Introduction to high
`performance liquid
`chromatography
`
`R. J. Hamilton
`and
`P. A Sewell
`
`Liverpool Polytechnic
`
`SECOND EDITION
`
`London New York
`CHAPMAN AND HALL
`
`FRESENIUS KABI 1023-0001
`
`

`
`
`
`o
`
`First published 1977
`by Chapman and Hall Ltd
`11 New Fetter Lane, London EC-4P 4E‘E
`Second edition 1982
`Published in the USA by
`Chapman and Hall
`in association with Methuen Inc.
`733 Third Avenue, New York, NY 10017
`©1977, 1982 RJ. Hamilton and RA. Sewell
`Printed in Great Britain by J. W. Arrowsmirh Ltd, Bristol
`ISBN 0 412 23430 0
`
`All rights reserved. No part of this book may be reprinted, or
`any form or by any electronic, mechanical
`reproduced or utilized in
`or other means, now known or hereafter invented, including
`photocopying and recording, or in any information storage or retrieval
`system, without permission in writing from the publisher.
`
`British Library Cataloging in Publication Data
`Hamilton, RJ. (Richard John)
`Introduction to high performance liquid
`chromatography.
`Includes index.
`I. Sewell,,P.A.
`I. Liquid chromatography.
`(Peter Alexis) H. Title.
`53420894
`8146840
`1981
`ISBNO—412-23430-0 AACR2
`
`QD79.C454H35
`
`Library of Congress Cataloging in Publication Data
`Hamilton, R. J. (Richard John)
`Introduction to high performance liquid
`chromatography.
`
`Includes index.
`I. Sewell, P. .4.
`1. Liquid chromatography.
`(Peter Alexis)
`II. Title.
`543'.0894
`é3‘1~I 6840
`QD7.9.C454H35 1981
`ISBN 0~41 2-23430-0
`AACR2
`
`FRESENIUS KABI 1023-0002
`FRESENIUS KABI 1023-0002
`
`

`
`Contents
`
`Preface
`
`Introduction to high performance liquid chromatography
`Introduction
`1.1
`1.2 Nomenclature
`1.3 Liquid Chromatography Modes
`1.4 Scope of Techniques
`References
`
`Chromatographic theory
`2
`2.1 The Process of Separation
`2.2 Retention in Liquid Chromatography
`2.3 Band Broadening-Origins
`2.4 Band Broadening and the Plate Height Equation
`2.5 Overall Plate Height Equation
`2.6 Comparison with Gas Chromatography
`2.7 Column Efficiency and Particle Diameter
`2.8 Reduced Plate Height and Reduced Velocity
`2.9 Extra-Column Band Broadening
`2.10 Resolution
`2.11 Resolution and Time for Analysis
`2.12 Theory of Exclusion Chromatography
`References
`Equipment
`Introduction
`3.2 Mobile Phase (Solvent) Reservoirs and Solvent Degassing
`3.3 Pumping Systems
`' 3.4 Flow Controllers
`3.5 Solvent Flow Programming Equipment
`3.6 Pulse Damping
`3.7 Pressure Measurement
`
`)
`
`3
`
`. 3.1
`
`xi
`
`2
`3
`11
`12
`
`13
`13
`13
`17
`20
`24
`25
`26
`27
`28
`29
`35
`37
`40
`42
`42
`42
`44
`49
`49
`53
`53
`
`FRESENIUS KABI 1023-0003
`
`

`
`4.5
`4.6
`4.7
`
`s.
`5.1
`
`5.4
`
`Introduction
`
`127
`127
`127
`
`134
`
`140
`141
`
`FRESENIUS KABI 1023-0004
`
`

`
`7.4 Packing of a Preparative Column
`7.5 Summary of Preparative HPLC
`7.6 Trace Analysis
`References
`
`8.
`Applications of high performance liquid chromatography
`8.1 Pharmaceuticals
`8.2 Biochemicals
`8 .3 Food Chemicals
`8.4 Heavy Industrial Chemicals
`8.5
`Inorganic
`8.6 Miscellaneous
`Subject Index
`Compound Index
`
`Contents I vii
`
`183
`184
`185
`188
`189
`190
`203
`218
`220
`232
`234
`237
`244
`
`FRESENIUS KABI 1023-0005
`
`

`
`6
`Developing a chromatogram
`
`In this chapter we shall discuss the approach to establish the best analytical
`conditions for a specific sample. However, most problems can be solved by more
`than one mode of liquid chromatography, and the final approach may depend
`on the availability of equipment, columns, or the personal preference of the
`chromatographer.
`
`6.1 Nature of the problem
`It is rare for the analyst to receive an 'unknown' sample; its origin, the use to
`which it is put, or even its physical form are important indications as to its
`nature. The more that is known about the sample the easier it will be to decide
`on the best approach for its analysis. The solubility characteristics of the sample
`must at least be known since it must be in solution to be injected. The nature of
`the solvent to be used will also affect the choice of stationary phase. A
`preliminary analysis by another technique may be necessary; e.g. analysis by i.r.
`or u.v. spectroscopy would indicate the nature of functional groups present, and
`a knowledge of the molecular structure will assist the analyst in his choice of
`detectors.
`It is also important to know the nature of the analysis required. Is there only
`a limited number of components of interest in a multicomponent mixture or is
`an analysis required of each component present? Would a 'fingerprint'
`chromatogram of the sample be sufficient or are the components to be separated
`and collected for further analysis? Is the analysis a one-off problem or will it
`becom~ a routine method for quality control? All these are questions the
`answers to which may affect the analyst's approach.
`
`6.2 Choice of Chromatographic Mode
`Having determined the extent of the analysis required and the nature of the
`sample, the analyst is in a position to select the chromatographic mode that is
`most likely to produce the desired results.
`
`FRESENIUS KABI 1023-0006
`
`

`
`146 I High performance I iquid chromatography
`A satisfactory choice of chromatographic mode requires an understanding of
`the mechanisms controlling retention in the different modes. These have been
`discussed in Section 1.3 but it is useful to summarize the main points again.
`
`Partition. Stationary phases are invariably of the chemically bonded type, i.e.
`the 'liquid' stationary phase is chemically bonded to the surface of a base particle,
`usually silica. Choice of the bonded liquid results. in phases of varying polarity.
`The mechanism of retention is complex but involves some form of partition be(cid:173)
`tween the stationary and mobile phases. The retention properties are similar to
`adsorption under certain conditions and to partition under others. Because of
`the wide variety of packing polarities and mobile phases which can be used with ..
`them, partition chromatography is the preferred choice in the majority of
`separations.
`
`Adsorption. Separation is the consequence of polar interactions between the
`active groups on the stationary phase and polar or polarizable functional groups
`on the solute molecules. The geometric arrangement and number of surface active
`groups determines the selectivity and the mode is best suited for separation into
`compound type (e.g. alcohols and esters) and for geometric isomers.
`
`Jon-exchange. Since this involves the substitution of one ionic group by
`another it is only suitable for ionic or ionizable compounds. The mobile phase
`is almost exclusively water and pH control is a major factor in selectivity.
`
`Ion-pairing. According to the nature of the system the predominant mode
`may either be partition or ion exchange. It is thus applicable to non-ionic and
`ionic or ionizable molecules.
`
`Exclusion. Solutes are separated by differences in molecular size and shape.
`It is most useful for high molecular weight materials (> 2000) and molecular
`weight differences of about 10% are required for separation. The mobile phase
`adopts a non-interactive role in exclusion chromatography.
`Bearing in mind the foregoing summary it should be possible to select the
`mode most likely to lead to a successful separation. Figure 6.1 illustrates how an
`initial choice may be made on the criteria of molecular weight, solubility and
`functional group character.
`
`6.2.1 Molecular Weight
`With an unknown sample a useful start is to determine its molecular weight range
`using exclusion chromatography; the resulting size fractions can then be subjected
`to further exclusion chromatography or they can be analysed by a different
`chromatographic mode.
`The choice of a molecular weight of 2000 is somewhat arbitrary since columns
`are available for molecular weight separations in the range 2 X 102 to 2 X 106 ,
`
`FRESENIUS KABI 1023-0007
`
`

`
`•
`
`1
`
`Water soluble
`I
`~----r---
`Ionic and
`Ionic
`I
`non - ionic
`I
`ION-PAIR
`
`I
`Non - ionic
`I
`I
`I
`ION EXCHANGE EXCLUSION REVERSE PHASE
`I
`r---... 1 -...
`PARTITION
`I
`I
`Acid ic
`Basic
`I
`I
`Anion
`Cation
`exchange
`exchange
`
`Aqueous
`mobile phose
`
`M. wt >2000
`
`Woter
`1insoluble
`, - - .----
`-1
`PARTITION
`ADSORPTION EXCLUSION
`I
`r
`---.
`Reverse Normal
`phose
`phose
`
`EXCLUSION
`I
`.---~ .
`Water Organic
`soluble
`soluble
`
`Fig. 6.1 Selection of chromatographic mode.
`
`"'Tl ;o
`m
`(f) m
`z
`c
`(f)
`
`~
`OJ
`
`.....l.
`
`0 "' w
`
`I
`0
`0
`0
`CX>
`
`

`
`148 I High performance liquid chromatography
`but generally speaking lower molecular weight materials are better separated by
`means other than exclusion chromatography. More important is the lipophilic/
`hydrophilic character of the sample since some exclusion packings are not suit(cid:173)
`able for use in aqueous mobile phases whilst others are not suitable for organic
`phases (see for example Table 4.12).
`
`6.2.2 Solubility
`Sample solubility is perhaps the most important guide to the selection of the
`best mode and column. Thus lipophilic molecules can be separated by reverse(cid:173)
`phase partition chromatography on a hydrocarbonaceous phase or by adsorption
`chromatography. For more polar molecules, soluble in methanol, there is a choice
`of both normal and reverse-phase partition chromatography though they may
`also be separated on adsorption packings if the mobile phase is sufficiently polar
`(e.g. alcohols or chlorinated hydrocarbons). Very polar water soluble molecules
`can be separated by reverse-partition, ion-exchange or ion-pair chromatography.
`
`6.2.3 Functional Group Character
`The presence of ionic or ionizable groups on the molecule indicates that ion(cid:173)
`exchange or ion-pair chromatography should be used. Strong hydrogen-bonding
`functional groups, e.g. alcohol or amine, are particularly amenable to adsorption
`chromatography especially where the molecules differ in the number and con(cid:173)
`figuration of the hydrogen-bonding groups whereas molecules which are pre(cid:173)
`dominantly aliphatic or aromatic where solubility differences can be exploited
`will best be separated by partition chromatography.
`A correlation between funCtional group characteristics and chromatographic
`mode is shown in Fig. 6.2. The choice between adsorption and a bonded phase
`packing may often be one of convenience and availability.
`
`Hydrocarbons and
`derivatives
`
`Oxygenated
`hydrocarbons
`
`Proton donors
`
`Ionic compounds
`
`Polarity of functional groups
`
`I
`I
`I
`I exchange
`1 ion pairing
`I Most
`Fig. 6.2 Correlation between packing and sample type (after Waters Associates).
`
`(f)
`
`/
`
`/ 1 Functional
`
`/
`
`/
`
`ill c
`E
`'(cid:173)c
`
`:; :;:
`
`0
`E
`g>
`~
`
`E
`
`lon
`
`polar compounds
`
`1f
`~'f:)f
`RH RX RN02/ ROR RC-OR RC-R RC-H RC-NHR RNH ROH H 0 ArOH RCO H Nucleotides NH -R-C06
`2
`R2NA
`/
`I
`Least polar
`R, N
`I
`compounds
`I
`j•
`I
`I
`I
`I
`
`e phose
`g[ Reverse
`~
`g
`~-c
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`0
`.1!
`~
`
`/
`
`/
`
`/
`
`Absorption
`
`_.....,L
`
`/
`
`group
`2 Functional
`~~
`3 Functional
`groups
`
`--::,.../""----- 4 Functional
`7
`groups
`
`Bonded
`phases
`
`5 or more
`functiona l
`groups
`
`FRESENIUS KABI 1023-0009
`
`

`
`Developing a chromatogram I 149
`6.3 Selection of Stationary Phase and Mobile Phase
`A successful separation is achieved when a proper balance is established between
`the intermolecular forces involving the sample, the mobile phase (solvent), and
`the stationary phase. The intermolecular forces may be measured in terms of the
`polarity of the molecules, and most good separations are achieved .12Y. matching .
`!._he _polarity of the sam le _'!_nd stationary phase and_ using a m~bile phase of
`~ifferef)t_ polarity. If the sample is too similar to the mobile phase in terms of
`polarity, the stationary phase is unable to compete successfully for the sample
`and there is little retention. In that case the polarity of the mobile phase or of
`the stationary phase or of both must be changed. When the stationary phase is
`more polar than the mobile phase the system is referred to as 'normal phase
`liquid chromatography'. In some cases the sample is so strongly retained by the
`stationary phase that even substantial changes in mobile phase polarity do not
`decrease the retention time sufficiently. In this situation it is appropriate to
`use 'reverse phase chromatography' in which the stationary phase is less polar
`than the mobile phase.
`Fig. 6.3 shows the general interactions between sample and mobile phase as a
`function of polarity. Thus in normal phase chromatography the least polar com(cid:173)
`ponent is eluted first and increasing the mobile phase polarity decreases the
`elution time, whereas in reverse phase chromatography the most polar component
`is eluted first and increasing the mobile phase polarity increases the elution time.
`6.3.1
`Stationary Phase
`The general nature of the stationary phase will be determined by the selected
`mode for the separation. But within a given mode there is still a considerable
`choice of stationary phase to be made: e.g. which molecular weight range exclusion
`packing to use; whether to use a weak or strong anion or cation exchange packing;
`the polarity of an adsorbent. Because of thU.Q!!lplex nature of the partition
`-J process the most difficuh choice is when using chemicaliy bonded partition pack(cid:173)
`ings, but this complexity also accounts for the variety of separations which may
`be achieved on these packings. The packings available range from those with polar
`functional groups -OH, -NH2 , - CN) to the apolar hydrocarbon types. The -CN
`type can be used to separate many of the polar compounds which may be separ(cid:173)
`ated by adsorption chromatography on silica. However the bonded phase is more
`suitable for gradient elution separations. The NH2 phase can operate in both the
`normal and reverse phase modes as well as a weak ion exchanger. Thus it can be
`used to separate polar compounds e.g. substituted anilines, esters and chlorinated
`pesticides in the normal phase mode, carbohydrates in the reversed phase mode
`and organic acids (e.g. dicarboxylic acids) in the ion-exchange mode.£!~cause of
`long equilibrium times, due to the polarity of the amino group~ it is not
`recomme~ded th~t a single column is used in all three modes but that a separate
`column should be kept for each mode.
`The simplest criteria for the selection of the stationary phase is that~
`has ~~ ~f!nity for like'. If the sample is soluble in hydrocarbon solvents, indicating
`
`FRESENIUS KABI1023-0010
`
`

`
`150 I High performance I iquid chromatography
`Increasing mobile phase
`polarity
`. - - - - -
`
`Medium polarity
`mobile phase
`1
`
`}U d
`
`'th
`se wr
`medrum
`to high
`polarity
`packrngs
`
`Low polarrty
`mobrle phase
`/
`
`~::::~:; B~
`
`sample
`polarity
`
`Component A f-- +--t-....;o.t
`Component B ~---.-J._,---=>~
`
`ln~~~~~~ng~
`
`polarity
`
`\ , \
`\
`
`'
`
`'
`
`Increasing mobile
`phase polarity
`
`' '
`
`High polarity
`mobile phase}
`.
`Used wrth
`low polarity
`packings
`
`'
`Medium polarity
`mobile phase
`Fig. 6.3 Interactions between sample and mobile phase as a function of polarity
`(after Waters Associates).
`
`its own low polarity, then an apolar stationary phase should be tried first. Con(cid:173)
`versely if the sample is soluble in polar solvents such as water, indicating a high
`polarity sample, then a polar stationary phase should be tried. This approach to
`column selection in partition chromatography using solubility in a hydrocarbon
`solvent, in alcohol and in water is exemplified in Table 6.1 which also lists
`typical mobile phases and gives examples of sample type.
`
`6.3.2 Mobile Phase
`In liquid chromatography the mobile phase may be any single liquid or com(cid:173)
`bination of liquids which are compatible with the sample, column and instru(cid:173)
`mentation. Physical characteristics, however, such as viscosity, volatility, com(cid:173)
`pressibility, refractive index and u.v. absorption can limit the choice of mobile
`phases.
`
`FRESENIUS KABI 1023-0011
`
`

`
`Table 6.1 Column selection for chemically bonded phases
`
`Column type Mobile phases
`-c,.
`
`Sample type
`
`Mode
`
`Low polarity-soluble Reversed
`in hydrocarbons
`phase
`
`Medium polarity-
`soluble in alcohol
`
`Normal
`phase
`
`Reverse
`phase
`
`High polarity
`soluble in water
`
`Reversed
`phase
`
`Cation exchange
`
`Anion exchange
`
`-Ion pair
`(reverse phase)
`
`"'Tl
`
`(f)
`
`:::0 m
`m z -c
`~
`OJ
`
`(f)
`
`.....lo.
`0
`I'V w
`I
`0
`0
`.....lo.
`I'V
`
`methanol/water,
`acetonitrile/water
`acetonitrile/tetrahydrofuran
`tetahydrofuran/methylene
`chloride
`acetonitrile, methylene,
`chlorides, hexane
`chloroform.
`hexane, methylene
`chloride, isopropanol.
`
`methanol, water,
`acetonitrile
`
`methanol, acetonitrile
`water, buffered water.
`
`water & aqueous buffers
`
`-CN
`
`-NH.
`
`-ODS
`-c.
`-CN
`-TMS
`-NH.
`- c.
`-CN
`-TMS
`-so;
`
`-NR;
`- NH2
`-ons,c.
`c.
`
`citrate, phosphate buffers
`pH 2-7
`water, methanol, aceton(cid:173)
`itrite and counter ions .
`
`Examples
`
`polynuclear aromatic hydrocarbons,
`triglycerides, lipids, esters, fat soluble vitamins,
`steroids, hydroquinones,
`sulphamides, alkaloids
`
`fat soluble vitamins, steroids, aromatic alcohols, amines,
`esters, lipids (class separation) analgesics
`
`aromatic amines, esters, chlorinated pesticides, carboxylic acids
`lipids (homologue separation) nucleotides, phthalic acids, poly(cid:173)
`nuclear aromatics
`steroids, alcohol soluble natural products
`
`vitamins, aromatic acids, xanthines
`antibiotics, anticonvulsants, food additives
`carbohydrates
`water soluble vitamins, amines, aromatic alcohols,
`analgesics, antibiotics,
`food additives
`inorganic cations, carbamates, vitamins, amino acids,
`catecholamines, nucleosides, glycosides
`nucleotides, inorganic anions, sugars
`organic acids, analgesics
`acids, sulphonated dyes, catecholamines
`
`

`
`152 I High performance liquid chromatography
`
`The role of the mobile phase may either be interactive, if a change in mobile
`phase composition produces a corresponding change in retention, or non-inter(cid:173)
`active if the retention is independent of mobile phase composition. Thus in
`adsorption, partition and ion-exchange the mobile phase is interactive, whereas
`in exclusion chromatography it is non-interactive. The ability to change the
`selectivity of the column by altering the mobile phase composition is a powerful
`variable and has made it possible to reduce the number of stationary phases
`necessary in liquid chromatography. However, fmding the best mobile phase
`system may lead to lengthy method development and it is in this area that fully
`programmable microprocessor controlled chromatographs can be invaluable.
`Mobile phase (solvent) strengths are usually measured in terms of the solvent
`polarity and are presented as the eluotropic series. A more fundamental measure
`of solvent polarity is defined by the Hildebrand solubility parameter o, and for
`normal phase chromatography the strength of the mobile phase increases with
`increasing o values. Solvents in the eluotropic series with comparable polarity
`rankings may have different solubility parameters. However, the eluotropic series
`is a useful means of ranking the relative polarities of solvents, and it is in
`common use. Fig. 6.4 shows the eluotropic series for many of the common solvents
`used in HPLC. Together with these are shown examples of various types of
`stationary phase. The full lines represent the range of solvents most frequently
`used with these phases.
`
`6.4 Choice of Detector
`The choice of detectors is still strictly limited. The refractometer is the only uni(cid:173)
`versal detector readily available but it suffers from lack oLsensitivity and the
`inability to cope with solvent programming other than of a stepwise nature. A
`variable wavelength u.v. detector offers the best choice for a large range of solutes
`but in specific cases the fluorescence or electrochemical detector can be used. Un(cid:173)
`less a solute system is reasonably well defined, whichever detector system is used,
`there will be a chance that solutes will not be detected, and a combination of
`detectors may be necessary.
`
`6.5 Chromatographic Separation
`Having considered the characteristics of the sample and decided on the mode of
`separation, the stationary phase, and the mobile phase, the analyst is now ready to
`make his first injection. An analytical column ( 4-5 mm i.d. x 25 em long) at room
`temperatlJre should be used for this purpose.
`Before. an injection is made the sample must be dissolved in a suitable sample
`solvent. Ideally the sample solvent should be the same as the mobile phase, but
`if the sample is not sufficiently soluble in the mobile phase another solvent can
`be used. This sample solvent must then be of high purity so that extraneous peaks
`are not introduced, be miscible with the mobile phase and should give a JUinimal
`detector response, otherwise peaks eluting just after the sample solvent may be
`
`FRESENIUS KABI 1023-0013
`
`

`
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`
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`
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`<t<{
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`CL.
`OG> mn.
`
`- methanol
`
`0·9
`
`1-
`_ethanol
`
`-iso-propanol, n-propanol
`
`- ;
`
`0·8
`
`- pyridine
`
`acetonitrile
`nltrQmethane
`dimethyl sulpt)oxide
`-
`Q!!lyl alcohQI
`__ methyl acetate
`-ethyl acetate
`-acetone/dioxane
`
`00·6
`....
`<
`
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`
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`- -ethylene dichloride
`
`-
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`-methylene chloride
`~ -chloroform
`~y~
`-ethyl bromide
`
`0·4
`
`0·3
`
`benzene
`_:"n -propyl chloride toluene
`r-
`- - -
`-Isopropyl chloride
`-di -isopropyl ether
`--xylene
`
`0.2
`
`1-
`-carbon tetrachloride
`
`. -carbon disulphide
`0· ,_
`
`-1-pentene
`cycloheptane
`-cyclohexane
`-n-heptane
`.. n-pentane
`
`0
`
`Fig. 6.4 Elutropic series and column packings.
`
`FRESENIUS KABI1023-0014
`
`

`
`154 I High performance liquid chromatography
`masked. Complex samples, e.g. body fluids, should be flltered or centrifuged
`before injection to remove particulate matter which may interfere with the proper
`functioning of pump check valves or block transfer lines.
`The chromatographic process must maintain the integrity of the sample, i.e.
`decomposition and/or degradation of the sample or irreversible adsorption of
`the sample must be avoided. Spurious or ghost peaks may be generated during a
`gradient elution when the mobile phase strength becomes sufficient to elute a
`previously retained sample component. It is therefore good practice to run a
`blank chromatogram before injecting the sample. The sample solvent, too, should
`be injected to check it for impurities. The initial chromatogram is unlikely to be
`completely successful; there may not be any resolution of the components, or
`some may be only poorly resolved, and the analysis time may be too long. How(cid:173)
`ever, by applying the simple theory presented in Chapter 2, the analyst can rapidly
`select the experimental conditions necessary to achieve the maximum separation
`in the minimum time.
`In considering the optimization of conditions for analysis, although the same
`general principles are applicable to all modes of chromatography, the means of
`achieving the desired result may be different. It will be best, therefore, to discuss
`the general principles and then to discuss them in a consideration of the different
`chromatographic modes.
`Two chromatographic parameters are important in the optimization to give
`maximum separation in the minimum time. Equation (2.20) relates the retention
`time (tR) to the capacity factor (k') thus:
`tR = !:_ (1 +k')
`
`(2.20)
`and Equation (2.58) relates the resolution Rs to the capacity factor (k'), relative
`retention (ex) and the number of theoretical plates (N) thus:
`1 (ex-1) ( k' )
`Rs=4 a
`1
`(2.58)
`l+k' N2
`As discussed in Section 2.10 the optimum value of k' lies between 1 and 10.
`Larger values of k' lead to longer retention times, and the chromatographic bands
`are eluted as wide, flat peaks which are difficult to detect. If k' lies in the optimum
`range, therefore, it is undesirable to change its value, and changes in resolution
`can be brought about only by increasing the number of theoretical plates (N) or
`the relative retention (ex).
`The capacity factor is the parameter which is most easily optimized since it
`usually only involves a change in the mobile phase strength. However, k' values
`may also be adjusted by a change in stationary phase polarity, although this
`approach is obviously not so convenient.
`The optimum value of ex lies between 1.05 and 10. A change in ex is achieved
`by altering the nature of the stationary phase and/or the mobile phase. However/
`in changing the mobile phase it is the composition rather than the solvent strength
`
`Vm
`
`FRESENIUS KABI 1023-0015
`
`

`
`Developing a chromatogram I 155
`
`which is the more important. The effect of changes in a: are more difficult to
`predict than are changes ink' and N, and in a multicomponent mixture changes
`in a: may merely result in a reshuffling of the elution order. In terms of analysis
`time, however, a change in a: is often the best way of improving R s.
`Since it is easier to achieve an increase in Rs by increasing N, this may be
`preferable. However, increasing Rs in this way generally results in longer
`retention times, since an increase in N is usually achieved by an increase in
`column length or a reduction in mobile phase flow rate. If a routine analytical
`method is being established it may be worth the extra effort to change a: to
`achieve the required resolution, there being a saving in time on subsequent
`analyses.
`An increase in N results in less band broadening and greater resolution, and
`can always be a<;hieved by increasing column length, increasing the pressure drop
`across the column, or increasing the analysis time. Unlike increases ink' ,N can
`be increased in theory without limit; however, there are practical limits set by
`the available pressure drop and also the packing characteristics of the stationary
`phase.
`The value of N for an LC column is dependent on a large number of
`variables. Some of these (choice of mobile phase and stationary phase) may
`already have been selected to optimize k' and a values. Others (particle size and
`temperature) can be optimized independently. This leaves the analyst with three
`interdependent parameters which affect the value of N: column length (L),
`pressure drop (t:..P), and analysis time (t). These parameters can be varied to
`increase N in three ways: (a) decrease t:..P holding L constant; (b) increase L
`holding t:..P constant; (c) increase L and t:..P holding t constant.
`Method (a) is obviously the simplest although it will result in longer analysis
`times. Method (b) may be preferred when a large number of analyses are to be
`performed; although time may be spent on preparing longer columns, the
`analysis time per sample will be less. Method (c) is only an option if the LC
`unit is already operating below its maximum pressure, or if a pumping unit with
`a higher maximum pressure is available.
`Although temperature may not have dran1atic effects it can be significant. An
`increase in temperature will give an increase in .. N by improving mass transfer;
`particularly in ion-exchange processes, and will also give smaller k' values; the
`effect on a: is difficult to predict.
`To a large extent then optimization of the separation is brought about by
`changes in the_mobile phase, the stationary phase having been selected in the first
`place to provide a suitable chromatographic mode for the analysis. This is not to
`imply that there is no choice, for in partition chomatography the choice is
`considerable.
`
`6.5.1 Adsorption
`The choice of adsorption packings is limited to silica and alumina with alumina
`having a greater selectivity toward unsaturated compounds. Since both of these
`
`FRESENIUS KABI1023-0016
`
`

`
`156 I High performance I iquid chromatography
`are strongly polar the problem is usually that of too high k' values rather than too
`small. Since they are both used in the normal phase mode, k' values may be de(cid:173)
`creased by using a more polar mobile phase. k' values can be increased by using a
`packing with a larger surface area. Most of the spherical silicas have surface areas
`"v 200-250 m2 g-1 , but the irregular ones tend to have higher values ( 400-500
`m2g - J ). In order to get reproducible k' values the water content of the mobile
`phase must be carefully controlled. This is most easily done by saturating the
`mobile phase with water.
`Selectivity may vary slightly with the pore diameter, but the use of a second(cid:173)
`ary solvent produces more predictable selectivity changes.
`
`6.5.2 Partition
`It is in the partition mode that the chomatographer has the widest choice of
`stationary phases and if conventional coated partition packings are included
`the choice is wider still. However, literature applications are almost exclusively
`with chemically bonded packings, which may be divided into those containing
`polar functional groups and the non-polar packings.
`Compared to silica the polar bonded phases respond more rapidly to mobile
`phase changes and show less peak tailing since the highly polar silanol groups
`are replaced by somewhat less polar groups. The weakly polar packings include
`diol, dimethyhimino or nitro groups. The diol is useful for very polar compounds,
`e.g. organic acids, whilst the nitro group shows a greater selectivity toward
`aromatic molecules. The dimethylamino function can also act as a weak anion
`exchanger.
`Moderately polar packings all contain a cyano function, either alone or as
`a cyanopropyl or cyano-amino grouping. The CN packings are an alternative to
`silica but they give lower k' vil.lues, although the selectivity is about the same. It
`is a very useful general purpose packing usually used in the normal phase mode.
`y The cyano-amino packing (Whatman. Partisil PAC) can be used in both normal
`and reverse phase mode. The secondary amine group shows a different selectivity
`to a primary amine and since it can also be protonated it can function as a weak
`anion exchanger. Its main application is for highly polar compounds where it is
`comparable in selectivity to conventional 'ether' and 'oxynitrile' phases.
`The most polar packings contain amino or aminopropyl functional groups.
`Since they are basic they give rise to quite different selectivities to silica when
`used in the -normal phase mode for the separation of polar compounds. In the
`reverse phase mode they can be used to separate mono, di, tri and polysaccharides
`using acetonitrile: water (80:20). They can also be used as anion exchangers for
`organic acids (e.g. carboxylic acids).
`The non-polar bonded phases used in a reverse-phase mode are the most
`frequently used packings. They are able to provide separation of non-ionic, ionic
`and ionizable solutes by the use of suitable mobile phases and the retention is
`predictable since the k' values usually decrease as the hydrophilic character of
`the solute increases. Column equilibration with mobile phase is very rapid so that
`
`,. I
`
`\
`
`~-
`
`FRESENIUS KABI1023-0017
`
`

`
`Developing a chromat ogram I 157
`
`gradient elution causes no problems.
`All the non-polar bonded phases contain a hydrocarbon or phenyl function.
`However, the method of preparation of the bonded phase can have a profound
`affect on the resulti