Petitioner
`Exhibit 1014 — 001
`
`Petitioner
`Exhibit 1014 - 001
`
`

`
`CHROMATOGRAPHIC
`
`ENANTIOSEPARATION:
`Methods and Applications
`
`STIG G. ALLENMARK
`Laboratory of Microbiol
`‘cal Chemis
`University of Go
`burg, Swe
`
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`3 1863 000 086 334
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`2501 N.
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`Publishers - Chichester
`Halsted Press: a division of
`JOHN WILEY & SONS
`New York - Chichesler - Brisbane -Toronto
`
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`
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`(V;
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`
`
`Petitioner
`Exhibit 1014 — 002
`
`Petitioner
`Exhibit 1014 - 002
`
`

`
`First published in 1988 by
`ELLIS HORWOOD LIMITED
`Market Cross House , Cooper Street,
`Chichester, West Sussex, P019 lEB, England
`The publisher's colophon is reproduced from James Gillison's drawing of the ancient Market Cross,
`Chichester.
`
`Distributors:
`Australia and New Zealand:
`JACARANDA WILEY LIMITED
`GPO Box 859, Brisbane , Queensland 4001, Australia
`Canada:
`JOHN WILEY & SONS CANADA LIMITED
`22 Worcester Road, Rexdale, Ontario, Canada
`Europe and Africa:
`JOHN WILEY & SONS LIMITED
`Baffins Lane, Chichester, West Sussex, England
`North and South America and the rest of the world:
`Halsted Press: a division of
`JOHN WILEY & SONS
`605 Third Avenue, New York, NY 10158, USA
`South-East Asia
`JOHN WILEY & SONS (SEA) PTE LIMITED
`37 Jalan Pemimpin # 05-04
`Block B , Union Industrial Building, Singapore 2057
`Indian Subcontinent
`WILEY EASTERN LIMITED
`4835/24 Ansari Road
`Daryaganj, New Delhi 110002, India
`© 1988 S. G. Allenmark/Ellis Horwood Limited
`
`British Library Cataloguing In Publication Data
`Allenmark, S. G . (Stig G.) , 1936-
`Chromatographic enamtioseparation.
`l . Chromatography
`I. Title
`543'.089
`Library of Congress Card No. 88-1092
`ISBN 0-84312-988-6 (Ellis Horwood Limited)
`ISBN 0-470-21080-X (Halsted Press)
`Typeset in Times by Ellis Horwood Limited
`Printed in Great Britain by Hartnolls, Bodmin
`
`COPYRIGHT NOTICE
`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 permission of Elhs Horwood Limited, Market Cross House, Cooper Street, Chichester, West
`Sussex, England.
`
`Petitioner
`Exhibit 1014 - 003
`
`

`
`Table of contents
`
`Preface . ... . .......... . ........ . ............... . ....... 9
`
`List of Symbols and Abbreviations ................ . ... . ......... 11
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`1 Introduction ..... .. .... .. .. .. . ....................... 13
`Bibliography .................... . ............. . ..... . 18
`References ..... . ....................... ... .... . . .. .. 18
`
`2 The development of modern stereochemical concepts
`2.1 Chirality and molecular structure ......................... 19
`2.1.1 Molecules with asymmetric atoms ...................... 19
`2.1.2 Other types of chiral molecular structures ..... . ........... 20
`2.2 Definitions and nomenclature .......... . ................ 23
`Bibliography ........................... . ...... . .. . . .. 26
`References . ....... . ................ .... .......... .. . 26
`
`3 Techniques used for studies of optically active compounds
`3.1 Determination of optical or enantiomeric purity ............... 27
`3.1. l Methods not involving separation ... .. ................. 27
`3.1. l.1 Polarimetry ................................. 27
`3.1.1.2 Nuclear magnetic resonance .. .. . .. .. . ...... . ..... . 29
`3.1.l.3 Isotope dilutio n .................. . ..... .. ..... 31
`3.1.l.4 Calorimetry .... . ... .. . .. ... . ................ 33
`3.1.1.5 Enzyme techniques ................... . ......... 33
`3.1. 2 Methods based on separation ......................... 34
`3.2 Determination of absolute configuration .................... 35
`3.2.l X-Ray crystallography with anomalous scattering ...... . .. ... 36
`3.2.2 Spectroscopic (ORD , CD) and chromatographic methods
`based on comparison ......... .. ..... .. ............ 37
`Bibliography ................... .. .. ...... .... ...... . .40
`References .. . . ... ............... . ................... 40
`
`Petitioner
`Exhibit 1014 - 004
`
`

`
`Tabie of contents
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`Modern chromatographic separation methods
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`4.1 A review of basic chromatographic theory .
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`4.2 Instrumentation .
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`4.2.1 Gas chromatographicinstrumentation .
`4.2.2 Liquid chromatographic instrumentation .
`' 4.3 Separation of enantiomers by means of covalent
`diastereomers — a survey .
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`4.3.1 Gas chromatography .
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`4.3.2 Liquid chromatography .
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`Theory of chiral chromatography for direct optical resolution
`5.1 The prerequisite for enantioselective interaction with the
`chiral stationary phase .
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`5.2 Some general aspects regarding chiral recognition models
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`5.2.1 Co-ordination to transition metals .
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`5.2.2 Charge-transfer interaction .
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`5.2.3 Inclusion phenomena .
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`5.3 Some thermodynamic and kinetic considerations .
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`5.3.1 Temperature effects .
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`5.3.2 Peak coalescence due to enantiomerization phenomena .
`Bibliography .
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`Chiral gas chromatography
`6. 1 The development of chiral stationary phases based on hydrogen bonding 75
`6.1.1 Amino-acid and oligopeptide derivatives .
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`6.2 Phases based on chiral metal complexes .
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`6.4 Relative merits of the various modes of chiral gas chromatography .
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`Chiral liquid chromatography .
`7.1 Chiral stationary phases based on naturally occurring and
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`7.1.1 Polysaccharides and derivatives .
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`7.1.1.1 Polysaccharides .
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`7.1.1.2 Polysaccharide derivatives .
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`7.1.2 Derivatives of polyacrylamide and similar synthetic polymers .
`7.1.2.1 Sorbents based on chirally substituted synthetic polymers .
`7.1.2.2 Sorbents based on isotactic linear poiymethacryiates of
`helical conformation .
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`Petitioner
`Exhibit 1014 — 005
`
`Petitioner
`Exhibit 1014 - 005
`
`

`
`Table of contents
`
`7.1.3 Synthetic polymers containing ‘grafted’ chiral cavities .
`7.1.4 Proteins
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`7.1.4.1 Immobilized albumin .
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`7.1.4.2 Immobilized C¥1'El.ClCl glycoprotein (orosomucoid)
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`7.2 Bonded synthetic chiral selectors .
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`7.2.1 Crown ethers {‘host—guest’ complexation) .
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`7.3 Techniques based on addition of chiral constituents to the mobile
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`7.3.1 Metal complexation .
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`7.3.2 Uncharged chiral mobile phase additives .
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`Analytical applications in academic research and industry
`8.1 Amino-acids .
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`8.1.1 The enantiomer labelling technique .
`8.1.2 Other applications of chromatographic amino-acid resolution .
`8.2 Stereochemical problems in natural product chemistry .
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`8.2.1 Pheromone stereochemistry .
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`8.3 Pharmaceutical applications .
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`8.4 Studies of microbial and enzymatic reactions .
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`8.4.1 Enzymatic and microbial alkene epoxidation .
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`8.5 Miscellaneous applications and techniques .
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`8.5.1 Determination of enantiomerization barriers .
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`8.5.2 Determination of configuration from chromatographic data .
`8.5.3 Evaluation of enantiomeric purity from chromatographic
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`. 183
`.185
`
`. 185
`
`.
`.
`.
`.
`.
`.
`.
`.
`.
`.
`.
`.
`.
`.
`.
`.
`.
`.
`.
`.
`
`.
`
`.
`
`.
`
`.
`
`Petitioner
`Exhibit 1014 — 006
`
`Petitioner
`Exhibit 1014 - 006
`
`

`
`8
`
`Table of contents
`
`Bibliography .................. . • ........ .. ..... . .. . .. 188
`References .. .... ... .. . .. .... . . ... ... .. .. .. . . .. .. .. . 188
`
`9 Preparative scale enantioseparations- need, progress and problems . . .. 192
`Bibliography . . .... . ..... . ............ . ... .. . .... .... 199
`References ... .. . . . . ... .. . ... . .... .. .... . .. . . .. ..... 199
`
`10 Future trends
`10.l New detector systems . .. .. .. .... . .. .. .... . .... . ... . .. 200
`10.2 Column improvements . . . . ... . .... . ... .. .... . ... . .... 203
`10.3 Supercritical fluid chromatography . . . . .... . . .. .. . ........ 206
`Bibliography .. . .... ... . .. .. ... ... .. ....... .. . . . ..... 206
`References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 206
`
`11 ~xperimental procedures for the synthesis of chiral sorbents
`11.1 Techniques for the preparation of chiral sorbents by
`derivatization of polysaccharides . .. .... ... .. .. . .. .. . . .. . 208
`11.1.1 Preparation of microcrystalline cellulose triacetate (MCTA) .... 208
`11.1 .2 Preparation of silica coated with cellulose triacetate .. .. . . .. . 209
`11.1.3 Preparation of silica coated with cellulose triphenylcarbamate ... 209
`11.2 Polymerization procedures used to obtain chiral synthetic
`polymer materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 209
`11.2.1 Preparation of poly[(S)-N-acryloylphenylalanineethyl ester] ... . 209
`11.2.2 Preparation of polycellulose(triphenylmethyl methacrylate) ... . 211
`11.3 Techniques used for the binding of chiral selectors to silica .. .. . ... 211
`11 .3.1 Preparation of 3-glycidoxypropyl-silica ... . ... ........ . . . 212
`11 .3.2 Large-scale preparation of (R)-N-(3,5-dinitrobenzoyl)-
`phenylglycine covalently silica-bound sorbent . .. .. .. . .. . .. 212
`11 .3.3 Preparation of (S)-(- )-~-N-(2-naphthyl)leucine .. .. .. ...... 213
`11 .3.4 Hydrosilylation of (R)-N-(10-undecenoyl)-~-(6,7-dimethyl-
`1-naphthyl) isobutylamine . ... ... . .. . . . . . . .. ... ... . . 213
`11.3.5 Preparation of silica-bonded (S) -1-(~-napththyl)ethylamine .... 213
`11.3.6 Preparation of silica-bound polyacrylamide and
`polymethacrylamide . . . .. . .... . ...... . ......... . . . 214
`Bibliography .... .. . ... ... .. .. . .. . . .. .. .. . .. .. . ... .. . 214
`References .... . ... . ... . ... ... ... . .. . ... . . .. ... . . ... 214
`
`Appendix: Commercial suppliers of chiral columns for GC and LC . . ....... 216
`
`Index . .. .. ....... .. .... . .... .. ...... .. . . ...... .. .. . . . 218
`
`Petitioner
`Exhibit 1014 - 007
`
`

`
`Chiral liquid chromatography
`
`Though the concept of chiral GC is quite clear, viz. that enantiomers are separable
`with a chiral stationary phase, the situation is more complex for chiral LC. Here we
`can distinguish between two fundamentally different cases, depending upon whether
`enantiodifferentiation takes place through a chiral recognition effect by the station-
`ary phase or by a chiral constituent of the mobile phase forming a diastereomeric
`complex in situ during the chromatographic process. From an experimental point of
`view, a clear distinction can be made between the use of a chiral column (i.e. a
`column containing a CSP) together with an achiral mobile phase, and the use of an
`achiral column together with a chiral mobile phase. In the latter case, however, the
`actual mode of chiral separation will depend on the relative affinity of the chiral
`constituent for the stationary phase and the analyte, respectively. In one extreme
`case, the chiral constituent may become strongly adsorbed on the stationary phase,
`thereby converting it into a CSP, and the separation process would then be regarded
`as a chiral recognition by the CSP thus generated. In the opposite case, the chiral
`constituent has a much lower affinity for the stationary phase than for the analyte.
`This means that diastereomeric complexes are generated in the mobile phase and the
`separation takes place as a normal LC separation of diastereomers. Between these
`two extreme cases there is probably a range of mixed retention modes. In other
`words: are diastereorneric complexes formed at the surface of the chromatographic
`sorbent, or in the mobile phase, or both?
`Chiral sorbents for LC may further be classified with respect to their general
`structural types. Some are based on synthetic or natural polymers and are totally and
`intrinsically chiral. Others consist of chiral selectors of low molecular weight which
`are bound to a hard, incompressible matrix, usually silica. There are also sorbents
`consisting of polymers anchored to silica in order to give improved column
`performance.
`A rough classification of the various modes of chiral LC is given in Table 7.1. This
`is mainly based on the general nature of the chromatographic sorbent, without
`regard to the type of retention process involved, which is often quite complex and
`difficult to evaluate in any detail.
`
`Petitioner
`Exhibit 1014 — 008
`
`Petitioner
`Exhibit 1014 - 008
`
`

`
`Sec. 7.1]
`
`Chiral stationary phases
`
`91
`
`Table 7 .1 - Summary of the main methods used for direct optical resolution by liquid chromatography
`
`Site of chiral
`selector
`
`Stationary phase
`(CSP)
`
`Stationary phase
`(CSP)
`
`Mobile phase
`(CMP)
`
`Basic principle
`
`Capacity
`
`Column efficiency
`
`Use of an intrinsically chiral, polymeric
`stationary phase, either of natural origin
`(polysaccharides and derivatives, pro(cid:173)
`teins) or synthetic (synthetic polymers
`with chiral substituents or 'grafted' chiral
`cavities)
`
`Use of bonded synthetic chiral selectors
`
`Addition of chiral constituents to the
`mobile phase system used. Column
`achiral , usually an alkyl-silica used in
`reversed-phase mode
`
`Analytical to
`preparative
`
`Low to moderate,
`depending on
`whether a support
`material is used or
`not
`
`Analytical to
`preparative
`
`Moderate to fairly
`high
`
`Analytical
`
`Moderate to fairly
`high
`
`7.1 CHIRAL STATIONARY PHASES BASED ON NATURALLY OCCURRING
`AND SYNTHETIC POLYMERS
`Owing to the early recognition of the chiral nature and ready availability of many
`natural products, particularly carbohydrates, such compounds were among the first
`to be tried as sorbents for optical resolution by LC. As early as 1938 a partial
`resolution of a racemic camphor derivative was obtained on a column packed with
`lactose [1]. Lactose remained a column material of interest for some years and was
`used with success in the first nearly complete chromatographic chiral resolution
`described in the literature, which took place in 1944, when Troger's base was
`resolved on a 0.9 m long lactose column [2]. The resolving capacity of a polysacchar(cid:173)
`ide, viz. cellulose, was first realized by the observation that a racemic amino-acid
`could occasionally give two spots in paper chromatography [3-5]. Dalgliesh
`advanced his three-point interaction theory in 1952 on the basis of results from paper
`chromatography of racemic amino-acids [6]. Other early findings on direct optical
`resolution of amino-acids by means of paper chromatography [7] and cellulose thin(cid:173)
`layer chromatography (TLC) (8] were reported. This led to further use of cellulose
`and cellulose derivatives, as well as investigations of starch and cyclodextrins for the
`purpose of chiral LC. At present, derivatives of a large number of natural polysac(cid:173)
`charides are under investigation as potential chiral sorbents.
`The principle of using chiral polymers has also been exploited with many
`different types of totally synthetic materials, by various approaches, and the results
`seem to be very promising.
`Further, the enantioselectivity of proteins, first observed by studies of binding
`equilibria in solution (for reviews see [9,10]), has been successfully utilized for
`analytical chiral LC.
`
`7 .1.1 Polysaccharides and derivatives
`7.1.1.1 Underivatized polysaccharides
`A. Cellulose
`The linear polysaccharide cellulose represents the most common organic compound
`of all. Its chemical constitution is that of a linear poly-~-D-1,4-glucoside (Fig. 7.la).
`
`Petitioner
`Exhibit 1014 - 009
`
`

`
`92
`
`Chiral liquid chromatography
`
`[Ch. 7
`
`(a)
`
`Ii near poly [1- 4-/3-D-glu cos e J
`OH
`Ho·-r--L~~OH H~-r--Cp
`~f ~
`o----
`
`OH
`
`0
`
`0
`
`0
`\
`
`(b)
`
`6-branched po1y[1-4-0'.-0-glu cose J
`
`(some 6 -p ositions are phospho rylated l
`
`Fig. 7.1-The chemical structure of (a) cellulose and (b) amylopectin, the main components of
`starch.
`
`It forms very long chains containing at least 1500 ( + )-D-glucose units per molecule.
`The molecular weight of cellulose ranges from 2.5 x 105 to 1 x 106 or more. In a
`cellulose fibre these long molecules are arranged in parallel bundles and held
`together by numerous hydrogen bonds between the hydroxyl groups . In the native
`state cellulose is therefore built up from partially crystalline regions. These are not
`regenerated on precipitation of cellulose from solution. As seen from Fig. 7 .la the
`( + )-D-glucose repetitive unit contains five chiral centres and three hydroxyl groups.
`All the ring substituents are equatorial.
`It has been found that partial hydrolysis of natural cellulose with dilute mineral
`acid can yield a material with a high degree of crystallinity because hydrolytic
`cleavage will take place preferentially in the amorphous regions. Such a material
`contains ca. 200 glucose units per chain and is usually called 'microcrystalline
`cellulose' [11]. It is marketed as 'Avicel' by several chemical companies.
`Although derivatives of cellulose have been used in most recent research efforts
`and successful resolutions, very good results have also been obtained by the use of
`unmodified cellulose and are therefore worth mentioning. The compounds resolved
`are, without exception, highly polar with multiple sites for hydrogen bond forma(cid:173)
`tion. Some typical results are summarized in Table7.2.
`In a recent work [22], it was found that on treatment with dilute alkali cellulose
`will lose its enantioselective properties owing to a transformation of the native,
`metastable form into a rearranged and stable amorphous form.
`
`Petitioner
`Exhibit 1014 - 010
`
`

`
`Sec. 7.1)
`
`Chiral stationary phases
`
`93
`
`Table 7.2- Examples of optical resolutions performed by liquid chromatography on cellulose
`
`Type of compound
`
`Amino-acids, amino-acid
`derivatives
`
`Diaminodicarboxylic acids
`Synthetic alkaloids
`Cathecins
`
`LC mode
`
`Paper
`Thin layer
`Column
`Column
`Column
`Column
`
`References
`
`(3-5,7,12]
`(8,13,14)
`(lS-17]
`(18)
`(19,20)
`(21]
`
`B. Starch
`The other widespread polysaccharide, also built from ( + )-o-glucose units, is starch.
`Its structure is more complex than that of cellulose. It is composed of ca. 20%
`amylase and 80% amylopectin, the latter being an insoluble fraction. Both are
`entirely composed of ( + )-o-glucose units , Jinked by tX-glucoside bonds. Whereas
`amylase is a linear polymer, amylopectin is branched by C1-C6 connections
`(Fig. 7.lb).
`Depending upon the source, there are different particle sizes of starch available.
`The material obtained from potatoes is relatively coarse (60-100 JLm) and has been
`favoured for column chromatography. Despite its ready availability and non(cid:173)
`swellable properties in aqueous media, which give good ftow properties, it has so far
`found very little use.
`As in the case of cellulose, starch appears to be most suitable for polar aromatic
`compounds. Its use for resolution of atropisomers with structures containing polar
`substituents has been particularly well documented [23-27]. These separations show
`a very pronounced dependence on the nature of the mobile phase, and are especially
`inftuenced by the ionic strength. Figure 7 .2 illustrates the chromatographic behav(cid:173)
`iour of a starch column after application of racemic 2,2' -dinitrodiphenic acid and
`elution with lM sodium citrate buffer, pH 7. 7, at 60°C. The separation factors
`obtained are quite satisfactory but the column efficiency is modest .
`
`C. Cyclodextrins
`As early as 1908 it was discovered by Schardinger [28] that new crystalline carbo(cid:173)
`hydrates, so-called dextrins, were formed if starch was subjected to degradation by a
`micro-organism, Bacillus macerans [29]. These compounds were found to be normal
`P-1 ,4-D-glucosides, but cyclized to rings of 6-12 units. Those with the three smallest
`rings (6-8 units) have been called a-, p-, and y-cyclodextrins (CD), respectively, and
`form inclusion complexes with various compounds of the correct size. The diameter
`of a P-CD ring is 8A and its volume is ca. 350A3
`. The stability of the inclusion
`complex is largely dependent on the hydrophobic and steric character of the guest.
`These phenomena make CDs, particularly P-CD, which is easily available, highly
`promising for use in chiral LC.
`The major development in chiral LC with cyclodextrins started with the tech(cid:173)
`nique of using them as mobile phase additives in TLC experiments [30-32]. This
`technique has also been applied with success to column chromatography and will be
`treated in Section 7.3. Earlier some efforts to use cross-linked cyclodextrin gels for
`
`Petitioner
`Exhibit 1014 - 011
`
`

`
`Chiral liquid chromatography
`
`to?» CUM
`
`ND;
`
`lM Na—citrete
`60“C
`
`3.70
`
`190
`
`Mu
`i ml 1 — -
`
`L35!
`
`nan
`
`b7[}
`
`Fig. 7.2 — Optical resolution of atropisomers on a starch column. (Reprinted, with permission.
`lrom H. Hess. G. Burger and H. Musso. Angew Charm. 1978, 90. 645. Copyright 1978, Verlag
`Chemie Gmbll)
`
`chromatographic purposes had also been made [33—36]. The first attempts to
`immobilize CDs on solid supports were made quite recently [37.38]. By an improve-
`ment in coupling techniques. a highly efficient B—CD silica-bonded phase column is
`now available [$59.40].
`Since the formation of inclusion complexes with CDs in aqueous systems is based
`mainly on hydrophobic interaction. it is logical that a CD column operates entirely in
`a reversed-phase mode. Consequently. the mobile phase systems normally used are
`the same as those used in ordinary reversed-phase LC, usually methanovwater or
`acetonitrile/water. This also means that buffers can be used to control the pH and
`possibly affect the retention of charged solutes.
`The rather special type of solute-CSP interaction present in the case of immobi-
`lized CD5 deserves particular attention. The inclusion complexes formed are of great
`interest. not only from a theoretical point of view. This field, which belongs to
`‘host-guest‘ chemistry (like the crown-ether complexes which will be treated in
`Section 7.2.1).
`is important in achieving better understanding of the role and
`function of ordered molecular complexes in biological systems.
`The conformation of a cyclodextrin in an aqueous system is generally assumed to
`approximate to a truncated cone. Fig. 7. 3. possessing a hydrophobic internal surface.
`Hydrophobic molecules such as benzene or hexane. which can diffuse in and out of
`the cavity, are reversibly adsorbed on this surface. Retention of a hydrophobic solute
`should largely be dependent on the efficiency of the contact with the interior of the
`cavity. Enantioselectivity is likely to be associated with the chiral structure at the
`entrance of the cavity, caused by the exposed 2- and 3-hydroxy groups in the glucose
`units. If the solute is of a suitable size . allowing good contact with the internal surface
`and hence restricted in movement. then a different interaction between the chiral
`
`Petitioner
`Exhibit 1014 - 012
`
`

`
`Chiral stationary phases
`
`Fig. 7.3 — The chemical structure of B-cyclodextrin and its assumed conformation in an
`aqueous solution.
`
`cavity entrance and the substituents of the two enantiomers may cause a difference in
`both the complexation constants and the chromatographic k’ values.
`The effect of the mobile phase on the enantioselectivity appears to be large. In
`general. both the k’ and or values tend to decrease with increasing content of organic
`modifier. In most cases, methanol as a retention modifier will decrease at to a lesser
`degree than acetonitrile. Retention on CD columns is markedly affected by the
`temperature, decreasing rapidly to zero between 60 and 80°C in most cases. This
`might be due to an increased conformational mobility of the ring with increased
`temperature.
`
`7.1.1.2 Polysacclurlde derlvatlv
`A. Cellulose triacetate
`
`In 1966 Lfittringhaus and co-workers [41.42] found that a partially acetylated
`cellulose (described as a 2.5 acetate) could be used with ethanol to achieve optical
`resolution in column chromatography. Some years later another German research
`group carefully investigated the heterogeneous acetylation of native (microcrystal-
`line) cellulose and found that a triacetate could be prepared with almost complete
`preservation of the microcrystallinity and excellent resolving properties [43]. They
`pointed out that the microcrystallinity was essential for the enantioselective proper-
`ties of the material. since the optical resolution power was totally lost on dissolution
`and reprecipitation. The metastable state of the material was evident from these
`experiments as the change was found to be irreversible. On the basis of the results
`from these investigations it was concluded that microcrystalline cellulose triacetate
`
`Petitioner
`Exhflwit 1014 - 013
`
`

`
`
`
`96
`
`Chiral liquid chromatography
`
`[Ch. 7
`
`(MCTA) gave retention by means of inclusion of the solute into molecular cavities in
`the chiral matrix. Therefore the term ‘inclusion chromatography’ was used [44,45].
`Owing to the availability and low cost of ‘microcrystalline’ cellulose (Avicel), the
`well described technique of its acetylation and the interesting properties of the
`product, MCTA has been the subject of extensive research during the last decade.
`Large columns can be packed with this cheap material and relatively large quantities
`of sample can be used, permitting preparative work. A typical example of a
`resolution on MCTA is shown in Fig. 7.4.
`
`Methylcyclohexylethyl-
`lJGl'bl‘lUl’lC acid
`
`l-l
`
`C2Hs
`
`O
`
`O
`
`HNYN\CH 3
`
`
`
`concentration———>
`
`500
`
`700
`eluoie (ml! *9
`
`900
`
`1‘lUU
`
`Fig. 7.4 — Separation of the enantiomers of 205 mg of (i)-methylcyclohexylethylbarbituric
`acid on 210g of MCTA. (Column 85 X 2.5 cm, ethanol 96%. flow-rate 50 mllhr). (Reprinted
`from G. Blaschke, J. Liquid Chromarog, 1986, 9, 341 by courtesy of Marcel Dekker, Inc.).
`
`It is very important that MCTA is allowed to swell in boiling ethanol before being
`packed into a column. Ethanol (95%) is a good medium for swelling, and does not
`dissolve MCTA.
`The inclusion mode of retention of solutes on MCTA is consistent with the very
`different chromatographic behaviour shown by benzene and mesitylene (1,3,5-
`trimethylbenzene), the first being much more strongly retained owing to better
`permeation into the cavities. 1,3,5-Tri-ten‘-butylbenzene is totally excluded and
`therefore of use for void volume determinations [46].
`Further evidence for the inclusion model of retention is the fact that very non-
`polar solutes, lacking functional groups, can be resolved. Thus, racemic mms-1,2-
`diphenylcyclopropane is easily resolved into its antipodes on MCTA [47]. Optical
`resolutions of a great number of structurally quite different compounds on MCTA
`
`
`
`Petitioner
`Exhibit 1014 — 014
`
`Petitioner
`Exhibit 1014 - 014
`
`

`
`Sec. 7.1]
`
`Chiral stationary phases
`
`97
`
`have been described to date. many on a preparative or semipreparative. scale. This
`will be treated further in Chapter 9.
`The main drawbacks of MCTA are its compressibility and relatively large,
`irregular and inhomogeneous particle size. This means that preparative columns can
`only be run at very low linear flow-rates (cf. the conditions used in Fig. 7.4). The
`latter problem can be partially solved by grinding and careful fan-sieving of the
`material. However. since MCTA requires a swollen state to function well, reduction
`of the compressibility is a more difficult problem.
`An exhaustive study of the influence of the supramolecular structure of cellulose
`triacetate on the chromatographic optical resolution of several racemates was
`recently made by Francotte er al. [48]. Their results confirmed the original ideas by
`Hesse and l-Iagcl [43—45] that the inclusion of low molecular-weight chiral molecules
`into a specific spatial arrangement of the glucose units of the polysaccharide chains is
`of fundamental importance for the chiral discrimination process.
`During experiments with deposition of CI‘A into silica gel particles. it was found
`by a Japanese research group [49,50] that, although the dissolved and reprecipitated
`polymer had apparently lost most of its microcrystalline structure. the new material
`still possessed sortie resolving capacity. The results presented in Table 7.3 are
`
`Table 7.3 — A comparison between microeryslalline (l) and reprecipituted (ll) cellulose triacela