High Performance Liquid Chromatography 9781483281292, 9780723608974

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High Performance Liquid Chromatography

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High Performance Liquid Chromatography Mary T. Gilbert formerly at the Wolson Liquid Chromatography Department of Chemistry University of Edinburgh

WRIGHT Bristol 1987

Unit

© IOP Publishing Limited. 1987 All Rights Reserved. No part of this publication may be reproduced, stored in a retrieval system, or transmitted in any form or by any means, electronic, mechanical, photocopying, recording or otherwise, without the prior permission of the Copyright owner.

Published under the Wright imprint by IOP Publishing Limited Techno House, Redcliffe Way, Bristol BS1 6NX

British Library Cataloguing in Publication Data Gilbert, Mary T. High performance liquid chromatography. 1. High performance liquid chromatography I. Title 543'.0894 QD79.C454 ISBN 0 7236 0897 0

Typeset by KEYTEC, Bridport, Dorset Printed in Great Britain by Adlard & Son Ltd, Dorking, Surrey

Preface

Since its inception twenty years ago High Performance Liquid C h r o m a t o g r a p h y ( H P L C ) has rapidly attained the status of the most widely used and versatile analytical technique available today. Progress is still continuing, particularly in the area of m i c r o - H P L C . T h e major developments have, however, been achieved and this is perhaps an o p p o r t u n e time to review the state-of-the-art in general use. My aim has been to provide a practical h a n d b o o k of current practices. With this end in view I have discussed e q u i p m e n t , packing materials and operating techniques in the various modes of m o d e r n H P L C . I have tried to select sufficient variety of examples from the vast applications literature to illustrate the topics discussed and to convey the versatility of the technique. It would be impossible to provide comprehensive coverage of all the areas in which H P L C has proved useful but hopefully I have succeeded in providing sufficient information to enable the reader to approach a separation p r o b l e m with confidence. M. T. G.

v

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Acknowledgements

I would like to thank D r J. V. Sorrentino and D r A . D . C o o p e r for allowing m e to use the library facilities at Richardson Vicks Inc., Shelton, Ct., U S A , without which preparation of this manuscript would have been much m o r e difficult. I am also indebted to my husband, D r J o h n Gilbert for reading the manuscript and for his many helpful suggestions. I would also like to t h a n k my typist Mrs Mary Kurinsky for her p r o m p t work and Mrs Betty Johnson for typing certain sections.

vii

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Contents

List of abbreviations

xii

Glossary of symbols

xiv

1

Introduction

1

2

Basic chromatographic theory

5

2.1 2.2 2.3 2.4

Column capacity ratio Efficiency Resolution Asymmetry

5 8 10 11

3

Equipment 3.1 Pumps and gradient systems 3.2 Injectors 3.3 Connectors 3.4 Detectors 3.5 D a t a systems

12 13 21 28 29 52

4

The 4.1 4.2 4.3 4.4

53 53 56 68 72

column Column design Packing materials Column packing and testing Practical considerations

ix

4.5 4.6

Coupling of columns Micro columns

78 87

5

Sample treatment 5.1 Clean-up procedures 5.2 Trace analysis 5.3 Derivatisation 5.4 Post-column reaction detectors

104 105 110 113 116

6

Separation methods 6.1 Selection/optimisation of conditions 6.2 Normal phase chromatography 6.3 Liquid-liquid partition chromatography 6.4 Reversed phase chromatography 6.5 Ion exchange chromatography 6.6 Size exclusion chromatography 6.7 Affinity chromatography 6.8 Inclusion chromatography 6.9 Chiral chromatography

125 125 135 158 164 180 202 216 224 225

7

Adjustment of selectivity by use of eluent additives 7.1 Ion pair partition chromatography 7.2 Chromatography on dynamically modified oxide gels 7.3 Metal complexation 7.4 Crown ethers

226 226 257 259 268

8

Preparative liquid chromatography 8.1 Separation strategy 8.2 E q u i p m e n t for preparative L C 8.3 Materials for preparative chromatography 8.4 Applications

273 273 280 285 286

9

Chiral chromatography 9.1 Enantiomeric resolution by preparation of diastereoisomer 9.2 Direct enantiomer resolution

291 292 295

10

Current trends in the practice of HPLC 10.1 Columns 10.2 Detectors 10.3 Packing materials 10.4 Supercritical fluid chromatography 10.5 Conclusions

313 313 314 315 316 316

References

x

317

Appendix I Packing materials for high performance liquid chromatography

346

Appendix II Properties of chromatographic solvents

360

Index

367

xi

List of

HPLC i.d. LC psi LOD S N UV SIT LDA IR FTIR AA ICP RI LC-MS GLC CI FAB DLI FDMS FIMS RDME xii

Abbreviations

high performance liquid chromatography internal diameter liquid chromatography p o u n d s per square inch limit of detection signal short-term noise level ultraviolet silicon-intensified linear diode array infrared Fourier transform infrared atomic absorption inductively coupled plasma refractive index liquid chromatography-mass spectrometry gas-liquid chromatography chemical ionisation fast atom b o m b a r d m e n t direct liquid introduction field desorption mass spectrometry field ionisation mass spectrometry rapidly dropping mercury electrode

BHT THF o.d. BOP DP PSD MW EESEAL HDL LDL VLDL BSA LDH CMC TLC GITC AITC a r rA G P SEC GC-MS

butylated hydroxytoluene tetrahydrofuran outside diameter /}, /J'-oxydipropionitrile degree of polymerisation p o r e size distribution molecular weight ethanol extracted steam-exploded aspen lignin high density lipoprotein low density lipoprotein very low density lipoprotein bovine serum albumin lactate dehydrogenase critical micelle concentration thin layer chromatography 2, 3, 4, 6-tetra-0-acetyl-/J-D-glucopyranosyl isothiocyanate 2, 3, 4-tri-O-acetyl-tf-D-arabinopyranosyl isothiocyanate a^-acid glycoprotein size exclusion chromatography gas chromatography-mass spectrometry

xiii

Glossary of Symbols

A,B,C A A'

c

c +/

C

C

C ' Cs"

c r

A

i, max

CN D D2 Dm E dP xiv

constants in Van D e e m t e r equation absorbance surface area of an adsorbant ( m 2 . g - 1) contribution to radial dispersion from stream splitting of the flow around the particles of column packing molecular surface area of solute molecule solute concentration counter ion equilibrium concentration of solute in the mobile and stationary phases, respectively ( g . m l - 1) concentration of adsorbate in moles per unit weight adsorbate concentration in moles per unit area of adsorbent surface available capacity of an adsorbent maximum injection concentration noise equivalent concentration ( g . m l - 1 or g . s - 1) equilibrium distribution coefficient for a solute between two phases linear portion of calibration curve in size exclusion chromatography diffusion coefficient of a solute in the mobile phase ( m 2 . s _ 1) separation impedance particle size of a packing material (/xm)

dc E F

/,

H

h h I, J K

f Ke

K0 Kads KD

\ogkw k' L

I M ^eluent

Mw Mn N NA,NB nA,nB "c P p+iP' AP PC PD

Q Qi, max

R

r RS RSP R

D

RL

s

column diameter (mm) extraction constant fluorescence intensity volumetric flow rate ( m l . m i n - 1) height equivalent to a theoretical plate (mm) reduced plate height incident light intensity transmitted light intensity solvent strength p a r a m e t e r efficiency of collection of fluorescence equilibrium distribution constant specific column permeability superficial adsorption coefficient ( m l . g - 1) adsorption coefficient ( m l . g - 1) distribution coefficient theoretical capacity factor solute capacity ratio column length ( m m ) path length of flow cell (cm) molar weight of b o n d e d group in a b o n d e d stationary phase eluent molecular weight (g) weight averaged molecular weight (SEC) n u m b e r averaged molecular weight (SEC) n u m b e r of theoretical plates mole fraction of solvent A / B in the mobile phase cross sectional area of molecule A / B n u m b e r of carbon atoms in a b o n d e d molecule partition coefficient pairing ion solvent polarity index pressure d r o p across the column (psi) (14.7 psi = 10 5 N m ~ 2 = 10 5 Pa = bar) measured carbon percentage of a b o n d e d phase polydispersity of a polymer q u a n t u m efficiency for fluorescence maximal sample load (g) quantity of solute in the mobile and stationary phases, respectively (g) flow resistance of system response index resolution between two bands specific resolution ( S E C ) dynamic range of a detector linear dynamic range of a detector solvent molecule xv

s° $Bet

T

Tf tR to U

v v

R m

Vs ^solute Vo

v

t

V v

i, max

W Wb,

Wf

W X a a P di>

$s> £>m

£

K

4> —2.0 V ) . T h e obvious advantage of this electrode is the provision of a constantly renewed surface which eliminates the problem of contamination. T h e major disadvantage of any electrode used in the reductive m o d e , however, is that oxygen must be excluded from the system to prevent interference ( M a c C r e h a n and May, 1984; Lloyd, 1983). Both dc (Debowski et al., 1982 b) and ac (Kutner, 1982) polarographic detection modes have been reported with this electrode. Solid working electrodes which allow the detection of electro-oxidisable compounds are generally preferred to the dropping mercury electrode. T h e cell design is simpler, noise levels are lower and greater sensitivity is usually achieved. A major advantage is the very small cell volume which can be achieved. Recently a cell volume of 1 nl was reported (Slais and Krejci, 1982). Two basic cell designs have been used, the thin layer cell (Kissinger et al., 1973) and the wall jet cell (Fleet and Little, 1974). These are illustrated in Fig. 3.17. T h e thin-layer cell is easier to construct and it has been found to be particularly useful for carbon paste working electrodes (Kissinger et al., 1973; Patthy et al., 1982). T h e use of a reticulated vitreous carbon electrode in a thin layer cell produced a three-fold improvement in the S/N ratio c o m p a r e d to a conventional thin-layer amperometric detector (Wang and D e w a l d , 1984; W a n g and Freiha, 1984). T h e design of coulometric detectors is very similar to that of amperometric detectors, except that larger surface areas of the working electrode are required to obtain complete electrolysis (Hagihara et al., 1983). T h e r e has been interest recently in using dual electrode cells to enhance detection capabilities. Increased selectivity can be obtained by operating each

EQUIPMENT

39

electrode at a different potential or by using o n e electrode to oxidise or reduce the constituents in the flow stream while the d o w n s t r e a m electrode detects the products of the reaction (Roston and Kissinger, 1982; Elchisak, 1983; Hiroshima et al., 1983; L u n t e and Kissinger, 1983; H a r o o n et al., 1984). Electrochemical detectors have found considerable application, particularly in trace analysis. This topic has recently been reviewed by Krull et al. (1983 a). T h e application of electrochemical detection to normal-phase liquid chromatography of phenols using n o n - a q u e o u s eluents of low dielectric constant has been reported (Gunasingham and Fleet, 1983).

Fig. 3.16. Flow through polarographic detector. 1: body; 2: detection passage; 3: waste solution capillary; 4: clamping sleeve to mercury connector tube; 5: solution inlet capillary; 6: inlet capillary fastening ring; 7: inlet capillary clamping sleeve; 8: rapidly dropping mercury electrode (RDME); 9: RDME fastening sleeve; 10: RDME clamping sleeve; 11: Ag/Ag CI reference electrode; 12: degassing passage; 13: waste solution capillary fastening sleeve. Reproduced with permission from Kutner et al., 1980.

40

H I G H P E R F O R M A N C E LIQUID C H R O M A T O G R A P H Y

fa)

(b) Fig. 3.17. Cells designed with solid working electrodes for use in amperometric detectors for HPLC. (a) Thin-layer design; (b) Wall jet design. In (a) the two blocks 1 and 2 are sandwiched together to form the flow cell via the hole cut in the PTFE spacer (3). The working carbon paste electrode (4) is contained in the well in block 2. The reference and auxiliary electrodes are contained in the overflow vessel (not shown). Reproduced with permission from Kissinger et al., 1973. In (b) 1: inlet nozzle; 2: glassy carbon disc working electrode; 3: adjustable working electrode body; 4: reference electrode 5: auxiliary electrode exit. Reproduced with permission from Fleet and Little, 1974.

C O N D U C T I V I T Y DETECTORS The

recent

considerable

upsurge

in

use

conductivity

of

interest

in

ion

chromatography

detectors.

These

has

measure

resulted

the

in

electrical

resistance of a conducting m e d i u m b e t w e e n two electrodes in t h e solution. Normally t h e specific c o n d u c t a n c e , K ( o h m " 1 . c m - 1 or m h o . c m - 1) is q u o t e d rather t h a n t h e specific resistance p , although strictly t h e circuitry m e a s u r e s resistance. T h e two are related by K =

lip

(3.7)

EQUIPMENT

41

The conductivity flow cell usually consists of a small c h a m b e r , less than 5 in volume, fitted with two electrodes. Generally platinum is used but stainless steel or gold can prove useful alternatives. In the older detector designs the resistance of the flow cell contents was measured by incorporating the cell in one arm of an ac Wheatstone bridge (Scott, 1977). T h e sensitivity of this detector is about 1 0 - 6 g . m l - 1 using an eluent of relatively high conductance. Reduction of the eluent conductance can increase the sensitivity by about two orders of magnitude. O n e of the major problems of this design is the detection of undesirable capacitance effects which restrict the conductance range of the detector. This problem can be overcome by using the dipolar pulse technique developed by Johnson and E n k e (1970). This technique consists of applying consecutive constant voltage pulses of equal magnitude but opposite polarity to a standard conductance cell. T h e current to voltage ratio is measured at the end of the second pulse. A n H P L C detector which uses this circuitry has been described (Keller, 1981) and found to show improved sensitivity. T h e detection of ionic species in H P L C eluents using conductivity detectors is very difficult because of the high background conductance of the eluent. T h e development of suppressed ion chromatographic systems (Small et al., 1975) obviated this p r o b l e m . Subsequently, non-suppressed ion chromatography with low capacity ion exchange resins and dilute eluents was proved feasible (Gjerde et al., 1979, 1980). This topic is discussed fully in Section 6.5.3.

A T O M I C ABSORPTION

A t o m i c absorption spectrometers offer excellent sensitivity and selectivity for the detection of a large n u m b e r of elements, and their combination with H P L C has generated considerable interest (Van L o o n , 1981; Jewett and Brinckman, 1983). These detectors are particularly suited to the determination of trace elements in biological and environmental samples because of their high specificity. T h e main p r o b l e m associated with the on-line coupling of the two techniques is the sample transfer. In classical absorption spectrometry the sample solution is aspirated into the flame via a pneumatic nebuliser and this would seem to be ideal for coupling with a liquid chromatographic separation technique. Unfortunately, the u p t a k e rate for most nebulisers (2-10 m l m i n - 1) is greater than the normal flow rates in H P L C and direct introduction leads to nebuliser starvation. Yoza and Ohashi (1973) described a m e t h o d of adding a m a k e - u p flow of water prior to the nebuliser but this leads to dilution of the sample. A n alternative approach, t e r m e d the "injection m e t h o d " (Berndt and Slavin, 1978) involves collecting the eluent from the column in a droplet former and allowing the droplets to fall into a P T F E funnel from where they are sucked into the flame. K o r o p c h a k and C o l e m a n (1980) have examined the effect of the nebuliser design on the transport efficiency. Withdrawal of the capillary tube within the nebuliser results in a back pressure across the tip and the flow rate from the chromatograph can then be used to control the aspiration rate. Favourable

42

HIGH PERFORMANCE LIQUID CHROMATOGRAPHY

results were obtained with this system, but unfortunately this was found to be a function of the design of the particular nebuliser used. Liquid chromatography directly coupled with flame atomic absorption detection has been used for the selective detection of various polyphosphates complexed with magnesium (Yoza et al., 1975; Kouchiyama et al., 1978), for the determination of organosilicons in industrial waste waters (Cassidy et al., 1976), for analysis of amino carboxylic acid-copper complexes (Jones and M a n a h a n , 1976a, b ) , and for the determination of tetralkyl lead c o m p o u n d s in gasoline (Messman and Rains, 1981). In the above cases the total eluent from the column is aspirated into the flame and the sample is therefore destroyed. A n alternative approach using the m o r e sensitive graphite furnace atomisation technique onl> uses a portion of the eluted sample for A A detection, allowing collection of the r e m a i n d e r for further analysis. Since the furnace process is m o r e time consuming (involving drying, charring and atomising cycles) it is not strictly an on-line procedure but the sampling and analysis has been fully a u t o m a t e d (Brinckman et al., 1977; Stockton and Irgolic, 1979). Brinckman et al. (1977) used two different sampling techniques. In the pulsed sampling m o d e the total column eluent was fed into a sampling well, from which the required volume was r e m o v e d at fixed time intervals and transferred into the graphite furnace t u b e . Overflow from the sampling well was continuously r e m o v e d by gentle suction. Alternatively, in the survey m o d e the eluent from the column was split and a portion fed directly into a fraction collector which was again automatically sampled at pre-set time intervals by the sampling pipette for the graphite furnace. T h e interface designed by Stockton and Irgolic (1979) used a pneumatically activated eight-port sampling valve to deliver samples of the eluent into the graphite cuvette of the furnace at pre-set times. Systems like this have been used to determine trace organometallic compounds (Brinckman et al., 1977), for the characterisation of organometallic copolymers by size exclusion chromatography (Parks et al., 1983), for the estimation of di- and tri-organotins in water (Jewett and Brinckman, 1981), for the analysis of metal containing macromolecules (Parks et al., 1979), for the determination of arsenic c o m p o u n d s in environmental samples (Stockton and Irgolic, 1979; Brinckman et al., 1980; Fish et al., 1982, 1983; Woolson et al., 1982), for the study of the complexation of copper by aquatic humic matter (Becher et al., 1983) and for the estimation of vanadyl c o m p o u n d s in heavy crude petroleums (Fish and Komlenic, 1984). In Fig. 3.18 the dual chromatograms obtained from the U V and A A analysis of a 1:1 solution of triphenyltin and tri-n-butyltin cations is illustrated, indicating the advantage of the technique. It is also possible to use atomic emission spectrophotometers to simultaneously monitor several elements (Fraley et al., 1981; Hausler and Taylor, 1981a; McCarthy et al., 1981; Bushee et al., 1982 a, b ) . F o r atomic emission to occur, the excitation source t e m p e r a t u r e must be considerably higher than in atomic absorption. B o t h flames and plasmas have been used as sources in spectrophoto-

EQUIPMENT

43

meters coupled with H P L C but the high t e m p e r a t u r e inert gas plasma m e t h o d suffers less from interference p r o b l e m s and therefore increases the sensitivity of the technique. B o t h direct current (Krull et al., 1983 b) and inductively coupled plasma (ICP) m e t h o d s have b e e n investigated, but t h e latter are preferred since no electrodes are required and therefore interferences from t h e source are minimised.

i 0

1

1

1 —

10 20 30 Time(min) Fig. 3.18. Dual chromatograms illustrating speciation of a 1:1 solution of triphenyltin and tri-Ai-butyltin cations compared in tin-specific graphite furnace atomic absorption (bottom) and UV (top) modes. Column packing, Partisil-10 SCX; column dimensions, 250 x 4.6 mm i.d.; eluent, 0.01 M ammonium acetate in methanol/water (70:30); flow rate, 1.00 ml.min - 1; furnace program: dry (80 °C), 10 s; char (100 °C), 10 s; atomise (3000 °C), 1 s and (2500 °C), 6 s; furnace purge gas, argon at 200 ml.min -1 stopped-flow mode; auto sampler: pipetting interval, 50 s; sample volume, 20 fi\; UV detection at 224.6 nm. Solutes, 1: triphenyl tin; 2: tri-w-butyltin.

Generally, t h e eluent from t h e H P L C column is directly aspirated into t h e plasma box. T h e influence of t h e flow rate and t h e n a t u r e of the mobile phase on the operation of t h e inductively coupled plasma source has b e e n examined (Gast et al., 1979). T h e r e are s o m e restrictions placed on t h e eluent, since t o o high an organic content m a y extinguish t h e plasma (Ibrahim et al., 1984). T h e effect of

44

HIGH PERFORMANCE LIQUID CHROMATOGRAPHY

spray chamber design (Hausler and Taylor, 1981 a) and position (Whaley et al., 1982) has also been investigated. A n u m b e r of recent applications of H P L C using inductively coupled plasma atomic emission detection are listed in Table 3.3. T h e comparison of detection limits for a n u m b e r of elements by atomic absorption and atomic emission spectrometry are given in Table 3.4. Table 3.3. Applications of HPLC with on-line inductively coupled plasma emission detection. Application

Reference

Phosphorus in nucleotides

Heine et al., 1982

Phosphorus in nucleotides

Yoshida et al., 1983a

Amino acid analysis by determination of carbon and sulphur

Yoshida et al., 1983b

Speciation of arsenic and cadmium compounds

Nisamaneepong et al., 1984

Tetraalkyl lead in petrol

Ibrahim et al., 1984

Protein analysis by determination of carbon, phosphorus and several metals

Morita et al., 1980

Metals in organometallics

Hausler and Taylor, 1981b

Metals in organometallics by micro HPLC

Jinno and Tsuchida, 1982

Determination of magnesium and calcium in lake and river water

Gardner et al., 1982

Phosphorus in phosphates

Morita and Uehiro, 1981

Arsenic in biological samples

Morita et al., 1981

Copper in chelates

Fraley et al., 1979

Arsenic and selenium

McCarthy et al., 1983

Carbon in organics by micro HPLC

Jinno and Nakanishi, 1983

R A D I O A C T I V I T Y MONITORS

T h e use of radioactivity detectors is very important in biological and biochemical fields, particularly in metabolism studies (e.g. Frey and Frey, 1982a). T h e monitoring of radioactivity in the eluent from a liquid chromatograph can be performed in either an off-line or on-line m o d e . T h e on-line m e t h o d shows some loss of counting efficiency but greatly improved analysis times (Everett, 1982; Frey and Frey, 1982b; Kessler, 1982). T h e r e are two types of on-line radioactivity detectors, namely heterogeneous and h o m o g e n e o u s systems. In the heterogeneous system the eluent passes through a cell which is packed with solid scintillator. With the h o m o g e n e o u s m e t h o d the scintillator is mixed with the eluent from the column prior to entering the flow cell. T h e h o m o g e n e o u s system generally has a higher counting 5 4 efficiency for the usual /^-emitters of interest (i.e. 3 S , 1C , 3H , 3 P2 ) . It is often desirable to use larger flow cells with radioactivity monitors than

EQUIPMENT

45

Table 3.4. Detection limits for some elements using AAS and AES. Detection limit fjig.mh1

Atomic absorption Flame

GFAAS

10-6 10-5

10-4

Mg

lO-3

Ag. Al. Be. Ca. Cd. Co. Cr. Cu. Fe. Mn. Na. Ni. Zn Ba. Pb. Sn. Sr

io-i

1 >1

Si. Tl. V Hg B.Th

Flame

Be. Cd. Cr. Fe. Mg. Mn. Zn Al. Ca. Co. Cu. Na. Pb. Ba. Ni. V Si. Ti

10-7

10-2

Atomic emission Plasma

Ca Ca. Na Ag. Ba. Cr. Mg. Mn. Sr

P

Al. Co. Cu. Fe. Ni Pb.Tl B. Sn

Sr B. Ba. Be. Cd. Co. Cu. Fe. Mg. Mn. Na Ag. Al. Cr. Hg. Ni. Pb. Sn. Th P. Si. Tl V Cd

Hg. Si. Zn

Reproduced with permission from White, 1984.

would be used in other H P L C detectors, since the response d e p e n d s on the total activity in the flow cell at any one time. M a x i m u m counting efficiency is obtained at low flow rates and flow rates near the optimum in the plate height curve should be used. Because of the necessity of using larger flow cells to achieve a d e q u a t e sensitivity, the radioactivity monitors are generally not suitable for use with narrow b o r e , high efficiency columns. Harding et al. (1982) developed a high resolution, high sensitivity flow cell. T h e important factors were high recovery of radiolabeled sample during preparation, reduction of instrument background count r a t e , and optimisation of cell design. B a b a et al. (1982) devised a synchronised accumulating detector using five counting cells. With the h o m o g e n e o u s counting m e t h o d , detection 4 limits were about 45 pCi for 1 C and about 270 pCi for 3H . y-detectors have also been used on-line with liquid chromatography. N e e d h a m and D e l a n e y (1983) d e m o n s t r a t e d the use of a cadmium telluride detector to monitor y-radiation from eluting radiopharmaceuticals. T h e purification of 1 2I5- l a b e l l e d c o m p o u n d s was achieved by reversed phase high performance liquid chromatography using a sodium iodide crystal as an on-line y-detector to m e a s u r e the specific activity of the purified samples (Von Stetten and Schlett, 1983). 3 A3

Bulk property

detectors—refractive

index

monitors

Bulk property detectors m e a s u r e a property of both the solute and the eluent and it is therefore very important to carefully control the value of the eluent

46

HIGH PERFORMANCE LIQUID CHROMATOGRAPHY

property to allow any degree of sensitivity. T h e commonest bulk property L C detector is the refractive index (RI) monitor and under properly controlled conditions this is virtually a universal detector, although it is less sensitive than the specific detectors. T h e detector response depends on the difference between the refractive index of the p u r e mobile phase and that of the mobile phase + solute eluting from the column. For this reason, all detector designs are based on a differential m o d e of operation. T h e r e are t h r e e c o m m o n detection m e t h o d s used in commercial refractive index monitors: 1) the deflection principle, 2) Fresnel's m e t h o d and 3) the interference principle. I.

DEFLECTION REFRACTOMETER

In the deflection refractometer light from the source passes through the cell, which is split into a sample and reference c o m p a r t m e n t . T h e incident light is reflected from a mirror at the rear of the cell back through the cell again to a photomultiplier. W h e n the composition of the eluent in the sample cell changes, the change in refractive index causes a deflection of the light b e a m detected by the photodetector. T h e detector produces a signal proportional to the position of the light. Deflection R I detectors have a wide range of linearity and only o n e cell is n e e d e d . 2.

FRESNEL REFRACTOMETER

H e r e incident light is passed through a prism to the flow cell and the a m o u n t of light reflected from the glass-liquid interface is measured. Since the a m o u n t of light reflected d e p e n d s on the angle of incidence and the refractive index of the liquid, the difference between the reflected light intensities from the sample and reference cells is a m e a s u r e of the solute refractive index. This type of refractometer, however, suffers from a limited linear range. Additionally, two different prisms are required to encompass the whole refractive index range of 1—1.75 refractive index units. T h e cell windows must also be kept very clean for good results. 3.

INTERFERENCE REFRACTOMETER

In the interference refractometer the light source b e a m is split by a b e a m splitter prior to passage through the sample and reference cells and then recombined using a second lens and b e a m splitter before detection. T h e refractive index difference between the sample and reference beams produces a difference in optical path length which is measured by the interferometer. This type of detector gives a linear response and it is m o r e sensitive than other types. F u r t h e r increase in sensitivity may be achieved using a laser light source (Woodruff and Y e u n g , 1982a,b). R I detectors require very careful thermostatting of the complete H P L C system, since they are very sensitive to t e m p e r a t u r e fluctuations. Despite the use

EQUIPMENT

47

of a reference cell it is not usually possible to use these detectors u n d e r gradient elution conditions, since it is very difficult to exactly match the two flows. E v e n under isocratic conditions the eluent composition must be carefully controlled. If mixed solvents are used, evaporation of o n e of the c o m p o n e n t s will cause a refractive index change resulting in detector drift. It is often desirable to stir the eluent in the reservoir to maintain homogeneity during a series of runs. Baseline drift may be severe when solvents are changed and stability will not b e obtained until all the previous solvent has been completely flushed from the system. Despite all the apparent drawbacks of this detection system it is frequently used and all manufacturers offer R I e q u i p m e n t .

3.4.4

Transport

detectors

T h e original transport detector (James et al., 1964; Scott and L a w r e n c e , 1970) employed a moving wire to collect the eluted solutes from the column and transport t h e m via an oven to evaporate the solvent to a combustion or pyrolysis chamber to volatilise the samples. T h e gaseous products were then carried in a stream of nitrogen to a flame ionisation detector. Unfortunately, problems associated with uniform coating of the wire, selective solvent removal, reproducibility and the very small proportion of the eluate which is deposited on the wire have m e a n t that this detector has not been as successful as first envisaged and it is rarely used today. O t h e r transport systems have been designed based on similar principals. These have been discussed in detail by Scott (1977).

3.4.5

Combined

liquid chromatography-mass

spectrometry

(LC-MS)

T h e technique of combined gas chromatography-mass spectrometry is undoubtedly the single most powerful analytical procedure available to the organic analyst. T h e potency of G C - M S is due to the fact that the individually sophisticated techniques of gas chromatography and mass spectrometry are both gas phase procedures which a r e , accordingly, eminently compatible. W h e n used as a chromatographic detector, mass spectrometry affords the kind of detailed structural information which cannot be surpassed by any single analytical technique. T h e combination of the remarkable versatility of H P L C with mass spectrometry is an obvious and potentially highly advantageous parallel to G C - M S . T h e r e has been considerable interest in LC-MS in recent years and several reviews have been published ( G a m e s , 1981; Curry, 1982; G a m e s , 1983; Guiochon and A r p i n o , 1983; A r p i n o , 1985; Bruins, 1985; Crowther et al., 1985; Niessen and P o p p e , 1985). Regrettably the use of a liquid phase separation system and a gas phase detection system operating at high vacuum are not (in contrast to G C - M S ) directly compatible. T h e r e are two principal problems associated with the combination of H P L C

48

HIGH PERFORMANCE LIQUID CHROMATOGRAPHY

with mass spectrometry. Firstly that of sample enrichment; t h e selective removal of mobile p h a s e , so well resolved by Ryhage with his jet separator for G C - M S , is not appropriate for combined L C - M S . Secondly, in contrast to G L C , H P L C readily permits t h e separation of solutes of both high molecular weight and/or high polarity. H o w e v e r , such substances, having negligible vapour pressures, frequently cannot be ionised by the conventional mass spectrometric ionisation techniques used in G C - M S . T h e former of these two problems has hitherto been that of most concern to investigators. T h r e e principal approaches have been used to directly interface the liquid chromatograph to a mass spectrometer: 1) moving belt, 2) direct liquid inlet and 3) thermospray. I.

M O V I N G BELT

This system was developed along the same lines as the moving wire detector (Scott et al., 1974). Better collection efficiency was obtained by replacing the wire with a polyimide belt ( M c F a d d e n et al., 1976). T h e first commercially available LC-MS system used this technique t o transport t h e solutes t o a flash vaporiser just prior to the ion source, as illustrated in Fig. 3.19. T h e next commercial system used a modification which allowed the moving belt interface to enter the ion source of the mass spectrometer (Yorke et al., 1981).

LC Effluent

Vac-locks

Flash vaporizer

Clean-up heater

Springloaded idler wheel

i

Drive wheels

Pump Pump Fig. 3.19. Schematic diagram of the moving belt LC-MS interface. Reproduced with permission of Finnigan-MAT Ltd.

T h e main disadvantages are the limitation of eluent flow rates to a m a x i m u m of about 1 m l . m i n - 1 to allow for almost complete solvent removal and the difficulties experienced with aqueous eluents. These systems can, however, b e combined with electron impact M S detection (this is not the case with o t h e r introduction modes) which provides much m o r e structural information.

EQUIPMENT 2.

49

D I R E C T LIQUID INTRODUCTION

Instead of using a moving belt it is possible to directly introduce the eluent from the column into the mass spectrometer and use the volatilised solvent as the reagent gas for chemical ionisation (CI) mass spectrometry (Baldwin and McLafferty, 1973; A r p i n o et al., 1974). This system is compatible with reversed phase liquid chromatography using up to 7 0 % water as eluent. The main problem associated with direct liquid introduction is that the flow rates from standard columns are normally too high to introduce completely into the mass spectrometer and some form of splitter must be used. This obviously reduces the sensitivity of the technique, since only 10% of the injected solute is transferred to the detector. This interface is, however, ideal for coupling to microbore columns since flow rates of 5-50 / x l . m i n -1 can be totally transferred to the mass spectrometer. Generally, the eluent from the microbore column passes through a pin-hole in a diaphragm directly into the ion source (Arpino et al., 1981). O n e of the problems sometimes associated with this system is plugging of the pin-hole and careful filtering of the eluent must be performed to avoid this ( M a u c h a m p and Krien, 1982). 3.

T H E R M O S P R A Y IONISATION

T h e thermospray system developed by Blakley and co-workers (1980) is a direct liquid introduction interface which can cope with conventional flows of 1-2 m l . m i n - 1. A typical interface is illustrated in Fig. 3.20 and it can be readily adapted to any commercial quadrupole MS equipped with a vacuum system suitable for CI operation. T h e only addition to the vacuum system is a single stage mechanical vacuum p u m p of m o d e r a t e capacity ( — 3 0 0 1 . m i n - 1) . Vapourisation of the eluent is achieved by rapid heating to produce a jet of vapour and an aerosol which allows analysis of non-volatiles. A n interesting aspect of this technique is that with dilute aqueous buffers, ions are created in the spray without the need for an external ionising source (Blakley and Vestal, 1983). For eluents which do not contain buffer salts, post-column addition of buffer may be used to produce this effect (Voyksner et al., 1984 b ) . T h e optimisation of thermospray conditions has been studied (Voyksner and H a n e y , 1985). Several recent applications of combined LC-MS are summarised in Table 3.5. Despite the intensive activity in the search for improved L C - M S interfaces much m o r e remains to be accomplished before the overall sample transfer efficiencies for LC-MS approach those obtainable in G C - M S . T h e second p r o b l e m with current LC-MS is to utilise ionisation systems which can do justice to the full range of solutes which may be separated by H P L C . A s indicated above, the ionisation techniques used for G C - M S (normally electron impact or chemical ionisation) do not permit the detection of solutes, such as polypeptides and polysaccharides, which can be very conveniently separated by HPLC.

50

HIGH PERFORMANCE LIQUID CHROMATOGRAPHY

Temperature control sensor

ii To trap and forepump

Filament Fig. 3.20. Schematic diagram of thermospray interface. Reproduced with permission of Finnigan-MAT Ltd.

Several mass spectrometric ionisation techniques have been developed for the determination of macromolecules (field ionisation, field desorption and fast atom b o m b a r d m e n t ( F A B ) ) . Of these techniques F A B appears most compatible with H P L C , at least if a moving belt sample transfer system is used. Stroh et al. (1985) have reported the use of an 'on line' L C - F A B - M S system for the identification and structural elucidation of a series of polypeptides with molecular weights u p to ca.1880 a.m.u.. Their interface is shown in Fig. 3.21.

HT insulator Variable angle jet

Tunnel seats Source housing

Probe aligning mechanism

FAB

Washbath Second stage

First stage Liquid N r | vacuum t Pr a a d vacuum

Fig. 3.21. Schematic diagram of a moving belt/FAB MS interface. Reproduced with permission from Stroh et al., 1985.

rive roller

Housing vacuum

EQUIPMENT

51

Table 3.5. Recent applications of combined LC-MS Application

Reference

Interface

Ion pairs

Solvent extraction/ moving belt

Kirbyet al., 1981

Polycyclic aromatic hydrocarbons and phenols

DLI 1

Schafer and Levsen, 1981

Explosives

DLI

Yinon and Hwang, 1983

Organophosphorus pesticides

DLI

Parker et al., 1982

Sulpha drugs in biological fluids

DLI

Henionetal., 1982

Perchloro cage compounds

Moving belt

Cairns et al., 1982

Phenylureas

DLI

Levsen etal., 1983

Hydrocarbon composition in high boiling coal liquids

Off-line LC-FIMS 2 Boduszynski et al., 1983

Measurement of endogenous leucine enkephalin in canine thalamus

Off-line LC-FDMS 3 Desiderio and Yamada, 1982

Nucleotides

Off-line LC-FDMS Seligeret al., 1982

Aliphatic acid determination in shale oil process water; valproic acid in human serum

DLI

Christensen et al., 1983

Corticosteroids in equine plasma and urine

DLI

Sugnaux et al., 1983; Skrabalak et al., 1984

Ion pairs of biogenic catecholamines

DLI

Milon and Bur, 1983

Pesticides

DLI

Voyksner et al., 1984a

Sequence analaysis of derivatised peptides

Moving belt

Yu et al., 1984

Ozonation products of phthalic acid in water

DLI

El Dine et al., 1984

Gossypol and derivatives

Moving belt

Matlin et al., 1984

Pesticides

Thermospray

Voyksner et al., 1984b

Phenols

Moving belt

Hayes et al., 1984

Pesticides

DLI

Voyksner and Bursey, 1984

Polycyclic aromatic hydrocarbons; acidic organic pesticides

Extraction/DLI

Apffel et al., 1984

Fusarium mycotoxins

Thermospray

Voyksner et al., 1985

Carbamate pesticides

Moving belt

Wright, 1982

Xanthine derivatives

Thermospray

Blakley and Vestal, 1983

Glucuronides

Thermospray

Polypeptides *DLI = FDMS= FIMS = 4 FAB = 2

3

Direct inlet introduction. Field desorption mass spectrometry. Field ionisation mass spectrometry. Fast atom bombardment mass spectrometry.

Moving belt/FAB

Liberato etal., 1983 4

Stroh et al., 1985

52

HIGH PERFORMANCE LIQUID CHROMATOGRAPHY

This work reflects a significant advance in LC-MS techniques. H o w e v e r , LC-MS is still a Cinderella technique (a very expensive one at t h a t ) , and will remain that way until one of the major mass spectrometer manufacturers has the courage to introduce a specifically designed LC-MS system and not, as is currently the case, advertise LC-MS as an interesting but problematic bolt-on to G C - M S . Many other detection systems have also been combined with liquid chromatography, e.g. r a m a n spectroscopy (Iriyama et al., 1983); nuclear magnetic resonance spectrometry (Bayer et al., 1982; H a w et al., 1983); light scattering (Stolyhwo et al., 1983, 1984; Lafosse et al., 1985); dielectric constant (Mowery, 1982); photoionisation (Locke et al., 1982); radioimmumoassay (Eibs and Schoneshofer, 1984; H e r m a n n et al., 1984); thermal lens effect (Buffet and Morris, 1982, 1983); permittivity (Alder et al., 1983, 1984) and r o o m temperature phosphorescence (Weinberger et al., 1982; D o n k e r b r o e k et al., 1983; Gooijer et al., 1984a, b ) . F u r t h e r information on these and other less-used detection systems can be obtained in the excellent review by White (1984). 3.5

Data systems

D a t a handling systems vary from the simple chart recorder which produces a chromatographic record of the elution profile to sophisticated computer systems which can calculate and print out final results such as retention times, efficiency and solute quantities. C o m p u t e r systems may also be used to control the complete liquid chromatograph to enable procedures such as eluent optimisation, automatic injection, column switching, etc. (Peichang and Xiaoming, 1984). Microprocessor systems are relatively inexpensive now and most routine analytical laboratories have systems which allow data acquisition and manipulation. These may often be interfaced with several chromatographs. A u t o m a t i o n considerably speeds up routine analytical work with much less o p e r a t o r handling and allows 24 hour utilisation of equipment.

Chapter 4

The Column

T h e most important feature of any c h r o m a t o g r a p h is the column. N o matter how good the e q u i p m e n t good resolution will not b e achieved without a column well packed with material of high quality. It should be stressed, however, that the resolution obtained from the most expertly packed column can very easily be lost if care is not paid to the following 1. T h e connecting tubing between the column and detector should be of minimum length and b o r e . 2. T h e r e should be no unswept dead volumes in the connecting unions at the top or b o t t o m of the column. For this reason the design of the column and connectors is important as well as the packing materials used and the technique of packing the column. 4.1 4.1.1

Column design Column

bore

With the increasing use of microparticulate packing materials with diameters of 3-10 /xm and the i m p r o v e m e n t s in column packing techniques with such materials, it b e c a m e a p p a r e n t that better efficiency could generally be obtained with columns of wider b o r e . T h e reasons for such effects arise from the fact that there is an area of packing close to the walls which is not as well packed as the 53

54

HIGH PERFORMANCE LIQUID CHROMATOGRAPHY

rest of the column (Knox and Parcher, 1969: Knox et al., 1976). W h e n a sample is applied to the top of a column as a point injection in the centre of the packed bed the solute band spreads across the bed at a rate much slower than that at which it is eluted through the column. For a centrally injected sample, the column bore should be large enough so that the bands elute before they reach the disturbed 'wall region'. T h e relationship between column length, bore and particle diameter has been given by Knox et al., (1976). ^ 16 (B/v + A r )

(4.1)

where dc is the column diameter, L is the column length, dp is the particle size and v is the reduced velocity of the eluent. B is the term from the Van D e e m t e r equation (Equation 2.19) representing axial dispersion and arises from molecular diffusion. Ar represents the contribution to radial dispersion from stream splitting of the flow around the particles of the packing. T h e recommended values of the constants are r ~ 30, B = 1.4 for impervious particles and 1.8 for porous particles and Ar = 0.060, which gives the equation for porous particles as

^ 16 (1.8/v + 0.060)

(4.2)

In practice, for 5 /xm particles the column bore should be about 5 m m to produce an infinite diameter' column. T h e foregoing discussion is only applicable where a point injection is used, which means syringe injection. Routinely, however, the use of syringes at high pressures can cause some problems, e.g. blockage of the syringe needle often occurs by extrusion of septum material. This can result in the splitting of the syringe barrel on depression of the plunger. T h e problem can often be avoided by the use of d o m e d syringe needles and pre-drilled septa, but for routine applications valve injectors have b e c o m e m o r e popular. In valve injection the sample is introduced to the top of the column in the stream of eluent and is therefore spread over the whole of the cross section of the column very quickly. Improved efficiencies with valve injectors may be obtained using a curtain flow technique. In this case the eluent stream to the top of the column is split. A portion of the eluent is passed through the loop of the valve injector onto the centre of the column, with the remainder of the flow being directly introduced to the top of the column. Adjustment of the ratio of the two flows is critical but provided this is correctly achieved very good efficiencies can be obtained. Such systems a r e , however, m o r e difficult to set up and maintain and in general the full bore of the column is used for valve injection. In this, the most c o m m o n case, it is necessary to minimise the column wall effects. This is achieved by reducing surface defects on the walls of the column which may result in a less well packed bed in that area of the column. T h e most c o m m o n m e t h o d of minimising such effects is to use stainless steel columns with polished inner

T H E COLUMN

55

walls (Karapetyan et al., 1983). A n o t h e r advantageous technique is the use of glass lined stainless steel tubes. Although such systems have been found to be efficient (Vigh et al., 1978: Von A r x , 1981), they are not routinely used. ( N . B . Merck have introduced a glass lined cartridge column system.) Waters Associates have introduced flexible walled columns and radial compression to produce homogeneously packed beds and reduce wall effects (Fallick and Rausch, 1979). Such columns have been used by several workers with good results (Kinberger et al., 1981; Kinberger and W a h r g r e n , 1982; Greenblatt et al., 1983; H a r t et al., 1984; Martin et al., 1984; Matlis and G r e e n b l a t t , 1984; Smith and M c D e r m o t t , 1984; Tarn et al., 1984). Landy and co-workers (1983) have evaluated radially compressed columns and conventional stainless steel columns for reversed phase H P L C . They concluded that the radially compressed columns were good but not necessarily m o r e efficient than a well packed stainless steel column. In general, the columns most commonly used in H P L C are 100-250 m m length stainless steel tubes of 4-5 m m internal diameter. T h e introduction of 3 /xm packing materials has required some modification to the standard column design because of the higher back pressures produced. With these materials shorter columns (30-100 m m length) are normally used (c.f. Section 4.2). T h e particular case of narrower bore columns, either microbore packed columns or capillary columns, is discussed in Section 4.6.

4.1.2

Column end fittings

Columns require a fitting at either end to retain the packing material. T h e r e are two types which are in general use: either a fine porosity frit or a stainless steel gauze. These can be fitted either into the column itself or into the coupling which completes the end fitting. T h e possible arrangements are illustrated in Fig. 4 . 1 . Frits are more readily blocked than gauzes and are also m o r e difficult to clean. In particular, frits inserted into the column cannot be removed easily and a blocked outlet frit has to be drilled out, causing considerable damage to the packed column bed. Fixed frits at the inlet end are also not r e c o m m e n d e d ; since again they cannot be easily removed to repair or top-up the column bed when efficiency loss is observed. W h e n syringe injection is to be used the injection should not be directly into the packed bed, since this causes disturbance of the packing after the first few injections. Instead, the packing should be protected by a gauze t o p p e d with a layer of glass beads or other suitable non-dispersive medium and the injection m a d e at a point just above the gauze. With valve injection the outlet from the valve (whether or not a curtain flow arrangement is used, see Section 3.2) should be just above the gauze. Many columns use end fittings which are based on commercial couplings (e.g. Fig. A.la and b). These commercial fittings need to be drilled out to eliminate dead volumes and once the coupling has been tightened against the tubing their position is fixed and the nuts are not usually interchangeable. This can be

56

H I G H P E R F O R M A N C E LIQUID C H R O M A T O G R A P H Y

disadvantageous, since after tightening and re-tightening several times the threads on the nuts may strip and cause the coupling to leak, rendering the column useless. For this reason great care must be t a k e n , when using this type of fitting, not to overtighten the coupling. It is much easier to use column designs of the type shown in Fig. 4.1c and d, which use hand-tightened nuts. These can be re-used over and over again with no problems. A n o t h e r commercial column design, first introduced by Brownlee Laboratories Inc., consists of a column holder fitted with a replaceable packed cartridge. T h e pre-packed cartridges are fitted with fixed frits at either e n d , and therefore can be neither repaired nor re-packed when efficiency is lost, but they are less expensive than other commercial pre-packed column systems and are becoming very popular.

^Seal - P T F E ring "Glass beads

j-Stainless steel I gauze

gauze

(a) Fig. 4.1.

4.2 4.2.1

(b)

(c)

(d)

Outline designs for columns showing various inlet and outlet fittings.

Packing materials Efficiency

and capacity of micr op articulate

materials

M o d e r n H P L C is performed on microparticulate materials usually of silica, or silica b o n d e d with an organic moiety. Alumina is occasionally used. T h e chromatographic silica gels generally consist of fused aggregates of spherical particles of colloidal silica having a surface area between 100 and 500 m 2 . g _ 1. (Her, 1979; U n g e r , 1979). P o r e sizes vary, usually in the range 5-10 n m ,

T H E COLUMN

57

although much wider pores u p to > 1 0 0 n m diameter can also be produced. These wider p o r e silica gels are increasingly being used in size-exclusion chromatography. It is important to avoid the presence of small pores ( < 3 nm diameter) which can trap molecules and produce poor p e a k shapes. T h e microparticles may be spherical or irregular in shape, depending on their method of preparation. It was thought that spherical particles should produce m o r e efficiently packed columns than irregular particles, but until recently there was little experimental evidence to support this (Laird et al., 1974; U n g e r et al., 1978; O h m a c h t and Halasz, 1981). W o r k with smaller particle sizes (2-5 /xm) does, however, indicate that spherical materials in this size range may indeed be m o r e efficient (Verzele et al., 1982; Verzele, 1984). It also appears that the permeability of columns packed with spherical particles may be better (Endele et al. 1974; U n g e r et al. 1978). T h e most important feature of the material, rather than the particle shape, is the range of particle sizes present in the batch. In particular the cut-off at the lower end of the range is critical, since the presence of fines can cause high back pressures and may result in blockages in the frit or gauze at the outlet end of the column with a concomitant reduction in flow rate (Dewaele and Verzele, 1983a). Column packing procedures have improved significantly over the years and it is now possible to efficiently pack small particle sizes of both adsorbents and b o n d e d phases (see Section 4.3). T h e most commonly used particle sizes are 10 and 5 /xm but 3 /xm materials are becoming increasingly popular. C h r o m a tography on these smaller particles is theoretically m o r e efficient but the back pressure generated is higher, leading to the use of shorter columns and faster analysis times (DiCesare et al., 1981a, b ; Katz and Scott, 1982b; D o n g and G a n t , 1984). T h e greater efficiency of the 3 /xm columns is illustrated in Table 4.1 by comparison of the column lengths required to produce 10 000 plates for 10 /xm, 5 /xm, and 3 /xm materials. T h e corresponding dispersion values are also given. A n o t h e r advantage of these materials is that the slope of the plate height curve is less (i.e. the V a n D e e m t e r C term decreases as particle size decreases) (Katz and Scott, 1982b; D e w a e l e and Verzele, 1983b; D o n g and G a n t , 1984). This is illustrated in Fig. 4.2. A s a result, higher flow rates can be used without great loss of efficiency. T h e 3 /xm materials packed in short columns ( 3 0 100 m m ) a r e , therefore, ideal for very fast analyses as exemplified in Fig. 4.3 by the resolution of several polycyclic aromatic hydrocarbons in 1 min. T h e major problem associated with columns packed with 3 /xm materials is the effect of extra-column band broadening. Peaks eluting from short 3 /xm columns can have a volume of the order of 30-50 /xl, whereas most current liquid chromatographs have instrumental bandwidths of 75-150/xl (DiCesare et al., 1981a). T h e instrumental bandwidth of a system can be measured by replacing the column with a zero dead volume union and recording the peak base width of a small volume injection. This is a measure of the dispersion produced by the injector, connecting tubing and the detector flow cell. T h e contribution of each component in the system to the overall dispersion has been studied (Kirkland et al., 1977; DiCesare et al., 1981b; Scott and Simpson, 1982; Wright et al., 1982)

58

H I G H P E R F O R M A N C E LIQUID C H R O M A T O G R A P H Y

and in general the most important factor is the flow cell. Typical instrument characteristics necessary for high speed L C using short columns have been proposed by D o n g and G a n t (1984) L C system: instrumental bandwidth < 2 0 /xl P u m p : pulse-free, low delay volume for fast gradients Injector: low bandwidth, flow bypass to eliminate pressure surges Tubing: short lengths of 0.005-0.007 in i.d. Detector: low bandwidth flow cell, response time < 2 0 0 ms; long path lengths to maintain sensitivity D a t a handling: data acquisition rates > 1 0 pt/s.

HETP( Mm)

Several instrument manufacturers are now producing instruments with fast L C in mind, but unfortunately where suitably modified detectors are not available standard equipment is not generally suitable for the reasons already outlined, and in this situation 5/xm columns are preferable ( C o o k e et al., 1982). O n e compromise which has evolved has been the production of wider short columns which have a similar volume to traditional chromatographic columns. These can be packed with 3 /xm particles and used with conventional equipment (Stout et al., 1983). These columns do not, however, have the advantage of the considerable saving in solvent produced by the narrower short columns.

0

0

2

6

4 Flow rate (ml. min

-1

Fig. 4.2. H vs u plots for (a) 10 (b) 5 fim and (c) 3 packing materials. Reproduced with permission from Dong and Gant, 1984.

8

T H E COLUMN

59

Table 4.1. Particle sizes and column lengths producing 10 000 plates with corresponding dispersion values (D). Conditions: columns of 4.6 mm i.d.; packing material, silica gel with Vm ~ / ml per 100 mm; k' = 10 for last peak of interest. Particle size (fim)

Column length (mm)

Retention volume for k' = 10 (ml)

B* for k' =2 M)

D** (id)

10 5 3 2

250 125 75 50

27.50 13.75 8.25 5.50

300 150 90 60

100 50 30 20

* B is the bandwidth given by B = 4VRV\^N. **D is the dispersion = B/k' + 4VJVN. Reproduced with permission from Verzele, 1984.

8 7

I -J 0

I 9

L 1

Time (min) Fig. 4.3. Separation of polycyclic aromatic hydrocarbons on 2 ^cm-packing material. Column packing, ROSIL-C l s-D (2 /im); column dimensions, 40 x 5 mm i.d.; eluent, acetonitrile/water (3:1); flow rate, 2 m l . m i n - 1; detection, UV at 254 nm. Solutes: 1: toluene; 2: naphthalene; 3: diphenyl; 4: fluorene; 5: phenanthrene; 6: anthracene; 7: fluroanthene; 8: pyrene; 9: tri-phenylene Reproduced with permission from Dewaele and Verzele, 1983(b).

T h e r e has b e e n considerable discussion of t h e dispersion p r o b l e m s associated with viscous heating in these c o l u m n s as a result of t h e flow of solvent t h r o u g h the p a c k e d b e d u n d e r these fast analysis conditions (Halasz et al., 1975; U n g e r

60

HIGH PERFORMANCE LIQUID CHROMATOGRAPHY

et al., 1978; Lin and H o r v a t h , 1981; P o p p e et al., 1981; C o o k e et al., 1982; Katz et al., 1983; P o p p e and K r a a k , 1983b). It would, however, a p p e a r that this effect does not present any major problems u n d e r normal operating conditions of moderate flow rates (2-4 m l . m i n " 1) (DiCesare et al., 1981a; Stout et al., 1983; Dewaele and Verzele, 1983b; D o n g and G a n t , 1984). T h e r e is some evidence that the stability of these small particle columns may be lower due to the dissolution of silica (Dewaele and Verzele, 1983b). This problem can be averted by including a silica saturating pre-column in the system (see Section 4.4.2; A t w o o d et al., 1979). Several applications using fast L C are summarised in Table 4.2. T h e capacity of a chromatographic material is taken to be the a m o u n t of solute which may be applied to the column before a 10 percent loss in efficiency is observed. It has been shown that the plate height is a linear function of sample load above 1 / x g . g - 1 for microparticulate silica adsorbents ( D o n e , 1976). T h e degree of d e p e n d e n c e is a function of both the k' of the solute and the surface area of the adsorbent. T h e capacity of reversed p h a s e , hydrocarbon b o n d e d materials is higher than that of the bare silica gels ( D o n e , 1976; Karch et al., 1976) and the capacity of the material increases with the chain length of the bonded hydrocarbon moiety (Karch et al., 1976). Reduction of particle size, however, reduces the capacity of a material so that for a preparative separation larger size materials may give the best results ( D o n e , 1976). Small sample volumes are o p t i m u m u n d e r analytical conditions but for preparative separations better efficiency is obtained when large volumes of dilute solutions are injected.

4.2.2

Preparation

and characterisation

of bonded

phases

Silica gel has been studied extensively in recent years with a view to understanding its properties as an adsorbent and as a starting material for the preparation of b o n d e d chromatographic materials (Her, 1979; U n g e r , 1979). The silica surface is covered with silanol groups which may be isolated or hydrogen b o n d e d to an adjacent group. T h e n u m b e r of these groups and their reactivity and physical accessibility depends on the m o d e of preparation of the silica. Usually there will be water associated with these hydroxy 1 groups and the surface is activated by heating between 150 and 200 °C to remove this physisorbed water. Heating above 200 °C can eliminate water between adjacent hydrogen b o n d e d silanol groups to produce a siloxane group which is much less reactive. In general, chromatographic silicas contain approximately five hydroxyl groups per n m 2 which corresponds to 8-9 / x m o l . m - 2. Several studies have shown that it is only possible to react about half of these groups with even the smallest reactive molecule (Unger et al., 1976; Colin and G u i o c h o n , 1977) and obviously even fewer react with much bulkier molecules. T h e silanol groups

Table 4.2 Applications of fast LC. Solutes

Eluent Iflowrate

Column

Analysis time

Ref.

afi acids in hop extract

40 x 5 mmi.d. 2 /xm RoSilC i 8D

Methanol/water/H 3P0 4 (9:1:0.05) 1 ml.min- 1

2 min

Dewaele and Verzele, 1983(b)

Polycyclic aromatic hydrocarbons

40 x 5 mmi.d. 2 /xm RoSilC i 8D

Acetonitrile/water (3:1) 2 m l . m i n -1

1 min

Dewaele and Verzele, 1983(b)

Leucineenkephalin and metabolites

100 x 4.6 mm i.d. 3 /xm RP-18

Gradient: 15%B to 50%B in 4 min A:0.005 M TBA phosphate buffer/methanol (90:10) B:methanol 1.5 m l . m i n -1

6 min

Mahyetal., 1982

Tricyclic antidepressants

100 x 4.6 mm Acetonitrile / methanol / NH 4OH(5%) (35:35:30) i.d. 3 /xm P-E/HS-3 3.0 m l . m i n -1 silica

Barbiturates

100 x 4.6 mm i.d. 3 /xm P-E/HS3 C 18

Methanol / 0.05 M ( N H 4) 2H P 0 4, pH8.0/ water, (55:20:25) 1.3 m l . m i n -1

3 min

Dong and DiCesare, 1982(a)

Polycyclic aromatic hydrocarbons

100 x 4.6 mm i.d. 3 /xm P-E/HS3 C 18

Gradient:60% acetonitrile / water —> 100% acetonitrile in 5.0 min 3.0 m l . m i n -1

6 min

Dong and DiCesare 1982(b)

Catecholamines, indoleamines and metabolites

3 /xm Ultrasphere ODS or P-E HC18

7 min

LinP.Y.T. et al. 1984

Serum theophylline

125 x 4.6 mm Acetonitrile / phosphate buffer, (9.5:90.5) i.d. 5/xmP.E. HC- 4.5 ml.min- 1/50°C 18

1 min

Kabra and Marton, 1982

Carbamazepine and phenytoin in serum

100 x 4.6 mm Methanol / water (68:32) 1.5 m l . m i n -1 i.d. 3/xmP.E. / H S 3 C 18


30000, using a column bore of 10/xm (Knox and Gilbert, 1979). O p t i m u m diameters between 1 and 3 /xm have been found in theory when

T H E COLUMN

99

limits are placed on the available pressure and analysis time (Jorgenson and G u t h r i e , 1983). H o w e v e r , these very small bores place even greater restrictions on injectors and detectors; detectors being limited very much by sensitivity as well as volume.

o

30 Time (mm)

60

Fig. 4.16. Resolution of polycyclic aromatic hydrocarbons on a packed capillary column. Column packing, 3 ^m ODS-silica; column dimensions, 850 x 0.24 mm i.d.; eluent, acetonitrile/water (85:15)^acetonitrile; initial flow rate, 1.12 /xl-min - 1; detection, UV at 265 nm. Solutes: 1: naphthalene; 2: fluorene; 3: acenaphthene; 4: anthracene; 5: fluoranthene; 6: 9-methylanthracene; 7: pyrene; 8: 9, 10-dimethylanthracene; 9: chrysene; 10: 1-methylpyrene. Reproduced with permission from Karlsson and Novotny, 1984.

O p e n tubular columns require lower operating pressures than packed microcapillaries because of their increased permeability and theoretically are more efficient, provided that optimum injector and detector volumes can be achieved. The capacity of these columns, is, however, much lower. Usually the columns are p r e p a r e d from soda lime glass capillaries which are either etched with acid or alkali in the liquid phase (Tesarik, 1980) or in the gas phase (Krejci et al., 1981). T h e resulting columns can be used directly for adsorption c h r o m a t o g r a p h y (Ishii et al., 1979; Krejci et al., 1980; Kucera and Guiochon, 1984), coated with a stationary phase for partition chromatography

100

H I G H P E R F O R M A N C E LIQUID C H R O M A T O G R A P H Y

CH3CN (%)

(Dolan and Snyder, 1979; Krejci et al., 1980; W a d a et al., 1983; Maskarinec et al., 1983; Takeuchi et al., 1983b), dynamically modified with long chain quaternary a m m o n i u m ions (Takeuchi and Ishii, 1983b; Takeuchi et al., 1983d) or chemically b o n d e d (Tsuda et al., 1978; Takeuchi et al., 1982; Jorgenson and G u t h r i e , 1983; Takeuchi and Ishii, 1983d; Ishii and Takeuchi, 1984). B o n d e d phases are preferred to the liquid stationary phases since the resulting columns are m o r e stable. Originally capillaries of 30-50 /xm bore were used, but as techniques have improved examples of the use of capillaries of 20 /xm bore (Tsuda et al., 1981) and less than 15 /xm bore (Krejci et al., 1981; Tijssen et al., 1981; Jorgenson and G u t h r i e , 1983; Tsuda and Nakagawa, 1983) have been obtained. Injection is usually achieved using some form of splitting device in which most of the sample is discarded. Nevertheless, such devices are effective (Tsuda and Novotny, 1978; Krejci et al., 1980; Jorgenson and G u t h r i e , 1983). A novel injection system which is compatible both with packed and open tubular

4

0 Time (h) Fig. 4.17. Continuous solvent-gradient separation of polynuclear aromatic hydrocarbons on an open tubular microcapillary column. Column, bonded ODS; column dimensions: 22 m x 31 /xm i.d.; eluent gradient from acetonitrile/water (40:60) to acetonitrile/water (70:30), gradient profile as indicated; flow rate, 0.52 /xl.min - 1; column temperature, 36 °C; detection, UV at 250 nm. Solutes: 1: benzene; 2: naphthalene; 3: biphenyl; 4: fluorene; 5: phenanthrene; 6: anthracene; 7: fluoranthene; 8: pyrene; 9: /7-terphenyl; 10: chrysene; 11: 9-phenylanthracene; 12: perylene; 13: 1,3,5-triphenylbenzene; 14: benzo [a] pyrene. Reproduced with permission from Takeuchi and Ishii, 1983(d).

T H E COLUMN

101

microcolumns has been developed by McGuffin and Novotny (1983a). A sample plug is introduced with a conventional valve injector and a 'heart-cut' fraction of the sample is diverted o n t o the microcolumn using sequentially timed valves. If the injection time and flow rates are varied, injection volumes from 1 nl to 1 /xl or m o r e can be delivered. A n o t h e r on-column injection system capable of injecting nanolitre quantities has been described by Tsuda and Nakagawa(1983). It sounds rather complicated for routine use, however. Detection is the principal problem because of the extremely small volumes (a few nanolitres) involved. If possible, on-column detection systems are preferred as these avoid the extra-column band broadening associated with connection to a post-column detector. Such systems have been described for U V absorption with microbore columns, but the capillary diameters have been fairly large (Yang, 1981). With columns of smaller diameter the optical path length will probably be too short for on-column U V absorption detection. T h e capillaries will also need

1 min

8 7

4

5

1

llfl

Fig. 4.18. Microcapillary LC separation of aromatic compounds by straight phase partitioning. Column, etched soft glass, coated with ODPN as stationary phase; column dimensions, 495 m x 12 /xm i.d.; eluent, isooctane (saturated with ODPN at 23 °C); flow rate, —; detection UV at 212 nm. Solutes: Ldiisoheptylphthalate; 2: dibutylphthalate; 3: dipropylphthalate; 4: 2,4,5-trimethylaniline; 5: N-methylaniline; 6: diethyl phthalate; 7: o-chloroaniline; 8: 2,3-dimethylaniline; 9: otoluidine; 10: /?-toluidine; 11: m-toluidine; 12: benzoquinone; 13: dimethyl phthalate; 14: aniline; 15: ra-chloroaniline; 16: p-chloroaniline. Reproduced with permission from Tijssen et al., 1981.

102

HIGH PERFORMANCE LIQUID CHROMATOGRAPHY

to be p r e p a r e d from borosilicate glass or fused silica to provide the necessary optical transparency. On-column fluorescence detectors have in fact been used with open-tubular columns (Guthrie and Jorgenson, 1984; Guthrie et al., 1984). Low cell volume electrochemical detectors (Slais and Krejci, 1982; Manz and Simon, 1983; Knecht et al., 1984) and flame based detectors (McGuffin and Novotny, 1981a, b , 1983b; Gluckman and Novotny, 1984) have also been developed and these may offer more potential. A low-dead-volume laser induced fluorescence detector has also been described (Gluckman et al., 1984). A n interesting development in L C has been the use of electro-osmotic flow in conjunction with open tubular columns. In such systems the eluent flows through the capillary under the influence of an applied electric field rather than being p u m p e d . Since the flow profile of electro-osmotic flow is much flatter than that of laminar flow (Pretorius et al., 1974) it has been suggested that capillary columns of slightly larger bore (i.e. 10 jiim instead of 2 ^ m ) can be used for maximum efficiency (Tsuda et al., 1982b). This is an obvious advantage of the 4

i

i

0

4

i 8 Time (min)

i 12

Fig. 4.19. Separation of phthalates on an open tubular column. Column, bonded y-aminopropylsilane; column dimensions, 8.3 m x 11 fjum i.d.; eluent, upper layer of hexane/acetonitrile/ dimethylsulphoxide (87:10:10); linear velocity, 2.3 c m . s - 1; detection, UV at 254 nm. Solutes: 1: dioctyl; 2: dibutyl; 3: dipropyl; 4: diethyl; 5: dimethyl phthalate. Reproduced with permission from Tsuda and Nakagawa, 1983.

T H E COLUMN

103

technique. H o w e v e r , Martin and Guiochon (1984) have concluded, that good performances will be obtained only if very large voltages (20-100 kV) can be used. A n o t h e r area of considerable interest recently is the use of capillaries in supercritical fluid chromatography. This technique is discussed in m o r e detail in Chapter 10. Capillary columns are not yet commercially available and they obviously require special handling and expertise. Although optimum conditions have not yet been achieved, the results obtained so far are very promising and advances are continuing to be m a d e . Fig. 4.17 illustrates the gradient separation of polynuclear aromatic hydrocarbons on a 22 m x 31 i.d. O D S - b o n d e d capillary. T h e uses of smaller bore capillaries are exemplified in Figs. 4.18 and 4.19. Fig. 4.18 shows the separation of several aromatic c o m p o u n d s by normal phase L-L partition chromatography on a 12 /xm capillary coated with /3/3'-oxydipropionitrile. T h e resolution of several phthalates on a y-aminopropyl silane column ( 8 . 3 m x l l / x m i.d.) is shown in Fig. 4.19. The efficiency measured for peak 5 (dimethylphthalate) is 50000 plates, corresponding to a reduced plate height of ~ 3.

Chapter S

Sample

treatment

Before samples can be analysed by H P L C they must be dissolved in a suitable solvent which is miscible and preferably similar to the elution system chosen. It is very important that the samples contain neither particulate matter which may clog frits, nor any components which might precipitate on contact with the eluent causing damage to the top of the column. Sometimes just the presence of a wide range of solute polarities can result in a build-up of non-eluted material at the top of the column and ultimately, reduced efficiency. Other problems may result from the components of interest being present as trace constituents in a complex matrix. In such a situation, purification and enrichment of the trace solutes is required. Alternatively, the sample may exhibit poor detection characteristics and require derivatisation prior to analysis. Derivatives may be formed either pre- or post-column. Pre-column derivatisation will alter the polarity of the solutes and hence their elution properties. Post-column reaction detectors present their own particular problems associated with maximising the reaction conditions while minimising band-dispersion. Normally some form of sample p r e t r e a t m e n t is required prior to L C analysis. T h e analyst likes to keep this treatment as simple and minimal as possible and whenever feasible automation of multi-sample preparation will be used. 104

SAMPLE T R E A T M E N T

5.1 5.1.1

105

Clean-up procedures Solvent

extraction

The commonest approach to solute enrichment prior to analysis is by solvent extraction. This technique may be used for removing soluble components from an insoluble matrix either by simply shaking with a solvent or by refluxing with the solvent in a soxhlet extraction procedure when a more exhaustive process is required. Further purification of complex samples may be achieved by partitioning between aqueous and organic phases. Additional steps may involve p H changes and back extraction techniques to separate ionic species. These are all standard analytical procedures. Finally the isolated extract is dried and concentrated by solvent evaporation prior to analysis. When the final extraction solvent is incompatible with the L C system, e.g. a hexane extract of a mixture to be analysed by reversed phase chromatography, complete solvent removal should be performed. T h e last of the solvent is normally carefully evaporated under a stream of dry nitrogen (care must be taken to avoid loss of volatile components) and the sample reconstituted in a more appropriate solvent. Ideally the eluent or an eluent c o m p o n e n t should be used but if this is not possible a weaker solvent must be used to avoid solute dispersion on injection. All samples should be filtered through a low porosity (0.2-0.5 /xm) filter prior to injection. Microfiltration units which can be used with syringes to filter small volume samples are commercially available (Millipore C o r p . ) .

5.1.2

Solid phase

extraction

A n o t h e r approach to the isolation of c o m p o u n d s from solution is the selective adsorption of the components of interest on a suitable solid matrix. This is best achieved by packing the adsorbent in a column and passing through the sample solution followed by several column volumes of eluent to remove unwanted material. A change in the eluent polarity, e.g. from water to methanol will then cause the components of interest to elute. T h e sample again usually requires concentration prior to analysis. This type of system may involve large classical L C columns packed with XAD-resins or silica to process litres of solvent. Alternatively, micro-scale extractions may be performed using small columns packed with silica or b o n d e d silica. Several small volume, disposable solid phase extraction systems are now commercially available (e.g. Sep Pak-Waters Assoc.; Baker-10 SPE System; Prep Sep-Fisher Scientific; Extrelut-Merck; Clin Elut-Analytichem International). These disposable columns are generally packed with 40 /xm material of high capacity and incorporate a sample reservoir above the packed bed. Solvents and samples are aspirated through the columns via syringes or vacuum manifolds.

106

HIGH PERFORMANCE LIQUID C H R O M A T O G R A P H Y

The use of a low-pressure multi-position valve to aid sample and solvent introduction is illustrated in Fig. 5.1.

Luer tip syringe

Top port (O) female luer/ fitting Detachable mounting arm

Side ports ( A B, C and D) male leur fittings

Bottom port (1) Universal S P E column adaptor

Baker-10 S P E ' 1column 1, 3 or 6 ml

Fig. 5.1. A low pressure multi-position valve designed for use with a sample clean up column. The side ports (A, B, C, D) will introduce conditioning, sample, wash or eluting solutions into the syringe or exit wastes from the syringe by positioning the indicating valve to the selected port. Reproduced with permission from Baker Chemical Co.

Solid phase extraction techniques are based on the processes used in liquid chromatography and the full range from liquid-solid adsorption to reversed phase or ion exchange may be used to selectively isolate the compounds of interest. Most commercial systems supply a complete range of packing materials. The type of system which is suitable for the isolation of a particular sample type depends on the solubility and ionic character of the sample. A typical scheme for selection of extraction conditions is illustrated in Fig. 5.2.

SAMPLE TREATMENT Separation Extraction mechanism' column' Polar ( m e t h a n o l , a c e t o n i t r i l e a n d e t h y l acet a t e soluble)

NPC

Dyoi(C lanO HN CO ) o(C )HH 777000892481 aC m i n o ( N ) 1 ,2 -Amino (NHN / H>7089

107

Elution solvent

Hhelxoaronfeorm C A Mceetthoanneol Hhexloaronfeorm C E theythlanacet 7086 M ol ate Silica gel Occtatydl(eC cy)l (C) 77008270 A Hecxetaonnee O H ) P h e n y l ( C 7 0 9 5 A Cyano (CN) 7021 W Mceaetthetorannoitrlile A nio-A (Nm Hin)o (NHN phosN phA ateHPb0uffers 1m ,2m /idH)770089780089A 0-0q1u-e0o-u1s M A r o a t i c s u l p h o n i c a c (CuH Q atS ern0aH ry)amine(N ) 7091 Dyoi(C lanO HN CO ) 77009241 Hexane C oo(C )HH Chcleotoronfoerm ) A m n i ( N 1"2,"-Amino(NH/NH) 70780988 A Methanol H helxyoarlonfeoacet rm Silica gel 7086 C E t h Methanol ate O yl) (C) 77002807 A Hecxetaonnee O ccettyandlyel(cC H ) 7 0 9 5 P h ( C A Cyano(CN) 7021 W Mceaetthetorannoitrlile aquaetoourss(t1h-8ioNuH A niom(N Sotrw onpH g chel reC a)I 1"2,m "-A inoH (N)H/NH) 70780988 L ?

2

Organic p- s o l v e n t soluble

• M o d e r a t e l y polar Non-polar c o m p o u n d s L(from aqueous solutions and hexane, heptane and chloroform soluble

Organic samples — • MW 2000

Organic

Size exclusion/ gel permeation

Fig. 6.1. Flow diagram for selection of liquid chromatographic conditions.

Snyder (1978) has classified the commonly used solvents according to their polarity ( P ' ) , which is an approximate measure of solvent strength, and their selectivity (x) based on their ability to interact as proton d o n o r s , proton acceptors or dipoles. Solvent strength P' and selectivity (x) values for solvents commonly used in liquid chromatography are listed in Table 6 . 1 . A plot of solvent selectivity using trilinear coordinates of a n d (%n) produces a solvent selectivity triangle (Fig. 6.2) (Snyder, 1978). Solvents at t h e apices of the

SEPARATION M E T H O D S

127

Table 6.1. Suggested solvents for LC. P' = Solvent polarity; xe = proton acceptor contribution; xd = proton donor contribution; xn = strong dipole contribution. xe + xd + xn = 1.00. Reproduced with permission from Glajch et al., 1980. P'

Normal phase Ethyl ether** Chloroform Methylene chloride rc-Hexane (carrier) Reversed phase Methanol Acetonitrile Tetrahydrofuran Water (carrier)

Xn

Solvent* group

2.8 4.1 3.1 0.1

0.53 0.25 0.29 -

0.13 0.41 0.18 -

0.34 0.33 0.53 -

I VIII V -

5.1 5.8 4.0 10.2

0.48 0.31 0.38 -

0.22 0.27 0.20 -

0.31 0.42 0.42 -

II VIb III -

*Solvent classification group, see Snyder, 1978. **More practical alternative: methyl tert-butyl ether. Proton Acceptor

Proton donor

Dipole interaction

Xn —

Fig. 6.2. Selectivity triangles for preferred solvents in reversed-phase and normal phase chromatography, reversed phase; , normal phase. Reproduced with permission from Glajch et al., 1980.

selectivity triangle will p r o d u c e t h e greatest differences in selectivity. C h r o m a t o g r a p h i c selectivity may also be varied by t h e addition of c o m p o n e n t s to the eluent such as ion pairing agents ( w h e n t h e solutes a r e ionisable) o r complexing

agents specific

for

some

of t h e solutes. Variations of p H

or

t e m p e r a t u r e may also effect selectivity changes. Finally, if o p t i m u m resolution can

still not

be

obtained,

the

selectivity

can

be

column-packing material ( A n t l e and Snyder, 1984).

varied

by changing

the

128

HIGH PERFORMANCE LIQUID CHROMATOGRAPHY

The final component in the resolution equation which should be optimised is the column plate n u m b e r (N). This can be achieved using basic chromatographic theory (see Chapter 2) by varying particle size, column length, column bore, and flow rate (Knox, 1978, plO; Katz et al., 1984; Snyder and A n t l e , 1985). 6.1.1

Mobile phase

optimisation

Eluent conditions are frequently chosen on a simple trial and error basis by using some knowledge of chromatographic variations caused by adjusting solvent strength. This procedure can, however, be very time consuming and predictions become more difficult when ternary or quaternary solvent systems are used. Gradient pumping systems can be used to rapidly scout eluent compositions (Schoenmakers et al., 1981) and simple graphic procedures can be used to determine the optimum composition (Issaq et al., 1983). These methods are generally limited to binary mobile phases. Fortunately, the development of computer technology has advanced to the stage that the optimisation of several simultaneous variables is now possible. T h e optimisation can often be achieved simply with the use of a programmable calculator (Gant et al., 1979; Berridge, 1980). The advent of microprocessor controlled L C systems makes the generation of data less operator time consuming and allows the whole optimisation scheme to be a u t o m a t e d (Berridge, 1982, 1984). Solute retention data for the range of mobile phases to be examined by the computer may be obtained in two distinct ways (Glajch and Kirkland, 1983; D'Agostino et al., 1985). T h e first approach, 'mixture design', involves the measurement of solute retention times for a limited n u m b e r of mobile phase compositions. These data are used as a basis for the theoretical generation of further data for other mobile phase compositions using known chromatographic relationships. The 'factorial design' m e t h o d involves measurement of retention data for a relatively large n u m b e r of eluent compositions covering the range of mobile phases to be searched. Further data points are obtained by extrapolation of the measured values rather than from theory. T h e main practical difference between the two methods is the n u m b e r of experimental data points which are needed to perform the optimisation. Many examples of computer optimisation schemes have been published and a number of these are summarised in Table 6.2. H a d d a d et al. (1983) have used a mixture design approach to optimise the p H and organic modifier content of the mobile phase for the separation of some closely related aromatic acids. Their method is based on a resolution criterion:

r = n** ,./[(2V,.)/(« - !>]" /•=1

+

/=1

1

i

where r is the resolution criterion and Rs , is the resolution factor for the adjacent peaks i and i + 1. This criterion reaches its maximum value when all Rs values are equal, i.e. when all peaks are distributed evenly over the chromatogram. This may not necessarily provide the most satisfactory chro-

SEPARATION METHODS

129

m a t o g r a m however, since the latter is o b t a i n e d by sequentially optimising t h e peak separation and t h e analysis t i m e . A value of Rs, min ^ 2 (the value of Rs for the least resolved p e a k pair in the c h r o m a t o g r a m ) is used to locate the o p t i m u m mobile phase compositions which are subsequently searched to provide the fastest analysis time. T h e optimisation scheme is illustrated in Fig. 6.3 for the organic modifiers m e t h a n o l , acetonitrile and tetrahydrofuran.

(a)

(b)

F

5-80

(c)

Fig. 6.3. Combined optimisation of mobile phase pH and organic modifier content by means of a sequential resolution-time procedure. Each three-dimensional surface illustrates the variation of \lt (where t is the retention time of the most retained peak). A value of Rs, min =sS2 was used. Chromatograms obtained with the selected optimal mobile phases (indicated by • ) are also shown. The organic modifiers used were (a) methanol, 0-40.0%; (b) acetonitrile, 0-28.0%; (c) tetrahydrofuran, 0-26.4%. Column packing, ODS-Hypersil (5 fxm); column dimensions 150 x 4.6 mm i.d.; detection, UV at 254 nm. Solutes: 1: 1,4-benzenedicarboxylic acid; 2: 4-aminobenzoic acid; 3: 4-hydrobenzoic acid; 4: 2-aminobenzoic acid; 5: benzoic acid. Reproduced with permission from Haddad et al., 1983.

Table 6.2.

Computer methods for the optimisation of liquid chromatographic separation.

Parameters optimised (a) Binary mobile phase composition and flow rate (b) Gradient elution profiles (c) Ternary mobile phase composition

Mode of chromatography RP

Optimisation scheme Mixture design

Solutes (a) 2-Substituted pyridines

Reference Berridge, 1982

(b) Phenolic antioxidants (c) Substituted phenols

Ternary mobile phase composition, flow rate and temperature

RP-ion pair

Mixture design

Concentration of ion pairing agent

RP-ion pair

Mixture design

Column design, temperature, solvent flow rate and composition

NPand RP

Mixture design

Phenothiazines

Bounine et al., 1984

Quaternary mobile phase composition

RP

Mixture design

Polar adrenal steroids

D'Agostino et al., 1984

Mobile phase pH

RP

Factorial design

Weak organic acids

Deming and Turoff, 1978

Mobile phase composition

RP

Mixture design

Substituted naphthalenes

Glajch et al., 1980

Mobile phase composition

NP

Mixture design

Substituted naphthalenes

Glajch et al., 1982a

Mobile and stationary phase composition

RP

Mixture design

Phenylthiohydantoin amino acids

Glajch et al., 1985

pH and ion pair reagent concentration

RP-ion pair

Mixture design

Cough/cold remedy and antidepressants

Goldberg and Nowakowska, 1984

pH

RP-ion pair

Mixture design

Basic samples

Goldberg et al., 1984

Ion chromatography

Mixture design

Inorganic anions

Haddad and Cowie, 1984

Mobile phase composition and pH

Organic bases and aromatic and heterocyclic acids

Berridge, 1984 Billietet al., 1984

Table 6.2 (contd.) Parameters optimised Mobile phase composition and pH

Mode of chromatography RP

Optimisation scheme Mixture design

NP

Solutes

Reference

Aromatic acids

Haddad et al., 1983

Carotenoids

Kester and Thompson, 1984

Multisolvent gradient elution

RP

Mixture design

Test samples which gave k' range ~ 100

Kirkland and Glajch, 1983

pH and ion pair concentration

RP-ion pair

Factorial design

Aromatic acids

Konget al., 1980

Mobile phase composition

NP

Mixture design

Isomers of retinol

Landers and Olson, 1984

Mobile phase composition, pH, elution strength and ionic strength

RP

Factorial design

Dibasic acids and bases, amino acid, dipeptides

Otto and Wegscheider, 1983

Mobile phase composition, pH and temperature

RP

Mixture design

Aromatic carboxylic acids

Patel et al., 1984

Mobile phase composition, temperature

RP

Factorial design

Nitroaromatics and flavone derivatives

Rafel, 1983

Flow, initial methanol concentration, concentration gradient and initial isocratic time

RP

Mixture design

2,4-Dinitrophenyl hydrazones

Sabate et al., 1983

pH and ion pair concentration

RP-ion pair

Factorial design

Aromatic acids

Sachok et al., 1980

Mobile phase composition and ion pair agent concentration

RP-ion pair

Factorial design

2,6—Di-substituted anilines

Sachok et al., 1981

RP

Mixture design

Saccharin, caffeine and benzoic acid

Weyland et al.,1982

Time and resolution

NP = normal phase chromatography; RP = reversed phase chromatography.

132

HIGH PERFORMANCE LIQUID CHROMATOGRAPHY

6.1.2

Optimisation

of column

selectivity

Changes in mobile phase composition normally produce larger effects on selectivity than can be obtained by changing the column packing material (but not the chromatographic m o d e ) . If, however, mobile phase optimisation proves inadequate it is then prudent to consider the use of alternative packing materials. Antle and Snyder (1984) have described a procedure for comparing column selectivity in reversed phase chromatography. A similar approach could also be applied to normal phase sytems. These workers defined a column strength p a r a m e t e r , / , based on the retention of samples on the test column relative to a standard C 8 column. In reversed phase chromatography column strength is generally found to increase with an increase in the amount and the surface area of the b o n d e d phase, and decrease with the introduction of polar functional groups. The solute capacity factor can, therefore, be obtained from log/;' = l o g K e + /

(6.2)

where Ke is an equilibrium distribution constant, and J is defined as the column

Table 6.3. Selectivity characteristics of various reversed-phase columns derived from Zorbax silica. Reproduced with permission from Antle and Snyder, 1984. Column

Source*

Group**

Pore size (mm)

Surface area of silica (m>.g-i)

Column strength f J %-org

Polarity •

(Pi)

Zorbax ODS

DP

-Ci8

1

330

0.26

+1

-0.55

Zorbax C18

DP

- c 8

6

330

0.00

(0)

0.00

res

—Ci8

15

128

0.04

Zorbax 150-C8

DP

- c 8

17

140

-0.32

-4

0.00

Zorbax C18

res

-Cis

30

45

-0.38

Zorbax Phenyl

DP

—C2—C 6H 5

9

330

-0.54

-6

1.24

Zorbax TMS

DP

- Q

8

330

-1.08

-13

0.96

Zorbax CN

DP

—C 3—CN

9

330

-1.64

-20

1.00

Zorbax c 8

*DP refers to commercial column sold by E. I. du Pont de Nemours & Co., res refers to a research packing. **Group refers to R in the silane structure —Si(CH3)2—R. tJ is the column strength parameter defined by Equation 6.2 in the text; %-org refers to change in mobile phase (%v acetonitrile) required to keep k' constant for average test solute.

SEPARATION M E T H O D S

133

strength p a r a m e t e r which combines the effects of the stationary phase volume, surface area and polarity. Columns with larger / values give larger k' values. Values of / for several b o n d e d phases are given in Table 6.3. Different selectivities may be obtained by using b o n d e d phases with the same group bonded on silicas of different surface areas, or by using packings containing different bonded groups (kf being held in the same range). These effects are illustrated in Figs. 6.4 and 6.5.

Absorbance (254 nm)

(a)

Time (min)

Absorbance (254 nm)

ib)

0

2

4 6 Time (min) Fig. 6.4. Changes in band spacing as a result of changes in column strength brought about by varying the packing surface area (k' range held constant). Column, (a) Zorbax C 8, (b) Zorbax 150-C8; eluent, (a) acetonitrile/water, (38:62), (b) acetonitrile/water (30:70); flow rate, 2.5 ml.min- 1; temperature, 50 °C; detection, UV at 254 nm. Solutes: 1: cortisone; 2: dexamethasone; 3: corticosterone; 4: orthonitrophenol; 5: fluorobenzene. Reproduced with permission from Antle and Snyder, 1984.

HIGH PERFORMANCE LIQUID (3

CHROMATOGRAPHY

Absorbance (254 nm)

134

(b

Absorbance (254 nm)

Time (min)

(c)

Absorbance (254 nm)

Time (min)

< I

i

i

0

2

1

1

x

4 6 8 Time (min) Fig. 6.5. Changes in band spacing as a result of changes in column strength brought about by varying bonded-phase functionality^' range held constant). Columns and eluents, (a) Zorbax ODS, acetonitrile/water (36:64); (b) Zorbax Phenyl, acetonitrile/water (32:68); and (c) Zorbax CN, acetonitrile/water (20:80); conditions and solutes as in Fig, 6.4. Reproduced with permission from Antle and Snyder, 1984.

6.1.3

Optimisation

ofN

Optimisation of N involves achieving the maximum n u m b e r of theoretical plates, in a minimum analysis time, within the pressure limitation of the equipment. A key equation in this respect is (6.3) where A P is the pressure drop across the column, 0' is the column resistance parameter, r] is the eluent viscosity, L is the column length, dp the particle size and t0 the retention time of an unretained solute. Maximising N then becomes a case of choosing the best column configuration (L and bore (dc)) packed with the right particles (dp) to give a minimum analysis

SEPARATION M E T H O D S

135

time (t0) within the pressure limitation ( A P ) . Schemes have been presented to optimise these variables assuming that column lengths and particle sizes are continuously variable (Katz et al., 1984; Bounine et al., 1984; Mellor, 1982). Perhaps it is m o r e realistic to place some restraints on the column dimensions based on configurations which are commonly available (Snyder and A n t l e , 1985). 6.2 6.2.1

Normal phase chromatography Adsorption

chromatography

on silica or alumina

Adsorption chromatography was introduced by Tswett (1903) and Day (1903). The classical technique was performed in large bore glass columns packed with large particles usually of silica or alumina, and eluted under gravity feed. This procedure changed little until the late 1960s with the introduction of pressurised systems in conjunction with the use of microparticulate materials, which led to vast improvements in the efficiency and the speed of analysis. T h e separation is based on the selective adsorption of solutes on the active sites on the surface of the adsorbent. T h e most commonly used adsorbent is silica, although much of the early experimental data on the thermodynamics of the adsorption process were based on work on alumina (Snyder, 1968). Fortunately both adsorbents are similar in behaviour and can be treated in the same m a n n e r .

S E P A R A T I O N MECHANISM

The active adsorption sites on silica are hydroxyl groups. In the a m o r p h o u s silicas which are used for chromatography the surface is covered with both isolated and vicinal hydroxyls which are capable of hydrogen bonding with each other (Her, 1979; U n g e r , 1979). The n u m b e r of hydrogen bonded hydroxyls present depends on the pore size of the material and on the heat treatment it has received. In particular, narrow pore silicas contain m o r e hydrogen-bonded hydroxyls. Heating the silica above 200 °C causes loss of water between the hydrogen bonded groups, resulting in the production of siloxane groups which are weaker adsorption sites. This reaction is reversible provided it takes place between two adjacent hydroxyl groups on the same surface. In a narrow pore silica, however, it is possible for two hydroxyl groups on adjacent surfaces to eliminate water to produce a siloxane. This reaction is irreversible and results in loss of surface area. Thermal treatment of silica above 600 °C results in sintering and loss of surface area. Activation of the silica surface is achieved by heating the material above 100 °C (but not usually in excess of 200 °C) to remove adsorbed water. The activity of the surface can then be controlled by the addition of known amounts of water or polar organic solvents to the eluent (Engelhardt, 1977). The active sites on the surface adsorb solvent molecules and adsorption chromatography takes place by the selective displacement of these

136

HIGH PERFORMANCE LIQUID CHROMATOGRAPHY

solvent molecules by solute molecules. Solute adsorption is determined by the number and type of functional groups present (Snyder, 1968), with aliphatic compounds being nonadsorbed and alcohols, phenols, amines and other polar compounds being strongly adsorbed. Solutes with strongly adsorbed functional groups will require stronger solvents for elution. Snyder (1968) has examined the thermodynamics of the adsorption process and his treatment produces a good description of the behaviour of simple solutes in adsorption systems using non-polar eluents. W h e n a packed column is conditioned with an eluent, solvent molecules, 5, are adsorbed on the surface forming a monolayer. W h e n a solute, X, is introduced competitive adsorption takes place between X and S and the active surface sites X + nSad ^± Z a d + nS where the energy of postulated properties

(6.4)

subscript 'ad' refers to an adsorbed species. Assuming that the net adsorption is greater than the energy of solution, Snyder (1968) an equation that relates the sample adsorption coefficient, Kads, to of the adsorbent, the solute and the eluent logio*** = logioV, + /3(S° - Ase°)

(6.5)

Kads has been defined in Chapter 2 (Equation 2.9) and is the ratio of the moles of solute adsorbed per gram of adsorbent to the moles of solute per ml of eluent. Va is the volume of an adsorbed monolayer of solvent per gram of adsorbent and (3 the surface activity of the adsorbent which is given the value of unity for a thermally activated adsorbent. Va and /? are properties of the adsorbent which control retention and are constant irrespective of solute or eluent. For an adsorbent that is completely free of adsorbed water, Va attains a maximum of VT ax = 0 . 0 0 0 3 5 . 5 ^ ,

(6.6)

where SBet is the specific surface area of the adsorbent. The factor 0.00035 corresponds to the average thickness of a monolayer of adsorbed solvent molecules. Va is reduced when water or some other polar compound is used to deactivate the surface, since these polar molecules are preferentially adsorbed on the surface in place of the adsorbed non-polar solvent molecules. Assuming that a volume of water replaces an equal volume of adsorbed solvent, the following relationship is obtained Va = 0.000 35SBet

- (ml water added per g)

(6.7)

The linear relationship between Va and the amount of surface coverage by water has been established (Snyder, 1968).

SEPARATION METHODS

137

T h e value of the dimensionless quantity /? d e p e n d s on the types of hydroxyl groups on the silica surface and on their reactivity. It is also affected by the specific surface area and the p o r e structure. T h e surface activity reaches a maximum value for a completely hydroxylated surface and it is considerably decreased by the adsorption of water, which occurs preferentially at reactive surface hydroxyl groups. Using a series of standard solutes, with a given adsorbent of constant water content, /? can be derived from the slope of the plot of k' versus (S° - Ase°) (Snyder, 1968). For adsorbents that have been sufficiently deactivated to be chromatographically useful, /3 is in the range 0.5-0.9. The factor in brackets (S° -Ase°) summarises the relevant properties of the eluent and the solute. S° is the relative adsorption energy of the solute interacting with the surface and As is the molecular surface area of the solute molecule. This is expressed in units of 0.085 n m 2 corresponding to \ th the area of an adsorbed benzene molecule or effectively the area of a 'single' aromatic carbon atom. e° is the relative adsorption energy of the eluent covering 0.0085 n m 2. T h u s As£ is the 'energy of adsorption' of the quantity of eluent required to cover the same area as the solute molecule. T h e quantities S° and As can be calculated as shown by Snyder (1968). e° represents the effect of solvent type on retention for a given adsorbent and solute and is therefore called the solvent strength. By convention e° is taken as zero for p e n t a n e . Values of e° for most other solvents are then positive and conveniently fall in the range 0 - 1 . Values are tabulated for single solvents as an eluotropic series, as shown in Table 6.4.

Table 6.4. Elutropic series for common solvents in adsorption chromatography. Reproduced with permission from Snyder and Kirkland, 1979. Solvent 3-M fluorochemical FC-78 /t-Pentane rt-Hexane j ^-Heptane / Isooctane JJ 1-Chlorobutane Chloroform Dichloromethane Isopropyl ether Ethyl acetate Tetrahydrofuran Propylamine Acetonitrile Methanol

Solvent strength e° Silica Alumina -0.2 0.00

0.25 0.00

0.01

0.01

0.20 0.26 0.32 0.34 0.38 0.44 -0.5 0.50 -0.7

0.26 0.40 0.42 0.28 0.58 0.57 0.65 0.95

138

Since

HIGH PERFORMANCE LIQUID CHROMATOGRAPHY

*'

-

Kads

(c.f. E q u a t i o n 2.9)

(6.8)

where W is the weight of adsorbent in the column and VM is the volume of eluent in the column, it follows from E q u a t i o n 6.5. that j8(S» - A S * > )

login*' = login

(6.9)

VAW is the volume of solvent forming a monolayer coverage on the total active adsorbent surface within the column and hence may be equated to VS, the volume of stationary phase in the column. Thus j8(S° -

l o g ^ = login)

Ass°)

Comparison with E q u a t i o n 2.6 indicates that the distribution between eluent and stationary (or surface) phase is given by log 1 0Z> = P(S° - A S * > )

(6.10) coefficient

(6.11)

Using Equation 6.10 it is possible to standardise an adsorption system using standard solutes of known S° and As with an eluent of known s°. Equation 6.10 leads to an expression for the variation of k' with mobile phase composition l o g i o ( * V * ' 2) = PM*2

'

£i)

(6-12)

where k\ and k'2 are k' values for a given solute X, using mobile phases 1 and 2, which can be pure solvents, or mixtures of two or m o r e solvents. In the case of mobile phases which are mixtures, this model allows the calculation of their combined solvent strength, e°, as a function of the chromatographic properties of the individual solvents. T h u s , for a mobile phase A + B, where solvent B is m o r e polar than solvent A and eB > eA (6.13)

£° = eA

Ke is the equilibrium constant for the displacement of adsorbed molecules A by molecules B, and is given by: K=10

-

P a t i e e t A)

(6.14)

NB is the mole fraction of solvent B in the mobile phase, and nb is the cross-sectional area of a molecule B. E q u a t i o n 6.13 can be generalised for

SEPARATION M E T H O D S

139

mobile phases which contain any n u m b e r of c o m p o n e n t solvents (Glajch and Snyder, 1981) E? = Ea + \og(NA/0A)/l3nb

(6.15)

H e r e , NA refers to the mole fraction of the weakest solvent A in the mixture (smallest e° value), and 6A is the fraction of the surface covered by molecules A. For a binary solvent mobile phase, NA = (1 - NB), and 0A = (1 - 6B), where 6B is the fraction of surface covered by molecules B. T h e fractional surface coverage by B is eB = NBK/(NA

+ NBK)

(6.16)

Equation 6.16 applies only to binary solvent mobile phases. Values of 6A which can be used in Equation 6.15 for mobile phases containing three or more solvents can also be calculated (Glajch and Snyder, 1981). As is apparent from Equation 6.13 e° is not a linear function of the composition of the binary mixture. Small amounts of polar solvents in less polar solvents considerably enhance e°. Most separations require a mixture of solvents to obtain the required e° for the separation, i.e. so that 1 ^ k' ^ 10. W h e n choosing a suitable eluent combination the most strongly adsorbed components will determine the choice of solvent strength. A useful rule to r e m e m b e r is that a change of e° of 0.05 units will change k' by a factor of 2-4 (Saunders, 1977). Once the approximate solvent strength has been chosen the differing solution energies of the various solvents can be used to optimise the resolution. Several combinations can be obtained with the same e° value but the selectivity of these different mixtures will not be identical. A simple graphical approach to choosing solvent combinations of similar solvent strength has been described (Saunders, 1977) and this is represented in Fig. 6.6. The fairly simple theory works well for eluents of low and medium strength. With increasing polarity of the eluent, however, solute-solvent interactions can no longer be neglected. Polar solvents may also displace adsorbed water from the silica surface. In such situations Equation 6.5 is no longer valid, and must be modified to take account of these secondary adsorbent activity effects. Polar solvent and solute molecules tend to become fixed or localised at the adsorption sites on the surface (Snyder et al., 1981b; Glajch and Snyder, 1981; Snyder and Glajch, 1982). W h e n the binary mobile phase contains a polar component (B) the molecules of the latter may be adsorbed with localisation when the concentration of the polar solvent in the mobile phase is small. U n d e r such conditions the effective solvent strength of B, e'B will be large. A s the surface becomes covered with B (6B ~ 0.75) already adsorbed molecules of B interfere with the localised adsorption of further molecules of B and further adsorption continues in a non-localised m a n n e r . This situation is referred to as restricted-access delocalisation of the solvent. T h e effective solvent strength of B

Solvent strength, ^ (silica) 0

0 05 1

l

0

5

0 10 1

0 15 1

10

50

'—1—I

1

10

•

1

1

0 1 3 1 i i i i

5

0 25 1

10 |

0 30 1

100

1 1 I I I ||

I

0 1 3 5 1 • ••

0 20 1

1 '

0 40 1

0-45 1

0-50 1

0 55

1

0-60 I

0 65 I

0 70 I

0 75 1

I

KIPrCljinH,

1 I 1

5 0 | 100 I III I

I

|

0 35 1

1 1

U | _ l p Xo n p

%MCinH x

SVT,

1

010

Mil——

5I 0

0

^

IPrCI, Isopropyl chloride MC, Methylene chloride Et 20, Ethyl ether ACN, Acetonitrile MeOH, Methanol

'i' I 50 100 1—j| | | | | I % Et 20 in H x i' ' so so 1 00 % MC in IPrCI

|

. ui

100

1 I I I j| I I I I I | % E t 20 in IPrCI 0

|

05

'—i

0

|

1

| 2

3

5

|i i

i

10

05

1 '

1

°20

0

60

0 1

1

0

1 % ACN in IPrCI

1—— |I

1

I

.° 5

30

1

2

3

5

'

1

1

«

0

10

20

1

30

,°

5

0

1

0

% MeOH in IPrCI

1—|

| | | | | I % E t 2O i n M C 0 1 23 5

P-u-J

Fig. 6.6. Graphical approach for obtaining the solvent strength of mixed solvents on silica. The solvent mixtures indicated by the dotted line all have the same solvent strength. Reproduced with permission from Saunders, 1977.

0

1

1

I 1 I11 I

00-5

|

| |

100 4

1

0

0

50

0

10 5

1 5 10

1

, 6° 8 0

21

3 2

1—i—i '—' 11

50 100 2 0 3 0 I 7 001 ,

• III

35

I|

% ACN in MC 50 50 5

0

10 2 0

1

110000

3 0 3 0 I 6 01701 801

% M e 0 H Ci n 11 1 1—| 1 |I |HI l| I %MeOHinEt 20 5 3 10

5.° 7 0

01

0

% ACN in Et 20 1 1 11 1 1 1—I I I I I % MeOH in ACN

M

SEPARATION M E T H O D S

141

for large values of 6B is, therefore, smaller: eB" < eB . The resultant solvent strength of B, eB, is a function of eB and eB depending on the overall localisation of B on the surface. Localisation of polar solvent molecules on the surface can also interfere with the localisation of adjacent adsorbed solute molecules, resulting in a reduction in the energy of interaction with the surface. This process is t e r m e d site-competition delocalisation. T h u s , a localising solvent will preferentially reduce the retention of a localising solute compared to a non-localising solute, effecting a change in selectivity. The contributions to solvent selectivity from solvent strength, solvent-solute localisation and solvent-solute hydrogen bonding have been discussed thoroughly by Snyder (1983a, b). In the 'sorption' model proposed by Scott and Kucera (Scott and Kucera, 1978, 1979d; Scott, 1980b; Scott and T r a i m a n , 1980) the activated surface in contact with low concentrations of a polar solvent is thought to be covered with a monolayer of solvent hydrogen b o n d e d to hydrated silanol groups. If the concentration of the polar solvent is increased, a bilayer is formed. W h e n low concentrations of polar solvent are used in the mobile phase moderately polar solutes (k' < 10) do not displace this solvent layer but associate directly with it. When m o r e polar solutes are chromatographed (k' > 20) they can displace the solvent layer and interact directly with the silanol groups. With high concentrations of polar solvent when such a 'bilayer' is formed, the solute molecule can displace a solvent molecule from the second layer and associate with the first layer. A detailed comparison of the two theories of retention has been m a d e (Snyder and P o p p e , 1980). The displacement model seems to work well in most situations and is generally accepted. A new model for monolocalised solute retention has been presented recently (Souteyrand et al., 1983, 1984). This is also based on a displacement mechanism. P R A C T I C A L CONSIDERATIONS

In order to obtain good, reproducible adsorption chromatography it is crucial to carefully control the activity of the adsorbent. Adsorbents are activated by heating under vacuum for several hours at temperatures near 200 °C. W h e n large particle sized materials were used the columns were conventionally dry-packed, and the adsorbent activity could be controlled by the addition of a known amount of water to the dried adsorbent. This is not possible using slurry packing procedures and the activity must be controlled by producing a dynamic equilibrium between the eluent and the adsorbent. W h e n water is used as a deactivator this procedure can be very lengthy because of the low solubility of the water in the non-polar eluents. The non-polar solvent is first dried by passage through a column of activated silica or alumina or a Linde molecular sieve. This process also cleans up the solvent by removal of U V absorbing or polar components. (Since dry solvents readily

142

HIGH PERFORMANCE LIQUID CHROMATOGRAPHY

absorb water they should be stored over molecular sieves in bottles which have been oven dried.) A portion of the dried solvent is then passed through a second column of water-saturated silica gel to produce 100% water-saturated solvent. The two solvents are mixed in the appropriate proportions to produce the required degree of water saturation. The column may be equilibrated by passage of the partially saturated eluent until the required volume of water has been adsorbed, but this can take several hundred column volumes of eluent. In practice, it is easier to equilibrate the column with a more polar eluent, such as ether saturated with water. Once the appropriate volume of other required to deactivate the adsorbent has been eluted the eluent can be changed to the partially saturated non-polar solvent. A series of isohydric solvents, i.e. solvents with water contents corresponding to the same adsorbent activity, has been reported (Thomas et al., 1977). This can still be a lengthy procedure and m o r e frequently polar organic modifiers are used instead of water. The most commonly used modifiers are methanol and acetonitrile, although other alcohols may also be used. The modifier is usually added in the concentration range 0 . 0 1 - 1 % v/v. A b o v e this concentration the polar component is no longer a modifier and a true solvent mixture is present (Engelhardt, 1977). T h e use of modifiers can improve efficiency and reduce peak tailing as well as reducing retention drift (Saunders, 1977). It is, however, only possible to use a modifier when the solutes to be separated are less strongly adsorbed than the modifier. Selection of appropriate solvents depends on the structure of the solutes to be analysed. T h e m o r e polar the sample the greater will be the solvent strength of the eluent required to elute it. A simple approach for determining the required solvent strength is a T L C spot test (Saunders, 1977). Several spots of the sample mixture are applied to a thin layer plate and each one is developed with a different solvent using a flat tipped hypodermic needle to apply the solvent. Several different solvents covering a wide polarity range should be used. Once the solvents have dried the migrated zones can be visualised with iodine vapour or destructive spray reagents. T h e appearance of narrow circular zones which have migrated away from the initial spot but not all the way to the solvent boundary indicates a solvent which will provide adequate retention. This spot test only takes a few minutes to perform and it can save considerable time in method development. A n optimisation procedure based on the use of isohydric solvents has been described (Thomas et al., 1979). Alternatively, a gradient elution system can be used to scout solvent compositions. Several step changes of solvent composition are required to cover the full range of sample polarity, since it is not r e c o m m e n d e d that a simple gradient from non-polar to very polar solvent be used because of demixing effects (Snyder and Saunders, 1969). If the components of interest elute over a narrow range of solvent strength (Ae° < 0.15) then isocratic elution can be used. Usually the isocratic solvent should have a solvent strength of 0 . 0 5 - 0 . 1 ^ units lower than that which elutes the same components under gradient conditions. If the required £° range is broad, gradient elution may be necessary. Gradient elution should, however,

SEPARATION M E T H O D S

143

only be used when column equilibration is rapid and this can be difficult to achieve in adsorption chromatography. Accordingly, isocratic conditions should be used whenever possible. Gradient elution may, however, be conveniently performed using isohydric solvents. Since the adsorbent activity remains constant, equilibration with the changing mobile phase is rapid and gradient techniques are therefore effective ( T h o m a s et al., 1977). A s already discussed in Section 6.1 with the availability of microprocessor controlled L C systems m o r e sophisticated m e t h o d s of optimisation can be used (Glajch et al., 1982a). Various microparticulate adsorbents which are commercially available are listed in the A p p e n d i x . S E L E C T E D APPLICATIONS

Selectivity in liquid adsorption chromatography has recently been reviewed (Yashin, 1982). Generally retention can be determined by specific or nonspecific intermolecular interactions both on the adsorbent surface and within the liquid mobile phase. T h e particular interactions and the selectivity thus produced are summarised in Table 6.5 for normal phase chromatography. Table 6.5. Classification of adsorption chromatography according to the main types of intermolecular interaction. Reproduced with permission from Yashin, 1982. Variant No.

Interaction determining substance retention

Retention characteristics

1

Substance-adsorbent SI

(1) (2) (3)

High selectivity towards compounds differing in polarity; high selectivity towards compounds differing in geometrical structure (isomers and others); group separation of homologous series is possible

2

Substance-adsorbent SI and substance-eluent Nl

Variations of retention order of aromatic alkyl derivatives

3

Substance-adsorbent SI and substance-eluent SI

Variation of retention order of polar compounds

SI = specific interaction; Nl = non-specific interaction.

The specific interactions between the solute and the adsorbent surface d e p e n d on dipole - dipole interactions which can be affected by the orientation of the molecule relative to the polar surface sites and the accessibility of the polar functional group. These interactions may produce a high selectivity towards the separation of isomers. Kiselev et al. (1980) have examined the separation of o-, m- and /7-isomers of benzene derivatives on silica using non-polar eluents. T h e retention order of isomers with different functional groups is influenced by several factors: (a) the nature of the substituents; (b) intra-molecular interactions (particularly when the substituents are in the o-position); (c) the influence of the substituents on the electron density distribution in the benzene ring and

144

H I G H P E R F O R M A N C E LIQUID C H R O M A T O G R A P H Y

(d) steric hindrance caused by non-polar substituents in the o-position to the formation of strong specific inter-molecular interactions between a polar entity of the molecule and the silanol groups on the adsorbent surface. Stancher and Z o n t a (1982) compared reversed phase chromatography and normal phase chromatography for the separation of retinol isomers. Better resolution was obtained in the normal phase system. By making small changes in the percentage of modifier (isopropanol) present at low concentrations (1.1-0.4%) in the eluent (^-hexane) it was possible to resolve all six isomers obtained by the photolysis of all-trans-retinal (Stancher and Z o n t a , 1984a). The suitability of the m e t h o d for resolving naturally occurring retinol and 3-dehydroretinol isomers was examined using cod liver oil samples and unsaponifiable fractions from fish livers and eyes (Stancher and Z o n t a , 1984a, b). T h e chromatogram obtained from a cod liver oil sample is shown in Fig. 6.7. A systematic approach to the optimisation of solvents for the separation of isomers of retinol has been described (Landers and Olson, 1984).

6

1

Time (min) Fig. 6.7. Chromatogram of retinol isomers in a cod-liver oil sample. Column packing, LiChrosorb Si-60 (5 /u,m); column dimensions, 250 x 4 mm i.d.; eluent, hexane/isopropanol (99.6:0.4); column temperature, 45 °C; detection, UV at 326 nm. Solutes: 1: 13-cw-retinol; 2: 13-ds-dehydroretinol; 3: 9,13-di-ds-retinol; 4: 9,13-di-dsdehydroretinol; 5: 9-ds-retinol; 6: all-Zrans-retinol; 7: all-frwzs-dehydroretinol. a: absorbance spectrum of all-rraRs-dehydroretinol (peak 7); b: absorbance spectrum of 13-ds-dehydroretinol (peak 2). Reproduced with permission from Stancher and Zonta, 1984(a).

The diastereoisomers of sumicidin (a pyrethroid insecticide) could not be resolved by reversed phase chromatography but were easily separated on a silica column as shown in Fig. 6.8 (Mourot et al., 1979).

SEPARATION M E T H O D S

145

(b)

1

2 3

I I*

(a) 2

I I 3

I

u I

1

r~

i

i 4

8 12 4 8 Time (min) Time (min) Fig. 6.8. HPLC of sumicidin in reversed- and normal-phase modes, (a) Column packing, LiChrosorb RP-8 (5 /am); column dimensions, 150 x 4.7 mm i.d.; eluent, acetonitrile/1% sulphuric acid (70:30); flow rate, 1.3 m l . m i n - 1; detection, UV at 235 nm. (b) Column packing, LiChrosorb Si-60 ( 5 / L t m ) ; column dimensions, 150 x 4.7 mm i.d.; eluent, anhydrous hexane/anhydrous isopropyl ether (90:10); other conditions as in (a). Solutes: 1: injection artefact; 2: diphenylamine (internal standard); 3,4: sumicidin isomers. Reproduced with permission from Mourot et al., 1979.

0

0

Normal phase chromatography is the m e t h o d of choice for the class separation of lipids and polynuclear aromatic hydrocarbons, since in such cases, the effect of carbonaceous side chains is minimal ( A b b o t , 1980). This technique has recently been applied to the separation of aromatic and polar c o m p o u n d s in fossil fuel liquids ( D a r k et al., 1977; Matsunaga, 1983; M e n e t et al., 1984) and t o the quantitative analysis of lipid classes (Phillips et al., 1982). O n e of the main problems associated with lipid analysis is the m e t h o d of detection. C h e n and Kou have examined both fluorescence and U V detection m e t h o d s for phospholipid analysis (Chen et al., 1981; C h e n and K o u , 1982a, b ; C h e n et al.,

146

HIGH PERFORMANCE LIQUID

CHROMATOGRAPHY

1983) and more recently have used deuterated solvents to permit infrared detection (Chen and Kou, 1984). Separations can also be obtained within a class of compounds when various polar substituents cause polarity differences. Fig. 6.9 shows the optimised separation of 13 substituted naphthalenes on a silica column using a quaternary mobile phase (Glajch et al., 1982a). The same separation has also been examined using l,l,2-trichloro-l,2,2-trifluorethane as the major eluent component instead of hexane (Glajch et al., 1982b). T h e two solvents exhibited similar elution characteristics. A quaternary solvent system has also been used for the optimised resolution of protected deoxyribonucleotide derivatives (Hara and O h k u m a , 1984). High performance liquid-solid adsorption chromatography has been used to separate uncoupled and coupled spiropyran-end-labelled polystyrenes from unlabelled polystyrenes (Mourey et al., 1984). Selective separations can be achieved according to molecular weight, end-group differences, or both using adsorbents of 6 and 50 nm p o r e diameters with mobile phases differing in elution selectivity. 900 800 700 600

| 500 o

S40-0 DC

300 200 100

~~00

50

100

150

200

250

300

350

Time (min) Fig. 6.9. Optimised separation of substituted naphthalenes on a silica column. Column packing, Zorbax SIL; column dimensions 300 x 4.6 mm i.d.; eluent, 50% water saturated hexane/ dichloromethane/acetonitrile/methyl tert-butyl ether (91.3:6:2:0.7); detection, UV at 254 nm. Solutes: 1: 2-methoxy naphthalene; 2: 1-nitro-naphthalene; 3: 1,2-dimethoxynaphthalene; 4: 1,5dinitro-naphthalene; 5: 1-naphthhaldehyde; 6: 2-carbomethoxy-naphthalene; 7: 1-carbomethoxynaphthalene; 8: 2-naphthaldehyde; 9: 1-cyanomethyl-naphthalene; 10: 1-hydroxy-naphthalene; 11: 1-methylcarbonyl-naphthalene; 12: 2-methylcarbonyl-naphthalene; 13: 2-hydroxy-naphthalene. Reproduced with permission from Glajch et al., 1982(a).

O n e area of interest which often requires the use of adsorption chromatography is the analysis of thermally or hydrolytically unstable compounds. This is often the case in organometallic chemistry and several examples of the use of normal phase chromatography in this field have been reported.

SEPARATION M E T H O D S

147

Examination of the separation of a series of Ni(II) bisdialkyl-dithiocarbamate complexes by adsorption chromatography on silica with chloroform-cyclohexane mixtures as the mobile phase showed that the size and shape (straight chain versus branched) of the N-alkyl substituent had a considerable influence on retention (Liska et al., 1979). T h e k' values for the complexes decreased with increasing n u m b e r of carbon atoms in the alkyl chains. T h e branching of the alkyl chain increased the polarity and hence the retention for the di-isopropyl complex. T h e di-isobutyl complex was retained less than the di-rt-butyl complex, probably because of steric hindrance. Both dithizone and diethyldithiocarbamate complexes have been used for the trace analysis of several metals in trade effluents by H P L C (Edward-Inatimi, 1983). Low t e m p e r a t u r e s ( - 3 0 °C) have been used to obtain the separation of thermally labile metal complexes ( H e n d e r s o n and Novak, 1982). A n investigation of the thermal rearrangements of tricarbonyl (phenylcycloheptatriene) iron isomers has been reported using normal phase H P L C (Pryde, 1978). T h e mild (dry, low temperatures) analysis conditions along with the fast analysis times made this technique suitable for the study of rearrangement kinetics. T h e chromatogram obtained from the products of the r e a r r a n g e m e n t of tricarbonyl (6-phenylcycloheptatriene) iron is shown in Fig. 6.10. T h e chromatographic conditions for the rapid separation of metallocarborane complexes on silica have also been investigated (Plzak et al., 1979). C h r o m a tography on silica often proved most effective for the separation of h o m o - and hetero-tetrametallic iron, ruthenium, osmium and nickel clusters (Casoli et al., 1984). The use of silica columns with polar eluents (usually aqueous methanol containing ammonia and a m m o n i u m salts) has become c o m m o n in recent years for the analysis of basic c o m p o u n d s . This is often referred to as reversed phase chromatography on silica, since the eluents are m o r e typically of the type used in reversed phase separations, but since retention will be caused by polar adsorptive interactions between the basic solute and the silica silanols a normal phase separation should result. This type of system was first introduced by J a n e (Jane and Taylor, 1975; J a n e , 1975) and it has been extensively used in the forensic analysis of drugs (Law et al., 1984). Applications include the analysis of tricyclic antidepressants (Watson and Stewart, 1975), morphine (Jane and Taylor, 1975; White, 1979), L S D (Christie et al., 1976), amiodarone (Flanagan et al., 1980) and its metabolite (Flanagan et al., 1982a), quinidine (Achari et al., 1978), nitrazepam (Kelly et al., 1982), antihistamines (Richardson and Bidlingmeyer 1984) and many other basic drugs (Achari and T h e i m e r , 1977; Sugden et al., 1978; Wheals, 1980; Bidlingmeyer et al., 1982; Flanagan et al., 1982b). Fig. 6.11 shows the separation obtained for several a m p h e t a m i n e analogues on a silica column with a mobile phase of m e t h a n o l / a m m o n i u m nitrate (Law et al., 1984). Silica has also been used as a cation exchanger for the separation of metal ions with an aqueous mobile phase (Smith and Pietrzyk, 1984). A l u m i n a , as a result of its amphoteric character, can be used either as an anion exchanger or a cation exchanger (Laurent et al., 1983a, b).

148

HIGH PERFORMANCE LIQUID CHROMATOGRAPHY

Time (min)

Retention time (min) Fig. 6.11

Fig. 6.10

Fig. 6.10. Chromatogram of the products of the rearrangement of tricarbonyl (6phenylcycloheptatriene) iron to equilibrium (69 h). Column packing, Hypersil silica (5 /mm); column dimensions, 125 x 5 mm i.d.; eluent, dry ^-hexane; column temperature, 20 °C; detection, UV at 254 nm. Solutes: 1: hydrocarbon decomposition products; 2: 3-isomer; 3: 5-isomer; 4: 6-isomer; 5: 1,2,5,6dibenzofluorene (internal standard). Reproduced with permission from Pryde, 1978. Fig. 6.11. Separation of amphetamine analogues on a silica column. Column packing, Spherisorb S5W; column dimensions, 250 x 5 mm i.d.; eluent, methanol/aqueous ammonium nitrate buffer, pH 10.1 (9:1); flow rate, 2 ml.min- 1; detection, UV at 254 nm. Solutes: 1: diethylpropion; 2: methylphenidate; 3: phenylpropanolamine; 4: amphetamine; 5: 4hydroxy-amphetamine; 6: 2-phenethylamine; 7: ephedrine; 8: methylamphetamine; 9: mephentermine. Reproduced with permission from Law et al., 1984.

6.2.2 Several

Polar bonded polar

phases

bonded

packing

materials

are

commercially

available

(see

A p p e n d i x ) . T h e most c o m m o n functional groups are c y a n o , n i t r o , a m i n o and diol. Solute interaction with t h e b o n d e d p h a s e may be by h y d r o g e n b o n d i n g with t h e polar g r o u p or t h r o u g h a specific interaction such as charge transfer. T h e

SEPARATION M E T H O D S

149

polar groups are b o n d e d onto the silica packing via an alkyl, arylalkyl or sometimes an alkyl ether spacer group. These spacer groups can obviously be involved in the retention mechanism. T h e bonding procedure will also produce differences in the selectivity of the packing material. Sometimes the required organic moiety is synthesised and then b o n d e d to the silica under conditions to give maximum coverage. This results in a surface concentration of between 2 and 4 / x m o l . m - 2 of the b o n d e d group. Alternatively, the b o n d e d phase may be prepared in two steps with an initial reaction bonding the alkyl or arylalkyl group to the surface and subsequent introduction of the polar group by a substitution reaction. In this case the concentration of polar groups may be less than the concentration of b o n d e d alkyl groups. T h e retention mechanism is, therefore, a result of the combination of interactions between the solute and the polar group, the hydrocarbon portion of the b o n d e d moiety, and any residual silanols. Exactly which interaction is principally responsible for a specific separation will depend on the polarity and p H of the eluent. For example, amine groups will be protonated at low p H , whereas residual silanol groups will tend to ionise at the same p H s . R E T E N T I O N MECHANISM

Although there are several polar b o n d e d phases available the most popular system is probably the amino b o n d e d phase. It is not surprising, therefore, that this has been the one which has been most extensively studied in attempts to elucidate the retention mechanism ( H a m m e r s et al., 1979; H e n n i o n et al., 1981; Hurtubise et al., 1981; Snyder and Schunk, 1982). Experimental data for amino phase packings has been generally interpreted in terms of the displacement model of Snyder (1968, 1983b) but solute-solvent interactions have also b e e n found to be important. A m i n o groups provide the adsorption sites in these materials and are assumed to extend above the silica surface. A competitive adsorption process (site competition delocalisation—see p . 141) similar to that observed on silica is proposed (Snyder and Schunk, 1982). T h e amino group is at the end of an alkyl chain, which renders the surface somewhat flexible with the amino groups being free to rearrange their relative positions. This is unlike the rigid silica or alumina surfaces. T h e concentration of amino groups on the surface of typical packings is 2 ^ t m o l . m ~ 2 (Majors, 1980a; O k a m o t o , 1980). This is about four times lower than the corresponding figure for silanol groups on the surface of silica (Unger, 1979; Her, 1979). It is, therefore, unlikely that adjacent adsorbed groups will interfere with each other to any extent (restricted-access delocalisation) and values of eB for a polar mobile phase c o m p o n e n t By should be constant and not vary with the surface coverage, 6B. R e t e n t i o n on an amino phase column is much weaker than on silica or alumina. T h e energy of interaction of solute or solvent molecules with the surface must, therefore, be smaller for amino phases. The corresponding interaction energies between solvent and solute molecules in the mobile phase will not vary with the adsorbent, which m e a n s that the relative

150

HIGH PERFORMANCE LIQUID C H R O M A T O G R A P H Y

importance of mobile phase versus stationary phase interactions will be greater for amino-bonded phases than on silica. Since the displacement model of Snyder (1968) ignores these mobile phase interactions, it will be less accurate for the amino phase system. However, Equations 6.12 and 6.13 still hold fairly well and they can be used to determine solvent strengths (Table 6.6). Table 6.6. Solvent strength €° of binary mobile phases A/B as a function of vol % B with amino phase packing. Experimental versus calculated values (Equation 6.13). Reproduced with permission from Snyder and Schunk, 1982. Values of e° (for indicated B solvent) CCl4 (eB = 0.069) Vol % 0.5 1.0 2.0 5.0 10.0 20.0 25.0 40.0 50.0 60.0 100.0 s.d. 0-60% 0-100%

Exptl

Calcd

0.011

0.013

0.026

0.028

0.046

0.046

0.081

0.069

Exptl

Calcd

0.004 0.008 0.011 0.021 0.028 0.045

0.002 0.004 0.007 0.016 0.029 0.048

0.074

0.074

0.096 ±0.0002 ±0.006

Ethyl acetate = 0.113)

THF (en = 0.111)

Exptl

Calcd

0.013 0.023 0.034

0.006 0.015 0.027

0.056

0.054

0.079

0.081

0.090 ±0.004

±0.006

(SB

CH2Cl2 = 0.130)

CHCl3 = 0.134)

Exptl

Calcd

Exptl

Calcd

0.025 0.041 0.063

0.022 0.039 0.063

0.028 0.044 0.063

0.022 0.039 0.063

0.088

0.092

0.092

0.093

0.112 0.150

0.110 0.130

0.112 0.170

0.111 0.134

±0.003 ±0.008

±0.004 ±0.015

SELECTIVITY O F POLAR B O N D E D PHASES

The selectivity of polar b o n d e d phases is generally different from that of silica and alumina and often specific interactions due to the nature of the polar group can be exploited to obtain selective separations. The bonded phases are weaker adsorbents than silica and alumina which gives them the advantage of m o r e rapid equilibration with water or polar solvents. They are accordingly m o r e suitable for use under gradient conditions. Such weak adsorbents are convenient for the analysis of very polar solutes which are strongly retained on silica and alumina. Sugars are a typical group of polar molecules which are difficult to analyse by liquid chromatographic methods. These are now routinely analysed using amino b o n d e d phase columns with acetonitrile/water eluents (Schwarzenbach, 1976; Jones et al., 1977; Kahle and Tesarik, 1980; Porsch, 1982; Nikolov et al., 1985). Fig. 6.12 illustrates the separation which can be obtained for malto-oligosaeeharides in syrups (Kahle and Tesarik, 1980). T h e resolution mechanism appears to be one of partition into a water-rich layer adsorbed on the amino surface (Verhaar and Kuster, 1982).

SEPARATION M E T H O D S (a)

(b)

1| |2

I

151

1|

I

I

I

I

I

0

5 10 0 5 10 Time (min) Time (min) Fig. 6.12. The separation of malto-oligosaccharides of (a) starch syrup and (b) maltose syrup. Column packing, Silasorb silica bonded with aminopropylsilyl groups (10 /xm); column dimensions, 100 x 4 mm i.d.; eluent, water/acetonitrile (40:60); flow rate, 0.9 m l . m i n - 1, column temperature, 20 °C; detection, refractive index. Solutes: 1: water, isopropanol; 2: glucose; 3: maltose; 4: maltotriose; 5: maltotetraose; 611: higher malto-oligosaccharides. Reproduced with permission from Kahle and Tesarik, 1980.

Sugar a n o m e r s can also be separated using acidic eluents and lower temperatures (Kahle and Tesarik, 1980;Moriyasu et al., 1984a). T h e resolution of arabinose anomers at 0 °C is shown in Fig. 6.13. A n o t h e r class of c o m p o u n d s which is often separated on amino-bonded columns is polycyclic aromatic hydrocarbons. T h e amino phase produces a group type separation based on the n u m b e r of aromatic rings present. This is complementary to the reversed phase separation where the size and nature of ring substituents has a large effect. T h e effect of alkyl substituents on the ring is even less on the polar b o n d e d phase than it is on silica or alumina, leading to an even more p r o n o u n c e d class separation (Wise et al., 1977). O n e type of polar b o n d e d phase which has been synthesised specially to p r o m o t e a specific interaction is the charge transfer p h a s e . These b o n d e d phases contain electron acceptor groups, usually nitroaromatic groups, and they are preferentially used for the separation of polynuclear aromatic hydrocarbons or azaarenes which are assumed to be electron donors. It is proposed that donor/acceptor complexes are formed between the b o n d e d phase and the solutes ( N o n d e k and Malek, 1978; H e m e t s b e r g e r et al., 1980; N o n d e k et al., 1979; Holstein and H e m e t s b e r g e r , 1982).

152

HIGH P E R F O R M A N C E LIQUID C H R O M A T O G R A P H Y 4

3

1

2

L_ 0

6 Time (min)

Fig. 6.13. The separation of arabinose anomers. Column packing, Aminopropylsilyl bonded Silasorb silica in the sulphate form; column dimensions, 100 x 4 mm i.d.; eluent, water/acetonitrile (20:80); flow rate, 1.2 ml.min- 1, column temperature, 0 °C; detection, refractive index. Solutes: 1: water; 2: arabinofuranoses; 3: /3-L-arabinose; 4: ar-L-arabinose. Reproduced with permission from Kahle and Tesarik, 1980.

There appear to be two c o m m o n types of charge transfer b o n d e d phase based on bonded nitrofluorene moieties (Lochmuller and A m o s s , 1975; Mikes et al., 1976; Hemetsberger et al., 1980) or nitrobenzene groups (Nondek and Malek, 1978; N o n d e k et al., 1979; Matlin et al., 1980; Grizzle and T h o m s o n , 1982; Eppert and Schinke, 1983; Felix and B e r t r a n d , 1984a, b). Lochmuller et al. (1979) examined four different nitrofluorenimine phases and demonstrated that the bonded phase selectivity for aromatic hydrocarbons increases with increasing number of nitro groups attached to the fluoreniminopropyl ligand. The position of nitro substitution in the aromatic ring also had an influence on the separation. Similar results have been obtained with the nitrobenzene type phases (Nondek and Ponec, 1984; Felix and B e r t r a n d , 1984b; Lochmuller,1980). H a m m e r s et al. (1982) have studied the chromatographic behaviour of 2,4-dinitroanilinopropyl ( D N A P ) , bis (3-nitrophenyl) sulphone and 2,4,7-trinitrofluoreniminopropyl ligands b o n d e d to silica. If a sufficiently dense layer of bonded groups covers the surface it appears that the ligands can self-associate in the presence of non-polar solvents. This can lead to restricted penetration of solute molecules into the b o n d e d layer, resulting in asymmetry of chromatographic peaks. A lengthy equilibration time may also be necessary when changing non-polar eluents. The equilibrium retention data on these b o n d e d phases can

SEPARATION M E T H O D S

153

1

4

Detector response (% f.s.)

be described using the basic equations of Snyder's adsorption model ( H a m m e r s et al., 1982). Nondek and Ponec (1984) have used molecular orbital theory to study the complex formation between the donor (solute) and acceptor (ligand). They showed that the complexing ability and selectivity of the bonded phase depended on the n u m b e r and position of the electron accepting substituents attached to the aromatic ring and also on the nature of the spacer group attaching the ring to the silica surface. T h e separation of some aromatic hydrocarbons on 2,4-dinitrobenzenesulphamidopropyl silica is shown in Fig. 6.14. This phase gave promising results with h ~ 4. Change transfer chromatography has also been used for the separation of biological molecules on acriflavin-silica (Small et al., 1982). This m e t h o d is an extension of earlier work on acriflavin b o n d e d Sephadex (Egly and Porath, 1979). The separation of flavin mononucleotide and riboflavin is illustrated in Fig. 6.15 (Small et al., 1982).

JUU 0

4

8

3

T i m e (min) Fig. 6.14

4

5

Time (min)

6

7

8

9

Fig. 6.15

Fig. 6.14. The separation of aromatic hydrocarbons on a charge transfer phase. Column packing, 2,4-dinitrobenzenesulphamidopropyl silica; column dimensions, 250 x 4 mm i.d.; eluent, nheptane/dichloromethane (10:1); flow rate, 1.5 m l . m i n - 1; detection, UV at 254 nm. Solutes: 1: benzene; 2: naphthalene; 3: phenanthrene; 4: anthracene. Reproduced with permission from Nondek and Ponec, 1984. Fig. 6.15. Resolution of flavins by high-performance charge-transfer chromatography. Column packing, acriflavin-LiChrosorb Si 60; column dimensions, 100 x 4.5 mm i.d.; eluent, water/ methanol (50:50); flow rate, 2 m l . m i n - 1; detection, UV at 266 nm. Solutes: 1: flavin mononucleotide; 2: riboflavin. Reproduced with permission from Small et al., 1982.

154

HIGH P E R F O R M A N C E L I Q U I D C H R O M A T O G R A P H Y

S E L E C T E D APPLICATIONS

Several recent applications which have been p e r f o r m e d on polar b o n d e d phases are listed in Table 6.7. Table 6.7. Applications on polar bonded phases. Packing material

Solute Phospholipids Styrene oligomers

Eluent

5 /xm DIOL or CN Spherisorb S5P (5 /xmphenyl)

Detection

Reference

UV, 203 nm

Andrews, 1984 Lai et al., 1984

Tetrahydrofuran/ water tetrahydrofuran/ hexane acetonitrile/water

UV, 260 nm

Inorganic and organic anions

Zorbax-NH 2

0.03 M phosphoric acid, pH 3.2

UV, 205 nm

Cortes, 1982

5-Triazine derivatives

LiChrosorbNH2

Pentane or heptane modified with alcohol

UV,230 or 235 nm

Dufek et al., 1980

Mono-alkyltin homologues

Cyanopropylsilica

Toluene/acetic acid/ methanol/ acetonitrile/ morin

Fluorescence

Langseth, 1984

Bis

Cyano-silica

Gradient 0-10% dichloromethane in hexane in 10 min

UV, 254 nm

Mazzo et al., 1983

Androgenic steroids

LiChrosorb DIOL (5 fxm)

«-Hexane/2propanol (9:1)

UV, 254 nm

Mancilla and Gil, 1984

Tri-alkyltin compounds

Cyanopropylsilica

Hexane/acetic acid or hexane/ethyl acetate

RI

Langseth, 1984

Polynuclear aromatic hydrocarbons

Various (see text)

Heptane

UV

Chmielowiec and George, 1980

(r/M, 2,3,4,5pentamethylcyclopentadienyl) titanium, zirconium and hafnium dichlorides

Chmielowiec and G e o r g e (1980) have e x a m i n e d several polar b o n d e d phases for

their

selectivity

in

separating

polycyclic

aromatic

hydrocarbons.

The

c h r o m a t o g r a m s o b t a i n e d on various polar b o n d e d phases are shown in Fig. 6.16. T h e selectivity of t h e various phases is obviously different with t h e d e g r e e of

SEPARATION METHODS Silica-CN n- Heptane

Silica-NH 2 n- Hexane

155

Silica-R(OH) 2 Silica-ROR n- Heptane n- Heptane

3

2,3

Absorbance (254 nm)

(a)

Time (min) Silica-(Diamine) 6%CH 2CL 2ln-Heptane

n- Heptane

Absorbance (254 nm)

(t>)

0

10

20

30

10

20

30

Retention volume (ml) Fig. 6.16. Chromatograms of aromatic hydrocarbons on polar phases bonded to silica. Column packing, as indicated; column dimensions, 250 x 4.6 mm i.d.; eluent, as indicated; flow rate, 2 ml.min - 1; column temperature ambient; detection, UV at 254 nm. Solutes: (a): 1: naphthalene; 2: phenanthrene; 3: pyrene; 4: chrysene, triphenylene; 5: perylene, anthanthrene; 6: dibenz [a,h]-anthracene; 7: dibenzo [e,h] pyrene; 8: coronene. (b): 1: benzene; 2: indene; 3: naphthalene; 4: 2,3,5-trimethylnaphthalene; 5: biphenyl; 6: azulene; 7: fluorene; 8: o-terphenyl; 9: anthracene; 10: phenanthrene; 11: 2-phenyl-naphthalene; 12: ra-terphenyl; 13: 9,10-diphenylanthracene; 14: fluoranthene; 15: pyrene; 16: benzo [b] fluorene, phenanthrene; 17: benz [a] anthracene, chrysene; 18: triphenylene; 19: benz [a] pyrene; 20: perylene; 21: anthanthrene; 22: benzo [ghi] perylene; 23: dibenz [a,h] anthracene; 24: dibenz [a,c] anthracene, coronene; 25: dibenzo [e,h] pyrene, dibenzo [a,h] pyrene. Reproduced with permission from Chmielowiec and George, 1980.

156

HIGH PERFORMANCE LIQUID C H R O M A T O G R A P H Y

resolution between the peaks varying significantly on different phases. T h e elution sequence is not always the same with coronene being eluted earlier on both the amino-phase and the ether phase. All the solutes were much m o r e retained on the diamine p h a s e , allowing much better resolution for the smaller polycyclic aromatic hydrocarbons (Fig. 6.16b). T h e higher ring structures were eluted with an eluent of h e p t a n e / m e t h y l e n e chloride (94:6). A s already discussed, normal phase chromatography is particularly suitable for the separation of isomeric lipophilic c o m p o u n d s . Fig. 6.17 shows an example of the resolution obtained for several androgenic steroids on a diol-bonded phase (Mancilla and Gil, 1984). These steroids are the major products of testosterone hydroxylation by rat liver microsomes. 1 2

0 0 0 2 au.f.s.

I

i

5 6

1

II

^—Injection

UV absorbance (254 nm)

4

0

5

10

15 20 25 30 Time (min) Fig. 6.17. Separation of androgenic steroids. Column packing, LiChrosorb Diol; column dimensions, 250 x 4.6 mm i.d.; eluent, n-hexane/2-propanol (9:1); flow rate, 1.3 m l . m i n - 1; detection, UV at 254 nm. Solutes: 1: androstenedione; 2: testosterone; 3: 2or-hydroxytestosterone; 4:6/3hydroxytestosterone; 5: 16ar-hydroxytestosterone; 6: 7a-hydroxytestosterone. Reproduced with permission from Mancilla and Gil, 1984.

Polar bonded phases may also be used for the analysis of organometallic compounds. Fig. 6.18 illustrates the complete resolution of bis (rj5-l,2,3,4,5pentamethyl cyclopentadienyl)-titanium, zirconium and hafnium dichlorides on a cyano-bonded silica. T h e separation of the zirconium and hafnium complexes could not be achieved on a silica column (Mazzo et al., 1983).

157

^Injection

SEPARATION M E T H O D S

4

6

Time (min) Fig. 6.18. Separation of bis (r/ 5-l,2,3,4,5-pentamethylcyclopentadienyl). Column packing, cyanobonded silica (5.9 fjum); column dimensions, 250 x 4.5 mm i.d.; eluent, gradient from 0 to 10% dichloromethane in hexane in 10 min; flow rate, 1.0 m l . m i n - 1; detection, UV at 254 nm. Solutes' 1: (PMCP) 2 ZrCl 2; 2: (PMCP) 2 Hf Cl 2; 3 (PMCP) 2 Ti Cl 2. Reproduced with permission from Mazzo et al., 1983.

A m i n o - b o n d e d phases can be used either as p o l a r b o n d e d phases or as w e a k ion exchangers. A n e x a m p l e of t h e use of an a m i n o b o n d e d p h a s e for t h e separation of inorganic a n d organic ions which can be d e t e c t e d by U V detection at low wavelength is given in Fig. 6.19 ( C o r t e s , 1982).

8 2

Detector response

7

1

0

1

2

3

4

5

6

7

8

10

1 1

Time (min) Fig. 6.19. Separation of organic and inorganic anions on an amino bonded phase. Column packing: Zorbax N H 2; column dimensions; 250 x 4.6 mm i.d.; eluent, 0.03 M phosphoric acid adjusted to pH 3.2 with sodium hydroxide; flow rate, 2.0 m l . m i n - 1; column temperature, 25 °C; detection, UV at 205 nm. Solutes: 1: acetate; 2: acrylate; 3: glycolate; 4: formate; 5: nitrite; 6: bromide; 7: nitrate; 8: iodate; 9: dichloroacetate. Reproduced with permission from Cortes, 1982.

158

6.3

HIGH P E R F O R M A N C E L I Q U I D C H R O M A T O G R A P H Y

Liquid-liquid partition chromatography

Liquid-liquid partition chromatography is rarely used today because of the problems associated with maintaining the stability of the system and the rapid growth in the popularity of the m o r e stable b o n d e d phase. It is, nevertheless, worthy of a brief mention. The technique was developed by Martin and Synge (1941) and it is based upon the components of a mixture partitioning between a mobile and a stationary phase. Separation is effected when the solutes have different distribution coefficients between the two phases.

6.3.1

Theory

The theory of liquid-liquid partition chromatography has not been as well defined as that for adsorption systems. T h e r e have, however, been equations derived to explain the distribution of a solute, /, between a stationary and a mobile phase (Karger et al., 1973). log/) =

m

- Sm)2

- (

mM N A D V 0 - 1 M pyruvate

"O O 1

Detector response (% f.s.) ^ ' •— • \ ..o., }

o

'

m o '

i

.

-L

•

1

1"°

—I C O

Enzyme activity (ju/ml) (-o-o-)

•—•

1

P ->

^ o

'

.

-0

BSA

0-5MKCI

*

~~ ~ ~~—

cn

i

'

co o

--_o LDH

> O

— —

1

N> 1

C O 1

Enzyme activity (ju/ml) (-o-o-)

i

u c

NH,

0

- 0 - S i - ( C H 2) 3O C H 2C H C H 20

CI

Fig. 6.74. Resolution of a mixture of bovine serum albumin (BSA) and pig heart lactate dehydrogenese (LDH) on a Procion Blue Mx-R silica column. Column packing, LiChrosorb Si 60 bonded with Prociom Blue MX-R as shown; column dimensions, 100 x 4.5 mm i.d.; eluent, 25 mM potassium phosphate, pH 7.0; strong eluent for elution (a) 0.5 M KC1 in 25 mM potassium phosphate, pH 7.0; (b) 0.1 mM NAD+-0.1 M pyruvate in 25 mM potassium phosphate, pH 7.0; flow rate, 1 m l . m i n - 1; detection UV at 210 nm. Reproduced with permission from Small et al., 1981.

Fig. 6.75. (a) Schematic diagram of an inclusion complex formed between /?-naphthol and a cyclodextrin molecule. (b) Schematic diagram showing the structure and relative size of the three most common cyclodextrin molecules, (i) ar-cyclodextrin (ii) /3-cyclodextrin (iii) y-cyclodextrin. Reproduced with permission from Armstrong and DeMond, 1984.

224

HIGH P E R F O R M A N C E LIQUID C H R O M A T O G R A P H Y

resolution of enantiomeric amino acids on bovine serum albumin bound phases (Allenmark et al., 1982, 1983, 1984), the separation of proteases using silica-bound soybean trypsin inhibitor (Kasche et al., 1981) and the isolation of various enzymes with silica-bound triazine dyes (Lowe et al., 1981; Small et al., 1981). Fig. 6.74 shows the resolution of a mixture of bovine serum albumin (BSA) and pig heart lactate dehydrogenase ( L D H ) on a silica immobilised triazine dye, Procion Blue M X - R (Small et al., 1981). T h e B S A was not held on the column and the L D H could be quantitatively displaced with a 200 fx\ pulse of either 200 m M KC1 or 0.1 m M N A D + / 0 . 1 M pyruvate.

6.8

Inclusion chromatography

Recently considerable interest has been generated in the use of cyclodextrin inclusion complexes to enhance chromatographic selectivity (SmolkovaKeulemansova, 1982). This can be achieved by using cyclodextrin as an eluent additive in reversed phase chromatography (Sybilska et al., 1982), or by using chemically bonded cyclodextrin phases (Armstrong and D e M o n d , 1984). There are three different size cyclodextrins available commercially and these are illustrated in Fig. 6.75. T h e interior of the cyclodextrin cavity is relatively hydrophobic and a wide variety of compounds can fit into the hole forming inclusion complexes as illustrated in Fig. 6.15a. With aqueous-organic eluents the retention mechanism is mainly due to inclusion complex formation and the separation depends on the varying strengths of the complexes formed with molecules of different sizes and geometrical shapes. Since the strength of the complex is very d e p e n d e n t on the geometrical structure of the molecule, this is a very selective chromatographic m e t h o d for the separation of isomers. T h e organic component of the eluent will also be included in the cavity and competes with the solute for the site. A n increase in the organic modifier concentration will, therefore, decrease the retention of the solutes. Organic solvents which form stronger inclusion complexes will also reduce retention. Cyclodextrin b o n d e d phases have been p r e p a r e d by reacting carboxylated silica with ethylenediamine mono-substituted cyclodextrin (Fujimura et al., 1983; Kawaguchi et al., 1983; T a n a k a et al., 1983). New phases which consist of cyclodextrin molecules linked to silica gel via a 6-10 atom spacer which does not contain nitrogen are thought to be m o r e hydrolytically stable (Armstrong and D e M o n d , 1984). These latter materials are now commercially available (Advanced Separation Technologies). The effect of acylation of the cyclodextrin moiety on the separation efficiency has also been examined (Tanaka et al., 1983, 1984a, b ) . Geometrical isomers which are difficult to separate by reversed phase chromatography can often be separated on cyclodextrin columns because of the unique selectivity of these materials. Since the cyclodextrin cavity is chiral even optical isomers can frequently be resolved (Armstrong et al., 1984, 1985). Fig. 6.76 illustrates the separation of dinitrobenzene isomers on a benzylated

SEPARATION M E T H O D S

225

/?-cyclodextrin phase and an acetylated /?-cyclodextrin phase (Tanaka et al.,

I-B

ll-B

6 12 18 0 12 24 36 48 Time (min) Time (mm) Fig. 6.76. Liquid chromatograms of dinitrobenzene isomers on (I) benzylated /3-cyclodextrin silica and II acetylated /3-cyclodextrin silica. Column packing, Develosil 60-3 (3 /xm) bonded with succinamidopropyl groups coupled to ethylenediamine substituted cyclodextrin; column dimensions, 100 x 4 mm i.d.; eluent, methanol/water (A) (40:60) and (B) (20:80); flow rate, 0.7 ml.min" 1; detection UV at 254 nm. Reproduced with permission from Tanaka et al., 1984(a).

6.9

Chiral chromatography

Chiral separations can be effected by either using chiral additives in the eluent or by using optically active bonded phases. B o n d e d phases of both the ligand exchange type or the Pirkle type have been shown to be effective This topic is discussed in depth in Chapter 9.

7

7.1

7.1.1

Adjustment of selectivity by use of eluent additives

Ion pair partition chromatography

Theory and separation

mechanism

The technique of ion pair partition whereby an ionic species can be extracted into an organic phase by association with a hydrophobic pairing ion has been used for many years. Much of the developmental work in both theory and practice was performed by Schill and co-workers who first applied the method to a chromatographic situation where the ion pairs were partitioned between a stationary and a mobile phase (Schill et al., 1965; Persson, 1968; Wahlund and Groningsson, 1970; Eksborg and Persson, 1971; Persson, 1971; Eksborg and Schill, 1973; Eksborg et al., 1973). When solute ions (S~) are added to a chromatographic system containing pairing ions (P+) and associated counter ions (C~) the degree of retention of (S~) depends on the following equilibrium +

^aq

with an extraction constant

^ a q

Esn = SP

— org $P

[^org]

—

rc-irn+i

(7*1) (7.2) v

/

Assuming that the concentration of the pairing ion in the aqueous phase is high compared to that of the solute ion, the distribution coefficient of S~ is given by 226

ADJUSTMENT O F SELECTIVITY BY USE O F E L U E N T A D D I T I V E S

Esp [^aq]

227

(7.3)

Since the capacity factor k' is proportional to IID in normal phase chromatography and to D in reversed phase chromatography it follows that k' in ion pair chromatography is inversely proportional to the pairing ion concentration in the normal phase situation but directly proportional in the reversed phase case. In the situation where the pairing ion is very hydrophobic P+ will be extracted into the organic phase along with its normal counter ion C~ according to Q

q

+ P a+q = CPOTg

(7.4)

Subtracting this from Equation 7.1 gives ^aq

C P o rg = SPQTg + C aq

(7*5)

This is very similar to ion exchange chromatography, with an equilibrium constant (7.6)

This gives

(7.7)

from which it follows that k' is inversely proportional to the concentration of the counter ion in the aqueous phase. The latter situation normally arises in the reversed phase m o d e where the hydrophobic pairing ion is adsorbed onto the bonded hydrocarbon of the packing material. W e can therefore envisage three distinct situations: 1. Normal phase ion pair partition chromatography where the support is coated with an aqueous stationary phase containing the pairing ion and the ion pairs are partitioned between this phase and an organic mobile phase. 2. Reversed phase ion pair partition chromatography where the liquid stationary phase is organic and the pairing ion is introduced in the aqueous mobile phase. 3. Reversed phase ion pair chromatography using a chemically b o n d e d stationary phase and a hydrophobic pairing ion in the aqueous mobile phase. W e shall consider each situation in m o r e detail. 7.1.2

Normal phase ion pair partition

chromatography

This is a liquid-liquid partition chromatographic system and it is important to avoid any interaction with the underlying support material. Initially, various

228

HIGH P E R F O R M A N C E LIQUID C H R O M A T O G R A P H Y

inert supports such as diatomaceous earth or cellulose were examined (Persson, 1968; Eksborg and Schill, 1973; Eksborg et al., 1973; Eksborg, 1975a, b ; Mellstrom and Eksborg, 1976; C r o m m e n et al., 1977). Although cellulose has the advantage of being stable over a wide p H range as well as being nonadsorptive the columns tended to show poor efficiency. This was partly a result of the low mechanical stability of the material which rendered it unsuitable for slurry packing at high pressure. In practice, the cellulose was normally loaded with the stationary phase prior to packing which was accomplished by tamping small portions at a time into the column with a rod. T h e systems were generally improved by the introduction of rigid porous silica supports which could be efficiently slurry packed, despite their adsorptive properties (Persson and Karger, 1974; Karger et al., 1974; Knox and Jurand, 1975; Fransson et al., 1976; Knox and J u r a n d , 1976; Persson and Lagerstrom, 1976; Su et al., 1976). In the search for increased p H stability columns packed with microparticulate ceria have also been examined (Gilbert and Wall, 1978) but the capacity of these systems was generally p o o r e r . The support is loaded with the aqueous stationary phase containing the pairing ion by one of three m e t h o d s . 1. Either the neat stationary phase or a concentrated solution of the stationary phase in acetone is p u m p e d through the packed column bed. The excess is then removed from the interparticle spaces by passage of eluent or hexane followed by eluent saturated with stationary phase until equilibrium is reached (Persson and Lagerstrom, 1976; Persson and Karger, 1974; Karger et al., 1974; Fransson et al., 1976; Mellstrom and Tybring, 1977). T h e excess phase is removed as bubbles in the eluent and it is important that the detector should be disconnected during this procedure to avoid contamination. Once the eluent emerging from the column is clear the detector can be connected and the eluent recycled. Equilibrium has been achieved when stable k' values are obtained for a series of representative solutes. This usually requires passage of several hundred millilitres of eluent. It may not always be desirable to p u m p the concentrated stationary phase through a chromatographic p u m p , since it can take a long time to remove all traces of the pairing ion from the system and some ions, e.g. perchlorate, can be corrosive at the concentrations required. In this case it may be more convenient to suck the plug of stationary phase through the column (Knox and J u r a n d , 1975, 1976). 2. Alternatively, the stationary phase can be loaded onto the column in several large plugs (0.1-1.0 ml) using a stopped-flow technique (Su et al., 1976; C r o m m e n et al., 1977). Equilibration is accomplished in the same way. 3. In the third approach the eluent which has been pre-equilibrated with the stationary phase is p u m p e d through the column until stable k' values are obtained (Gilbert and Wall, 1978; Mellstrom and Braithwaite, 1978). The stationary phase is adsorbed onto the support surface but at equilibrium the pores of the support are not completely filled as they are in the heavily loaded columns obtained by the first two m e t h o d s . Equilibration in this case can be a lengthy procedure but the columns which are obtained are stable and

ADJUSTMENT O F SELECTIVITY BY USE O F E L U E N T ADDITIVES

229

reproducible. In order to obtain equilibrium, it is necessary that the mobile phase is saturated with the stationary phase. This is achieved firstly by equilibrating a quantity of mobile phase with stationary phase and then filtering the separated organic layer through glass wool to remove water droplets. The eluent is subsequently passed through a pre-column loaded with stationary phase prior to the chromatographic column and recycled to maintain equilibrium. It is also very important to maintain the constant t e m p e r a t u r e of the system by thermostatting both the pre-column and column. Ideally, the total system should be enclosed in a thermostatted oven, but if this is not available all interconnecting tubing should be lagged and the detector should be carefully thermostatted to prevent stationary phase separating out and contaminating the cell. As indicated by Equation 7.3, the k' of a solute is inversely proportional to the pairing ion concentration in the normal phase situation. Unfortunately, because the pairing ion is in the stationary phase this concentration is not readily changed except by stripping the phase completely and reloading with a new concentration of pairing ion. For this reason retention is normally controlled by modification of the eluent. Usually hydrocarbon or chlorinated hydrocarbon solvents are used in conjunction with a small percentage of alcohol as modifier. Variation of the concentration or nature of the alcohol can produce the required changes in retention or selectivity (Su et al., 1976; Knox and J u r a n d , 1976). Since the columns are in an equilibrium situation it is obviously not possible to perform gradient elution chromatography. Normal phase ion pair partition chromatography has been applied to the separation of quaternary a m m o n i u m c o m p o u n d s (Eksborg et al., 1973; Eksborg and Schill, 1973; E k s b o r g , 1975a, b ; Gilbert and Wall, 1978; C r o m m e n , 1980), biogenic amines (Persson and Karger, 1974; Persson and Lagerstrom, 1976; Knox and J u r a n d , 1976), carboxylic acids (Eksborg et al., 1973; Persson and Lagerstrom, 1976; Lagerstrom 1976; Fransson et al., 1976) sulpha drugs (Karger et al., 1974; Su et al., 1976), amino acids (Persson, 1971; C r o m m e n et al., 1977; Persson and Karger, 1974) divalent amines (Westerlund et al., 1979, 1981) and tricyclic amines (Knox and J u r a n d , 1975; Lagerstrom et al., 1976). A n example of the separation of a series of sulpha drugs is illustrated in Fig. 7 . 1 . Unfortunately, very high efficiencies have not usually been achieved with these systems and the advantage of the reversed phase m o d e , where the pairing ion concentration can be easily be altered, has led to its almost total take-over in the ion pair field. T h e normal phase system has one advantage which until recently could not be exploited so easily in the reversed phase m o d e , viz the use of UV-absorbing pairing ions to enhance or enable the detection of the solutes. For example, picrate (Eksborg and Persson, 1971; E k s b o r g et al., 1973; Eksborg and Schill 1973; H u e n et al., 1978; Gilbert and Wall, 1978) and naphthalene sulphonate (Eksborg et al., 1973; Santi et al., 1975; E k s b o r g , 1975a, b ; C r o m m e n et al., 1977; C r o m m e n , 1979a, 1980; Hackzell and Schill, 1981) have been used to enable the determination of non-UV-absorbing bases and tricyclic tranquilliser ions have enhanced the detection of weakly absorbing carboxylic acids (Eksborg et al., 1973; Lagerstrom, 1975; Lagerstrom and T h e o d o r s e n ,

230

HIGH PERFORMANCE LIQUID CHROMATOGRAPHY

1975; Hackzell et al., 1981). Recently, however, the use of very low concentrations of pairing ion in the mobile phase has allowed this technique to be used in the reversed phase m o d e (e.g. D e n k e r t et al., 1981). This will be discussed in Section 7.1.4.

11

7 10 12

13

A 30

45

60

75

90

Time (min) Fig. 7.1. Separation of sulphonamides by normal phase ion pair partition chromatography. Column packing, LiChrospher SHOO (10 /im) loaded with 0.3 M TBA H S 0 4 in 0.1 M phosphate buffer, pH 6.8; column dimensions, 250 x 3.2 mm i.d.; eluent, heptane//t-butanol (75:25); flow rate, 0.4 m l . m i n - 1; column temperature, 27 ± 0.5 °C; detection, UV at 254 nm. Solutes: 1: phthalyl sulphathiazole; 2: sulphabenzamide; 3: sulphisoxazole; 4: sulphacetamide; 5: sulphadimethoxine; 6: sulphachloropyridazine; 7: sulphadiazine; 8: sulphaquinoxaline; 9: sulphamerazine; 10: sulphamethoxypyridazine; 11: sulphathiazole; 12: sulphamethazine; 13: sulphapyridine. Reproduced with permission from Su et al., 1976.

7.1.3

Reversed phase ion pair partition

chromatography

Many of the above points mentioned with respect to equilibrium and maintenance of equilibrium also apply h e r e , although pre-columns are not generally used. Initial experiments in reversed phase ion pair liquid-liquid chromatography were performed by Wahlund and Groningsson (1970), Eksborg et al. (1973) and Kraak and H u b e r (1974). T h e first really successful system was achieved by Wahlund (1975). Most commonly, pentanol or butyronitrile is used as the stationary phase loaded onto a hydrophobic support such as silanised silica (Wahlund, 1975; Wahlund and L u n d , 1976; Johansson and W a h l u n d , 1977) or a longer chain hydrocarbon b o n d e d silica (Fransson et al., 1976; Wahlund and Sokolowski, 1978; Johansson et al., 1978; Wahlund and Beijersten, 1978, 1982). Column equilibration is normally achieved by priming with mobile phase saturated with stationary phase. T h e time taken for equilibration depends on the

ADJUSTMENT O F SELECTIVITY BY USE O F E L U E N T A D D I T I V E S

231

hydrophobicity of the support, but Wahlund and Beijersten(1978) found that the coating of pentanol on a hydrocarbon b o n d e d silica took no longer than 2 h at a flow rate of approximately 1 m l . m i n - 1. Once again, the loading procedure is followed chromatographically by measuring the retention volume of various solutes. After equilibrium is reached, it is maintained by recycling the eluent. Accurate t e m p e r a t u r e control is of course very important. Several factors can be varied in reversed phase ion pair chromatography to regulate the retention of the solutes. 1. k' increases with the hydrophobicity of the pairing ion. Accordingly for hydrophilic solutes, hydrophobic pairing ions are chosen and vice versa. Since the pairing ion is added in the mobile p h a s e , it is fairly easy to test two or three possible ions of different hydrophobicities before selecting the best conditions. 2. k' increases linearly with pairing ion concentration, and since it is relatively easy to alter this concentration in the mobile phase it is possible to vary the degree of retention of the solutes after selection of the pairing ion. Alternatively, gradient elution can be performed by decreasing the pairing ion concentration (Wahlund, 1975). 3. T h e choice of the organic phase will influence the selectivity of the system. In practice, however, only pentanol and butyronitrile systems have been successfully used. Johansson et al. (1978) attempted to use methylene chloride as the stationary phase but they found the system to be unstable over a period of time even under carefully thermostatted conditions. They also found evidence for ion pair adsorption on the solid phase under the conditions studied. Evidence of adsorption was also observed in the pentanol system when hydrophobic amines were used as solutes (Wahlund and Sokolowski, 1978). T h e poor peak shape obtained for the amines was thought to be caused by their adsorption on residual silanol groups on the surface of the packing material. It was found possible to avoid this by the addition of an alkylammonium c o m p o u n d to the eluent. This topic will be discussed m o r e fully in Section 7.1.4. The ability to adjust conditions relatively easily has led to reversed phase systems being the ion pair m e t h o d of choice and many applications have been published. Some of these are summarised in Table 7 . 1 . Since the mobile phase is an aqueous phase, the analysis of clinical samples in body fluids is possible without prior extraction or extensive clean-up with the result that this technique is widely used in clinical chemistry, pharmacology, forensic science, etc. T h e efficiency which can be obtained is illustrated in Fig. 7.2 by the separation of several aromatic acids.

7.1.4 Reversed phase ion pair chromatography stationary phase

using chemically

bonded

By far the most versatile, and therefore the most commonly used, form of reversed phase ion pair chromatography is the one employing a hydrophobic pairing ion in an aqueous mobile phase in conjunction with a hydrogen bonded

Table 1.1 Applications of reversed phase liquid-liquid ion pair chromatography. Solute

Packing material

Stationary phase

Mobile phase

Pairing ion

Reference

Benzoic and benzenesulphonic acids

LiChrosorb RP-2 or Bondapak C18/ Porasil B

1-Pentanol

Aqueous soln of pairing ion

TBA

Wahlund, 1975

Benzoic and benzenesulphonic acids

LiChrosorb RP-2 or RP-18

1-Pentanol

Aqueous soln of pairing ion

TBA

Fransson et al., 1976

Acidic catecholamine metabolites

LiChrosorb RP-2

1-Pentanol

Aqueous soln of pairing ion

TBA

Wahlund and Lund, 1976

Acidic metabolites of biogenic amines

LiChrosorb RP-2 or RP-18

1 -Pentanol

Aqueous soln of pairing ion

TBA

Fransson et al., 1976

Phenylacetic acids

LiChrosorb RP-2

1-Pentanol

Aqueous soln of pairing ion

TBA

Wahlund and Lund, 1976

Nicotinic acid and related pyridine derivatives

LiChrosorb RP-2

1-Pentanol

Aqueous soln of pairing ion

TBA

Wahlund and Lund, 1976

Aromatic carboxylic acids

LiChrosorb RP-8

1-Pentanol

Aqueous soln of pairing ion

TBA,TPrA

Wahlund and Beijersten, 1978

Sulphonamides

LiChrosorb RP-2 or RP-18

1-Pentanol, butyronitrile

Aqueous soln of pairing ion

TBA

Fransson et al., 1976

Sulphonamides

LiChrosorb RP-2

1-Pentanol, butyronitrile

Aqueous soln of pairing ion

TBA

Johansson and Wahlund, 1977

Barbiturates

LiChrosorb RP-2 or RP-18

1-Pentanol, butyronitrile

Aqueous soln of pairing ion

TBA

Fransson et al., 1976

Table 7.1

(contd.) Solute

Packing material

Stationary phase

Mobile phase

Pairing ion

Reference

Barbiturates

LiChrosorb RP-2

1-Pentanol, butyronitrile

Aqueous soln of pairing ionTBA

Johansson and Wahlund, 1977

Phenylethylamines

LiChrosorb RP-18

1-Pentanol

Phosphate buffer (pH 3.0)

Octylsulphate Cyclohexylsulphamate

Johansson et al., 1978

Tricyclic ammonium compounds

LiChrosorb RP-8

1-Pentanol

Phosphate buffer containing TMNA (0.036 M)

h 2p q 4

Johansson et al., 1978

Antidepressive and neuroleptic amines

LiChrosorb RP-8

1-Pentanol

Phosphate buffer containing long chain ammonium ion

H 2P 0 4 BrCyclohexylsulphamate

Wahlund and Sokolowski, 1978

Quaternary ammonium compounds

LiChrosorb RP-!

1-Pentanol

Phosphate buffer containing long chain ammonium ion

H 2P 0 4 Br" Cyclohexylsulphamate

Wahlund and Sokolowski, 1978

Catecholamines

LiChrosorb RP-8

Tri-/i-butyl phosphate

0.05 M phosphate buffer (pH2.10)

Perchlorate

Janssen et al., 1980

Serotonin and derivatives

LiChrosorb RP-8

Tri-rc-butyl phosphate

Water/methanol

Perchlorate

De Jong et al., 1983

TBA = tetrabutylammonium TPrA = tetrapropylammonium TMNA = trimethylnonylammonium

234

HIGH PERFORMANCE LIQUID

CHROMATOGRAPHY

i 20

i 10

i 0

Time (min) Fig. 7.2. Separation of aromatic carboxylic and sulphonic acids by reversed phase ion pair partition chromatography. Column packing, LiChrosorb RP-2 (5 jum) loaded with 1-pentanol; column dimensions, 200 x 4.5 mm i.d.; eluent, aqueous TBA, 0.03 M, pH 7.4; flow rate, 1.7 mm/s; pressure 95 bar; detection, UV at 254 nm. Solutes: 1: 4-amino benzoic acid; 2: 3-amino benzoic acid; 3: 4-hydroxy benzoic acid; 4: 3-hydroxy benzoic acid; 5: benzene sulphonic acid; 6: benzoic acid; 7: toluene-4-sulphonic acid. Reproduced with permission from Fransson et al., 1976.

silica packing material as the stationary phase. This technique has been variously called soap chromatography (Knox and Laird, 1976), solvent generated (dynamic) ion exchange (Kraak et al., 1977), hetaeric chromatography (Horvath et al., 1977b), solvophobic-ion chromatography (Hoffman and Liao, 1977), ion interaction chromatography (Bidlingmeyer et al., 1979) and 'chromatography on sorbed ionic sites' (Nilsson and Samuelson, 1981, 1982), since its development almost simultaneously by H a n e y and co-workers (Wittmer et al., 1975; Sood et al., 1976) and Knox and Laird (1976). Both groups independently found that the addition of a hydrophobic quaternary a m m o n i u m salt to the eluent in a reversed phase analysis of sulphonic acid dyestuffs greatly improved the efficiency of the system and caused enhanced retention of the acids. T h e r e has been much discussion about the mechanism of separation, hence the many names for the technique (Horvath et al., 1977b; Kissinger, 1977; Terweij-Groen et al., 1978; Van de Venne et al., 1978; Bidlingmeyer et al., 1979; D e e l d e r et al., 1979; Riley et al., 1979; Tilly-Melin et al., 1979a, b ; Tomlinson et al., 1979; H u n g and Taylor, 1980; Melander and H o r v a t h , 1980; Melander et al., 1980; H u n g and Taylor, 1981; Knox and Hartwick, 1981; Stranahan and D e m i n g , 1982). It is now fairly well agreed that the hydrophobic ion is adsorbed onto the surface of the stationary phase. Several people have measured adsorption isotherms for

ADJUSTMENT OF SELECTIVITY BY USE OF ELUENT ADDITIVES

235

(a)

(b)

(c)

Fig. 7.3. Proposed mechanism for production of dynamic ion exchange capacity by adsorption of a hydrophobic ion from the eluent into the stationary phase.

various anionic and cationic pairing agents (Knox and Laird, 1976; TerweijG r o e n et al., 1978; Van de V e n n e et al., 1978; D e e l d e r et al., 1979; Scott and Kucera, 1979b; Tilly-Melin et al., 1979a, b ; D e e l d e r and Van den Berg, 1981; H u n g and Taylor, 1980, 1981; Knox and Hartwick, 1981; Jansson et al., 1981; Bartha and Vigh, 1983a). It is assumed that the pairing ion is adsorbed onto the hydrocarbon bonded phase by hydrophobic attraction between the two long hydrocarbon chains as shown in Fig. 7.3a. In this way the polar end of the molecule will extend into the mobile phase, presenting an 'ion exchange' like surface to the solute. T h e a m o u n t of pairing agent adsorbed on the surface of the packing material d e p e n d s on the hydrophobicity of the agent. For very hydrophobic pairing ions the surface will be saturated at equilibrium and the system will behave like an ion exchange system. For example, the addition of competing counter ions to the eluent causes a reduction in the retention of the solute ions (Knox and J u r a n d , 1978; Van de V e n n e et al., 1978; C r o m b e e n et al., 1978; Van R o o j et al., 1979; Nilsson and Samuelson, 1981; Knox and Hartwick, 1981; Low et al., 1983a; Bartha et al., 1984). U n d e r many conditions, however, especially with short chain pairing ions (such as the commonly used tetrabutylammonium) the surface will be far from saturated with pairing ion and the effect of added counter ions on the solute retention is minor (Knox and J u r a n d , 1978). Following the observations of amine adsorption o n the packing material by Wahlund and Sokolowski (1978), during the chromatography of hydrophobic amine ion pairs, there has been some discussion about the

236

HIGH PERFORMANCE LIQUID CHROMATOGRAPHY

adsorption sites on the bonded phase surface. Studies indicate that there are two types of adsorption site present on the surface (Wahlund and Sokolowski, 1978; Tilly-Melin et al., 1979a; Jansson et al., 1981; Van der H o u w e n et al., 1981), one of which adsorbs amino c o m p o u n d s . Improved chromatographic efficiency is obtained for amine ion pairs by blocking these sites by inclusion in the eluent of a long chain amine (Sokolowski and W a h l u n d , 1980; H u n g et al., 1982). It seems likely that these acidic adsorption sites could be unreacted silanols on the surface of the bonded material (Tilly-Melin et al., 1979a; Melander et al., 1979b; Jansson et al., 1981). The presence of these sites will lead to an increase in the amount of amine ion pairing agent adsorbed via the mechanisms illustrated in Fig. 7.3b without increasing the ion exchange capacity of the stationary phase, unless a bilayer is proposed as shown in Fig. 7.3c. Such bilayers have been observed when dynamic modification of silica gel surfaces by surfactants is examined (cf. Section 7.2; Wall, 1980). Unfortunately, the amount of these unreacted silanol groups will vary between materials from different manufacturers and possibly even between batches of the same material. This could lead to differences in retention characteristics on transferring methods from one material to another. Slight adjustment of the mobile phase composition is usually all that is necessary to 're-tune' the separation. Columns are prepared by equilibrating the stationary bonded phase with the mobile phase containing the pairing ion. Very hydrophobic pairing ions have such a great effect on the retention of the ionised species that they are used at a very low concentration ( ~ 1 0 - 5 M) and equilibration may take a very long time (several hours) (Knox and J u r a n d , 1978). In this case a deloading technique starting with a more concentrated eluent composition is probably preferred. Less hydrophobic pairing ions are added at a slightly higher concentration ( 1 0 - 4- 1 0 - 3 M) and equilibrium is reached much faster (typically 1-2 h at a flow rate of 1 m l . m i n - 1) . Knox and Hartwick (1981) have illustrated this for three alkyl sulphates on a 100 mm column packed with 5 /mm particles of O D S Hypersil containing a total surface area of 100 m 2. Examination of Fig. 7.4a shows the extremely long equilibrium time required for lauryl sulphate ( C 1 2) at a concentration of 0.1 m M (1.5 1 = 25 h at a flow rate of 1.0 m l . m i n - 1) compared to octyl sulphate ( C 8) (100 ml = < 2 h) at the same concentration. A further consequence of the hydrophobicity of the pairing ion is the slow rate of desorption of the adsorbed pairing ion by elution with mobile phase containing no pairing ion. A s Fig. 7Ab illustrates, strongly adsorbed pairing ions require very large volumes of eluent for complete desorption and it has in fact been possible to continue ion pair chromatography for some time with no pairing ion in the eluent (Terweij-Groen et al., 1978; Knox and J u r a n d , 1978). In practice, when very hydrophobic pairing ions are used the columns are irreversibly altered, since the ions can never be completely removed. W h e n e v e r the pairing ion concentration in the mobile phase is altered the column must be re-equilibrated and this can be monitored by following the change in k' of a suitable solute on repeated injection. T h e r e are no problems associated with having the detector connected under these conditions. Once equilibrium is

ADJUSTMENT O F SELECTIVITY BY USE O F E L U E N T ADDITIVES

237

r

/ 0 - 1 mM

(I)

Cs (jitmol • m~~2)

Cs (jiimol • m~2)

(a)

(b) Volume of eluent (I) Fig. 1 A. (a) Breakthrough volumes for alkyl sulphates at various mobile phase concentrations as indicated, (b) Desorption of alkylsulphates by standard eluent containing no alkyl sulphate. Column packing, ODS-Hypersil (5/xm); column dimensions, 100 x 5 mm i.d.; eluent, water/methanol (80:20) containing 0.018 M K H 2P 0 4 and 0.002 M N a H P 0 4, pH 6.0; Vj, = breakthrough volume, Cs = surface coverage. Reproduced with permission from Knox and Hartwick, 1981.

reached the columns are stable a n d can b e used for several m o n t h s . It is r e c o m m e n d e d , h o w e v e r , that t h e system be flushed with eluent free from ion pairing agent at t h e e n d of each working day to p r e v e n t either clogging fine tubing or disturbance of t h e column b e d by t h e precipitation of salts. It is not

238

HIGH P E R F O R M A N C E LIQUID C H R O M A T O G R A P H Y

usually necessary to use a pre-column. T e m p e r a t u r e effects are still important, but careful thermostatting of the systems as described previously is unnecessary under most routine situations. A s already explained in Section 7.1.3 solute retention can be altered easily by varying 1. The hydrophobicity of the pairing ion: k' increases with hydrophobicity (chain length). 2. The mobile phase concentration of pairing ion: k' increases with [P+]. 3. The organic content of the mobile phase: k' decreases with increasing organic content. 4. The addition of counter ions: k' decreases with [C~]. This flexibility is one of the greatest attractions of the technique (Riley et al., 1980). It is possible to analyse complex mixtures of solutes, whether ionised or not. The presence of pairing ion will increase the retention of ionised solutes of opposite charge, decrease the retention of solutes of like charge and slightly decrease the retention of neutral solutes by removal of available adsorption sites on the surface (Bidlingmeyer et al., 1979; G r a h a m and Rogers, 1980; Knox and Hartwick, 1981; Bartha and Vigh, 1983b). It is also possible to perform gradient elution in this m o d e (Gloor and Johnson, 1977) either by reducing the concentration of the pairing ion or by increasing the ionic strength of the mobile phase to decrease retention. Alternatively, the organic content of the mobile phase can be increased to produce a gradient system (Fiedler, 1981a; H a r t et al., 1981). As a result of its versatility, reversed phase ion pair chromatography with hydrophobic pairing ions has become one of the most widely used techniques in modern liquid chromatography, as indicated by the vast numbers of publications appearing in the applications literature. It would be impossible to give a comprehensive list of references h e r e , but several relevant applications are summarised in Tables 7.2-7.5 and a few interesting examples are discussed more fully below. For further information several relevant reviews have been published (Tomlinson et al., 1978; H e a r n , 1980; Schill, 1981; Tomlinson, 1983; Karger et al., 1980; Bidlingmeyer, 1980).

7.1.5

Selected

applications

T H E USE O F U V - A B S O R B I N G O R F L U O R E S C E N T ION PAIRING AGENTS T O E N H A N C E OR PERMIT DETECTION

As discussed already in Section 7.1.2 UV-absorbing ion pairing agents may be used in the stationary phase to permit the detection of non-absorbing solutes. For some time it was assumed that a similar approach could not be used in the reversed phase m o d e , since the presence of the UV-absorbing component in the eluent would cause too high a background absorbance. Recently, the use of low concentrations of hydrophobic pairing ions and more sophisticated spec-

ADJUSTMENT OF SELECTIVITY BY USE OF ELUENT ADDITIVES

239

trophotometers which allow compensation for this background absorption have permitted the application of these techniques to the m o r e favourable reversed phase situation. High background absorbances (above 0.5), however, decrease the sensitivity of the system and should be avoided ( D e n k e r t et al., 1981; Hackzell et al., 1983). The UV-absorbing or fluorescent c o m p o n e n t in the mobile phase will be distributed to the solid phase as explained in Section 7.1.4 and an equilibrium situation will be attained. T h e injected solutes will influence the distribution of the detectable ion either by binding or displacement, causing a change in the equilibrium and thus giving rise to a detector response. This response can be positive or negative depending on the retention and charge of the solute relative to the absorbing pairing ion. In general, when the sample and detectable ion are of opposite charge, the response will be positive whenever the sample elutes after the peak due to the reagent itself (system p e a k ) . Conversely, if the sample elutes before the system peak a negative peak will be obtained. T h e opposite situation holds when the sample and pairing ion are of the same charge (Hackzell et al., 1983; W a r r e n and Bidlingmeyer, 1984). Detection sensitivity is dependent on the retention ratio between the solute and the detectable mobile phase component. For the best sensitivity, the UV-absorbing or fluorescent ion pairing agent should have high molar absorptivity or fluorescence and it should have a retention similar to the solute with k' in the range 5-10. In particular, the detection sensitivity of c o m p o u n d s with retention times shorter than that of the system peak is generally rather low (Bidlingmeyer and W a r r e n , 1982; D e n k e r t et al., 1981; Hackzell and Schill, 1982; Barber and Carr, 1983; Hackzell et al., 1983). The retention ratio may be altered according to the usual ion pairing rules by varying the concentration of non-detectable ions in the mobile phase. It is also important to choose the most suitable stationary phase based on the hydrophobicity of the pairing ion, since it is not as easy to alter the retention of the system peak. Various b o n d e d phase systems have been examined and in general the m o r e polar types (phenyl and cyanopropyl) appear most suitable for the detection of moderately to highly hydrophobic ions (Hackzell et al., 1983; H e l b o e , 1983), whereas m o r e hydrophilic ions are better c h r o m a t o g r a p h e d on a hydrophobic adsorbent (Hackzell et al., 1983). Detection of the solute ions can be achieved by using an absorbing pairing ion of either similar or opposite charge, since either an increase or a decrease of the pairing ion concentration in the eluent will produce a detector response. Use of a pairing ion of opposite charge to the solute, however, allows m o r e flexibility in tuning the system to maximum sensitivity using the techniques mentioned in Section 7.1.4. and summarised below. 1. A n increase in the mobile phase concentration of a non-absorbing ion of like charge to the solute will compete with the solute for the pairing ion and decrease the retention of the solutes. O p t i m u m detection sensitivity occurs when the retention of solutes and pairing ion are close. 2. Highest detection sensitivities are obtained when other ions of like charge to the pairing ion (e.g. buffer ions) are hydrophobic and present in low concentration.

Table 7.2

Biochemical applications of bonded phase ion pair chromatography.

Solute

Pairing ion

Concn

Eluent

Packing material

Reference

Lincomycin in fermentation beers

SDS

0.01 M

Acetonitrile/ammonium phosphate buffer (pH 6)

Zorbax C8

Asmusetal. 1983

Gentamicin sulphate

PANS

0.015 M

Aqueous soln of pairing ion, 0.2 M sodium sulphate and 0 . 1 % acetic acid

/ot-Bondapak C18

Getek et al., 1983

Nikkomycins

HPTS

0.001 M

Ammonium formate

LiChrosorb RP-8

Fiedler, 1981a

Basic, hydrophilic peptides

HXS

Spherisorb C18

Fransson et al., 1982

Tri-to heptapeptides

Phosphoric acid HXS SDS

/i-Bondapak alkylphenyl

Hancock et al., 1978

Di-to pentapeptides

Tetradecapeptide somatostatin

buffer, pH 4.7

Polyamines in red blood Catecholamines Biogenic amines and metabolites

-0.01 M 0.005M

Phosphate buffer/ethanol

Tetraalkylammonium ions

0.002 M

Methanol/water

/A-Bondapak alkylphenyl

Hancock et al., 1979

PANS

0.03 M

Acetonitrile/phosphate

LiChrosorb RP-8

Abrahamsson and Groningsson, 1980

/A-Bondapak C18

Brown et al., 1982

Ultrasphere I. P. C18

Martinet al. 1983

Ultraspherel. P. C18

Sailer and Salama, 1984

Methanol/water

buffer (pH 4.5) HPTS

0.02 M

Acetonitrile/water

SDS

0.015 M

OCTS.

0.001 M

Acetonitrile or methanol/ buffer Acetonitrile/0.1 M sodium phosphate, 1 mM EDTA

Table 1.2

(Contd.)

Solute

Pairing ion

Concn

Eluent

Packing material

Reference

Norepinephrine, dopamine 5-hydroxytryptamine and metabolites

OCTS

0.0005 M

Methanol/0.1 M citric acid, 0.1 M sodium acetate, 0.15 mM EDTA, I m M dibutylamine

5 fxm C i 8- R C M 100

Warnhoff, 1984

Tyrosine, tryptophan and related monoamines

Sodium octyl sulphate

0.001 M

Acetonitrile/water

Biophase-ODS 5 ^tm C18

Lasley et al., 1984

Biogenic amines

SLS

0.02 M

Acetonitrile/phosphate buffer, 0.5 mM EDTA

ODS-Hypersil

Taylor et al., 1983

Morphine alkaloids

CAMS

0.005 M

Methanol/phosphate buffer

/x-Bondapak C18

Lindberget al., 1981

Opium alkaloids

HPTS

0.005 M

Methanol/water

fx Bondapak C18

Olieman etal., 1977

Nucleobases and nucleosides

SDS

0.004 M

0.1 M HC10 4/ethanol

ODS-Hypersil

Nucleosides and bases

TBA

0.001 M

Phosphate buffer, pH 5.7

Partisil PXS 10/25 C8

Perrone and Brown, 1984

Nucleotides

DTMA

0.0005 M

Methanol/0.1 M phosphate buffer (pH 6.0)

ODS-Hypersil

Gilbert, 1980

5-Alkyluracils and purine bases

Octylsulphate

0.0010.01 M

Methanol/phosphate buffer

ODS-Hypersil

Csarnyi etal., 1981

Biogenic amine metabolites

TEA

0.03 M

Phosphate buffer

ODS-Hypersil

Taylor et al., 1983

Indole-3-acetic acid

TBA

0.01 M

Methanol/phosphate buffer

RP-18

Mitchell et al., 1984

Neopterin phosphates

TEA

Isopropanol/phosphate buffer

LiChrosorb RP-8

Blau et al., 1982

Kraak et al., 1981

Table 7.2

(Contd.)

Solute

Pairing ion

Concn

Eluent

Packing material

Reference

Veratric acid and metabolites

TBA

0.015 M

Methanol or acetonitrile/ water

LiChrosorb-C18

Suprynowicz et al., 1984

Bile acids

TBA

0.005 M

Acetonitrile or methanol/ phosphate buffer (0.005 M)

Nucleosil C18 and C8

Lu et al., 1983

Conjugated bile acids

TBA

-0.005 M

Acetonitrile/water

Ultrasphere I. P. C18

Wildgrube et al., 1983

Free and conjugated bile acids

Hyamine 1622

0.002 M

Methanol/phosphate buffer

Chromegabond C

Parris, 1979a

Steroid 3-sulphates and free steroids

Ammonium sulphate

0.02 M

Methanol/water

Apex-ODS or Spherisorb S5-ODS

Simoniam and Capp, 1984

Haem compounds

TBA

0.001 M

Methanol/water

Supelcosil LC-18

Tangeras, 1984

Doxorubicin and 4'epidoxorubicin

SDS

0.039 M

Acetonitrile/phosphate buffer

ODS-Hypersil

Thomas et al., 1984

DTMA= decyltrimethyl ammonium TEA = tetraethyl ammonium TBA = tetrabutyl ammonium SDS = sodium dodecyl sulphate PANS = pentane sulphonate HXS = hexane sulphonate HPTS = heptane sulphonate OCTS = octane sulphonate SLS = sodium lauryl sulphate CAMS= camphor sulphonic acid

Table 7.3

Application of bonded phase ion pair chromatography to pharmaceutical analysis.

Solute

Pairing ion

Concn

Eluent

Packing material

Reference

Sulphinpyrazone

TBA

0.005 M

Methanol/water

fx Bondapak C18

Patelet al., 1982

Phenylephrine HC1 PANS phenylpropanolamine HC1 quaifenesin

0.005 M

Methanol/water

\x Bondapak C18 or Partisil 10-C8

Schieffer and Hughes, 1983

Isoxicam in plasma and urine

HPTS

0.005 M

Tetrahydrofuran/water/ acetic acid or acetonitrile/water/ acetic acid

IJL Bondapak C18

Daftsioset al., 1984

Primaquine in plasma and urine

OCTS

0.0005 M

Acetonitrile/methanol/ water

Partisil ODS-III

Ward etal., 1984

Ouinidine analogues

OCTS

0.001 M

Methanol/water

IJL Bondapak C18

Al-Kaysietal., 1984

Norfloxacin in plasma and urine

HXS OCTS

-0.0001 M

Methanol/phosphate buffer

/JL Bondapak C18

Pauliukonis et al., 1984

Ritodrine HC1

OCTS

0.0003 M

Methanol/phosphate buffer

fx Bondapak C18

LinL. S.et al.,1984

Chloroprocaine HC1

HPTS

-0.0003 M

Acetonitrile/methanol/ acetic acid

\x Bondapak C18

Menonet al., 1984

Chloroquine acetylated metabolite in plasma and urine

HPTS

0.02 M

Acetonitrile/water

/x Bondapak C18

Brown etal., 1984b

Penicillin complexes

HPTS

0.03Acetonitrile/phosphate 0.035 M buffer

ODS-Hypersil

Irwin etal., 1984

Orphenadrine HC1

SLS

0.04 M

ODS-Hypersil

Selkirk et al., 1984

Acetonitrile/phosphate buffer, pH 4.0

Table 1.3

(Contd.)

Solute

Pairing ion

Concn

Eluent

Packing material

Reference

Diltiazem and desacetyldiltiazem in plasma

HPTS

0.008 M

Acetonitrile/water

fx Bondapak C18

Wiens etal., 1984

7-R-O-methylnogarol

SDS

0.005 M

Water/acetonitrile tetrahydrofuran/glacial acetic acid

Zorbax C8

Zoutendam and Ryan, 1984

Phencyclidine

PANS, HXS, OCTS, MAS

0.005 M

Methanol or acetonitrile/ water glacial acetic acid, pH3.5

tt-Bondapak CN, C18, Brownlee RP-8

Jones et al., 1982

Tricyclic antidepressants

SLS

0.08 M

Acetonitrile/phosphate buffer, pH 2.0 + organic amine

ODS-Hypersil

Hung et al., 1982

Paracetamol metabolites

TBA

0.005 M

Methanol/water containing 0.01 MTris and 0.005 M E D T A pH7.2

it-Bondapak C18

Hart etal., 1981

Resoln of R,S-propanolol diastereomeric derivatives in human plasma

N,N-DMOA

Acetonitrile/phosphate buffer

LiChrosorb RP-18

Hermansson, 1982

Impurities and degradn products of penicillins

TBA

0.008 M

Acetonitrile/phosphate buffer, pH 7.5

Ultrasphere ODS

Ghebre-Sellassie et al. 1982

Degradn products of tetracyclines

TrPrA or N,NDMOA

0.010.02 M

Acetonitrile/phosphate buffer, pH 8.0

LiChrosorb RP-8

Hermansson and Andersson, 1982

Table 7.3

(Contd.)

Solute

Pairing ion

Concn

Eluent

Packing material

Reference

Chlorophacinone residues in animal tissues

TBA

0.005 M

Methanol/water

ODS-Hypersil

Hunter, 1984

Tetracyclines

1-hydroxy2,3-diisobutyl benzene sulphonic acid

0.004 M

Acetonitrile/phosphate buffer

LiChrosorb N H 2

Eksborg, 1981

TBA = tetrabutyl ammonium N,N-DMOA= N,N-dimethyloctylamine TrPrA = tripropylamine Tris = tris (hydroxymethyl) aminomethane EDTA = ethylene diamine tetraacetic acid PANS = pentane sulphonate HXS = hexane sulphonate OCTS = octane sulphonate MAS = methane sulphonate HPTS = heptane sulphonate SLS = sodium lauryl sulphate

Table 1.4 Application of bonded phase ion pair chromatography to organic analysis. Solute

Pairing Ion

Concn

Eluent

Packing material

Reference

Naphthalene acetic acids

TPrA

0.010.05 M 0.010.04 M

Methanol or acetonitrile/phosphate buffer

LiChrosorb RP8 or Spherisorb ODS

Westerlund and Theodorsen, 1977

Naphthalene and anthraquinone sulphonic acids

TMA, TEA, TBA

0.0050.01 M

Methanol/water

Silasorb C18 Silasorb C8

Jandera et al., 1983

Aromatic sulphonic acids

TBA

-0.001 M

Methanol/phosphate buffer

LiChrosorb RP8

Prandi and Venturini, 1981

Fluorescent whitening agents in detergents

TEA

0.005 M

Methanol/acetate buffer

Parathion metabolites

TBA

0.005 M

Acetonitrile/water (40 °C)

RSilC18

Rosenberg and Nakatsugawa, 1984

4(5)-Methylimidazole in foodstuffs

SDS

0.005 M

Methanol/phosphate buffer

Nucleosil 5 C8

Thomsen and Willumsen 1981

Anthracyclines

SDS

0.010.06 M

Acetonitrile/phosphate buffer

Spherisorb S5 ODS

VanLanckeret al., 1983

2,6-Disubstituted anilines

OCTS

0.01 M

Methanol/water

/JL Bondapak C18

Stranahanetal., 1980

HI-6 [4-carbamoyl-2' hydroxyiminomethyl1,1-oxydimethylendi(pyridinium chloride)]

HPTS

0.01 M

Acetonitrile/water

JJL Bondapak C18

Brown et al., 1984a

Flavonol sulphates

TBA

0.01 M

Water buffered to pH2.2 with acetic acid/formic acid

Spherisorb S5-ODS-2

Harborne and Boardley 1984

Micalietal., 1984

Table 1.5 Application of bonded phase ion pair chromatography to inorganic analysis. Packing material

Reference

LiChrosorb RP-18

Molnar et al., 1980

Methanol/phosphate buffer

Sil 60-D 10-CN

Reeve,1979

0.00010.001 M

Methanol or acetonitrile/water or water + buffer

Supelcosil LC-18; Partisil PXS 10/25 ODS-3; MCH-10; PRP-1

Cassidy and Elchuk, 1982

Cetylpyridinium

0.001 M

TMA or TBA salicylate in acetonitrile/water

PRP-1 Supelcosil LC-18 Radial Pak C18

Cassidy and Elchuk, 1983b

Anions

TBA

0.0010.002 M

Phosphate buffer

Silasorb 300 ODS

Vespalec et al., 1984

Iodide

Dibutylamine phosphate

0.01 M

Methanol/water

LiChrosorb RP-18

Miyashita etal., 1984

Eluent

Solute

Pairing Ion

Concn

Inorganic cations/anions

HPTS/TBA

Aqueous soln of pairing 0.005 M/ 0.002 M ion, pH 2.0/phosphate buffer pH6.7

Main group anions

Cetrimide

Cations/anions

Q 2 , C20Sulphate C 6, C 8 Sulphonate TEA, TBA, TrOMA, TOA, TrDDMA

Anions

TPrA TBA TMA TEA SDS OCTS

= = = = = =

tetrapropylammmonium tetrabutyl ammonium tetramethyl ammonium tetraethyl ammonium sodium dodecyl sulphate octane sulphonate

-0.003 M

Table 7.5

(Contd.)

Solute

Pairing Ion

Concn

Eluent

Packing material

Reference

Heteroborane anions

C i 2o r

0.0027 M

Methanol/water

Q-bonded phase; Separon SI Q ; Separon SI C18

Plzak et al., 1981

C 6-alkylamine Boron (as chromotropic acid complex)

TBA

0.011 M

Methanol/phosphate buffer

TSK LS-410K (ODS)

Motomizu et al., 1983

Pentaamine cobalt(III) complexes

Toluenesulphonate

0.025 M

Methanol/water

it-Bondapak C18

Buckingham et al., 1983

Palladium(II)-thioether peptide complexes

Cetrimide TrOMA

0.002 M

Methanol/phosphate buffer, pH 3.0

ju-Bondapak C18

Lam-Thanh et al., 1982

Iron, cobalt, nickel chelates with 4-hydroxy-3-(2pyridylazo) naphthalene-1sulphonic acid

TBA

0.025 M

Acetonitrile/water

Silica-ODS

Wada et al., 1984

139

OCTS

0.0050.01 M

ar-Hydroxy isobutyric acid/water

Supelcosil C18

Knight et al., 1984

Trioctylphosphine oxide and impurities

TOA TDA TDDA Cetrimide

0.0020.003 M

Methanol/phosphate buffer, pH 8/methylene chloride

LiChrosorb RP-8 Hypersil C8

Kolosky et al., 1984

Inorganic and organic anions

N-OA

0.01 M

Water, pH 6-6.5

Partisil 10/25 ODS, ODS-2, ODS-3 and C8; LiChrosorb RP-18, RP8

Skelly, 1982

1,10-Phenanthroline complexes of Fe(II), Ru(II),and Ni(II)

MAS HPTS

0.015 M 0.00010.01 M

Methanol or acetonitrile/water glacial acetic acid

fx Bondapak C18 or CN

L a in fission products

O'Laughlin and Hanson, 1980

Table 1.5

(Contd.)

Solute

Pairing Ion

Concn

1-10-Phenanthroline complexes of Fe(II), Ru(II),Ni(II),Zn(II), Co(II),Cd(II) and Cu(II)

Perchlorate

0.00010.02 M

TBA - tetrabutyl ammonium Cetrimide = cetyltrimethyl ammonium TEA = tetraethyl ammonium TrOMA = trioctylmethyl ammonium TOA = tetraoctyl ammonium TrDDMA= tridodecylmethyl ammonium TDA = tetradecyl ammonium TDDA = tetradodecyl ammonium N-OA = N-octylamine HPTS = heptane sulphonate OCTS = octane sulphonate MAS = methane sulphonate

Eluent Acetonitrile/water

Packing material

Reference

/x-Partisil SCX PRP-1

O'Laughlin, 1982

250

HIGH P E R F O R M A N C E L I Q U I D C H R O M A T O G R A P H Y

3. Addition of a non-ionic modifier, e.g. methanol, is a very useful way of decreasing the retention of both system and sample peaks without changing the relative elution order; however, detection sensitivity may also be reduced. 4. The detection sensitivity for compounds of low retention can be improved if the mobile phase contains two UV-absorbing ions with opposite charges and hydrophobicities but both having absorptivities in the same wavelength range (Hackzell and Schill, 1982). Because systems of this type involve so many variables which can affect the sensitivity of detection, it is crucial that quantitive analysis be performed under very carefully controlled conditions to ensure adequate reproducibility and precision (Bidlingmeyer and W a r r e n , 1982; Barber and Carr, 1983; Hackzell et al., 1983; W a r r e n and Bidlingmeyer, 1984). U V or fluorescent visualisation techniques like these have been applied to the detection of free and conjugated bile acids (Parris, 1979a), surfactants (Parris, 1980), amino acids, dipeptides (Denkert et al., 1981) sulphonic and carboxylic acids ( D e n k e r t et al., 1981; Petterson and N o , 1983), alkylammonium compounds ( D e n k e r t et al., 1981; H e l b o e , 1983) alkylsulphonates ( D e n k e r t et al., 1981; Sachok et al., 1982) and inorganic ions (Dreux et al., 1982; Barber and Carr, 1983). The usefulness of this technique for the quantitative analysis of inorganic ions is illustrated in Fig. 7.5. Similar approaches have been used to enhance the detection of non-ionic species such as aliphatic alcohols ( G n a n a s a m b a n d a n and Freiser, 1981, 1982a; Parkin, 1984a) monosaccharides ( G n a n a s a m b a n d a n and Freiser 1982b) and ketones ( G n a n a s a m b a n d a n and Freiser, 1981).

R E S O L U T I O N O F ENANTIOMERS BY ION PAIR C H R O M A T O G R A P H Y

Resolution of enantiomers by H P L C can be achieved in several different ways as discussed in Chapter 9. H o w e v e r , only in recent years has it proved possible to resolve enantiomers directly by the inclusion of a chiral pairing ion in the eluent (Yoneda, 1979; Pettersson and Schill, 1981, 1982; Pettersson and N o , 1983). Resolution by ion pair chromatography is assumed to result from the interaction of the chiral pairing ion with the enantiomers to give two diastereomeric ion pairs. Studies have indicated that there are certain structural criteria necessary to obtain stereoselectivity. This corresponds to the three-point interaction necessary for chromatographic enantiomeric resolution obtained in all previous systems (see Chapter 9). A simultaneous electrostatic interaction and hydrogen bonding between the ions appears vital for the separation, and a third interaction will occur between the 'diastereomeric ion pair' and the stationary phase. The intermolecular distance between the two functional groups which produce the necessary interactions is therefore important. For example, Pettersson and Schill (1982) found that only amino alcohols having an alkyl chain of two carbons between the hydroxy group and the amine function could be resolved with (+)-10-camphorsulphonic acid as pairing ion. The structure of the pairing ion is also important, and improved stereoselectivity is observed

251

UV detector response

ADJUSTMENT OF SELECTIVITY BY USE OF ELUENT ADDITIVES

4 VR(m\)

Fig. 7.5. Detector response and retention volume for various concentrations of injected sample: 50/xl injection of 1, 0.65, 0.3 and 0.1 mM each of C I - , N 0 2 - , B r - and N O 3 - . Column packing, Supelco LC18DB; column dimensions 50 mm x4.6 mm i.d.; eluent, 0.25 mM hexane sulphonate, 4 mM a-naphthylmethyl tributylammonium and 10 mM acetic acid/sodium acetate in water at pH 4.75; flow rate, 1 m l . m i n - 1; column temperature, 25 ± 0.1 °C; detection, UV at 316 nm, background absorbance of approximately 1.1 a.u. Reproduced with permission from Barber and Carr, 1983.

when t h e pairing ion contains bulky, rigid groups in t h e vicinity of t h e chiral centre (Pettersson and N o , 1983). This presumably enhances t h e sterochemical differences between the diastereomeric ion pairs. Similar results have been observed when diastereomeric derivatives a r e used t o resolve enantiomers by either G L C or H P L C (see Chapter 9). The particular advantages of using large fairly rigid cyclic molecules such as quinine and quinidine (see Fig. 7.6) as pairing ions are indicated by t h e efficient resolution of various carboxylic a n d sulphonic acid enantiomers (Pettersson a n d N o , 1983). These pairing ions can also be used to enhance the U V detection of weakly absorbing solutes. This is well illustrated in Fig. 1.1 by t h e resolution and detection of (±)-o>tertbutoxycarbonylphenylalanine using quinidine as pairing ion. A novel approach to chiral resolution employs two electrostatic interactions for the necessary two-point interaction between solute and pairing ion by using a

252

HIGH PERFORMANCE LIQUID CHROMATOGRAPHY 4

(a)

(b)

Fig. 7.6. Structures of rigid pairing ions suitable for enantiometric resolutions, (a) quinine, x = OCH 3[3(R), 4(S), 8(S), 9(R)], or cinchonidine, x = H[3(R), 4(S), 8(S), 9(R)]; (b) quinidine, 3(R), 4(S), 8(R), 9(S).

(+)

H|

i

i

i

10

5

0

Time (min) Fig. 1.1. Enantiomer resolution of (±)-N-tert-butoxycarbonyl-phenylalanine by ion pair chromatography. Column packing, LiChrosorb DIOL (5/xm); column dimensions, 150 x 3 mm i.d., eluent, 3.5 x 10~ 4 M quinidine and 3.5 x 10~ 4 M acetic acid in dichloromethane/l-pentanol (99:1); column temperature, 25 °C; detection, UV at 337 nm. S = system peak. Reproduced with permission from Pettersson and No, 1983.

ADJUSTMENT OF SELECTIVITY BY USE OF ELUENT ADDITIVES

253

chiral pairing zwitterion with a zwitterionic solute (Knox and J u r a n d , 1982). Using this technique the enantiomeric resolution of tryptophan and glycylphenylalanine has been achieved with L-leucyl-L-leucyl-L-leucine as pairing ion. This is discussed m o r e fully in the following section. The topic of enantiomer resolution and the structural requirements necessary for resolution are considered further in C h a p t e r 9. 7.1.6

Zwitterion

pair

chromatography

A further development of ion pair chromatography has been the elution of dipolar solutes in association with a zwitterionic pairing agent. This was first discovered by Knox and J u r a n d (1979) during work on the liquid chromatography of tetracyclines in the presence of E D T A . Maximal retention of each tetracycline occurred at a p H intermediate between the isoelectric points of the tetracycline and E D T A and it was proposed that this was d u e to the formation of zwitterion pairs between the two dipolar species S± + P + ^ aqueous phase

{S± + P } organic phase

Subsequent work using 11-amino undecanoic acid as pairing agent (Knox and Jurand, 1981a, b , c) has substantiated this hypothesis. Very good separations for nucleotides (Knox and J u r a n d , 1981a,b), ampicillin, lysergic acid and tryptophan (Knox and J u r a n d , 1981c) have been achieved but the retention is very closely d e p e n d e n t on p H as shown in Fig. 7.8. A very slight change in p H from 5.35 to 5.65 results in considerably reduced retention for the nucleotides. As mentioned previously, use of this technique with a chiral pairing zwitterion, L-leucyl-L-leucyl-L-leucine has m a d e it possible to resolve the enantiomers of tryptophan and glycylphenylalanine (Knox and J u r a n d , 1982). Very careful control of the eluent composition was found to be necessary, since the separation was destroyed by the presence of metal ions. E x t r e m e care must be taken in reproducing all the eluent conditions in zwitterion pair chromatography, since very slight changes can produce very large effects on retention and resolution. So far, this m e t h o d has not been widely exploited but it has potential for further development.

7.1.7

Mice liar

chromatography

The preceding discussion has concerned the addition of surface active agents below their critical micelle concentration ( C M C ) . Recently, however, there has also been some interest in the use of these agents at concentrations at which they will form micelles. This form of chromatography, where a reversed phase column is used in conjunction with an a q u e o u s micellar eluent, has been referred

254

HIGH PERFORMANCE LIQUID CHROMATOGRAPHY pH 5-65

11 3+4

10

i

20

10

0

30

20

10

0

Time (min) Time (min) Fig. 7.8 Effect of pH on the separation of nucleotides by zwitterion-pair chromatography. Column packing, ODS-Hypersil (5/xm); column dimensions, 100 x 5 mm i.d.; eluent, water/methanol (88:12) made 75 mM in phosphate and 1.25 mM in 11-amino undecanoic acid; column temperature, 25 °C; detection, U V at 254 nm. Solutes: 1: inosine monophosphate; 2: guanosine monophosphate, 3: adenosine monophosphate; 4: deoxyguanosine monophosphate; 5: deoxyadenosine monophosphate; 6: guanosine diphosphate; 7: adenosine diphosphate; 8: 3',5'-cyclic adenosine monophosphate; 9: NADH; 10: guanosine triphosphate; 11: adenosine triphosphate. Reproduced with permission from Knox and Jurand, 1981a.

is

Micelle

Bulk water

Stationary phase Fig. 7.9. A schematic representation of the three 'phase' model for micellar chromatography. K mw = partition coefficient between micelle and water. K sw = partition coefficient between stationary phase and water. K sm = partition coefficient between stationary phase and micelle. Reproduced with permission from Armstrong and Nome, 1981.

ADJUSTMENT OF SELECTIVITY BY USE OF ELUENT ADDITIVES

255

to as pseudophase chromatography (Armstrong, 1980). In this situation, the solute partitions between the stationary phase and water, between the stationary phase and the micelle, and between the micelle and water, and the elution characteristics of the solute depend on the combined effects of the three partition coefficients as illustrated in Fig. 7.9. A r m s t r o n g and N o m e (1981) have derived equations to determine these partition coefficients. A three-phase equilibrium model relating capacity factor to micellar mobile phase concentration has been proposed by A r u n y a n a r t and Love (1984). This approach assumes that the micelle-stationary phase c o m p o n e n t can be neglected, and allows calculation of micelle-solute equilibrium constants. If the equilibrium constants can be obtained from independent m e t h o d s , the equations can be used to predict capacity factors for the solutes. The net effect of the addition of the micelle to the eluent is to cause a reduction in the retention of the solute compared to that obtained with neat water as eluent, i.e. it acts as an organic modifier ( G r a h a m and Rogers, 1980; Armstrong and H e n r y , 1980; A r m s t r o n g and N o m e , 1981; Y a r m c h u k et al., 1982). T h e retention of all solutes decreases with increasing surfactant concentration but since the rate of decrease varies depending on the relative strengths of the solute-micelle association and the solute-stationary phase interaction, retention order reversals can be observed with increasing concentration of surfactant. This effect can be exploited to control selectivity. O n e important consideration is the effect of the electrostatic interaction when the surfactant and solutes are ionised. A s already discussed for surfactants below their C M C , this can be a repulsive or attractive effect depending on the relative charges of the surfactant and solute. Fig. 7.10 shows the improvement in resolution obtained for nitrobenzene, 2-naphthol and toluene by changing the surfactant from anionic sodium lauryl sulphate to cationic decyltrimethylammonium bromide. The efficiencies obtained in micellar chromatography were initially less than those of conventional reversed phase ion pair chromatography, due primarily to the large mass transfer term involving the micelles (Yarmchuk et al., 1984). It has now been shown, however, that the efficiency can be optimised by use of elevated t e m p e r a t u r e s , low flow rates and minimal micelle concentration (Yarmchuk et al., 1984) or by improving the wetting of the stationary phase surface by addition of a small percentage of organic modifier to the eluent in conjunction with elevated t e m p e r a t u r e s (Dorsey et al, 1983). T h e excellent efficiencies which can be achieved under these conditions are illustrated in Fig. 7.11. T h e rapid re-equilibration of the system when the micellar concentration is changed means that this form of chromatography is ideal for gradient programming (Dorsey et al., 1984; Landy and Dorsey, 1984). The technique of micellar chromatography has been combined with micellestabilised room t e m p e r a t u r e phosphorescence for the detection and quantitation of aromatic molecules ( A r m s t r o n g et al., 1981; Weinberger et al., 1982). The mobile phase of sodium thallium lauryl sulphate could be used both for chromatography and detection. T h e use of cetyltrimethylammonium bromide above its C M C has been effective for the resolution of dithiocarbamates

HIGH PERFORMANCE LIQUID CHROMATOGRAPHY

Absorbance

256

I ime (mm)

Fig. 7.10. Anionic and cationic micellar chromatograms. Column packing, Supelcosil LC-1 (5 /xm); column dimensions, 150 x 4.6 mm i.d.; eluent, (a): 0.05 M sodium lauryl sulphate, (b): 0.05 M dodecyltrimethyl ammonium bromide; column temperature, 25 °C; detection, UV at 254 nm. (a) 0.02 AUFS, (b) 0.01 AUFS. Solutes: 1: nitrobenzene; 2: 2-naphthol; 3: toluene Reproduced with permission from Yarmchuk et al., 1982.

2

I

Absorbance

3 I

I

4 5

8

16

Time (min)

25

Fig. 7.11. Separation by micellar chromatography. Column packing, Ultrasphere ODS; column dimension, 250 x 4.6 mm i.d., eluent, propanol/water (10:90) + 1 x lO" 1 M sodium dodecyl sulphate, flow rate, 1.5 m l . m i n - 1, column temperature 40 °C; detection, UV at 254 nm; solutes: 1: phenol; 2: acetophenone; 3: nitrobenzene; 4: benzene; 5: toluene. Reproduced with permission from Dorsey et al., 1983.

ADJUSTMENT OF SELECTIVITY BY USE OF ELUENT ADDITIVES

257

(Kirkbright and Mullins, 1984) and micellar chromatography with hexadecyltrimethylammonium chloride has allowed the separation and determination of inorganic anions (Mullins and Kirkbright, 1984). Recently a non-ionic ethoxylated alcohol surfactant has been used in a phosphate buffer eluent to separate proteins (Barford and Sliwinski, 1984).

7.2

Chromatography on dynamically modified oxide gels

An obvious development arising from the observation that hydrophobic amines were adsorbed on the surface silanols of silica gel was the intentional adsorption of a long chain amine from an aqueous mobile phase onto a silica gel surface to produce a dynamic reversed phase material (Knox and Laird, 1976; G h a e m i and Wall, 1979; C r o m m e n , 1979b; H a n s e n , 1981a). T h e technique was extended by Ghaemi and Wall to included the use of non-ionic surfactants ( G h a e m i and Wall, 1980), anionic surfactants in conjunction with basic aluminium oxide gels (Ghaemi et al., 1981) and the co-adsorption of mixtures of non-ionic and ionic surfactants to produce dynamic ion exchange systems (Wall, 1980; G h a e m i and Wall, 1981). Detailed studies of the silica gel/cationic surfactant system have been m a d e by H a n s e n and co-workers ( H a n s e n , 1981a; H a n s e n et al., 1981, 1982,1983a, b ; H a n s e n and H e l b o e , 1984). It seems p r o b a b l e that a mono-layer of surfactant is adsorbed on the silica surface via electrostatic association of the amino group with the acidic silanol or by hydrogen bonding of the non-ionic surfactant with the surface silanols. Subsequently, a partial bilayer is formed more slowly by hydrophobic interaction between the alkyl chains of the b o u n d surfactant and those of the surfactant dissolved in the eluent (Fig. 7.12). The columns are p r e p a r e d in a m a n n e r similar to that described in Section 7.1.4, by equilibration with eluent containing the surfactant. W h e r e low concentrations of surfactant are desired equilibration can take a long time and initial loading from a m o r e concentrated eluent is advised. Equilibration can again be followed by monitoring k' values for suitable solutes, until stable values are obtained. The efficiency of the dynamic system is similar to that obtained with b o n d e d reversed phases, but these systems have the advantage of using cheaper packing materials. Retention and selectivity may be controlled by varying the concentration or nature of the surfactant, the ionic strength and the p H of the buffer, or by changing the concentration or nature of the organic modifier in the mobile phase. A n examination of these effects has been reported for eluents containing cetyltrimethylammonium bromide (Hansen et al., 1983b; Hansen and H e l b o e , 1984). In the case of co-adsorption of two surfactants on silica, the retention of solutes is a function of the concentrations of both surface-active agents in the eluents. Greatest selectivity in control of relative retention is, however, achieved by variation of the concentration of the ionic rather than the non-ionic surfactant. This technique has been used extensively for the chromatography of urinary

258

HIGH PERFORMANCE LIQUID CHROMATOGRAPHY 0

I - + .

.Si-O

NR3

H

,0

(a) reversed phase

\

- o n

.Si-o

+

NR;

\

^ N R 3

H {b) anion exchange

O \

X H 2O S 0 3"

H—(X I ^ ( C H 2C H 20 ) ^ - C H

-O-Si-0 \ / /O H

^O \ I - 0 - S i - 0 - ( C H 2C H 20 ) ^ - C H

Fig. 7.12.

C H 2O S 0 3~

(c) cation exchange Proposed mechanism for production of dynamic stationary phases.

Time (min) Fig. 7.13. Separation of tyrosinyl peptides by dynamic cation exchange chromatography. Column packing, Hypersil (5 /im); column dimensions, 113 x 5 mm i.d.; eluent, water/methanol (60:40), pH 3.08, containing 5 x 10~ 4 M Tween 40 and 5 x 10~ 3 M sodium dodecyl sulphate; flow rate, 0.6 m l . m i n _ 1; detection, UV at 275 nm. Solutes: 1: alanine-tyrosine; 2: valine-tyrosine; 3: valine-tyrosine-valine; 4: leucine-tyrosine; 5: glycine-leucine-tyrosine. Reproduced with permission from Wall, 1980.

ADJUSTMENT OF SELECTIVITY BY USE OF ELUENT ADDITIVES

259

acids and their metabolites ( H a n s e n , 1981b; H a n s e n and Doessing, 1982) and amines (Svendsen and G r e i b r o k k , 1981; Bidlingmeyer et al., 1982; H e l b o e , 1982; Jansson et al., 1982). Fig. 7.13 shows the separation of tyrosinyl peptides by dynamic ion exchange chromatography (Wall, 1980). Recently Takeuchi and co-workers (Takeuchi and Ishii, 1983b; Takeuchi et al., 1983d) have reported the advantage and ease of preparation of open-tubular columns dynamically modified with long chain a m m o n i u m ions c o m p a r e d to the difficulties involved in the preparation of narrow bore immobilised or chemically b o n d e d opentubular columns.

7.3

Metal complexation

O n e major area of liquid chromatography involving metal complexation is the use of ligand exchange chromatography for the resolution of enantiometric amino acids. This topic is discussed in C h a p t e r 9 and the present section is concerned only with cases where the metal is used as a mobile phase additive. The metal ions may be added to the mobile phase directly along with anions of weak complexing capacity or as complexes with various chelating agents. Both situations will be considered. 7.3.1

Direct addition of metal to the mobile

phase

Although this section is primarily concerned with the reversed phase use of metal ions in the mobile phase it is perhaps pertinent to mention the evolution of this technique from classical column chromatography utilizing the complexing abilities of metal salts adsorbed on suitable solid supports ( G u h a and J a n a k , 1972). The most commonly used system utilised the 77-election complexing abilities of adsorbed silver ions and, not suprisingly, this was the first to be transferred to m o d e r n H P L C (Vivilecchia et al. 1972; Mikes et al., 1973; H e a t h et al., 1975; Houx and V o e r m a n , 1976; Aigner et al., 1976; H e a t h et al., 1977; Scholfield, 1980; Joyce and U d e n , 1983). Similar systems were also investigated with rhodium (Mikes et al., 1973), cadium (Kunzru and Frei, 1974) and thallium (Siouffi et al., 1977) impregnated silica columns. The first p a p e r on reversed phase argentation chromatography was published in 1975 by Schomburg and Zegarski, and several publications have followed (Tscherne and C a p i t a n o , 1977; Vonach and Schomburg, 1978; Phelan and Miller, 1981). O t h e r metals which have been used in reversed phase systems are Ni(II) (Sternson and d e W i t t e , 1977; Cooke et al., 1978; Smith et al., 1980, 1981), Z n ( I I ) (Cooke et al., 1978), Cu(II) (Grushka et al., 1982, 1983b) and Co(II) (Smith et al., 1980, 1981). Recently d'Avila et al. (1983) have examined several metals for their ability to complex with azaarenes and have concluded that silver is probably the best choice. Equilibration with the eluent containing the metal ion is very rapid, allowing alteration of the elution characteristics by changing the metal ion concentration and the possibility of gradient elution. Metal ions are normally added in the

260

HIGH PERFORMANCE LIQUID CHROMATOGRAPHY

range 10~ 5 - 10~ 2 M. There is no evidence for metal adsorption on the stationary phase and the formation of the metal-solute complex can be written as follows Sm + nMm \[KX Ss

^

S M n >m \\K2 S M n, s

where m and s represent the stationary and mobile phase, respectively. Km, K± and K2 are the formation constants of the complex (SM) in the mobile phase and the partition constants of the solute (S) and the complex, respectively. The capacity factor is given by (7.8) where 0 is the phase ratio. By substitution of the expressions for the equilibrium constants it follows that (7.9) which becomes (7.10) when k'0 and k'c are the values of k' when [ M ] m = 0 and [ M ] m —» °o respectively. It is obvious from Equation 7.10 that the change in k' for a given solute when a metal ion is added to the system depends on the formation constant of the complex and the concentration of the metal ion present. The stronger the complex the more quickly is the solute eluted in a reversed system. A knowledge of the tendency of metals to form complexes can enable the right choice of metal to be m a d e for a particular separation. T h e choice of metal is restricted, however, by other criteria such as the solubility of the salt in the commonly used eluent system, and the compatibility of the metal ion with the equipment and the detection system. Generally, the oxidation potential of the mechanical parts of the chromatographic system must be lower than the oxidation potentials of any metals added to the eluent to avoid problems from metal deposition in the system. A n o t h e r important consideration is the toxicity of the metal salt to be added. From a chromatographic point of view the kinetic aspects of the metal-solute complexation process are important, since these determine the degree of band broadening. Rapid association-dissociation processes correspond to symmetrical peaks and high efficiencies (Karger et al., 1980). W h e n the complexation rates

ADJUSTMENT OF SELECTIVITY BY USE OF ELUENT ADDITIVES

261

are slow, broad and asymmetrical bands are obtained if the free and complexed species have different migration rates. This situation may b e improved by m e a n s of temperature control or by altering the solvent ionic strength. Usually the best solution is to change the complex (Kohler and Schomburg, 1981). The irreversible formation of the complex (no dissociation during the migration through the column) is similar to pre-column derivatisation, provided there is enough metal to complex all solute molecules, and results in good efficiencies and peak shapes (Smith et al., 1980, 1981). A n extension of this technique is the preparation of the complexes prior to chromatography, thus avoiding the problems associated with the presence of the metal ions in the eluent (Kohler and Schomburg, 1981, 1983). Various applications of direct metal complexation in the mobile phases are summarised in Table 7.6 and the technique has been reviewed by Smith (1984a). Fig. 7.14 illustrates the separation of several polynuclear N-heterocyclic compounds by complexation with silver ions in the mobile phase.

10 min Fig. 7.14. Separation of polynuclear N-heterocyclics using silver complexation. Column packing, Nucleosil 5 C18 (5 /xm): column dimensions 150 x 4 mm i.d.; eluent, (a) methanol/water (1:1 v/v); (b) A + 10" 2 M AgC10 4; flow rate, 0.5 ml.min- 1; column temperature, 24 °C; detection, UV at 254 nm. Solutes: 1: quinoline; 2: isoquinoline; 3: benzene; 4: phenazine; 5: acridine; 6: 5,6-benzoquinoline; 7: 7,8-benzoquinoline; 8: naphthalene. Reproduced with permission from Vonach and Schomburg, 1978.

Table 7.6

Applications using metal complexation in the mobile phase.

Solute

Packing material

Mobile phase

Metal ion

Reference

Mono- and polyunsaturated hydrocarbons

Nucleosil5C18

Methanol/water

Ag+

Vonach and Schomburg, 1978

Unsaturated hydrocarbons

LiChrosorb RP8

Isopropanol/water

Ag+

Schomburg and Zegarski, 1975

+

Heterocyclic hydrocarbons

Nucleosil5C18

Methanol/water

Ag

Azaarenes

Nucleosil5C18 LiChrosorb RP18 ODS-Hypersil

Methanol/water

Various

d'Avilaetal., 1983

Unsaturated fatty acid esters and triglycerides

Nucleosil5C18

Methanol/water

Ag+

Vonach and Schomburg, 1978

Unsaturated fatty acid esters

LiChrosorb RP18

Isopropanol/water

Ag+

Schomburg and Zegarski, 1975

D-vitamins

ix Bondapak C18

Methanol/water

Ag+

Tscherne and Capitano, 1977

+

Tscherne and Capitano, 1977

Estrogenic compounds

ix Bondapak C18

Methanol/water

Ag

Insect pheromones

fx Bondapak C18

Methanol/water

Ag+ +

Vonach and Schomburg, 1978

Phelan and Miller, 1981 Sternson and deWitte, 1977

Aniline and metabolites

ix Bondapak C18

Methanol/water

Ni

Amino sugars and peptides

600-NH amino (Alltech)

Acetonitrile/2,2,2trifluoroethanol/CdS04 or zinc acetate

Cd+ + , Zn+

Dua and Bush, 1982

Amino acids

Partisil 10 ODS

Water

Cu+ +

Grushkaet al., 1982

N-methylamino acids

ODS

Water

Cu+ +

Grushka et al., 1983b

N-alkyl and NN-dialkyldithiocarbamates

ODS-Hypersil

Methanol/water

Co+ + ,Ni+

Smith et al., 1980, 1981

Tab lei.6

(contd.)

Solute

Packing material

Mobile phase

Metal ion

Reference

Sulpha drugs

LiChrosorb RP8

Acetonitrile/water

Zn+ + ,Ni+ +

Cooke et al., 1978

Dipeptides

LiChrosorb RP8

Acetonitrile/water

Zn+ + ,Ni+ +

Cooke etal., 1978

Isomeric amino benzoic acids

IX Bondapak C18

Methanol/phosphate buffer

Zn+ +

Walters and Raghavan, 1979

Cisltrans isomers of fatty acid esters

Partisil-10-ODS Spherisorb-S5-ODS

Methanol/water

Ag+

Chan and Levett, 1978

Nucleotides

Dithiocarbamate bonded Partisil-10

Phosphate buffer

Mg+ +

Grushka and Chow, 1980

264

7.3.2

HIGH PERFORMANCE LIQUID CHROMATOGRAPHY

Addition

of metal-chelate

complex

A n alternative to the direct addition of metal ions as weak salts to the mobile phase is the addition of a metal along with a hydrophobic chelating agent. This method was developed by C o o k e et al. (1978) using 4-dodecyldiethylenetriamine ( C 1 2- d i e n ) as the chelating agent and Z n ( I I ) as the metal ion. The hydrophobic portion of the chelating agent is adsorbed on the stationary phase in a m a n n e r similar to a hydrophobic ion pairing agent. Association of the metal ions with the chelate produces a dynamic ligand exchange system. In such a situation the stronger the complex formed between the solute and the metal-chelate the longer is the retention of the solute. C o o k e et al. found these systems to be very efficient for the separation of sulpha drugs, aromatic acids, dansyl amino acids and dipeptides. T h e resolution of several sulpha drugs is illustrated in Fig. 7.15. They showed that the metal-solute association occurred

3,4

0

4

8 12 Time (min)

16

20

Fig. 7.15. Separation of sulpha drugs by Ci2-dien-Zn(II) chromatography. Column packing: LiChrosorb C8 (10 /xm); column dimensions: 250 x 4.6 mm i.d.; eluent, acetonitrile/water (35:65), containing 1 0 - 3 M Z n S 0 4, 0.025% Ci2-dien, 1% ammonium acetate; column temperature, 30 °C; detection, UV at 254 nm. Solutes: 1: sulphacetamide; 2: sulphanilamide; 3: sulphabenzamide; 4: sulphisoxazole; 5: sulphaquinoxaline; 6: sulphapyridine; 7: sulphadiazine; 8: sulphamerazine; 9: sulphamethazine; 10: sulphathiazole; 11: sulphadimethoxine. Reproduced with permission from Cooke et al., 1978.

ADJUSTMENT O F SELECTIVITY BY U S E O F E L U E N T A D D I T I V E S

265

through an outer-sphere complex, i.e. without disruption of the inner co-ordination sphere of the metal, and since this association-dissociation is usually a fast process it results in good efficiency and p e a k shape. T h e metal and chelating agent should be a d d e d in 1:1 stoichiometric ratio for m a x i m u m efficiency and selectivity. After equilibration the solute retention can be adjusted by varying the ionic strength of the eluent (kf decreases with increasing ionic strength) and the organic content of the mobile phase (k' decreases with increasing organic c o n t e n t ) . T h e columns have b e e n found to be stable and reproducible provided care is taken in the p r e p a r a t i o n of the mobile phases. W h e n chiral metal chelates of the type shown in Fig. 7.16 are used as eluent additives the enantiomeric resolution of the dansyl derivatives of all c o m m o n protein amino acids except proline, can be achieved (Le Page et al., 1979; Lindner et al., 1979). T h e separation of several D,L-dansyl amino acids using L-2 isopropyl-4-octyl-diethylenetriamine-Zn(II) complexation is shown in Fig. 7.17. The role of the metal ion in the separation has b e e n examined using N i ( I I ) , C d ( I I ) , C u ( I I ) , H g ( I I ) and Z n ( I I ) complexes for comparison (Lindner et al., 1979) and the results are summarised in Table 7.7.

+2

R C H 3— ( C H 2) 7— N - M : .

NH2 .NH9

Fig. 7.16. Structure of chiral metal chelate L-2-R-4-octyldiethylene triamine-M(II). R = ethyl, isopropyl, isobutyl: M = Zn, Cd, Ni, Cu, Hg.

Table 1.1 Influence of metal on chromatographic chiral separation. 0.8 mM Cj-Cg-dienmetal(II), pH 9, 30 °C, flow rate 2.0 ml/min, acetonitrile-water (35:65, v/v). Column: 15 cm x 4.6 mm, 5-/mm Hypersil Cg. Metal ion

Dansyl amino acid

a=

Ni(II)

a-A\a Thr ar-Ala Thr ar-Ala Thr mx depends on two terms; the a maximal injection volume, V i > mx aand t h e maximal injection concentration in this v o l u m e , Ciymax

(Coq et al., 1979b) Qi,max

~

Ci max .Vi

xm

a

(°^1)

If the injected volume Vt is large enough (V/ > 8cr, where a is t h e standard deviation of t h e Gaussian peak) it can be shown (Reilley et al., 1962) that the peak width at the base AV is given by AV

=

Vt

+ 2.5a

(8.2)

Thus, under volume overload conditions, t h e plot of t h e band broadening AV versus volume Vt is linear. Resolution Rs between two solutes 1 a n d 2 is given by (8.3)

PREPARATIVE LIQUID CHROMATOGRAPHY

275

where 1 and 2 refer to the first and second eluted solutes, respectively. Recovery of products with a definite purity requires a minimum resolution Rsm\n for which the injection volume becomes maximum Vv

i, max

= V

n

v

(8.4)

where k\ is the capacity factor of the first eluted solute; oris the selectivity factor; Vm the column void volume and N the theoretical plate n u m b e r of the column. The maximum injection volume, therefore, depends on the chromatographic parameters (k', arand N) and is directly proportional to the column void volume, H u p e and L a u e r (1981) have discussed optimising a preparative system with respect to time, solvent consumption and the smallest possible dilution of the sample to obtain the optimal production rate. They propose that the column length should be chosen to be —twice as long as is needed to perform the separation with an infinitely small injection volume. T h e injection volume should then be increased until the apparent plate n u m b e r drops to that minimum value required for resolution, thereby making the dispersion contribution of the column and the injection volume of about equal size. O n c e the required length has been chosen an increase of the column cross-section will increase the capacity of the system and hence the rate of throughput. Several investigations have discussed the 'loadability' of chromatographic columns and have defined various measures of the column capacity. Snyder (1968) defined the linear capacity of an adsorbent as the a m o u n t of solute which could be applied to a column before a reduction of 10% in the capacity factor (kf) or plate n u m b e r (N) occurred. D o n e (1976) subsequently showed that efficiency is linearly d e p e n d e n t on sample load down to very low values ( ~ 1 /mg of sample per g of support) and proposed instead the term 'relative capacity' defined as the sample weight per g of material which increases H for the column by 0.1 m m from its minimum value. Recently, P o p p e and Kraak (1983a) used data from numerical simulations to propose that the loadability be defined as the number of micrograms of solute per g of adsorbent in one theoretical plate. Alternatively, Gareil et al. (1984) have defined the available capacity, CA, as the amount of solute, injected in the eluent, that saturates the column stationary phase under a given set of elution conditions. This can be expressed as moles of solute per g of stationary phase. These concepts are all very theoretical and in practice it is not possible to generalise about the a m o u n t of sample which can be loaded on a column to effect a separation. Various factors such as the complexity of the mixture and the resolution between the peaks of interest render each separation unique and conditions must be optimised for each particular problem. In preparative separations the capacity of the column is not limited by a change in peak shape or k' value but the column loading may be continued until the resolution between adjacent peaks is no longer a d e q u a t e to achieve the required degree of solute purity. This does not necessarily m e a n that there

276

HIGH PERFORMANCE LIQUID CHROMATOGRAPHY

should be no overlap of adjacent peaks, since collection of fractions through the peak envelope can result in the isolation of pure material. This is illustrated in F i g . 8.1 for two adjacent peaks which are overlapping under the overloaded conditions chosen. Fractions should be collected and subsequently monitored by another technique, e.g. T L C , analytical H P L C or G L C , to indicate their purity. The fractions isolated prior to tx can be pooled to give pure component A while those collected after t2 will give pure component B . T h e fractions collected between t\ and t2 may be pooled and re-injected if further material is heeded. Obviously, it would save considerable time if positions tx and t2 could be calculated and the intermediate portion automatically recycled for further fractionation when needed. Coq et al. (1982) have derived equations for the calculation of the injection volume and the optimal cut elution volume as a function of the resolution when a given degree of purity and recovery ratio for the sample to be collected are chosen. In practice, the positions of tx and t2 are usually estimated from experience, particularly when the recycle technique is employed. It is better to err on the conservative side to avoid contamination of the isolated material. Care must be taken, however, when recycling to ensure that band broadening does not occur to the extent that the front of the first peak on the next elution overlaps with the tail of the second peak, thereby causing re-mixing of the solutes. This may limit the n u m b e r of recycles which can be performed effectively unless sufficient purified material is isolated from each cycle to counteract the band broadening.

A f

/

/

/

B

/

/

\

N

\

\

\

\

y

Fig. 8.1 Preparative chromatogram of two adjacent overlapping peaks indicating fractionation to isolate pure material.

Care must also be taken to allow for the effects of peak tailing. F i g . 8.2 illustrates the preparative separation of a mixture of diamantanols under heavily overloaded conditions (Kriz et al., 1982). Analysis of the isolated fractions indicates that c o m p o n e n t A can be obtained in a p u r e state but component B is always contaminated with A because of the tailing of the peaks. If both components are required in a purified state replicate analysis should be performed at a lower loading until sufficient material has been collected.

PREPARATIVE LIQUID CHROMATOGRAPHY

277

Generally in overload, peaks on normal phase columns tail while under reversed phase conditions pre-tailing occurs (Nettleton, 1981). This should be taken into account when deciding where cuts should be m a d e .

i

i

i

40

60

80

Time (min)

i

i

1

100

0

10

i

20

Time (min)

Fig. 8.2 Preparative separation of a mixture of diamantanols and GLC analyses of some fractions. Column packing, irregular silica (10-20/xm); column dimensions, 270 x 40 mm i.d. axially compressed; eluent, /i-pentane/2-propanol (98:2); flow rate, 22 m l . m i n - 1; detection, refractive index; sample loading, 5 g of mixture introduced in the solid state. GLC conditions, column, 1200 x 3 mm i.d., packed with Chromaton N AW DMCS coated with 3% XF 1150; temperature, 190 °C. Reproduced with permission from Kfiz et al., 1982.

Additional problems may arise when components of longer retention are present in the mixture, since a long elution time may be required to remove such substances from the column. T h e eluent may be changed immediately after the desired fractions have b e e n collected, in order to r e m o v e any further material from the column. This does, however, necessitate a re-equilibration of the column with the initial mobile phase prior to further injections. With large preparative columns these procedures usually involve the consumption of very large volumes of solvent, as well as being time consuming. A n interesting method which has been used to overcome this problem during the isolation of poly (ethylene glycols) and poly (oxyethylene) m o n o - a n d dialkyl esters in fatty acid ethoxylates by preparative reversed phase chromatography is column back flushing (Kudoh et al., 1984). T h e c h r o m a t o g r a m obtained for the lauric acid ethoxylate is illustrated in Fig. 8.3. T h e poly (ethylene glycols) and poly (oxyethylene) mono-lauryl esters were easily separated using an eluent of

278

HIGH PERFORMANCE LIQUID CHROMATOGRAPHY

acetone/water (70:30) but under these conditions the poly (oxyethylene) di-lauryl esters were very strongly retained. Back flushing the column after elution of the monolauryl esters reduced the analysis time and also helped to effect the accurate determination of the dialkylesters by reducing peak broadening.

PEG

1 Monolauryl ester

—i

0

1

60

1

120

Time (min) Fig. 8.3 Chromatogram of lauric acid ethoxylate (average 10EO units). Column packing, LiChroprep RP-18; column dimensions, 500 x 20 mm i.d.; eluent, acetone/water (70:30); flow rate, 10 m l . m i n - 1, detection, refractive index; sample loading, 7.87 ml of sample solutions of 10-20% in eluent, were injected. Reproduced with permission from Kudoh et al., 1984.

A more c o m m o n approach for removing unwanted but strongly retained material is to use a sample clean-up p r o c e d u r e . This pre-fractionation can be performed on the same column u n d e r grossly overloaded conditions by isolating a heart-cut fraction which contains the c o m p o n e n t s of interest (see Fig. 8.4). Immediately after elution of these components the solvent strength can be increased to elute the solutes with longer retention. T h e isolated fraction may then be concentrated and re-injected in smaller a m o u n t s , once the column has been re-equilibrated with the initial eluent, and the solutes of interest separated to yield pure components. Several injections may be necessary at this stage to isolate sufficient material if large quantities are required. For maximum throughput repeat injections may be performed before elution of the desired solutes, especially when c o m p o n e n t s of longer retention have previously been removed as described above. Careful timing of this procedure is obviously necessary to avoid overlapping of bands. Prefractionation may also be performed on-line, using a column switching technique (see Section 4.5.2) to transfer the desired fraction to the preparative column while the remainder of the sample is diverted to waste. A n example of the use of this technique in the isolation of the sweetener stevioside from dried

PREPARATIVE LIQUID CHROMATOGRAPHY

(a)

(b)

279

(c)

Fig. 8.4 Pre-fractionation of a complex mixture using a heart-cut technique to clean up the sample. (a) Analytical chromatogram. (b) Heart-cut under gross-overload conditions. (c) Isolation of component of interest.

leaves is shown in Fig. 8.5 (Little and Stahel, 1984). In Fig. 8.5a an analytical chromatogram of the leaf extract is shown indicating the presence of components of very long retention. T h e preparative separation is shown in Fig. 8.5b with the zone cut indicated being automatically transferred to the second column for further separation and purification. A second zone cut is then taken and collected as fraction F 2 which contains pure stevioside as indicated by the analytical c h r o m a t o g r a m shown in Fig. 8.5c. While this second fractionation is proceeding, the first column is cleaned by changing the eluent to methanol and subsequently re-equilibrated with mobile p h a s e , ready for the next injection. A slightly different approach is required when the c o m p o n e n t of interest is not the main peak in the sample. In this case overloading of the column can cause the peak to be completely swamped by the bulk of the other sample constituents, as shown in Fig. 8.6. Judicious collection of fractions, however, in the area where the peak would be expected to occur results in considerable enhancement in the peak of interest (Fig. 8.6b). After analysis of the fractions, those which are richest in the desired c o m p o n e n t should be pooled for further chromatography to effect the purification and isolation of material of interest (Fig. 8.6c).

280

HIGH PERFORMANCE LIQUID CHROMATOGRAPHY

Time (min)

Time (min)

(a)

Time (min)

(b)

Time (min)

(c)

Fig. 8.5 Use of a column switching technique to enable the rapid isolation of stevioside from an extract of Stevia rebaudiana leaves. (a) Analytical chromatogram of a leaf extract. Column packing, Spherisorb S5-ODS; column dimensions, 250 x 4.6 mm i.d.; eluent, methanol/water, 70:30; flow rate, 2 m l . m i n - 1; detection, UV at 214 nm. (b) Preparative separation of stevioside under conditions of gross overloading using a column switching procedure to link the two columns. Column packing, Spherisorb S10-ODS; column dimensions, 250 mm x 3/8 in.; eluent, methanol/water, 70:30; flov rate, 2 m l . m i n - 1; detection, UV at 220 nm. Throughput of stevioside, 30-50 mg per run. (c) Analytical chromatogram of isolated stevioside. Conditions as in (a). Reproduced with permission from Little and Stahel, 1984.

8.2

Equipment for preparative LC

Several commercial preparative L C systems are available with fully integrated pumping systems, column holders and detectors, e.g. Waters Prep LC/System 500 and System 500A; Instruments S A (Jobin Yvon) C h r o m a t o s p a c and Miniprep systems; Separations Technology; Elf A q u i t a i n e ; Varex C o r p . Additional features may include solvent recycle capability or a u t o m a t e d sample injection and collection. By far the most commonly used commercial systems are those from Waters and Jobin Yvon; these have also been available for the longest period of time. Since many manufacturers provide columns of suitable dimensions for semi-preparative or preparative separations chromatographers frequently build their own P r e p - L C systems from equipment already available. Pumps for preparative work must be capable of delivering high flow rates (up

PREPARATIVE LIQUID CHROMATOGRAPHY

[a)

(b)

281

(c)

Fig. 8.6 Preparative isolation of a minor component.

to —100 m l . m i n - 1) to function effectively with the large bore columns. Reciprocating and pneumatic amplifier p u m p s are usually used because of their high flow capabilities. High pressure capability ( > 3000 psi) is not normally necessary, since larger particles are commonly used in preparative work. Pulsation from reciprocating p u m p s is not such a problem as it is in analytical H P L C since detectors are usually operated at low sensitivity settings. Sample introduction may be performed using a valve injector with a very large volume loop. Syringe injection may be used, but for the large sample volumes involved a stopped-flow technique should be employed. For very large volumes of sample ( > 100 ml) the sample solution may be p u m p e d onto the column using a system such as that illustrated in Fig. 8.7.

282

HIGH PERFORMANCE LIQUID CHROMATOGRAPHY C

Fig. 8.7 Injection device for large sample volume, a = pump; b = column; c = adjustable constriction; d = 6-way valve; e = solvent tank; / = sample tank; , injection phase; , elution phase. Reproduced with permission from Coq et al., 1979(b).

Ideally, to obtain minimum band broadening a plug injection should be achieved (Coq et al., 1981). With a loop injector, extra band broadening due to the injector can be avoided by using an interrupted injection technique (Coq et al., 1979a; Gazda and Kowalczyk, 1983) or by using a packed sampling loop (Coq et al., 1981). In the interrupted injection technique the valve is returned to the load position at the end of the injection (i.e. when 9 9 % of the sample has been transferred to the column). This prevents the sample tail inside the loop slowly bleeding onto the column (see Fig. 3.10). A n almost rectangular concentration distribution of sample being introduced onto the column was also found to result from using a p u m p to supply the sample solution (Gazda and Kowalczyk, 1983). In preparative chromatography the sample should be evenly distributed over the whole of the top of the column to avoid local overloading, and also to m a k e use of the full capacity of the column. Several possible injection configurations which have been examined are illustrated in Fig. 8.8. Central injection as illustrated in (1A) is not ideal, since maximum use is not m a d e of the capacity of the column. A better injection profile is obtained by using a stainless steel frit to spread the sample across the column. T h e best arrangement to obtain maximum loading of the column appeared to be the curtain-flow system in combination with a solute sprinkler to distribute the sample over the column bore (Fig. 8.84B) (Coq et al., 1979b). Stainless steel columns of 10 m m and 22 m m diameter are available from most column manufacturers for semi-preparative or preparative applications. Column lengths of 250 and 500 m m are commonly used. Larger b o r e columns are also available for very large scale separations, e.g. W h a t m a n produce a column of

PREPARATIVE LIQUID CHROMATOGRAPHY

283

48 mm i.d. (Magnum 40). Large bore glass columns are still sometimes used under low pressures or even with gravity feed. T h e advantages of the pressurised systems in speeding up separations and improving efficiencies, however, have resulted in the decline in use of these systems and this is likely to continue even further.

Type 3

'A

Type 4

V//A

3 4A Fig. 8.8 Schematic representation of different injection devices. ® sampling valve; 1 M i l l stainless steel frit; | l dead volume. Reproduced with permission from Coq et al., 1979(b).

Several specialised column designs have been developed with a view to improving efficiency. Varian Associates supply 8 m m i.d. stainless steel columns

284

HIGH PERFORMANCE LIQUID CHROMATOGRAPHY

with tapered inlets and W h a t m a n ' s semi-preparative columns have tapered outlets. These columns are, however, m o r e expensive to manufacture than the straight columns and there is no clear evidence to indicate that they are more efficient. A n o t h e r technique which has been used to improve column efficiencies is compression of the packed bed. Waters Associates supply flexible walled columns which are radially compressed by hydraulic pressure in a specialised chamber. This system can only be used with the cartridges supplied, which are 300 x 57 m m i.d. for the Prep 500 and 100 x 13 m m i.d. for semi-preparative work with the Radial Compression Module. T h e Prep 500A has been modified to accept a 1 in. diameter stainless steel column as an alternative to the radially compressed cartridges. Alternatively, a few column designs provide axial compression of the packed bed. T h e Chromatospac unit (Instruments SA) will take either of two compression systems with 1 m columns of 40 m m (Prep 10 unit) or 80 m m (Prep 100 unit) i.d. Compression is achieved by a piston moving upward in the cylindrical chamber against a slurry of the packing. Excess solvent passes out at both ends, the lower frit being located in the top of the piston. The piston is moved by application of gas pressure up to 150 psi and the effective column height is determined by the a m o u n t of packing used. T h e Miniprep system (Instruments SA) works on the same principal but the columns are 0.5 mm in length and 20 m m i.d. and the piston is driven by an oil-based hydraulic jacking system. T h e Elf Aquitaine (Elf Aquitaine D e v e l o p m e n t , N V ) preparative L C system is also supplied with axially compressed columns of 40 m m or 80 m m i.d. Rainin Instruments ( W o b u r n , M A ) supply modular preparative H P L C columns with re-usable end fittings which can be tightened by hand to exert an axial force on an inner piston, thus compressing the column packing. These are available as 21.4 m m and 10 m m i.d. columns packed with 8 /xm silica or C 1 8 bonded silica. The most commonly used detectors in preparative separations are refractive index and U V monitors. Sensitivity is not normally a problem and usually low sensitivity settings are used. If a variable wavelength U V spectrophotometer is used the wavelength is generally set away from the maximum absorbance of the major component when the column is overloaded. This avoids detector overload which may otherwise result in broad poorly resolved peaks which can be mistaken for column overload. If overloading is observed it is worthwhile switching to another wavelength to check if the column is really overloaded, or just the detector. Sometimes additional column capacity may be found by this simple procedure. Frequently R I and U V detectors are used in series for preparative work and the results can be helpful. T h e specificity of the U V detector has obvious advantages yet it may not give an accurate picture of the relative amounts of components present in a mixture, especially if some of these are devoid of a U V chromophore. In such situations the I R detector may be m o r e informative. The use of microprocessors allows the development of automated preparative H P L C systems which are capable of performing automatic injection and fraction

PREPARATIVE LIQUID CHROMATOGRAPHY

285

collection as well as sample recycling and column switching. Berger and Gilliard (1981) designed a microprocessor-controlled instrument that has facilities to select buffers, load samples, generate linear gradients and modulate the programmed elution processes by positive feed-back interaction with the detector. Sugnaux and Djerassi (1982) a u t o m a t e d their preparative liquid chromatograph through intelligent fraction collection, with unlimited injection volume and repetitive collection of separated solute peaks. Similar techniques have been used to convert analytical chromatographs into a u t o m a t e d semipreparative units (Hadfield et al., 1983; T e h r a n i , 1985b). Colin et al. (1985) have described the design and performance of an a u t o m a t e d preparative scale system which is available from the Varex Corporation.

8.3

Materials for preparative chromatography

Packing materials similar to those used in analytical separations are employed for preparative separations. Totally p o r o u s materials should always be used, since they have a higher capacity than the pellicular materials. Pellicular materials are very rarely used now, even for analytical work. Usually larger particle sized material is used for preparative chromatography, since some efficiency may be traded off against the use of lower pressures and higher flow rates to achieve a good throughput of material. O n e of the main drawbacks against using smaller particle sized material is the cost, particularly if b o n d e d phases are required! In general 10-20 /xm materials are used to pack the 10 m m and 22 m m i.d. columns. With larger bore columns larger particle sizes are generally used. W h a t m a n provides Partisil P r e p 40 materials of 53 /xm size. These are available as silica, O D S or C 8- b o n d e d phases for use with the M a g n u m 40 column of 48 m m i.d. Similarly, the W a t e r s P r e p L C systems is supplied with columns pre-packed with 70-75 /xm material. Packing procedures are similar to those for analytical columns (except for very large ( > 20 /xm) particle sizes which can be dry-packed) and reduced plate heights of —2 should be obtained from a well packed column (Verzele and D e w a e l e , 1985). Packing m e t h o d s for the axially compressed Instruments SA system have been described in several papers (Gasparrini et al., 1980; Matlin and C h a n , 1984). Solvents for use in preparative H P L C should be free of contaminants (see Section 4.4.1 for clean-up procedures), since collection and concentration of large volumes will cause concentration of non-volatile impurities resulting in contamination of the isolated fractions. T h e selection of eluent conditions is performed in the same way as for an analytical separation. E l u e n t additives a r e , however, generally avoided unless they can be easily removed after fractions have been collected. T h e recovery of purified material involves the removal of the solvent at the end of the p r o c e d u r e . This process is obviously helped by the use of volatile

286

HIGH PERFORMANCE LIQUID CHROMATOGRAPHY

solvents. F o r small fractions e v a p o r a t i o n u n d e r a s t r e a m of nitrogen with gentle heating is possible. L a r g e r volumes m a y be r e m o v e d by rotary e v a p o r a t i o n . Freeze drying m a y b e used t o eliminate water. In s o m e cases, with reversed phase solvents, removal of t h e organic c o m p o n e n t will result in t h e precipitation of the purified solute from the w a t e r residue ( B e r a n et al., 1983).

8.4

Applications

Several recent examples of p r e p a r a t i v e separations a r e given in Table 8 . 1 . T h e d e v e l o p m e n t of a p r e p a r a t i v e separation is discussed in m o r e detail below using t h e example of t h e purification of a synthetic p e p t i d e synthesised by the solid phase methodology of Merrifield (Rivier et a l . , 1984). T h e gradient analysis of crude amidated h u m a n pancreatic t u m o u r growth h o r m o n e releasing factor (hp G R F ( 1 - 4 0 ) — N H 2) is shown in Fig. 8.9. T h e preparative purification of 2.1 g of Table 8.1.

Selected applications of preparative HPLC. Solute type

Acetogenin mevalonates (Eucalyptus globulus Labill) Caffeoyl esters Soybean isoflavones Hydroxycinnamic acid esters Alternaria metabolites Chlorophylls and pheophytins Steroid alcohols la- and 7^-methyl-17/?-acetoxy-3-oxoandrost-4-enes Corticosteroids Steroids Steroidal lactones Nucleosides and nucleotides Nucleic acid fragments Monensin A and B sodium salts Ferricrocin (microbial product) Polyenic antibiotic Linear gramicidins ^-Substituted tetraphenylporphyrins Phospholipids Juvenile hormone acids Marine lipid extracts Labdadiene and labdatriene Farnesol isomers Apolipoproteins Myelin basic protein peptide 43-88 Proteins Amino acids and peptides Amino acids Amino alcohols Synthetic peptides

Reference Amano et al., 1981 Chappie and Ellis, 1984 Farmakilidis and Murphy, 1984 Brandl and Herrmann, 1983 Griffin et al., 1983 Watanabe et al., 1984 Allenmark and Boren, 1981 Gasparrini et al., 1980 Kalasz and Horvath, 1982 Matlin and Chan, 1984 Megges and Grobe, 1982 Aoyagi et al., 1982 Larson et al., 1979 Beran et al., 1983 Fiedler, 1981(b) Gareil etal., 1981 Koeppe and Weiss, 1981 Bayer and Seelman-Eggebert, 1984 Geurts van Kessel et al., 1981 Goodman and Adams, 1984 Hennion et al., 1983 Mohanraj and Herz, 1981 Warthen, 1980 Wehr et al., 1982 Gilliom et al., 1983 Pearson and Regnier, 1983(b); Janson and Hedman, 1982 Pietrzyk etal., 1982 LeMaster and Richards, 1982 Musso and Mehta, 1981 Rivier et al., 1984; Vivelle et al., 1983

PREPARATIVE LIQUID CHROMATOGRAPHY Table HA.

287

(Contd.) Solute type

Reference Tyler and Rosenblatt, 1983 Hadfield et al., 1983 Hicks et al., 1983; Kondo et al., 1981 Smith and Weber, 1984 Belliardo et al., 1983 Dixon et al., 1984 Miyashitaet al., 1983 Tiebach and Schramm, 1980 Evershed et al., 1982 Holstein and Severin, 1982 Kohler et al., 1984 Pei et al., 1983 Ruedi et al., 1981 Schou et al., 1983 Mintaset al., 1981 Salvaetal., 1982 Bailey, 1984 Pirkle and Finn, 1982

A 2 1(a.u.f.s.) 0 o

Parathyroid hormone antagonist Benzyl a- /3-glucofuranosides and glucopyranosides Saccharides Amygdalin Epimeric guaianolides Drug glucuronide conjugates Monoiodocarnosine Food ingredients Alkene geometric isomers Coal liquefaction product Olefins Lubricating oil 3,3-Dimethyl-2-acetylnorborn-5-ene Styrene oligomers Diaziridines Alkaloids Dyestuffs D & C Red No 33 Racemates

Time (min) Analytical separation of lyophilised hp GRF (l-40)-NH 2 from HF cleavage (20/xl, ca 20/Ltg). Column packing, Vydac Q 8 (5 /xm); column dimensions, 250 x 4.6 mm i.d.; eluent, A = 0.1% TFA in water; B = 0.1% TFA in acetonitrile/water (60:40); gradient: 40%-20'-65% (3')-l'-95%B; flow rate, 2.0 ml.min-1; detector UV at 210 nm. Reproduced with permission from Rivier et al., 1984. Fig. 8.9

HIGH PERFORMANCE LIQUID CHROMATOGRAPHY

A 2 28 (a.u.f.s.)

288

A230 (a.u.f.s.)

Time (min) Fig. 8.10 Preparative separation of lyophilised hpGRF (l-40)-NH 2 from HF cleavage (400 ml, 2.1 g). Column packing, Vydac Q 8 (17/xm); column dimensions, Prep Pak 500 cartridge^ 300 x 50 mm i.d.; 600 psi radial compression; eluent, A = TEAP 2.25; B = acetonitrile/A (60:40), gradient: 35% (load)-60'-55%-l'-95%B; flow rate, 100 ml.min-i; detector, UV at 228 nm. Reproduced with permission from Rivier et al., 1984.

_i 0

i 10

i 20

i 30

l j

40

i min

50

i 60

Time (min) Fig. 8.11 Further purification of hpGRF(l-40)-NH 2 3 x fraction 8 (300 ml, 0.2 g) from the preparative purification shown in Fig. 8.10. Column packing, Vydac C 4 (17 /xm); column dimensions, Prep Pak 500 cartridge, 300 x 50 mm i.d.; 600 psi radial compression; eluent as in Fig. 8.10; gradient: 37% (load)-60'-57%B; flow rate, 100ml.min" 1; detector, UV at 230 nm. Reproduced with permission from Rivier et al., 1984.

PREPARATIVE LIQUID CHROMATOGRAPHY

289

the crude peptide was performed on a Vydac C 1 8, 17 fxm particle size radially compressed cartridge using phosphoric acid/triethylamine (TEAP)/acetronitile buffer (Fig. 8.10). A total of 6.3 g of crude peptide was purified in three preparative runs. Fractions were collected approximately every 75 ml and analysed isocratically in TEAP/acetonitrile buffer. The appropriately enriched fractions from each of the three runs were combined and re-fractionated on a Vydac C 4 , 17 j L t m , cartridge in T E A P 2.25/acetonitrile. T h e chromatogram obtained from fraction 8 is shown in Fig. 8.11. These purified fractions were then combined, diluted with water and re-chromatographed with desalting with 0 . 1 % trifluoroacetic acid/acetonitrile (Fig. 8.12). T h e finally purified protein was collected as three fractions which were subsequently lyophilised. T h e gradient analysis of these three fractions are shown in Fig. 8.13. The overall procedure yielded approximately 540 mg of protein with a purity of around 9 8 % .

2

r

i100

A 2 3(a.u.f.s.) 0

67

1h

00.

37

•-/A

T

AB

2 3

-30

0

10

20

30

Time (min) Fig. 8.12. Desalting of combined hpGRF (l-40)-NH 2 fractions from five preparative C 4 purifications (3000 ml ~ 500 mg) shown in Fig. 8.11. Column packing, Vydac C 4 (17 itm); column dimensions, as in Fig. 8.11.; eluent, A = 0.1% trifluoroacetic acid (TFA) in water; B = 0.1% TFA in acetonitrile/water (60:40); gradient: 37% (load + 5 ')-30'-67%B; flow rate, 100ml.min" 1; detector, UV at 230 nm. Reproduced with permission from Rivier et al., 1984.

290

HIGH PERFORMANCE LIQUID CHROMATOGRAPHY

100

A 2 0] (a.u.f.s.) o 6 en

(Mr

60

60

005 45

45

0 10

-i100

0L 1

-JO

20

10

"20

Time (min)

Time (min)

100

(Mr

A 2 I0 (a.u.f.s.)

DO

60 005

45

as 00

c

10 Time (min)

20

Fig. 8.13 Analytical chromatograms of fractions 1-3 (20 /xl ~ 20 /xg) from the preparative desalting of hpGRF (l-40)-NH 2 shown in Fig. 8.12. Column packing, Vydac C 18 (5 /xm); column dimensions, 250 x 4.6 mm i.d.; eluent, A = 0.1% TFA in water, B = 0.1% TFA in acentonitrile/water (60:40); gradient: 45%-20'-60%B; flow rate, 1.8 ml.min- 1; detector, UV at 210 nm. Reproduced with permission from Rivier et al., 1984.

Chapter 9

Chiral

chromatography

O n e specialist area of chromatography which is of particular interest is the resolution of optical enantiomers. This topic is important in many fields but it is of particular interest currently in the pharmaceutical industry, since many of the most widely prescribed drugs contain chiral centres. T h e r e is increasing evidence of significant differences in the bioavailability and metabolic properties of enantiomers, with a consequential interest in the use of chiral analytical techniques for their determination. In liquid chromatography there are three distinct approaches to enantiomer resolution 1. Preparation of diastereoisomers using chiral derivatising agents. Since diastereoisomers exhibit different physical properties they can usually be separated quite easily by standard chromatographic procedures. 2. Use of chiral mobile phase additives. Association of the enantiomers with a chiral additive in the mobile phase can result in diastereomeric ion pairs or complexes resulting in resolution of the enantiomers. 3. Chiral stationary phases. Alternatively, diastereomeric-like associations can occur between enantiomers in the mobile phase and chiral molecules coated on or bound to the stationary p h a s e , which may effect resolution. All three approaches have been used successfully. 291

292

HIGH PERFORMANCE LIQUID CHROMATOGRAPHY

9.1

Enantiomeric resolution by preparation of diastereoisomers

Derivatisation with a chiral reagent followed by either normal or reversed phase chromatography of the diastereomeric products is a c o m m o n procedure for enantiomeric resolution. Several suitable chiral reagents for the resolution of hydroxy or amino compounds or carboxylic acids have been developed (Tamegai et al., 1979b). Unfortunately, these are not always commercially available. Some recent applications are summarised in Table 9 . 1 , giving an indication of the variety of reagents which are used. T h e r e a r e , however, some basic rules governing the choice of reagent. Standard derivatisation procedures are employed, but, since it is important to avoid racemisation during the reaction, mild reaction conditions are preferred. For quantitative analysis it is critical that the chiral reagent be enantiomerically pure and that the reaction goes to completion. C o m m o n reagents are acyl chlorides or acid anhydrides which react with enantiomeric alcohols or amines to form diastereomeric esters

i

006-

I2 r

3

4

5

•

o 6

Absorbance (250 nm)

c c

004

10 Time (min)

20

Fig. 9.1 Separation of thiourea derivatives formed from amino acids with AITC. Column packing, Develosil ODS-5; column dimensions, 150 x 4.6 mm i.d.; eluent, methanol/10 mM phosphate buffer, pH 2.1 (50:50); flow rate, 0.9 m l . m i n - 1; detection UV at 250 nm. Solutes, 1: alanine; 2: tyrosine; 3: valine; 4: leucine; 5: phenylalanine. Reproduced with permission from Kinoshita et al., 1981.

CHIRAL CHROMATOGRAPHY

293

or amides. Alternatively, isothiocyanates and isocyanates are frequently used to produce urea derivatives. Chiral amines may be used as reagents for the derivatisation of chiral carboxylic acids and vice versa. The structure of the derivatising agent is important in effecting chromatographic resolution. Usually the presence of bulky groups close to the chiral centre enhances the differences between the diastereomers resulting in better resolution. T h e polar group which reacts to form the derivative (with the - O H , N H 2 or C O O H ) should also be close to the chiral centre for maximum effect, since this bonded linkage is often involved in hydrogen bonding with t h e stationary phase. For detection purposes it can also be useful to employ a chiral reagent which exhibits a U V or fluorescent c h r o m o p h o r e . W h e n amino acid enantiomers are to be chromatographed it is usually necessary to protect the amino or carboxyl group which is not involved in the chiral derivatisation. This was, however, found not to be necessary when either 2,3,4,6-tetra-O-acetyl-jS-D-glucopyranosyl isothiocyanate ( G I T C ) or 2,3,4-tri-Oacetyl-