at a given SP, change in column temperature can be effectively used to control the retention. In this context, the CCP composition of the polymer under study on given SP is the key information that can be found in literature [10–14] or by EGIC [15–19]. Ideally, eluent for a TGIC should be thermodynamically good solvent while promoting sorption of the polymer on the SP. However, a good solvent often promotes desorption and vice versa. Hence, often relatively poor solvent combination that allows adsorption of polymers at initial temperature is used as an eluent for TGIC. The use of different injection solvent from the eluent may lead to breakthrough peaks that should be avoided [20– 25]. Hence, the use of same solvent combination both as injection solvent and eluent is recommended. Isocratic eluent composition in TGIC render application of traditional LC detectors such as refractive index detectors, light scattering detectors, and viscosity detectors [26]. The sample capacity of IC is typically large compared to SEC despite similar concentrations of injection samples are used [27].

11.2 Applications of TGIC in Polymer Analysis 11.2.1 Molar Mass Separation of Homopolymers Molar mass analysis of polymers by SEC is a universal approach, however, it suffers from limited resolution. It is near to impossible to avoid overlapping of peaks even in case of narrow molar mass standards due to band broadening [28–30]. On the other hand, TGIC separation of polymers is not universal and require an optimized set of eluent-SP-temperature gradient program. However, an unprecedented resolution can be achieved resulting in fully resolved peaks of polymer standards differ only by a factor of 1.5. Molar mass analysis of several polymers by TGIC method is reported such as poly(ethylene oxide) (PEO) [9], poly(dimethyl siloxane) (PDMS) [31], poly(methyl methacrylate) (PMMA) [32], poly(2-vinyl pyridine) (P2VP) [33], polyisoprene (PI) [34], polystyrene (PS) [34] and poly(vinyl chloride) (PVC) [35]. As a typical example, a TGIC analysis of most widely used narrow molar mass polymer standards, polystyrene (PS), is demonstrated in Fig. 11.2A [1]. Eluent was CH2 Cl2 /CH3 CN (57/43 v/v) that allows weak interaction of PS with the RP ODS SP. A temperature gradient in a range 10–40 °C in 40 min promote desorption and elution of PS standards in order of increasing length (molar mass), having excellent resolution compared to SEC. Moreover, true and small values of dispersity (M w /M n ) are obtained by TGIC compared to SEC [36, 37]. The lower values of M w /M n as obtained by TGIC follows the Poisson distribution as per theoretical predictions. In another study, Park and Chang reported characterization of P2VP homopolymers by temperature gradient interaction chromatography (TGIC) in order to separate high molecular weight samples [33]. Moreover, application of TGIC to polyolefins has also attained significant attention recently. Polyolefins are soluble at temperatures higher than their melting points

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Fig. 11.2 TGIC elugrams of mixture of narrow molar mass homopolymer standard, molar masses are mentioned at the top of the peaks (A) PS on a RP ODS column in CH2 Cl2 /CH3 CN (57/43, v/ v) using temperature gradient of 5 → 40 °C in 40 min [1]. → Copyright 2021, Elsevier; (B) PE on a Hypercarb column in 1-decanol/TCB (47.5/52.5 v/v) using temperature gradient of 80 170 °C in 100 min [38]. Copyright 2021, Elsevier

in high boiling solvents. As mentioned in the previous chapters, 1-decanol is an adsorption promoting solvent while TCB is a desorption promoting solvent for polyolefins on Hypercarb SP. An eluent composition near to CCP is required for any TGIC run. In this context, a mixture of 1-decanol/TCB (47.5/52.5 v/v) is employed as an isocratic eluent [38]. The dissolution of the polyolefins is assured by keeping it at 160 °C for two hrs. Temperature of the system was then lowered to 80 °C and a linear temperature gradient started that reaches 170 °C in 100 min. The elution profile is shown in Fig. 11.2B where 0 mL refers to the starting point of temperature gradient. The method renders an excellent separation of linear PE in a broad range of 1–1050 kg/mol. Similarly, separation of isotactic PP with regard to molar mass is also shown in the same publication.

11.2.2 Polymer Blends LCCC and EGIC can be employed for separation of polymer blends. However, sensitivity, reproducibility, and detection issues are some of the associated limitations. In this context, TGIC is more reproducible, and can be performed using typical HPLC detectors. Moreover, separation of blends of polymers can be achieved with regard to molar mass of both components. A molar mass-based separation of polymer blends is reported for PS/PMMA [39], PI/PS and PI/PMMA [40]. For separation of polymer blends, eluent must be strong enough to promote exclusion of one component while promoting weak enthalpic interaction of the other component at the initial temperature of the system. The interaction strength should be weak enough to allow desorption and elution of the second polymer component by increasing temperature. Hence, an appropriate eluent selection is imperative for each polymer blend. As a typical case, separation with regard to molar mass of PMMA and PS blends is demonstrated

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Fig. 11.3 TGIC elugram of a mixture of 10 polystyrene (a–k: 1.7 k, 5.1 k, 11.6 k, 22.0 k, 37.3 k, 68.0 k, 114 k, 208 k, 502 k, 1090 k, 2890 k) and 5 poly(methyl methacrylate) (1–5: 1500 k, 501 k, 77.5 k, 8.5 k, 2.0 k) standards on a set of RP ODS columns in CH2 Cl2 /CH3 CN (57/43 v/v) using temperature gradient of 0 → 50 °C in 60 min [39]. Copyright 1996, American Chemical Society

in Fig. 11.3 [39]. A temperature gradient in a range of 0–45 °C while using eluent composed of CH2 Cl2 /CH3 CN 57/43 (v/v) on a set of three ODS columns render excellent separation of PS/PMMA blends with regard to molar mass of both components. PMMAs are excluded from the porous SP at the initial temperature of the system while PS interacted with SP and did not elute. Therefore, PMMAs elute in SEC regime before the dead volume of the column set. Subsequently, a continuous increase in temperature resulted in desorption and elution of PS in order of increasing molar mass.

11.2.3 End-Functionalized Linear Homopolymers A distinctly different end-group from the repeat unit in the polymer chain is referred as end-functionalization. End-functionality is especially important for telechelics or intermediate polymers for subsequent reactions. SEC separations are not sensitive to these important functionalities. LCCC is the most widely employed methods for separation with regard to functionality based on its blindness to the molar mass disparity of the critical polymer [41–50]. Molar mass independent elution enhances the effect of other variables in the structure (functional end-groups). Any IC approach requires stronger interaction of the end-group with the SP compared to the repeat

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Fig. 11.4 TGIC elugrams of PS of different molar masses and end-groups (A) RP-TGIC, RP ODS column in CH2 Cl2 /CH3 CN: 57/43; (B) NP-TGIC, NP Si column in isooctane/THF: 55/45 [34]. Copyright 2001, Elsevier

unit. For instance, NP columns are required for polymers having polar functional endgroups while RP for the polymer with non-polar functional end-groups. However, the resolution with regard to end-group functionality progressively decrease with an increase in the molar mass of the polymer. TGIC provides an alternative platform that can separate according to both molar mass and end-group functionality vis-avis. In this context, typical examples include separation of hydrogen and hydroxyl terminated PS [34], methoxy, carboxylic acid, and dipolystyryl ether terminated PS [51], silyl, hydroxyl, and bromide terminated PS and PB [52]. Importance of selection of appropriate chromatographic system (SP, eluent, conditions) for TGIC is demonstrated by Lee et al. [34, 53–56]. RP-TGIC was unable to differentiate between hydrogen and hydroxyl terminated PS of similar molar mass, Fig. 11.4A [34]. PS having molar mass of 11 and 105 k eluted as a single peak without any differentiation of their end-group. On the contrary, same polymers were separated into four fully resolved peaks on a NP silica column. First two peaks in the range of 1–2 mL correspond to PS 11 k but differing in their end-group, late eluting peak has hydroxyl end-group that interact strongly with the NP SP compared to the repeat unit. In general, the effect of end-group minimizes with increase in the molar mass, however, in this case PS of molar mass 105 k was also fully resolved into two distinct peaks in a range of 4–6 mL. Third peak in the elugram before 5 mL corresponds to hydrogen terminated PS 105 k while hydroxyl terminated PS 105 k retained more and eluted after 5 mL.

11.2.4 Branched Polymers Branched polymers are widely used in rheological studies where their application is very much dependent upon the extent of branching. Modern controlled polymerization methods allow synthesis of these model polymers [57, 58], however, formation of byproducts cannot be avoided. TGIC has ability to resolve these byproducts far better compared to SEC. Main contrast between TGIC and SEC in the separation

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of branched polymers is that SEC separates the polymers by hydrodynamic size while TGIC separates the polymers mainly by MW (interaction strength). Most of side-products in the model branched polymers are imperfect branched structures. For example, star polymers with missing one or more arms have similar hydrodynamic volume with the perfect star and SEC hardly resolves those while TGIC can resolve them well according to their MW. A better understanding of structure-rheological properties correlation can be developed through high resolution TGIC [25, 57–66]. TGIC peaks have low band broadening compared to SEC and its isocratic nature allows triple detection similar to SEC [67, 68]. The resolution can be further improved by coupling of TGIC in first dimension with SEC-TD in the second dimension [59, 68–71]. Linear and branched polymers of similar molar mass have substantially different mechanical and processing properties that opens numerous avenues for their potential applications [57, 58, 72]. SEC has been employed for branching analysis based on concept of chain contraction i.e., the hydrodynamic size of branched polymer is smaller compared to its linear analogue [73]. This allows estimation of chain branching of polymers of same molar mass which is further strengthened by SECtriple detection (concentration, light scattering, and differential viscosity). Linear and branched polymers are compared for their concentration (amount), light scattering (molar mass), and intrinsic viscosity (chain size) at each point of SEC elution profile [74–76]. However, SEC separation is based on hydrodynamic size and not strictly on the molar mass. Therefore, a narrow SEC fraction may have different molar masses and architectures (branching) despite same hydrodynamic size and elution volume. The discrepancy is further deepened by position of branching that may lead to different hydrodynamic sizes of polymers having same number of branches but only differing in their position. On the contrary, separation in any IC method is based on enthalpic interaction of the polymeric analyte with the SP. Therefore, sensitivity of TGIC to chain architecture is less compared to SEC. TGIC render separation of polymers according to number of interacting repeat units (molar mass) rather than hydrodynamic volume. Although, IC render better selectivity for separation of branched polymers according to molar mass compared to SEC, it is still not a complete solution for analysis of branched polymers. Three differently branched PS varying in the length of the backbone and branches were synthesized by introducing PS branch to chloromethylated PS backbone [68]. As a representative example, branched PS polymer having backbone of 140 k and branches of 47 k is compared for their SEC-TD and TGIC-TD analysis in Fig. 11.5. The product is heterogeneous with regard to number of branches and their position. Broad and multimodal SEC profile confirms the heterogeneity of the samples. Unreacted branches elute as a sharp peak at higher elution volume while the early eluting peak of branched PS have significant broadness that may be attributed to different number of branches. The molar mass (Mw ) as obtained by light scattering as a function of elution time confirms that molar mass decreases with increase in the retention time, Fig. 11.5A. A step-wise molar mass curve at higher retention time of the major peak endorses narrow molar mass distributions of different constituents of the sample. The separation of the same sample by TGIC-TD has significantly higher

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Fig. 11.5 Comparative analysis of comb-shaped polystyrene (A) SEC on mixed bed Polypore using THF; (B) TGIC on RP ODS column in diethyl malonate using temperature gradient of 27 → 37 °C in 80 min [68]. Copyright 2017, John Wiley and Sons

resolution, Fig. 11.5B. Unreacted branches elute first followed by branched PS as per number of branches based on their molar mass. A stair-like molar mass curve for TGIC-TD further confirms the better resolved constituents with regard to molar mass that has direct correlation with the number of branches. The separation by TGIC is based on increase in number of repeat units with branches according to molar mass while SEC separation is not enough to separate since the hydrodynamic size does not change much with increase in the number of arms. Another relevant example is analysis of branching based on number of functional end-groups [49, 77–80]. Bivariate branched PS samples, having distribution both in molar mass and number of branches, have rather broad but unimodal peaks in SEC. The separation of the branched PS samples according to number of branches is improved by TGIC. In a representative study, anionically polymerized PS are subsequently linked with p-(chlorodimethylsilyl) styrene (CDMSS). The number of CDMSS-terminated branches increase with increase in the concentration of CDMSS. The silyl group at the branch site and the n-butyl group at the chain end have stronger enthalpic interaction with the RP ODS SP. These branched polymers have two overlapping distributions namely molar mass and number of branches. The resolution of number of branches by TGIC is directly connected to the broadness of the molar mass. Broad multimodal peaks are obtained for branched PS prepared by n-butyl anions which render broad MMD, Fig. 11.6A [49]. Conversely, narrow molar mass distribution of branched PS as prepared by sec-butyl Li resulted in well-resolved branched PS peaks with regard to number of branches, Fig. 11.6B [77]. In both cases, ODS SP was used with an eluent constituted CH2 Cl2 /CH3 CN. However, the branched PS analyzed in the first case have broad MMD compared to the branched PS in the latter case. It is pertinent to mention here that LCCC render suppression of molar mass distribution and is the most appropriate choice for separation of linear and branched polymers according to number of functional end-groups [44, 49, 77–87].

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Fig. 11.6 TGIC elugrams of branched PSs on RP ODS column in CH2 Cl2 /CH3 CN (55/45, v/ v) using shown temperature gradient (A) broad molar mass branched PSs [49]. Copyright 2006, Elsevier; (B) narrow molar mass branched PSs [77]. Copyright 2004, American Chemical Society

In this context, H-shaped polybutadienes (PBD) were synthesized and characterized by TGIC [63, 88]. TGIC separates according to number of interacting repeat units (molar mass) and not as sensitive to architecture of polymers as SEC. These samples are basically H-shaped polymers having four arms and one crossbar. The samples naming refers to length of the arm and crossbar. For instance, HA30B40 correspond to H-shaped PBD with four arms of 30 kg/mol and crossbar of 40 kg/mol [63]. A low resolution of SEC profile for the analyzed products is clearly visible in Fig. 11.7A. However, a multimodal SEC profile indicates the presence of polymer chains that may differ in molar mass and/or extent of branching. TGIC profiles for the same products on an ODS column using 1,4-dioxane as eluent is shown in Fig. 11.7B. TGIC analysis revealed the presence of three or more distinctly different components that are excellently separated from each other. The separation in TGIC is based on number of repeat units that are interacting instead of hydrodynamic volume. TGIC is less sensitive to architecture and reveal much better separation compared to SEC. The molar mass increment by addition of branches can be correlated to the number of arms of H-shaped star polymer. TGIC has also been recently employed for analysis of polyolefins at elevated temperatures. Polyolefins produced by chain-walking catalysis can be of diverse structures depending upon the used catalyst. On a PGC using ODCB and TCB as eluent, different topologies can be separated using temperature gradient, Fig. 11.8 [89]. The sample Ni1 has lowest branching content and is retained more and eluted

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Fig. 11.7 Analysis of star-shaped polybutadiene (A) SEC; (B) TGIC on an RP ODS column in 1,4-dioxane [63]. Copyright 2011, American Chemical Society

at higher temperatures in both eluents. The sample Ni2 has a broad bimodal elution profile. The polymers eluting at low elution temperature have more branching compared to those eluting at a bit higher elution temperature. The elution profile indicates topology/branching heterogeneity of the sample. Moreover, the dendritic nature of Pd1 and Pd2 samples hinder their enthalpic interactions with the flat PGC surface that resulted in their early elution. Different topological orientation of the samples Pd1 and Pd2 render their different elution profiles despite similar branching content (as obtained by NMR). Pd1 and Pd2 samples were not retained in the TCB and eluted before the start of the temperature gradient. However, Pd1 sample retained and eluted after start of temperature gradient while using ODCB as eluent. It is pertinent to mention here that SEC resolution was not sufficient to differentiate among the above-mentioned products. (A)

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Fig. 11.8 HT-TGIC profiles of linear, branched, and dendritic polyethylene on Hypercarb column using eluent (A) ODCB; (B) TCB [89]. Copyright 2020, American Chemical Society

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11.2.5 Star-Shaped Polymers Star polymers is the term applied broadly for branched polymers in which all the branches root from a single center point [90]. There can be further sub-divisions such as star homopolymers, miktoarm star copolymers, star block copolymers, statistical copolymer star etc. Star-shaped polymeric architectures have been employed for myriad of peculiar applications in diverse fields [90–92]. Separation and characterization of branched and star homopolymers with regard to number of arms is a challenging task. Similar value for distribution coefficient for linear and star polymers at the CCP of the repeat unit is predicted by statistical theory of polymer chromatography [78, 93, 94]. Applications of TGIC for analysis of star-shaped polymers have been demonstrated by Chang and his team [60, 61, 67, 69, 83, 95]. The strength of TGIC-TD for the analysis of number of arms of star-shaped polybutadiene (PB) compared to SEC-TD is shown in Fig. 11.9 [67]. Size exclusion chromatography has serious limitations for such analysis since it separates according to the hydrodynamic volume that doesn’t change much compared to linear polymers. SEC-TD only provides average value of number of branches for the SEC fractions. As per Zimm-Stockmayer theory, linear polymer is considered as two arm star that render a deviation of fg from fM for linear precursor, Fig. 11.9A. Hence, branching analysis by SEC-TD seems to be quite successful. However, low resolution of SEC impedes the determination of detailed branch distribution. On the other hand, TGIC-TD analysis of the same sample perfectly resolved stars with different number of branches based on their molar mass, Fig. 11.9B. The temperature of the effluent of TGIC was carefully kept constant in order to get reliable viscosity measurement. The molar mass of fractions with different number of branches perfectly matched the expected values. (A)

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Fig. 11.9 Analysis of PB star with arm length of 35 k, (A) SEC-TD; (B) TGIC-TD on a RP ODS column using 1,4-dioxane as eluent [67]. Copyright 2017, Elsevier

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11.2.6 Statistical Copolymers Polymerization of two or more monomers simultaneously by same mechanism results in copolymers that have different arrangement of these monomers in the chain. The composition of the synthesized copolymers depends upon several factors such as relative reactivity of the monomers, feed ratio, and reaction conditions. Different reactivities of the involved monomers at the experimental conditions results in a drift in the chemical composition of the polymer chains that are produced at different reaction times. Therefore, a drift in the chemical composition of statistical copolymers is not easy to avoid. Determination of distribution of chemical composition is imperative for development of structure–property correlations. Chromatographic behavior of statistical copolymers is similar to homopolymers, a relatively longer segment (composed of 2–4 monomeric units) behaves as a pseudo repeat unit [96–98]. The polymer chains produced at different reaction times may have different composition and subsequently different pseudo repeat unit. The polymer chains will elute at the corresponding CCP of the longer repeat unit independent of the molar mass [18, 97]. Any TGIC chromatography run starts with an eluent/temperature combination that allows enthalpic interaction of the repeat units with the SP. In EGIC, eluent composition is changed during run at constant temperature while temperature variations are used to weaken the interaction strength of the repeat unit with the SP using isocratic eluent in TGIC. While LCCC is not a suitable technique for analysis of statistical copolymers, EGIC has been widely used for analysis of copolymers according to chemical composition (see Chap. 10). TGIC at elevated temperatures has been employed for chemical composition analysis of statistical polyolefins [99– 107]. However, TGIC provides a limited window for manipulating the interaction strength of the polymer with SP [1]. In this context, TGIC analysis of ethylene-octene (EO) copolymers according to chemical composition is presented on a Hypercarb column in ODCB [107]. In this study, authors combined the strengths of dynamic cooling process of CEF [108] with chromatography. TGIC run is divided into three distinct steps namely; introduction of polymer solution into column at constant temperature, retention of polymers on column by reducing temperature, and elution of polymers by raising temperature, Fig. 11.10A. The dynamic cooling processes help in improvement of the resolution of ethylene-octene (EO) copolymers with regard to chemical composition. As is known from EGIC of polyolefins on PGC, linear polymer chains like PE have the strongest interaction with the SP. Introduction of small chain branching due to incorporation of α-olefin sterically hinder the interactions with the SP. The elution of EO copolymers using ODCB as eluent on a PGC column as a function of the elution temperature is demonstrated in Fig. 11.10B. EO having 50% octene eluted at the start of the temperature gradient while subsequent EO copolymers eluted in order of decreasing content of octene as a function of increase in temperature. Finally linear PE eluted at temperature close to 160 °C.

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Fig. 11.10 HT-TGIC of polyolefin copolymer (A) Temperature gradient scheme, (B) elugrams of ethylene-octene (EO) copolymers with different content of octene as given on top of peaks on a Hypercarb column using Ortho-dichlorobenzene (ODCB) as eluent [107]. Copyright 2011, American Chemical Society

11.2.7 Polymers Varying in Tacticity/Microstructure Relative stereochemistry of adjacent chiral center in polymer chains is termed as tacticity. Tacticity of polymers have significant influence on their chemical and physical properties similar to molar mass, architecture, and chemical composition. Although hydrodynamic volumes of polymers with different tacticity are slightly different, it is not enough to separate them by size exclusion chromatography. Similar

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Fig. 11.11 TGIC elugrams of polymers varying in tacticity (A) poly(ethyl methacrylates) and reanalysis of their fractions on a RP ODS column using CH2 Cl2 /CH3 CN (30/70, v/v) as eluent [109]. → Copyright 2002, American Chemical Society; (B) Blend of equal weight ratios of aPP, iPP and HDPE on a Hypercarb column using Ortho-dichlorobenzene (ODCB) as eluent, temperature program: 140 °C → 40 °C → 175 °C at 6 °C/min and 3 °C/min, respectively [102]. Copyright 2009, John Wiley and Sons

to other techniques of IC (LCCC, EGIC), TGIC has also been employed for analysis of polymers with regard tacticity [101, 102, 109, 110]. A pioneering study in this context is reported by Chang and coworkers for separation of three samples of poly(ethyl methacrylates) (PEMA) differing in tacticity [109]. The tacticity of three PEMA samples, defined as rr triad content, were 0 (i-PEMA), 53 (h-PEMA), and 91% (s-PEMA), where s, h, i refer to syndiotactic heterotactic, and isotactic respectively. A mixture of CH2 Cl2 and CH3 CN was taken as eluent and temperature gradient ranged from 30–50 °C. Highly syndiotactic PEMA was least retained compared to i-PEMA that retained most strongly while h-PEMA eluted after s-PEMA and before i-PEMA, Fig. 11.11A-top. The bottom three TGIC elugrams show re-analysis of the fractions of different samples having same molar mass, as confirmed by their MALDI-TOF MS analysis. The analysis confirmed small band broadening of TGIC method compared to SEC. In context of polyolefins, crystallization behavior of semi-crystalline polymers has direct correlation with the tacticity distribution. Hypercarb columns allow selective adsorption and desorption of polyolefins that render new possibilities of analysis of polyolefins beyond the crystallization behavior. A comparison of TGIC and crystallization elution fractionation (CEF) separation of PE, aPP, and iPP is demonstrated in Fig. 11.11B [102]. In case of TGIC, atactic PP eluted first at an elution temperature of 40 °C followed by iPP that eluted at 120 °C. HDPE eluted at the last at a temperature of 160 °C. On the other hand, iPP eluted after HDPE in CEF analysis of the same blend. Late elution of HDPE and improved resolution of separated components of the blend confirms that separation in TGIC is based on enthalpic interactions rather than only crystallization behaviors as typical for CEF.

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11.2.8 Graft Copolymers Graft copolymers are usually used in food packaging, metal adsorption, drug delivery, and dye removal [111, 112]. Graft copolymer samples contain several by-products majorly un-grafted backbone and free grafts along with target graft copolymer. The performance properties of graft copolymers are strongly influenced by presence of these by-products. Moreover, the graft density and length of the grafts are important analytical questions. Analysis of graft copolymers is a challenging task in context of above-mentioned questions. SEC is generally incapable of discriminating these side products and distribution of graft density due to its inherent low resolution. An exact polystyrene-g-polyisoprene is rigorously characterized by TGIC and compared with the SEC [59]. As prepared graft copolymer contains several by-products. These by-products eluted on both sides of the main peak in SEC. These by-products were even better resolved by TGIC (see the original reference). The chemical composition distribution for as-prepared PS-g-PI by TGIC seems not to be reliable due to interference of by-products that render inconsistent overlap of detector signals. Main SEC peaks are fractionated and analyzed by SEC and TGIC for analysis with regard to grafting density. Analysis of SEC-fractionated PS-g-PI eluted as a narrow monomodal peak, Fig. 11.12A. As per SEC analysis, the fraction contains narrowly distributed PS-g-PI which was further confirmed by decent match of signals of different detectors. TGIC analysis of SEC-fractionated PS-g-PI was performed at the interaction conditions for PI while SEC conditions for PS. Hence, an increase in the content of PI renders an increased retention time. TGIC chromatogram of SEC-fractionated PS-g-PI is resolved into several different species varying in molar mass of PI, Fig. 11.12B. A narrowly distributed SEC profile of the same clearly shows the limitations of SEC for complex polymeric systems. The possibility of applications of various different detectors for TGIC is one of the major advantages of TGIC over EGIC. An excellent separation of PS-g-PI with regard to number of grafts by TGIC reveal the information that was not possible by SEC or any other technique. Another (A)

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Fig. 11.12 Comparison of resolution of SEC and TGIC for PS-g-PI (A) SEC; (B) TGIC on a RP ODS column in 1,4-dioxane [59]. Copyright 2017, American Chemical Society

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study reports TGIC analysis of comb-shaped polystyrene having hydrogenated PS as backbone while deuterated PS as grafts on both NP and RP SP [25].

11.2.9 Block Copolymers Retention in LC of polymer is strongly affected by the monomer sequence. Chromatographic behavior of statistical copolymer is completely different from that of a block copolymer of similar chemical composition and molar mass [98]. Statistical copolymers elute between the CCPs of homopolymers of constituent monomers near the CCP of the composition of statistical copolymer while block copolymers elute very near to the CCP of the strongly interacting block. Hence, statistical copolymers elute earlier than the block copolymer of same chemical composition and molar mass [16–18, 96–98]. Interaction strength in TGIC is controlled by changes in temperature during analysis. TGIC has been employed for characterization of hydrogenous PS-b-deuterated PS [113], PS-b-PB [114], PS-b-PB, PS-b-PB-b-PS, and PB-b-PSb-PB [115], oligomer separation of low molar mass PS-b-PI [116, 117], PS-b-PI, PS-b-PI-b-PS, and st-PS-b-PI [118], PS-b-P2VP and P2VP-b-PS-b-P2VP [119]. PS-b-P2VP and P2VP-b-PS-b-P2VP of similar overall molar mass but varying in chemical composition were synthesized by sequential anionic polymerization [119]. Chromatographic analysis of P2VP suffers from several limitations due to presence of lone pair of electrons on nitrogen of the pyridine moiety. Amine group strongly interact with many SPs and additives such as lithium chloride (LiCl), triethylamine (TEA) etc. are required to overcome these unwanted interactions even in the noninteractive SPs for SEC analysis [33, 120]. P2VP Analysis of the synthesized polymers was conducted by TGIC on a Hypersil ASP-1 amine-bonded silica gel using THF/ACN (53/47, v/v) with 0.005 M LiCl as an eluent. PS is excluded from the porous SP in the used eluent at 5 °C prior to the start of the temperature gradient while P2VP strongly interacted with the SP and eluted in order of increasing molar mass with increase in the temperature of the column. Figure 11.13A demonstrate the separation of PS-b-P2VP varying in the composition from 10 to 90% P2VP. Retention of PS-b-P2VP having less than 20% P2VP content is dominated by excluded PS block that render their elution before the start of the temperature gradient. Moreover, retention of PS-b-P2VP with more than 30% P2VP was dominated by strongly interacting block that resulted in their elution in order of increasing content of P2VP. Similar trend was shown by P2VP-b-PS-b-P2VP, triblock copolymers having less than 20% P2VP eluted in SEC regime before the start of temperature gradient due to dominance of longer PS block under current conditions. Strong interaction of P2VP in P2VP-b-PS-b-P2VP having more than 30% P2VP render dominance of interaction over exclusion. Seven triblock copolymers are separated with regard to P2VP content, Fig. 11.13B. Furthermore, it has been shown that retention in TGIC is also influenced by position of the block. P2VP-b-PS-b-P2VP and PS-b-P2VP having similar molar mass and chemical composition have different retention time. Tri-block copolymer having interacting block on both sides of the excluded block (PS) had more effective

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(A)

(B)

Fig. 11.13 TGIC analysis of block polymers of PS and P2VP on amin-bonded silica column in THF/ACN: 27/43 v/v with 0.005 M LiCl (A) PS-b-P2VP copolymers varying in relative content of PS and P2VP (top), PS100k (bottom); (B) PS-b-P2VP-b-P2VP copolymers varying in relative content of PS and P2VP (top), effect of position of blocks on elution volume (bottom) [119]. Copyright 2005, American Chemical Society

interaction with the SP in TGIC compared to di-block copolymer. In another study, same group compared the retention behavior of PS-b-PB, PS-b-PB-b-PS, and PB-bPS-b-PB of similar molar mass and chemical composition [115]. They conclude that strongly interacting block in the center of a tri-block copolymer render early elution compared to di-block or tri-block having strongly interacting block at ends.

11.3 Limitations and Opportunities TGIC is an excellent technique that provides a high-resolution molar mass separation of polymers owing to its low band broadening compared to SEC. However, TGIC is not a universal method like SEC. It is especially useful for analysis of polymer blends, branched polymers, star-shaped polymers, statistical copolymers, block copolymers, graft copolymers, polymers varying in tacticity, and end-functionalized polymers. TGIC is an inherently isocratic mode of chromatography that provides an opportunity of using common differential HPLC detectors and multi-detection (concentration, viscosity, and light scattering) similar to SEC while taking advantage of the manipulation of interaction strength during the chromatographic run. However, liberty of manipulation of interaction strength in TGIC is rather limited compared to EGIC. Hence, prior selection of appropriate eluent is necessary for an TGIC experiment. The eluent must allow weak interaction of the polymers with the SP that is weakened by changing temperature rendering switch from interaction to exclusion through CCP during the chromatographic run. Thus, chromatographic conditions such as eluent, flow rate, temperature gradient, and stationary phase must be optimized for each polymer sample. This is a rather tedious process especially for the polymers without any history of chromatographic analysis. Despite the above-mentioned limitations,

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TGIC can provide deep insight into the composition of the complex polymers which is not possible by using any other technique or chromatographic approach.

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107. Cong R, deGroot W, Parrott A, Yau W, Hazlitt L, Brown R, Miller M, Zhou Z (2011) A new technique for characterizing comonomer distribution in polyolefins: high-temperature thermal gradient interaction chromatography (HT-TGIC). Macromolecules 44(8):3062–3072. https:/ /doi.org/10.1021/ma200304e 108. Monrabal B, Sancho-Tello J, Mayo N, Romero L (2007) crystallization elution fractionation. A new separation process for polyolefin resins. Macromol Symp 257(1):71–79. https://doi. org/10.1002/masy.200751106 109. Cho D, Park S, Chang T, Ute K, Ii F, Kitayama T (2002) Temperature gradient interaction chromatography and MALDI-TOF mass spectrometry analysis of stereoregular poly(ethyl methacrylate)s. Anal Chem 74(8):1928–1931. https://doi.org/10.1021/ac010995j 110. Macko T, Pasch H, Wang Y (2009) Liquid chromatographic separation of olefin oligomers and its relation to separation of polyolefins—an overview. Macromol Symp 282(1):93–100. https://doi.org/10.1002/masy.200950810 111. Feng C, Li Y, Yang D, Hu J, Zhang X, Huang X (2011) Well-defined graft copolymers: from controlled synthesis to multipurpose applications. Chem Soc Rev 40(3):1282–1295. https:// doi.org/10.1039/B921358A 112. Kumar D, Gihar S, Shrivash MK, Kumar P, Kundu PP (2020) A review on the synthesis of graft copolymers of chitosan and their potential applications. Int J Bio Macromol 163:2097–2112. https://doi.org/10.1016/j.ijbiomac.2020.09.060 113. Lee S, Lee H, Thieu L, Jeong Y, Chang T, Fu C, Zhu Y, Wang Y (2013) HPLC characterization of hydrogenous polystyrene-block-deuterated polystyrene utilizing the isotope effect. Macromolecules 46(22):9114–9121. https://doi.org/10.1021/ma4018247 114. Lee S, Choi H, Chang T, Staal B (2018) Two-dimensional liquid chromatography analysis of polystyrene/polybutadiene block copolymers. Anal Chem 90(10):6259–6266. https://doi. org/10.1021/acs.analchem.8b00913 115. Park I, Park S, Cho D, Chang T, Kim E, Lee K, Kim YJ (2003) Effect of block copolymer chain architecture on chromatographic retention. Macromolecules 36(22):8539–8543. https:/ /doi.org/10.1021/ma035033o 116. Im K, Park H-W, Kim Y, Chung B, Ree M, Chang T (2007) Comprehensive two-dimensional liquid chromatography analysis of a block copolymer. Anal Chem 79(3):1067–1072. https:// doi.org/10.1021/ac061738n 117. Park S, Cho D, Ryu J, Kwon K, Lee W, Chang T (2002) Fractionation of block copolymers prepared by anionic polymerization into fractions exhibiting three different morphologies. Macromolecules 35(15):5974–5979 118. Kumar S, Shah PN, Kang B-G, Min J-K, Hwang WS, Sung IK, Shah SR, Murthy CN, Ahn S, Chang T, Lee J-S (2010) Facile one-pot synthesis of linear and radial block copolymers of styrene and isoprene through a novel coupling agent by living anionic polymerization. J Polym Sci, Part A: Polym Chem 48(12):2636–2641. https://doi.org/10.1002/pola.24044 119. Cho D, Noro A, Takano A, Matsushita Y (2005) TGIC Separation of PS-b-P2VP Diblock and P2VP-b-PS-b-P2VP triblock copolymers according to chemical composition. Macromolecules 38(7):3033–3036. https://doi.org/10.1021/ma0474877 120. Malik MI, Mahboob T, Ahmed S (2014) Characterization of poly(2-vinylpyridine)-blockpoly(methyl methacrylate) copolymers and blends of their homopolymers by liquid chromatography at critical conditions. Anal Bioanal Chem 406(25):6311–6317. https://doi.org/ 10.1007/s00216-014-8075-2

Chapter 12

Two-Dimensional Liquid Chromatography

Polymers are complex materials that possess multiple distributed properties. Homopolymers, made from a single monomer, have molecules with different number of repeat units that is the basic and unavoidable heterogeneity of any polymer sample. Introduction of a different end-group to some chains introduces another type of heterogeneity to these rather simple polymers. Other heterogeneities in this context could be chain architecture (linear, cyclic, branched etc.), and microstructure. Situation is more complex for copolymers where molar mass and chemical composition are two obvious distributed properties that are overlapping. Additionally, repeat units may be arranged in different sequences that originates the monomer sequence distribution. Polymers having any distribution beyond only molar mass are termed as complex polymers. For proper characterization of complex polymers, analysis of one distributed property as a function of the other distributed property (mainly molar mass) is imperative. Hence, the evaluation of the interdependence of any other distributed property such as chemical composition, molecular topology, functionality, branching, microstructure etc. and the molar mass is the ultimate target of comprehensive polymer analysis. The performance properties of the polymers depend not only on the average of these individual properties but also on their distributions as a function of each other. It is pertinent to mention here that multiple detector SEC is not enough for the comprehensive analysis of the molecular heterogeneity of complex polymers. For this purpose, multi-dimensional analytical approaches are required. Obviously, accurate characterization of ‘n’ independent properties of polymers requires n-dimensional analytical techniques.

12.1 Overlapping Distributed Properties of Polymers A copolymer, consisting of two repeat units A and B, have molar mass and chemical composition as the inherent distributed properties, Fig. 12.1. A hypothetical molar mass distribution of AB copolymer is presented on x-axis where m is the © The Author(s), under exclusive license to Springer Nature Switzerland AG 2023 M. I. Malik and D. Berek, Liquid Chromatography of Synthetic Polymers, Physical Chemistry in Action, https://doi.org/10.1007/978-3-031-34835-8_12

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molar mass at the peak max while mi and mf are the polymer molecules with lowest and highest molar masses, respectively. On the other hand, y-axis demonstrates the distribution with regard to the chemical composition (% A content). Peak max a at the y-axis represents the chemical composition of highest number of polymer molecules while ai and af represent polymer molecules having lowest and highest A content of the copolymer molecules. Hence, any point of the peaks at x- or y-axis may have polymer molecules having many different values for the other axis. For instance, polymer molecules having molar mass m may have any composition in a range from ai to af, and vice versa. Hence, complete characterization of such samples requires analysis of both distributions as a function of each other and also monomer sequence distribution. Same is true for all other distributed properties. Any polymer sample may exhibit a molar mass distribution, a chemical composition distribution, a molecular architecture distribution, an individual block length distribution, monomer sequence distribution, a functionality type distribution (end group distribution) and so on. Individual separation of polymer sample by any method yields fractions that are homogeneous (at least have narrow distribution) with regard to that particular distributed property but may have different values for the other properties. In this context, coupling of different modes of HPLC of polymers provide an opportunity for separation of polymers with regard to two independent properties prior to detection. Fig. 12.1 Molecular heterogeneity of AB copolymer [1]. Copyright 2021, Elsevier

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12.2 Approaches of Two-Dimensional Analysis Multi-dimensional analytical approaches are required for detailed insight into the complex polymer composition. Obviously, accurate characterization of ‘n’ independent properties requires n-dimensional analytical methods. In this context, HPLC is a useful, flexible, non-destructive, and versatile separation technique. Samples are subjected to two independent separation mechanisms prior to detection in a twodimensional liquid chromatography setup. Different modes of HPLC of polymers, that separate with regard to different distributed property, can be coupled/hyphenated with each other or with spectroscopic techniques (such as NMR, MS, FTIR etc.). These hyphenations can be either off-line or on-line/comprehensive. Presence of polymer molecules of different molar masses in any polymer sample introduce the molar mass distributions. The molar mass distributions are expressed as averages such as number-average molar mass (Mn ), weight-average molar mass (Mw ), etc. Another relative term in this context is the molar mass dispersity (Ð) that defines the broadness of molar mass distribution and is calculated as a ratio of weight-average and number-average molar masses (Mw /Mn ). The most widely used method for analysis of molar mass distribution is size exclusion chromatography [2]. Functional groups at the ends of the polymer chain generates a heterogeneity with regard to end-group functionality that is particularly important for oligomers and telechelics. Direct spectroscopic analysis can reveal only average values while a two-dimensional chromatographic separation may reveal functionality distribution as a function of molar mass distribution [3]. Polymers can be separated with regard to functional groups by LCCC, EGIC, and TGIC as 1st dimension that is subsequently coupled to SEC in the 2nd dimension for molar mass analysis of the separated fractions in the 1st dimension. Moreover, situation gets more complex for copolymers where there is a chemical composition distribution along with molar mass distribution. Additional heterogeneities in this context could be total molar mass distributions, monomer sequence distribution, presence of homopolymers in a copolymer, branching distribution etc. Direct spectroscopic analysis by NMR and FTIR could only reveal the average chemical composition of such samples. Furthermore, differentiation of copolymers and a mixture of homopolymers is typically not possible by spectroscopic techniques. Different modes of LC of polymers namely LCCC, EGIC, and TGIC can provide deep insight into the complex composition of copolymers. Copolymers can be separated with regard to any of the above-mentioned distribution by LCCC, EGIC, or TGIC in the 1st dimension that can be coupled with SEC in the 2nd dimension for molar mass analysis of the separated fractions. Ideally, each separation technique should be able to selectively separate with regard to one type of heterogeneity that is then separated in 2nd dimension with regard to the other type of heterogeneity in order to have a mapping of their interdependence. Hence, two independent and selective separation methods can be coupled to each other or with spectroscopic techniques for evaluation of interdependence of different distributed properties.

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Experimental protocols of coupling different modes of polymer liquid chromatography may vary. Initially, collected fractions of SEC were analyzed by HPLC in an off-line mode [4]. Due to poor resolution and selectivity of SEC, different mode of IC (LCCC, EGIC, TGIC) are now generally preferred as 1st dimension followed by analysis of the separated fraction by SEC in the 2nd dimension. An obvious advantage of using IC as first dimension is its higher sample capacity compared to SEC. The off-line approach is based on collection of fractions by 1st dimension separation using a fraction collector. The solvent of the collected fractions may be evaporated, followed by its re-dissolution in the MP for the 2nd dimension analysis. Off-line approach allows performing both analysis at their optimum conditions. Detailed analysis of the collected fractions is possible by using multiple detector SEC [5–7]. Off-line approach is laborious, tedious, and more prone to contamination. Moreover, this is not a comprehensive analysis and only peaks (regions) of interests are analyzed in the 2nd dimension. This type of two-dimensional analysis weather it is conducted on-line or off-line is referred as “heart-cut”. Off-line “heart-cut” method was mostly practiced for 2D-LC in the past [7–9]. On the other hand, on-line/comprehensive two-dimensional analysis refers to direct transfer of aliquots from the 1st dimension LC system to the 2nd dimension LC system in a repetitive and sequential manner [10–13]. Theoretical simulations of the options in two-dimensional analysis of block copolymers unveiled its merits and limitations [14]. The major issue for on-line 2D-LC is the synchronization of the two separation systems. In principle, 2nd dimension analysis must be fast while 1st dimension analysis must be slow, both far from van Demeter optimum. SEC analysis in the 2nd dimension is often preferred due to possibility of high-speed analysis without much compromise on resolution. Both on-line and off-line 2D-LC analysis have their peculiar advantages and limitations [5]. Off-line approach is flexible, laborious and provides opportunity of 2nd dimension analysis by any technique at the optimum conditions. However, collected fractions are prone to contamination during sample preparation for 2nd dimension analysis. On the contrary, on-line 2D-LC setup is automated with minimal operator involvement but synchronization of two LC systems is challenging with regard to time and eluent compatibility [10–13, 15–17].

12.3 Experimental Setup of Orthogonal Chromatography Experimental setup for orthogonal chromatography is challenging. The major challenge is synchronization of the timeline of two independent chromatographic analysis. Initially, off-line or stop-flow modes were used due to technical limitations, mostly SEC as a 1st dimension and EGIC as a 2nd dimension [4, 18]. In some cases, collected EGIC fractions of copolymers were analyzed by SEC in the 2nd dimension in an off-line mode [19]. On-line coupling of different modes of LC of polymers with SEC in 2nd dimension in a fully automated chromatographic setup is the most feasible and modern approach

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[20, 21]. Two independent chromatographs are connected through two-storage loop system, Fig. 12.2A [3, 8]. The sample collected from the 1st dimension effluent in the first storage loop is analyzed in the 2nd dimension in the time required to fill the other loop. A switching valve operates after pre-decided time intervals that directs the flow of 1st dimension eluent from one loop to the other. Accurate and judicious time synchronization is crucial for an efficient comprehensive analysis. For instance, the storage loop of 100 μL takes 2 min to fill at a flow rate of 0.05 mL/min. Therefore, 2 min is the maximum available time for the 2nd dimension analysis in this setup. SEC analysis on high-speed columns is possible at fairly high flow rate rendering a fairly acceptable resolution. Another important issue in this context, though to lesser extent compared to IC as 2nd dimension, is compatibility of the eluents of both dimensions. In this well-established approach, sufficient solubility of the analyte in both eluents and their miscibility is the minimum requirement [17, 22–28]. The analyte (polymer) in the fraction from the 1st dimension leaves the solvent plug as soon as it is injected in the 2nd dimension column due to its exclusion from the porous SP and hence has minimal effect on the 2nd dimension analysis. This is not the case while using any mode of IC in the 2nd dimension. While SEC analysis can be completed swiftly using high flow rates on a short and high-speed column, this is not generally the case for IC. In some reports, TGIC separation is coupled with LCCC in the 2nd dimension, which is obviously a good configuration for orthogonal chromatography, using the same experimental setup as shown in Fig. 12.2A [28, 29]. This is a peculiar situation where same eluent and SP are used for both dimensions that additionally avoid the compatibility problems. However, situation is complicated if 2nd dimension analysis has to be completed through IC using a different separation mechanism (NP or RP). Any reasonable analysis in the 2nd dimension by IC, with exception of LCCC, is not possible in the available short time that is required to fill the typical sample loop. A weak solvent can be added in the effluent from the 1st dimension prior to the 2nd dimension analysis

Fig. 12.2 (A) Schematic representation of an automated two-dimensional HPLC-SEC chromatograph [41]. Copyright 2014, American Chemical Society; (B) Experimental setup of coupling two HPLC separations through multiple trapping with two 10-port switching valves [32]. Copyright 2007, American Chemical Society

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[30, 31]. Moreover, a more reliable approach is use of an interface equipped with a multiple trapping system that requires three independent pumps, Fig. 12.2B [32– 34]. The 1st dimension analysis is conducted at lowest possible flow rate similar to typical two-loop storage system. On the same line, the first valve switches every two minutes if connected to 100 μL storage loops at a 1st dimension flow rate of 0.05 mL/ min. After two minutes first valve switches the effluent from the 1st dimension to the second loop while first loop is being flushed with a chromatographically weak eluent on a short trapping column of the same nature as the 2nd dimension column. This process is repeated several times as per requirement of the time required for the 2nd dimension analysis. For instance, if 2nd dimension analysis requires 10 min, then five times trapping is appropriate provided each trapping step takes two minutes. Chromatographically weak solvent ensures trapping of analyte from the 1st dimension separation at the start of the trapping column. After multiple trappings, second switching valve starts and the trapped fraction get analyzed by pumping eluent for 2nd dimension analysis by a third pump. Multiple trapping system ensures the availability of sufficient time for 2nd dimension analysis along with high concentration of the analyte. Proper synchronization of the timeline of two chromatographs is essential for an efficient 2D-LC system. The challenge of compatibility of all the involved solvent systems is a multifaceted issue for this type of setup. The eluent for 1st dimension analysis may be strong for the 2nd dimension SP and hinder efficient trapping (adsorption) at the start of the column that results in a so-called “breakthrough” phenomenon [35]. The solvent pumped for trapping by the second pump must be so weak that it does not render movement of the adsorbed fractions in the process of multiple trapping [36]. Finally, the eluent for the 2nd dimension should be able to complete the analysis in the available time. Detection is one of the major problems for 2D-LC systems since different solvent systems are mixed that impede the use of common HPLC detectors. Detector is used for the effluent of the 2nd dimension analyses and ELSD is the detector of choice for 2D-LC due to its ability to nullify the solvent effect. For SEC in second dimension, multiple detection (concentration, light scattering and viscosity) can also be applied but it has not been practiced much [26]. ELSD is literally a universal detector that can detect any non-volatile components of the eluate [37, 38]. Nebulization of eluate results in the evaporation of the solvent and formation of particles of the non-volatile components of the droplets that subsequently scatter the incident light beam in a photodiode cell. Nullification of the eluent effect is an obvious advantage of ELSD for IC and LCCC. However, a poor linearity and dependance of ELSD signal on the molar mass and chemical composition makes quantification a complex issue. The intensity of the scattered light is influenced by number, size, and refractive index of the particles. The operating conditions such as nebulizer temperature and gas flow rate along with the chemical nature of the sample have significant effect on the size of the formed droplets. Furthermore, experimental parameters such as oven temperature, flow rate of the carrier gas and eluate, physical properties of eluate like viscosity and surface tension etc. strongly effect the ELSD signal. Hence, a careful calibration of ELSD is imperative for reliable results [39, 40]. Furthermore, ELSD does not reveal any information on the polymer itself.

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12.4 Applications of Two-Dimensional Liquid Chromatography in Polymer Analysis Multivariate distributions of polymers necessitate their multi-dimensional analysis where one dimension can selectively analyze (ideally) with regard to one particular heterogeneity. An appropriate 2D analysis unveils the interdependence of different distributed properties of the polymers. As mentioned in the previous section, two different experimental setups can be applied depending upon the technique used in the 2nd dimension. If the 2nd dimension analysis can be conducted within the time required to fill the storage loop such as SEC and LCCC, the experimental setup shown in Fig. 12.2A is the appropriate choice. However, the experimental setup depicted in Fig. 12.2B is the appropriate choice for the 2nd dimension analysis that takes more time. This setup resolves compatibility problems between eluents of both dimensions in order to avoid breakthrough issues. Additionally, it helps in time management for synchronization of the two dimensions. In the following section, applications of 2D-LC are divided on the basis of techniques used for the 2nd dimension analysis namely SEC and IC. Collection of fractions and the subsequent off-line analysis of these fractions by SEC and other modes of liquid chromatography was the initially opted approach which is still a suitable option for systems where on-line setup is not convenient [1, 6, 7, 9, 22].

12.4.1 SEC as Second Dimension As iterated multiple times, molar mass is the inherent and basic distributed property of polymers. Generally, interdependence of other type of heterogeneity as a function of molar mass is sought. For this purpose, SEC analysis is appropriate and it can be conducted on the well-established setup for 2D-LC, Fig. 12.2A. Further sub-sectioning of 2D-LC with SEC as second dimension is based on the technique used for 1st dimension analysis. Data handling and operating software for 2D-LC is commercially available from Polymer Standards Service [42].

12.4.1.1

LCCC-SEC X SEC

LCCC-SEC is the most widely employed approach for LCCC analysis of high polymers. This refers to the situation when one of the segments is at critical condition while the non-critical segment follows the SEC elution mode [1, 3, 8, 17, 43, 44]. LCCC-SEC has been employed for analysis of polymer blends [45–48], block copolymers [29, 46, 49–51], star-shaped copolymers [52–54], graft copolymers [55–58], polymers varying in tacticity/microstructure [59–62], statistical copolymers [63–67] etc. The approach is applicable to high molar mass polymers having fairly high individual segment lengths. On-dimensional analysis reveals the presence of

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critical homopolymers and estimation of the individual block length of the noncritical segment of the copolymer. However, non-critical homopolymers are difficult to differentiate from the target copolymer by independent LCCC-SEC analysis in most of the cases. Coupling of LCCC-SEC to SEC in second dimension additionally reveals the molar mass of critical homopolymers, total molar mass of the copolymers, and also may help differentiate non-critical homopolymers from copolymers based on the overall molar mass [8, 17, 43]. As mentioned earlier, LCCC-SEC x SEC is a well-established method for characterization of a variety of complex polymer samples [1, 3, 8, 68]. 2D-LC has great potential to elucidate the heterogeneity of complex polymers compared to independent analysis by both techniques. Herein, we take two typical examples to demonstrate the strength of 2D-LC compared to independent analysis by these techniques. 2D-LC analysis of PS-bPDMS copolymers is reported by using LCCC(PS)-SEC x SEC and LCCC(PDMS)SEC x SEC [22]. The 2D contour plot of PS-b-PDMS copolymer using LCCC(PS)SEC x SEC is shown in Fig. 12.3A, where LCCC(PS) was established on a NP silica column using n-hexane/THF: 55/45 (v/v). Under these conditions PDMS is excluded from the porous SP making this method LCCC-SEC for PS-b-PDMS copolymers. The independent analysis of PS-b-PDMS copolymers under these conditions successfully separated PS homopolymers from the rest of the sample. The block copolymer peak eluted earlier than the PS and have a shoulder that indicates presence of unwanted other species in the sample (possibly PDMS homopolymer) which are also excluded under these experimental conditions. However, these two species are not fully resolved in most of the cases. It is pertinent to mention here that independent SEC analysis of this product was unable to discriminate such a heterogeneity of the sample. 2D-LC having a synergistic combination of two separations based on different mechanism render a complete resolution of all three species in the sample. In the 1st dimension, PS homopolymer eluted at its CCP while the block copolymer is excluded as per length of the PDMS block. At the same time, PDMS homopolymer also eluted with PS-b-PDMS in most of the real sample. This is often the case for analysis of the complex polymers by LCCC. Y-axis demonstrates the separation at the CCP of PS (1st dimension) while x-axis show the molar mass of the separated fractions by SEC relative to PS standards (2nd dimension), Fig. 12.3A. The shoulder in the LCCC-PS analysis of the early eluting species (PS-b-PDMS and PDMS) get better resolved due to higher total molar mass of block copolymer compared to PDMS homopolymer. 2D-LC reveals separation of all three species in the sample namely PS, PDMS, and targeted PS-b-PDMS and the PS equivalent molar mass of the separated species. Similarly, graft copolymer samples may also contain several different species that are not possible to identify by independent analysis by different techniques. In this context, a graft copolymer was synthesized by grafting butyl acrylate onto polystyrene-b-polybutadiene [58]. The grafting reaction render a complex sample that contains poly(butyl acrylate) (PBA), ungrafted polystyrene-b-polybutadiene (PS-b-PB), along with targeted polystyrene-b-(polybutadiene-g-polybutyl acrylate) PS-b-(PB-g-PBA). Grafting reaction occurs at the double bond of the polybutadiene block. SEC analysis reveals presence of two populations with varying UV

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Fig. 12.3 LCCC-SEC X SEC (A) PDMS-b-PS 1st dimension: LCCC(PS) on a NP silica column using n-hexane/THF: 55/45 (v/v), 2nd dimension: SEC on a PSS Linear SDV column using THF as eluent and PS calibration, detection: ELSD [22]. Copyright 2012, Springer Nature; (B) PS-b(PB-g-PBA) 1st dimension: LCCC(PBA) on a NP silica column in THF/cyclohexane: 15.5/84.5 v/ v, 2nd dimension: SEC on a SDV column using THF as eluent and PS calibration, detection: ELSD [58]. Copyright 2000, Elsevier

activity. Low molar mass peak has lowest UV-absorption and is assigned to PBA homopolymer when ELSD and UV are normalized at peak maximum of the main peak. Chromatographic critical point (CCP) of PBA was established on a NP silica column in THF/cyclohexane: 15.5/84.5 v/v. Under these conditions PB eluted in SEC regime while PS in IC regime. LCCC-PBA of graft polymer have tri-modal distribution with varying UV absorbance (see original reference). If UV and ELSD traces of lowest eluting peak are normalized, the peak eluting second has lowest UV absorbance while higher UV absorbance is again shown by the high elution volume peak. Since the middle peak is eluting at the CCP of PBA, it is assumed to be PBA homopolymer while first eluting peak may be assigned as grafted and ungrafted block copolymer. Moreover, the third eluting peak rather tail may be graft and ungrafted block copolymer with partially degraded PB block. The chemical heterogeneity of the graft copolymer sample is further exposed by 2D-LC which reveal six different populations, Fig. 12.3B. The assignment of the contours is based on comparative response of ELSD and UV detectors, UV detector response is not shown. The most intense peak with highest molar mass is assigned to the main product PS-b-PB-gPBA while the peak with slightly less molar mass and higher elution volume in 1st dimension is assigned to ungrafted PS-b-PB. The highest elution volume peak of 1st dimension (y-axis) having similar molar mass to that of target grafted product (x-axis) is assigned to PS-b-(PB-g-PBA) with partially degraded PB block. The third intense peak appearing at the elution volume of app. 5 mL in 1st dimension and having a fairly low molar mass is assigned to PBA homopolymer. While a minor fraction with similar molar mass to PBA homopolymer but higher 1st dimension elution volume is assigned to maleic anhydride (MA) incorporated PBA homopolymer or graft product. The lowest molar mass minor fraction eluting at the same elution volume of PBA homopolymers is identified as additive. The molar mass as given on the x-axis in both cases is relevant to linear PS standards.

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Hence, LCCC-SEC x SEC is a powerful combination for detailed insight into heterogeneity of complex polymers which are not possible to reveal by independent analysis by both techniques.

12.4.1.2

LCCC-IC X SEC

LCCC-IC refers to the chromatographic conditions when one of the segments of the copolymer is at its critical point while the other segment has preferential enthalpic interaction with the SP and elute later than the critical point. As mentioned previously, the conditions are more applicable to functional polymers, polymers with varying architecture, and oligomer separation of low molar mass copolymers [1, 3, 8, 17, 43, 44]. Although LCCC-IC is one of the first approach used in LCCC analysis, there are not many examples of coupling of LCCC-IC with SEC in 2nd dimension. LCCC-IC has been used for separation of polymer blends [32, 33, 49, 50, 69, 70], endfunctionalized homopolymers [23, 71–77], oligomer separation of block copolymers [7, 23, 32, 33, 49, 50, 69, 70, 78–80], star-shaped polymers [72, 75, 81–85], branched polymers [28, 72, 75, 81, 82, 86], linear and cyclic copolymers [82, 87–93] etc. However, coupling of LCCC-IC with SEC in 2nd dimension is not a commonly applied approach. In this context, polycaprolactones (PCL) varying in number of arms are synthesized by ring-opening polymerization of ε-caprolactone using n-hexyl, diethylene glycol, glycerol, and pentaerythritol as initiator [72]. The initiators have one, two, three and four hydroxyl groups in the same order. Hence, the synthesized polycaprolactones can be categorized as linear with hydroxyl group at one end, linear with hydroxyl groups at both ends, tri-arm stars with three hydroxyl groups, and tetra-arm stars with four hydroxyl groups. Thin films of these polymers varying only in architecture have shown significant morphological selectivity [94]. PCL of similar molar mass but varying in number of arms could not be differentiated by SEC or LCCC(CL) on a RP column. LCCC(CL) on a NP column using 1,4-dioxane/ n-hexane: 52/48 v/v resulted elution of PCL of similar molar mass but varying in number of interacting hydroxyl groups in order of increasing number of hydroxyl groups. However, the hydroxyl groups are interacting strongly with the SP under these conditions. An eluent gradient after 50 min rendered a baseline separation of a four-component blend, that is composed of PCL of similar molar mass but differing in number of hydroxyl groups, in a reasonable time frame. The 1st dimension separation method is subsequently coupled with SEC in the 2nd dimension for estimation of molar mass of the separated components of the polymer mixture, Fig. 12.4A. LCCCNP followed by EGIC separated polymer mixture according to number of arms and also from cyclic polycaprolactone (by-product) that doesn’t contain any hydroxyl group (y-axis). Similar molar mass of all separated fractions from 1st dimension is confirmed by their similar elution volume in the 2nd dimension (x-axis). Only cyclic PCL eluted later in the 2nd dimension, that indicates its low molar mass, since it is a by-product and cyclization of cationic polymerization can happen any time that stops further increase in the chain length. Moreover, higher concentration of

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the fraction eluting after 100 min in 1st dimension is due to presence of PCL-diol in all samples that originates by unwanted initiation of polymerization by the moisture content. Similarly, branched polymers can be separated by LCCC according to number of branches [28, 95]. Another example of coupling of LCCC-IC with SEC is presented for analysis of branched polystyrene [28]. Linear PS eluted at the same elution volume irrespective of its molar mass on a ODS column at its CCP in CH2 Cl2 / CH3 CN: 53/47 v/v at 53.3 °C. Under LCCC-RP of linear PS conditions, branched PS eluted in order on increasing number of branches. The functionality in this case was the silyl group at the branch point that is more interactive with RP SP. Retention volume of LCCC-RP of branched PS is depicted on y-axis while x-axis show the SEC elution volume of the 2nd dimension. The contour plot demonstrates the increase in retention by LCCC (y-axis) with increase in branches that is further supported by decrease in SEC elution volume (x-axis), Fig. 12.4B, C.

Fig. 12.4 (A) LCCC-IC to EGIC x SEC for separation of star-shaped polycaprolactones varying in number of arms 1st dimension: NP silica column using LCCC(CL) eluent 1,4-dioxane/n-hexane: 52/48 v/v for 50 min followed by EGIC to 100% 1,4-dioxane in 200 min, 2nd dimension: SEC on a SDV column in THF [72]. Copyright 2016, Royal Society of Chemistry; (B, C) LCCC-IC x SEC analysis of branched polystyrene 1st dimension: LCCC-RP at CCP of linear PS on a RP ODS column in CH2 Cl2 /CH3 CN: 53/47 v/v at 53.3 °C, 2nd dimension: SEC on SDV column in THF [28]. Copyright 2006, Elsevier

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EGIC X SEC

EGIC has been extensively used for analysis of complex polymers such as oligomer separation of low molar mass homopolymers [6, 7, 33, 96], homopolymer blends [64, 97–108], end-functionalized homopolymers [72, 109–111], statistical copolymers [112–126], star-shaped polymers [25], block copolymers [127–129], graft copolymers [56, 130–132], separation according to microstructure [129, 133–139], dendrimers [140], branched polymers [113] etc. Molar mass analysis of separated fractions by EGIC is of great interest for the development of structure–property correlations [1, 3, 8, 25, 27, 56, 84, 131, 141–145]. In the proceeding discussion, we take three different examples to demonstrate the strength of EGIC x SEC coupling for unveiling the heterogeneity of complex polymers. As iterated in previous chapters, EGIC is a flexible technique that allow adjustment of various experimental parameters of gradient such as composition of initial and final eluents, gradient shape and steepness etc. for the achievement of the required separation. Moreover, online coupling of EGIC with SEC render a twodimensional mapping with a reasonable orthogonality. Four PS standards (PS1-4: 1,060 k, 200 k, 50 k & 10 k), four PMMA standards (PM1-4: 870 k, 200 k, 53 k & 4.9 k), and four poly(styrene-co-methyl methacrylate) (SM1-4: 185 k/95 st%, 152 k/ 80 st%, 153 k/65 st% & 240 k/19 st%) are mixed and separated on a RP column using THF/CH3 CN: 10/90 → 60/40 in 200 min at 0.015 mL/min while using SEC on a SDV column in THF in the 2nd dimension [144]. PMMA elute in SEC regime in the initial eluent composition as depicted by elution order on the y-axis from PM1 to PM4, Fig. 12.5A. The SEC separation of PMMA in the 1st dimension is confirmed by 2nd dimension analysis as shown on x-axis. SEC resolution of 2nd dimension is higher compared to 1st dimension that render completely separated spots in the contour plot. On the other hand, PS are strongly retained in the initial eluent composition and elute only with the higher content of THF at the later end of the 1st dimension chromatogram in order of increasing molar mass (IC regime). Well-separated spots of four PS standards at the upper part of the plot demonstrate the strength of 2D-LC. Moreover, poly(styrene-co-methyl methacrylate) samples get separated between the PMMA and PS standards on y-axis as per PS content despite having similar molar mass (note similar elution volume of the x-axis). Independent EGIC analysis render baseline separation of copolymers but resolution for PMAA and PS was insufficient (y-axis). Similarly, independent SEC analysis was unable to differentiate among copolymers due to similar molar masses. However, SEC separated homopolymers as per their molar mass with a sufficient resolution. A synergistic combination of EGIC x SEC render completely separated spots for all the components of the mixture which was not possible by independent analysis by both techniques. This represents a good example of near orthogonal 2D-LC (composition x chain size). In the meantime, 2D-LC chromatograph working at elevated temperature (~150) is also developed for analysis for polyolefins (HT 2D-LC). Ethylene–vinyl acetate copolymers (EVA) are important commercial polymers whose applications are very much dependent on their chemical composition and molar mass. Hence, determination of molar mass dependence of chemical composition is imperative. A 2-D contour

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Fig. 12.5 EGIC X SEC (A) blend of 4 PS standards (PS1-4: 1,060 k, 200 k, 50 k & 10 k), 4 poly(styrene-co-methyl methacrylate) (SM1-4: 185 k/95 st%, 152 k/80 st%, 153 k/65 st% & 240 k/ 19 st%), and 4 PMMA standards (PM1-4: 870 k, 200 k, 53 k & 4.9 k), 1st-D: ODS column, THF/ CH3 CN: 10/90 → 60/40 in 200 min at 0.015 mL/min, 2nd D: SEC on a SDV column in THF [144]. →→ Copyright 2021, Elsevier; (B) Blend of PE (1.18 kg mol−1 ), EVA (6.5 mol% of VA), EVA (20 mol% of VA), EVA (57 mol% of VA) and PVAc (37 kg mol−1 ) 1st-D: NP Si column using TCB cyclohexanone linear gradient at flow rate of 0.1 ml/, 2nd D: SEC: column PL Rapide H in TCB at a flow rate of 2.5 mL/min., system temperature: 160 °C [146]. Copyright 2010, Elsevier; (C) Blend of iPP (1.1 kg/mol), iPP (60 kg/mol), aPP (211 kg/mol), sPP (196 kg/mol), PE (1.18 kg/ mol), PE (126 kg/mol) and EP copolymer (81.3 wt.% of ethylene, M w = 164 kg/mol, PDI = 3.01), 1st D: Hypercarb® column using a 1-decanol 1,2,4-trichlorobenzene gradient at 0.1 mL/min, 2nd D: SEC: column PL Rapide H in TCB at a flow rate of 2.5 mL/min., system temperature: 160 °C [147]. Copyright 2011, Elsevier

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plot of a blend of PE, PVAc and three EVA copolymers varying in the content of VA is shown in Fig. 12.5B [146]. First dimension is depicted on y-axis that corresponds to EGIC, TCB → cyclohexanone, on a NP silica column while x-axis corresponds to SEC. On a NP column, the least polar component of the blend PE eluted first while most polar component PVAc eluted at the end. In between these two spots, three EVA copolymers elute in order of increasing VA content as shown on y-axis. The 1st dimension EGIC separation is supplemented by 2nd dimension SEC separation that render interdependent molar mass and chemical composition mapping. In another study, EGIC x SEC of a blend of iPP (1.1 kg/mol), iPP (60 kg/mol), aPP (211 kg/mol), sPP (196 kg/mol), PE (1.18 kg/mol), PE (126 kg/mol), and EP copolymer (81.3 wt.% of ethylene, M w = 164 kg/mol, PDI = 3.01) is demonstrated [147]. EGIC separation is conducted on a Hypercarb column in 1-decanol → 1,2,4-trichlorobenzene gradient as shown on y-axis while x-axis corresponds to SEC. All seven components of the model blend get excellently separated from each other owing to synergistic combination of EGIC and SEC. Hence, EGIC x SEC provides a practical opportunity to unveil the heterogeneity of complex polymer samples that is not possible by individual analysis by these techniques.

12.4.1.4

TGIC X SEC

TGIC has been employed for analysis of variety of complex polymers such as molar mass separation of homopolymers [144, 148–152], polymer blends [153–155], endfunctionalized linear homopolymers [150, 156–158], branched polymers [28, 95, 159], star-shaped polymers [140, 160, 161], statistical copolymers [121, 162–166], separation according to microstructure/stereoregularity [108, 163, 164, 167], graft copolymers [34, 168], block copolymers [29, 32, 78, 86, 169–171] etc. Obviously, hyphenation of TGIC with SEC is of great interest for detailed insight into the heterogeneity of the complex polymers. Although, application of multiple detection for any isothermal and isocratic elution of second dimension is possible in principle, the practical examples include mostly TGIC x SEC [26, 169, 172]. SECTD is the most widely employed method for the branching analysis that estimates the branch number based on contraction of the chain size due to chain branching in comparison to linear polymer of the same molar mass [2, 173]. As is well-known, SEC separates according to hydrodynamic volume and a SEC fraction may contain various polymer species varying in molar mass and architecture, termed as local polydispersity [174, 175]. For the accurate branching analysis, an appropriate approach could be separation by IC as per molar mass followed by SEC-TD analysis of homogeneous molar mass fractions. SEC-TD and TGIC x SEC-TD analysis of a model mixture of linear and star-shaped PS is compared by Chang and coworkers [26]. SEC was unable to differentiate linear PS from star-shaped PS owing to coelution of linear PS of lower molar mass with star PS of higher molar mass. The coelution of different molar mass species leads to inaccurate branching analysis [174, 175]. TGIC can separate polymers according to their molar mass independent of chain architecture that resulted in strict separation of the model mixture of linear and star

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PS with regard to molar mass [160, 176]. Direct use of triple detection with TGIC is possible, however, the fractions are homogeneous with regard to molar mass but not to architecture. Hence, an average branch number of each fraction can be obtained but a full mapping was still not possible. Coupling of TGIC with SEC-TD render an interdependent mapping of chain size and branch structure. A contour map of TGIC x SEC-TD of the model PS mixture is demonstrated in Fig. 12.6 using light scattering (A), RI (B), and viscosity detection (C). The shapes of contour maps are similar; however, they differ in their intensity distribution. LS show MW x concentration distribution (A), RI show the concentration distribution (B), and viscosity detector show MWα x concentration distribution (C). Unreacted arms and stars with small number of arms get resolved as distinct peaks by TGIC in a range of 350– 450 min (y-axis). The analysis of individual TGIC x SEC-TD chromatograms by three detectors confirmed similar molar mass of 1st dimension TGIC separation that has different branched content. An increase in the number of branches resulted in an improved 2nd dimension SEC separation due to increase in the difference of size with number of branching compared to their linear counterpart. Analysis of all SEC-TD chromatograms of the 2nd dimension based on chain contraction factor revealed a clear mapping of molar mass versus branch number, Fig. 12.6D.

12.4.2 Interaction Chromatography in Second Dimension IC in the 2nd dimension of any 2D-LC setup is more challenging compared to SEC. The major challenge for using SEC as 2nd dimension is the synchronization of the timeline of both dimension analyses unless analyte is soluble in both solvent systems and solvent systems of both dimensions are compatible with each other [9, 17, 22– 28]. The analyte (polymer) in the fraction from 1st dimension leaves the solvent plug as soon as it is injected in the 2nd dimension column due to its exclusion from the pores, hence, has minimal effect on the 2nd dimension analysis. This is not the case while using any mode of IC in the 2nd dimension. Only exception in this case is LCCC as 2nd dimension analysis technique when it is coupled to TGIC in which same SP and eluent can be used for both dimensions [28, 29]. Experimental setup shown in Fig. 12.2A can be used to perform these 2-dimensional analyses. There are multifaceted challenges in coupling of IC methods if different SP, MP have to be used for both dimensions. This is especially the case in coupling of RP separation with NP separation. An acceptable IC separation method requires fairly long time than the time available for the 2nd dimension analysis in typical setup shown in Fig. 12.2A. For this purpose, multiple trapping approach (an interface) with an additional pump and a short trapping column is required, Fig. 12.2B [32–34]. The additional challenges for this type of experimental setup are related to eluent used for both dimensions and trapping. The eluent for 1st dimension analysis may be strong for the 2nd dimension SP and hinder efficient trapping (adsorption) at the start of the column that results in a so-called “breakthrough” phenomenon [35]. The solvent pumped for trapping by second pump must be so weak that it does not render movement of the adsorbed

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Fig. 12.6 (A) Contour plots of RP-TGIC × SEC 2D-LC chromatograms of the mixture of linear and star-shaped PS 1st D: RP-TGIC on an ODS column in CH2 Cl2 /CH3 CN (57/43, v/v) at a flow rate of 0.01 mL/min temperature program:, 2nd D: SEC on a SDV column in THF at a flow rate of 1.2 mL/min; column temperature: 60 °C using triple detection (A) light scattering, (B) refractive index, (C) viscosity; (D) Plot of branch number vs MW for PS mixture. Solid lines are for starshaped PS (red line) and linear PS (blue line). Black dotted line is the 1D-SEC analysis result [26]. Copyright 2012, American Chemical Society.

fractions in the process of multiple trapping [36]. Finally, the eluent for the 2nd dimension should be able to complete the analysis in the available time. Moreover, off-line coupling can be an approach which is laborious and prone to contamination, for instance, coupling of LCCC-SEC with LCCC-IC for analysis of block length of low molar mass block copolymers [6, 7].

12.4.2.1

LCCC-IC X LCCC-IC

LCCC-IC refers to the situation where one segment is at critical conditions while other is interacting with the SP. The method is generally applicable for polymers

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having a monodisperse interacting segment at the CCP of the repeat unit. Although LCCC-IC is one of the first approach used in LCCC analysis, there are not many examples of coupling of LCCC-IC with SEC in 2nd dimension. LCCC-IC has been used for separation of polymer blends [32, 33, 49, 50, 69, 70], end-functionalized homopolymers [23, 71–77], oligomer separation of block copolymers [7, 23, 32, 33, 49, 50, 69, 70, 78–80], star-shaped polymers [72, 75, 81–85], branched polymers [28, 72, 75, 81, 82, 86], linear and cyclic copolymers [82, 87–93] etc. For copolymers, LCCC-IC is used for oligomer separation of one block at the CCP of the other block. A very specific combination of CCP of one block and interaction strength of the other block is required for such separation which is only found for few polymers such as PEO-b-PLA [50, 69], PS-b-PI [32, 78, 156], PEO-b-PS [177] and industrially important EO-PO block polymers, also called poloxamers [6, 7, 23, 33, 49, 70, 79, 80, 96, 178, 179]. Since critical conditions refer to a fine balance of experimental parameters, deviations from these experimental parameters results in loss of the critical behavior. In order to strictly have critical conditions in both dimensions, off-line approach may be used [6, 7, 9, 22, 180]. The coupling of a LCCC-IC method with another LCCC-IC method for simultaneous molar mass and chemical composition mapping is rather challenging and could only be accomplished on the experimental setup shown in Fig. 12.2B. In this context, separation of PO11 -EO27 -PO11 with regard to both EO and PO oligomers at the CCP of the other block is shown in Chap. 8 (Fig. 8.7) at the optimum experimental conditions [33]. There are certain experimental variables that determine the success of any 2D-LC IC x IC system which includes miscibility of the eluents, trapping efficiency of the trapping interface, strength of 1st dimensional eluent for the 2nd dimension analysis, and speed of the 2nd dimension analysis with acceptable resolution etc. Hence, the experimental conditions applied for both dimensions are generally far from optimum. First dimension analysis has to be very slow while 2nd dimension analysis must be given sufficient time for acceptable resolution. Since the time available for 2nd dimension analysis in typical 2D-LC setup (Fig. 12.2A) is not enough for the minimum time required for 2nd dimension analysis by IC. An interface is required that can hold the 1st dimension analyte for multiple times that can be analyzed subsequently by 2nd dimension in a repetitive and comprehensive manner using an experimental set-up shown in Fig. 12.2B. Moreover, trapping efficiency of the interface is a crucial factor for an acceptable 2D-LC analysis. In this case, first dimension was conducted on CCP of PPO on a NP Si column using ACN/water (84/16 v/v) as eluent at a flow rate of 0.05 mL/min. Second dimension analysis was conducted on a comparatively short ODS column at the CCP of PEO using methanol/ water: 87/13 v/v at a flow rate of 1.0 mL/min. The trapping interface consisted of two short ODS columns (30 mm) of using methanol/water: 50/50 v/v at a flow rate of 1.0 mL/min. Both chromatographic conditions can be adjusted as per block lengths of the interacting block while keeping the critical block fairly at critical conditions. In the 1st dimension, PO11 -EO27 -PO11 get separated with regard to PEO block while PPO is at CCP (y-axis). Analysis of six trapped fractions from 1st dimensions on the trapping column is accomplished on a RP column at the CCP of PEO while revealing

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oligomer separation of PPO block in a comprehensive and repetitive manner. The contour plot of LCCC-PPO and IC-PEO with LCCC-PEO and IC-PPO is depicted in Fig. 12.7. PPO homopolymers eluted before 100 min as a sharp peak at its CCP (yaxis) get subsequently separated into individual oligomers in the 2nd dimension (xaxis). The oligomer number at the corresponding axis was obtained by collection of fractions and its off-line analysis by MALDI-TO MS. Hence, the relative abundance of EO and PO repeat units can be elucidated by the contour plot. There are only very few reports on comprehensive 2-dimesional analysis of polymers while coupling two IC methods [32–34, 181–185]. Moreover, Trathnigg and coworkers demonstrated the coupling of LCCC with LEAC in the second dimension for the analysis of nonionic surfactants [186, 187]. Transfer of fractions into 2nd dimension using full adsorption–desorption interface rendered analysis with regard to end-groups. Moreover, coupling of IC with another IC method on columns of different natures for comprehensive analysis of commercial non-ionic surfactants is also reported [5, 181, 188].

Fig. 12.7 D NPLC x RPLC contour plots of PO11 -EO27 -PO11 , 1st dimension: LCCC-ICPPO on a NP silica 300 mm column in acetonitrile/water: 84/16 v/v at 0.05 mL/min (y-axis), trapping interface: on a RP ODS 30 mm column using methanol/water: 50/50 v/v at a flow rate of 1.0 mL/ min, 2nd dimension: LCCC-ICPEO on RP ODS 100 mm column in methanol/water: 87/13 at 1.0 mL/ min (x-axis) [33]. Copyright 2016, Elsevier.

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TGIC X LCCC

Another opportunity for comprehensive analysis of complex polymers is the application of LCCC in the 2nd dimension [28, 29, 168, 169]. It is clear from the elaborated discussion throughout this monograph, LCCC is a fine balance of entropy and enthalpy compensation that is very sensitive to eluent composition and temperature. LCCC is a swift method that makes it applicable for 2nd dimension analysis. However, sensitivity of the critical conditions is a caveat while applying it in the 2nd dimension for 2D-LC. Second dimension analysis must be completed and equilibrated back to the initial conditions in the time to fill the other loop of the interface. The use of same isocratic eluent for both dimensions and maneuvering the elution pattern by change in temperature in the first dimension (TGIC) while keeping 2nd dimension temperature that corresponds to the CCP provides an appropriate combination. The peculiar situation is only possible while using same SP for both dimensions [81]. In this context, isotopically different block copolymers consists of hydrogenous polystyrene (hPS) and deuterated polystyrene (dPS) were synthesized and comprehensively analyzed by 2D-LC [29]. Several different products were synthesized with a fixed length of h-PS (18 kg/mol) while length of d-PS block was varied from 17 to 80 kg/mol. CCP of dPS on the RP ODS column while using CH2 Cl2 /CH3 CN: 57/ 43 (v/v) as eluent was found to be at 33.6 °C. These conditions were selected for 2nd dimension analysis. Under these CCP conditions, hPS elutes in IC regime. On the other hand, a TGIC analysis starting from low initial temperature (10 °C) on the same chromatographic system (SP & MP) render elution of hPS-b-dPS in order on increasing overall molar mass. Although, hPS is retained slightly more than dPS, all the target products and byproducts get separated with regard to overall molar mass (y-axis). TGIC separation is coupled to LCCC in the 2nd dimension for comprehensive analysis. In this case, the eluent compatibility issues are evaded due to use of same eluent and SP for both dimensions. TGIC analysis revealed overall molar mass (y-axis) while individual block length of dPS block is unveiled by 2nd dimension LCCC analysis (x-axis), Fig. 12.8A. At CCP of dPS (x-axis), hPS-b-dPS elutes in IC regime due to stronger interaction of hPS. However, despite having same hPS block, hPS-b-dPS elute in order of decreasing block length of dPS in a SEC like retention but after the dead volume of the column. The study demonstrates the incomplete invisibility of the critical block on the retention of interacting block. Target polymer species and side products are clearly separated and identified in the contour plot. In another study, same group analyzed branched polystyrene with regard to number of branches by coupling TGIC with LCCC [28]. CCP of PS on an ODS column was realized in CH2 Cl2 /CH3 CN: 57/43 (v/v) at 53.3 °C. Under these conditions, branched PS get separated with regard to number of branches in IC regime owing to presence of silyl end-group despite broad MMDs of the products. Same products were not separated well by TGIC due to simultaneous influence of molar mass and branch number (that also has broad MMD) on the retention. Coupling of TGIC with LCCC render a contour plot that has TGIC separation on y-axis while LCCC separation on x-axis, Fig. 12.8B. TGIC separation is dominated by molar mass

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Fig. 12.8 TGIC-LCCC contour plots (A) TGIC × LCCC-SEC of a mixture of three as-prepared hPS-b-dPS (18 k – 17 k, 18 k – 38 k, 18 k – 80 k) 1st dimension: TGIC on a RP ODS column in CH2 Cl2 /CH3 CN: 57/43 (v/v), temperature program: 0–30 min at 10 °C, followed by a linear increase to 20 °C in next 20 min, and subsequently to 35 °C in next 180 min, 2nd dimension: LCCC-dPS on a RP ODS column at 33.6 °C in CH2 Cl2 /CH3 CN: 57/43 (v/v) [29]. Copyright 2013, American Chemical Society; (B) TGIC x LCCC-IC of branched PS 1st dimension: RP-TGIC on an ODS column in CH2 Cl2 /CH3 CN: 53/47 (v/v) at a flow rate of 0.02 mL/min, temperature program: 0–4 min at 10 °C followed by a linear temperature increase to 55 °C in next 16 min, 2nd dimension: LCCC-IC on an ODS column in CH2 Cl2 /CH3 CN: 53/47 (v/v) at a flow rate of 1.2 mL/ min, temperature: 53.3 °C [28]. Copyright 2006, Elsevier

while LCCC separation is based on number of branches owing to presence of silyl end-group. Another example is separation of PS/PB block copolymer (Styrolux). Styrolux is a complex sample having different star-shaped block copolymer species differing in molar mass, architecture, and chemical composition. TGIC on a NP column separates according to the PS block length (1st dimension) while LCCC at the CCP of PS separates with regard to PB block length in the SEC regime (2nd dimension). NP-TGIC x NP-LCCC separated complex Styrolux sample into numerous different species which was not possible to attain by independent analysis by any technique [169].

12.4.2.3

EGIC/TGIC X IC

Hyphenation of any IC method with another IC method in the 2nd dimension is challenging as shown in previous sections. Due to multiple complications, off-line

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approach is often employed for such an analysis [6, 7, 9, 22, 180]. For on-line (comprehensive) analysis, the most viable approach is performing 2nd dimension analysis under isocratic and isothermal conditions in order to save any extra time required for reconditioning of the column for the next run. In this context, separation of a blend of fatty alcohol ethoxylates with regard to both end-group and chain length is reported by coupling of EGIC on a RP column with isocratic IC on a NP column [141]. EGIC on a RP column using a gradient of methanol/water (80/20) to 100% methanol render baseline separation of the blend of fatty alcohol ethoxylates with regard to alkyl end-group. Same sample analyzed on a NP Si column provides a separation with regard to the number of repeat units of ethylene oxide using isopropanol/water: 88/12 v/v as an isocratic eluent. Hyphenation of RP-EGIC with NP-IC produced an excellent mapping of the blend with regard to both end-group and ethylene oxide repeat units, Fig. 12.9A. Distribution of alkyl end-group is depicted on y-axis while x-axis represents the oligomer distribution. The analysis was performed on the experimental setup shown in Fig. 12.2A. Each contour in the plot represents a monodisperse fraction with regard to both endgroup and oligomer number. Interestingly, a very different ethylene oxide oligomer distribution for different end-groups is revealed. In another study, Chang and coworkers hyphenated NP-TGIC with RPLC for rigorous characterization of comb-shaped polystyrene composed of hydrogenous backbone and deuterated side chains [34]. NP-TGIC separated with regard to molar mass irrespective of the isotope composition. On the other hand, RPLC separated these samples with regard to isotope composition. IC separations require comparatively more time for obtaining satisfactory resolution, hence, the experimental setup

Fig. 12.9 (A) EGIC x LC of fatty alcohol ethoxylates blend, 1st dimension: EGIC on a RP ODS column using a gradient of methanol/water: 80/20 v/v → methanol 100% at a flow rate of 0.025 mL/ min; 2nd dimension: isocratic IC on a NP Si column in isopropanol/water: 88/12 v/v at a flow rate of 1.5 mL/min [141]. → Copyright 2010, John Wiley & Sons; (B) NP-TGIC x RPLC for characterization of comb-shaped polystyrene (hydrogenous backbone while deuterated side chains), 1st dimension: NP Si column using isooctane/THF (53/48 v/v) at a flow rate of 0.5 mL/min, temperature gradient: 5–32 °C in 300 min, 2nd dimension: RP ODS column using eluent gradient from CH2 Cl2 /CH3 CN (57/43 v/v) CH2 Cl2 /CH3 CN (59/41 v/v) in 6 min at a flow rate of 1.5 mL/ min [34]. Copyright 2011, American Chemical Society

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shown in Fig. 12.2B was used for this analysis. The eluate from the 1st dimension is trapped multiple times in the trapping column before analysis in the 2nd dimension. Separation with regard to molar mass on NP column using a TGIC method is not an appropriate choice for 2nd dimension since it requires significant amount of time to re-equilibrate the column for the next run. On the other hand, RPLC using a shallow gradient with only 2% change in the eluent composition in 6 min is more suitable for 2nd dimension analysis. Hence. NP-TGIC is coupled with RP-EGIC in the 2nd dimension that render an excellent mapping of the isotope composition of combshaped PS as a function of molar mass, Fig. 12.9B. Free hydrogenated backbone and deuterated branches get separated from the target comb-shaped PS. Furthermore, comb-shaped polymers get separated with regard to both molar mass and number of branches.

12.5 Limitations and Opportunities Coupling of two different modes of LC can unveil many important information which is not possible to attain by individual analysis by any technique. Applications of SEC as 2nd dimension technique for 2D-LC has now become a standard technique with commercially available instrument/software for running and data processing. Coupling of different modes of IC with SEC in 2nd dimension can provide mapping of any distributed property (chemical composition, branching etc.) as a function of molar mass. The only major challenge in this context is the synchronization of the analysis time of two independent chromatographic analysis that has now been successfully resolved owing to developments in the hardware and software. In principle, 1st dimension analysis has to be very slow while 2nd dimension analysis has to be fast, both away from Van Demeter optimum. The 2nd dimension SEC analysis is not generally influenced by 1st dimension eluent provided they are miscible. Analyte immediately leaves the 1st dimension solvent plug and get separated as per hydrodynamic volume in the 2nd dimension eluent. In principle, any IC technique can be coupled with SEC in 2nd dimension owing to its robustness and swiftness. However, even in this case the resolution is much worse than 1D-SEC. Probably, the new UPLC type SEC can improve the 2nd dimension SEC resolution if it is coupled properly. Moreover, in principle, IC x SEC 2D-LC rarely provides deep insight more than two separate 1D-LC analyses. Most of them appearing in the literature is no more than nice show-ups (particularly ELSD detection). Nonetheless, in the industry, it can be useful for the quality control of the polymeric materials since IC x SEC is not very difficult, not much time consuming and provides better finger print than 1D-SEC. On the contrary, coupling of an IC method with another IC method has multifaceted additional challenges. The first and foremost is the synchronization of analysis time of two chromatographs. IC separations of polymers generally takes fairly long time compared to SEC since separations are based on the interaction strength of the analyte with the SP that render its elution later than the dead volume of the column

References

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in contrary to SEC where analytes elute before the dead volume of the column. To address the time synchronization, multiple trapping interface is proposed that has an additional pump and two short columns of similar nature as that of 2nd dimension chromatograph. The 1st dimension effluent can be trapped multiple times as per time required for the 2nd dimension analysis. Next important aspect is the strength of 1st dimension eluent for the 2nd dimension system. First dimension eluent should be weak enough for the interface column to allow efficient trapping of the analyte. The eluent for trapping interface must be sufficiently weak to impede the movement of adsorbed analytes during multiple trappings. Moreover, a strong eluent for 2nd dimension is required that render an acceptable 2nd dimension analysis. The 2DLC based on different modes of IC in both dimensions is a specific and peculiar approach that is applicable to only few polymers. Unlike SEC, IC methods are very sensitive to slight changes in the eluent. The trapping interface not only help in time synchronization but also in removal of 1st dimension eluent that may influence the 2nd analysis otherwise. In principle, multiple detection of isocratic SEC effluent is possible that can provide a much-detailed insight into the polymer composition. However, ELSD is almost exclusively used for 2D-LC that can provide only limited information regarding the polymers under investigation. However, triple detection system has been successfully used for TGIC x SEC. The use of triple detection to a 2D-LC can reveal many additional information of complex polymers. Like SEC, LCCC analysis can also be accomplished swiftly. However, the LCCC conditions are very sensitive to slight changes in the eluent composition and temperature. LCCC can be used in 2nd dimension analysis on the commercially available 2D-LC system without requiring an additional interface. The use same eluent for both dimensions on columns of similar nature is a preferred choice for this. In the 1st dimension, temperature gradient (TGIC) can be used to achieve the required separation while the analysis can be performed on critical conditions by maintaining temperature of the 2nd dimension column. Hence, multiple options of coupling different modes of LC of polymers are possible that render different information. The primary selection criterion of the coupling method is the target information that may differ from case to case such as branching, chemical composition etc.

References 1. Malik MI, Pasch H (2021) Chapter 1— Basic principles of size exclusion and liquid interaction chromatography of polymers. In: Malik MI, Mays J, Shah MR (eds) Molecular characterization of polymers. Elsevier, pp 1–59. https://doi.org/10.1016/B978-0-12-819768-4.000 07-5 2. Striegel AM, Yau WW, Kirkland JJ, Bly DD (2009) Modern size-exclusion liquid chromatography: practice of gel permeation and gel filtration chromatography, vol 2. Wiley, Hoboken, New jersey. https://doi.org/10.1002/9780470442876

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113. Apel N, Uliyanchenko E, Moyses S, Rommens S, Wold C, Macko T, Brüll R (2018) Separation of branched poly(bisphenol A carbonate) structures by solvent gradient at near-critical conditions and two-dimensional liquid chromatography. Anal Chem 90(8):5422–5429. https:/ /doi.org/10.1021/acs.analchem.8b00618 114. Ndiripo A, Pornwilard MM, Pathaweeisariyakul T, Pasch H (2019) Multidimensional chromatographic analysis of carboxylic acid-functionalized polyethylene. Polym Chem 10(43):5859–5869. https://doi.org/10.1039/C9PY01191A 115. Chitta R, Macko T, Brüll R, Kalies G (2010) Elution behavior of polyethylene and polypropylene standards on carbon sorbents. J Chromatograph A 1217(49):7717–7722. https://doi.org/ 10.1016/j.chroma.2010.10.036 116. Roy A, Miller MD, Meunier DM, deGroot AW, Winniford WL, Van Damme FA, Pell RJ, Lyons JW (2010) Development of comprehensive two-dimensional high temperature liquid chromatography × gel permeation chromatography for characterization of polyolefins. Macromolecules 43(8):3710–3720. https://doi.org/10.1021/ma100010e 117. Magagula SI, Ndiripo A, van Reenen AJ (2020) Heterophasic ethylene-propylene copolymers: New insights on complex microstructure by combined molar mass fractionation and high temperature liquid chromatography. Polym Degrad Stab 171:109022. https://doi.org/10.1016/ j.polymdegradstab.2019.109022 118. Lee D, Li Pi Shan C, Meunier DM, Lyons JW, Cong R, deGroot AW (2014) Toward absolute chemical composition distribution measurement of polyolefins by high-temperature liquid chromatography hyphenated with infrared absorbance and light scattering detectors. Anal Chem 86(17):8649–8656. https://doi.org/10.1021/ac501477a 119. Macko T, Ginzburg A, Remerie K, Bruell R (2012) Separation of high-impact polypropylene using interactive liquid chromatography. Macromol Chem Phy 213(9):937–944. https://doi. org/10.1002/macp.201200017 120. Prabhu KN, Brüll R, Macko T, Remerie K, Tacx J, Garg P, Ginzburg A (2015) Separation of bimodal high density polyethylene using multidimensional high temperature liquid chromatography. J Chromatogr A 1419:67–80. https://doi.org/10.1016/j.chroma.2015.09.078 121. Arndt JH, Brüll R, Macko T, Garg P, Tacx JCJF (2018) Characterization of the chemical composition distribution of polyolefin plastomers/elastomers (ethylene/1-octene copolymers) and comparison to theoretical predictions. Polymer 156:214–-221. https://doi.org/10.1016/j. polymer.2018.09.059 122. Arndt JH, Brüll R, Macko T, Garg P, Tacx JCJF (2020) In-depth characterization of polyolefin plastomers/elastomers (ethylene/1-octene copolymers) through hyphenated chromatographic techniques. J Chromatogr A 1621:461081. https://doi.org/10.1016/j.chroma.2020.461081 123. Arndt JH, Brüll R, Macko T, Garg P, Tacx JCJF (2019) High performance liquid chromatography of polyolefin plastomers/elastomers (ethylene/1-octene copolymers)–Comparison of different solvent systems. J Chromatogr A 1593:73–80. https://doi.org/10.1016/j.chroma. 2019.01.067 124. Mekap D, Macko T, Brüll R, Cong R, deGroot W, Parrott A, Yau W (2014) Multiple-injection method in high-temperature two-dimensional liquid chromatography (2D HT-LC). Macromol Chem Phy 215(4):314–319. https://doi.org/10.1002/macp.201300649 125. Macko T, Brüll R, Alamo RG, Thomann Y, Grumel V (2009) Separation of propene/1-alkene and ethylene/1-alkene copolymers by high-temperature adsorption liquid chromatography. Polymer 50(23):5443–5448. https://doi.org/10.1016/j.polymer.2009.09.057 126. Macko T, Brüll R, Alamo RG, Stadler FJ, Losio S (2011) Separation of short-chain branched polyolefins by high-temperature gradient adsorption liquid chromatography. Anal Bioanal Chem 399(4):1547–1556. https://doi.org/10.1007/s00216-010-4335-y 127. Radke W (2019) The retention behavior of diblock copolymers in gradient chromatography; Similarities of diblock copolymers and homopolymers. J Chromatogr A 1593:17–23. https:/ /doi.org/10.1016/j.chroma.2019.01.043 128. Descour C, Macko T, Schreur-Piet I, Pepels MPF, Duchateau R (2015) In situ compatibilisation of alkenyl-terminated polymer blends using cross metathesis. RSC Advances 5(13):9658– 9666. https://doi.org/10.1039/C4RA11056K

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Chapter 13

Hyphenation of Liquid Chromatography with Spectroscopy

Compositional changes as a function of molar mass are important for evaluation of the properties and performance of the polymers. Direct analysis of polymers by spectroscopic techniques such as FTIR and NMR provides only average values of the composition. Moreover, direct spectroscopic analysis cannot determine if the sample is mixture of two structures or these structures are joined with each other. For instance, analysis of a styrene-butadiene rubber (SBR) by NMR or FTIR reveal the averages number of both units in the sample. However, it is not possible to determine if the sample is a mixture of polystyrene (PS) and polybutadiene (PB), a copolymer of PS-PB, or an admixture of copolymer and homopolymers. The hyphenation of LC with the spectroscopic techniques (such as NMR, FTIR, MS etc.) may reveal compositional changes as a function of molar mass [1–4]. This chapter focuses on the strategies of hyphenation of LC with spectroscopy for polymer analysis.

13.1 Hyphenation of Liquid Chromatography with Fourier Transform Infrared Spectroscopy FTIR (Fourier Transform Infrared Spectroscopy) is an excellent technique for swift analysis of the composition of any unknown complex polymer. In parallel, SEC can provide relative MMD of the unknown sample. However, no information on chemical composition as a function of molar mass is obtained by independent analysis by both techniques. The obtained information is extremely important for the development of any sophisticated separation technique such as LCCC, EGIC, TGIC etc. SEC-FTIR is indeed a comparatively established method for unraveling the chemical composition as a function of MMD especially for polyolefins [5–7]. Hyphenation of SEC with FTIR is possible through a flow cell [8, 9] or a solvent elimination interface [10–13]. Flow cell and solvent elimination interface have their peculiar advantages and limitations, hence, can be preferred over one another as per application requirements, Table 13.1. Solvent elimination interface consist of a deposition and an optic © The Author(s), under exclusive license to Springer Nature Switzerland AG 2023 M. I. Malik and D. Berek, Liquid Chromatography of Synthetic Polymers, Physical Chemistry in Action, https://doi.org/10.1007/978-3-031-34835-8_13

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module [14, 15] that makes it comparatively complex compared flow cell [16]. In the deposition module, eluate from LC is sprayed on a disk rotating at a fixed speed. After the complete deposition of the analyte as a function of LC retention time, disk is transferred to optics module to record the FTIR spectra while rotating the disk at the same speed, Fig. 13.1. The set-up allows removal of the eluent and spectra of each position of disk are recorded without any interference. The LC-transform was originally developed by Lab Connections Inc. [17, 18]. In the deposition module, some part of LC effluent is directed to the heated nebulizer that is located above the rotating disk. The volatile eluent immediately evaporates, leaving behind a focused track of the analyte on the disk. Subsequently, disk has analyte deposited at different positions that has a correlation with the LC retention time. The prepared disk is then transferred to the optic module for analysis of the composition of the analyte as a function of LC retention time. Hence, a complete FTIR spectrum of each LC fraction is obtained. LC-transform system has now been widely accepted for various applications [5–7, 19, 20]. The morphology of the deposited film has the key role in the quality of the SEC-FTIR results especially for quantification [21]. Hyphenation of SEC with FTIR through a Germanium disk interface has now become a standard technique for Polyolefin analysis that is also employed to other polymers [12, 14, 15, 17, 22–26]. Coupling of SEC with viscometer along with FTIR reveals concentration and composition of copolymer as a function of absolute molar mass [23]. Moreover, analysis of collected fractions of LCCC and IC by FTIR can unveil quantitation of different block copolymer [14, 27–29], high impact polypropylene [30], and styrene-butyl acrylate copolymers [31] etc. An HPLC instrument additionally equipped with a built-in FTIR is developed by Spectra-Analysis Table 13.1 Characteristics of LC/FTIR on-line flow cell and off-line solvent elimination interface [15]. Copyright 2003, Elsevier

Condition

Flow Cell Interface

Solvent Elimination Interface

Eluent gradient No

Yes

Qualitative information

Limited, eluent dependent

Yes

Quantitative information

Excellent

Limited

Sensitivity

Moderate

Excellent

Limit of detection

Low, eluent dependent High

Spectral S/N ratio

Moderate, spectra collection on the fly

High, post-run scanning possible

Peak asymmetry

Not affected

Not affected

Ease of operation

User friendly

Time consuming optimization

Application area

SEC

SEC, gradient HPLC

13.1 Hyphenation of Liquid Chromatography with Fourier Transform …

267

Fig. 13.1 Scheme of the principle of coupled LC-FTIR using the LC-transform interface [12]. Copyright 2012, Elsevier

[17, 32]. SEC-FTIR has been used for analysis of chemical composition as a function of molar masses of polyolefins [33–44]. On-line coupling of SEC with FTIR is impeded by strong absorption of LC solvents in the mid-IR region. However, the most widely used solvent for SEC of polyolefins, TCB, is sufficiently transparent in the range of 2700–3000 cm−1 that allows efficient polyolefin detection. HT-SEC-FTIR has been employed for analysis for chain branching by on-flow SEC-FTIR [45–47]. The spectrum of pure MP was used as background while recording transmittance spectra of the eluate. Beskers et al. reported an online SEC-FTIR-UV-DRI system [48]. The mathematical treatment for subtraction of solvent signals of the recorded spectra of the SEC eluate is conducted for several polymer samples. SEC-FTIR-UV-DRI analysis of a blend of PMMA (Mw = 90 600 g mol−1 ) and PS (Mw = 8900 g mol−1 ) is demonstrated as a typical example. The contour plot as obtained by spectral detection for the polymer blend is shown in Fig. 13.2A. The characteristic signals used for detection are listed as: carbonyl stretching of PMMA (1735 cm−1 ), PMMA (1150 cm−1 ), and phenyl ring vibration of PS (700 cm−1 ). Several other peaks can also be extracted for detection of PS and PMMA, Fig. 13.2B. The analysis of a PS-b-PMMA copolymer by SEC-FTIR reveals chemical composition as a function of molar mass, Fig. 13.2C. FTIR has also been used as detector for LCCC x SEC 2D-LC system [49]. Polystyrene-b-(polybutadiene-g-poly butyl acrylate) PS-b-(PB-g-PBA) is separated into several different fractions, such as target graft product and backbone, by LCCC in 1st dimension that is then coupled with SEC in the 2nd dimension for molar mass analysis while using FTIR as quasi on-line detector. The major limitation in this context is the low analyte concentration (ng range) due to significant dilution in the

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Fig. 13.2 SEC-FTIR on-line detection of (A) 2D spectral chromatogram of a blend of PS and PMMA; (B) extracted spectra after solvent suppression; (C) Chemical composition of PS-b-PMMA copolymer as a function of SEC elution profile [48]. Copyright 2015, Royal Society of Chemistry

2nd dimension. Arrangement of FTIR data “waterfall diagram” indicates changes in the chemical composition as a function of 2D-LC run-time. As a typical case, FTIR analysis of a specific SEC inject (inject 20) is demonstrated in Fig. 13.3. SEC inject #20 corresponds to elution volume of 4 mL in the 1st dimension that is a graft copolymer fraction. The SEC peak is homogeneous for ELSD, UV and also for Gram–Schmidt plot. Gram–Schmidt plot presents the concentration profile as obtained by summation of all peak intensities for all frequencies while chemigrams are based on one specific peak intensity for that particular component. The fraction is homogeneous with regard to the distribution PS, PB, and PBA. Similar analysis of all the 2nd dimension injects accumulates the chemical composition as a function of 1st dimension elution volume.

13.2 Hyphenation of Liquid Chromatography with Mass Spectrometry

269

Fig. 13.3 LCCC x SEC—FTIR analysis of polystyrene-b-(polybutadiene-g-polybutyl acrylate) Gram–Schmidt diagram and chemigrams for PB, PS and PBA (top) and FTIR spectra of inject 20 of the 2D separation [49]. Copyright 2000, Elsevier

13.2 Hyphenation of Liquid Chromatography with Mass Spectrometry The importance of hyphenation of chromatography with Mass Spectrometry (MS) has been realized from early days of its development. Hyphenation of GC with MS was natural and easy owing to vapor phase nature of both techniques. However, coupling of LC with MS took significantly long time to develop due to different nature of two techniques. The major hinderance is associated with vaporization of the LC effluent. A typical LC flow rate of 1 mL/min translates into approximately 1000 mL/ min in the vapor phase. However, commercial interfaces have been developed that allow broad applicability of HPLC–MS hyphenation [12, 13, 50–55]. Introduction of liquid LC effluent into MS can be realized on the basis of several principles [50, 51, 54]. Atmospheric pressure ionization interface (APCI) is based on pneumatic nebulization of the column effluent in the heated tube that ensures complete solvent evaporation. APCI is initiated by electrons from the corona discharge needle [56]. The generated ions are subsequently introduced into high vacuum environment of the MS. In electrospray interface, high electric field is used to nebulize the column effluent that is created by potential difference between spray capillary and the counter electrode [57, 58]. The fine threads of effluent emerging from the capillary disintegrate into tiny droplets.

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13 Hyphenation of Liquid Chromatography with Spectroscopy

In a thermospray interface, a vapor jet and tiny droplets are generated out of the heated tube while nebulization occurs due to disruption of the liquid by expanding vapors [59]. A considerable amount of heat is transferred to the solvent inside the tube prior to evaporation. The desolvation of droplets in low pressure region is facilitated by transferred heat to the solvent molecules in the tube. These tiny droplets are now transferred directly to the vacuum system of MS. Solvent-mediated chemical ionization and ion evaporation process ensures ionization of the analyte. In a particle beam interface, nebulization of column effluent is done either pneumatically or through thermo-spray into an atmospheric pressure desolavation chamber [60]. A momentum separator directs high-mass analytes to MS while low-mass analytes are sent to waste. The collision of analyte molecules with the heated source wall of a conventional ion source results in their disintegration. The gaseous molecules are subsequently ionized by chemical ionization or electron impact. The application of MS as a detector to HPLC is a captivating approach. However, the above-mentioned ionization techniques can only be applied to a low molar mass range (