Chiral Separations - ACS Symposium Series (ACS Publications)

Chapter 1

Chiral Separations An Overview

Downloaded by UNIV OF ILLINOIS CHICAGO on May 25, 2012 | http://pubs.acs.org Publication Date: August 14, 1991 | doi: 10.1021/bk-1991-0471.ch001

Satinder Ahuja Development Department, Pharmaceuticals Division, CIBA-GEIGY Corporation, Suffern, NY 10901

Optically active compounds have attracted great attention because living systems are chiral. Proteins, nucleic acids, and polysaccharides possess chiral characteristic structures that are related closely to their functions. Because of chirality, living organisms usually show different biological responses to one of a pair of enantiomers (optical isomers) i n drugs, pesticides, or waste compounds. For example, sodium L-(+)glutamate tastes good, whereas i t s mirror image D-(-)glutamate tastes bitter or f l a t , depending on the taster (1). Molecules that relate to each other as an object and i t s mirror image that i s not superimposable are enantiomers or chiral (from the Greek word, cheiro, meaning hand); they are like a pair of hands. Stereoisomers are isomeric molecules with identical constitution but a different spatial arrangement of atoms. The symmetry factor classifies stereoisomers as either enantiomers, as defined above, or diastereoisomers. A pair of enantiomers i s possible forallmolecules containing a single chiral carbon atom (one with four different groups attached). Diastereoisomers or diastereomers, are basically stereoisomers that are not enantiomers of each other. Although a molecule may have only one enantiomer, it may have several diastereoisomers. However, two stereoisomers cannot be both enantiomers and diastereoisomers of each other simultaneously. Stereoisomerism can result from a variety of sources, including the single chiral carbon (or chiral center), for example, a chiral atom that i s a tetrahedral atom with four different substituents. I t i s not necessary for a molecule to have a chiral carbon i n order to exist i n enantiomeric forms, but i t i s necessary that the molecule as a whole be chiral. Detailed discussion on these topics may be found i n several books and review articles (2-9). Enantiomers have identical physical properties except for a plus or minus sign of the optical rotation. A racemate, a mixture consisting of equal amounts of enantiomers i s obtained experimentally by chemi0097-6156/91/0471-0001$07.50/0 © 1991 American Chemical Society In Chiral Separations by Liquid Chromatography; Ahuja, S.; ACS Symposium Series; American Chemical Society: Washington, DC, 1991.

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CHIRAL SEPARATIONS BY LIQUID C H R O M A T O G R A P H Y

c a l reactions c a r r i e d out i n an a c h i r a l environment. separation of an enantiomeric mixture, or o p t i c a l necessary f o r y i e l d i n g o p t i c a l l y pure species.

Therefore, the resolution, i s


I t should be noted that the R,S convention f o r nomenclature i s not necessarily connected with the d i r e c t i o n of o p t i c a l r o t a t i o n , i . e . , the r o t a t i o n that solutions of c e r t a i n molecules impart to the plane of polarized l i g h t . This c l e a r l y indicates that a molecule's c h i r a l i t y , evidenced by the d i r e c t i o n or magnitude of s p e c i f i c r o t a t i o n , must be experimentally determined (although some empirical c o r r e l a tions e x i s t ) regardless of i t s absolute configuration. Accurate assessment of the isomeric p u r i t y of substances i s c r i t i c a l since isomeric impurities may have unwanted t o x i c o l o g i c , pharmacolog i c , or other e f f e c t s . Such impurities may be carried through a synthesis and p r e f e r e n t i a l l y react at one or more steps and y i e l d an undesirable l e v e l of another impurity. Frequently one isomer of a series may produce a desired e f f e c t , while another may be i n a c t i v e or even produce some undesired e f f e c t . Large differences i n a c t i v i t y between stereoisomers point out the need to accurately assess i s o ~ meric p u r i t y of pharmaceutical, a g r i c u l t u r a l , or other chemical e n t i t i e s . Often these differences e x i s t between enantiomers, the most d i f f i c u l t stereoisomers to separate. Some examples of a c t i v i t y d i f ferences between stereoisomers are noted i n Table 1.1. Many factors may be responsible f o r the extent of interactions of stereoisomeric molecules i n any environment: e l e c t r o s t a t i c forces; inductive e f f e c t s ; dipole-dipole i n t e r a c t i o n s ; ion-dipole i n t e r a c t i o n s ; hydrogen bonding; hydrophobic bonding; resonance i n t e r a c t i o n s / s t a b i l i z a t i o n ; van der Waals forces; s t r u c t u r a l rigidity/conformat i o n a l f l e x i b i l i t y ; s t e r i c interference, that i s , s i z e , o r i e n t a t i o n , and spacing of groups; s o l u b i l i t i e s ; pKa d i f f e r e n c e s — e x t e n t of i o n i z a t i o n ; p a r t i t i o n c o e f f i c i e n t differences; ligand formation; and temperature. The nature and e f f e c t s of some of these factors as they influence chromatography of stereoisomers i s of great importance. The importance of determining the stereoisomeric composition of chemical compounds, e s p e c i a l l y those of pharmaceutical importance, cannot be overemphasized (1). Dextromethorphan provides a dramatic example i n that i t i s an over-the-counter a n t i t u s s i v e , whereas levomethorphan, i t s stereoisomer, i s a controlled n a r c o t i c . Likewise, i t has been reported that the teratogenic a c t i v i t y of thalidomide may reside e x c l u s i v e l y i n the (S)-enantiomer (10). Less dramatic examples abound; 12 of the 20 most prescribed drugs i n the USA and 114 of the top 200 possess one or more asymmetric centers i n the drug molecule (11). About h a l f of the 2050 drugs l i s t e d i n the U.S. Pharmacopeial Dictionary of Drug Names contain at l e a s t one asymmetric center, and 400 of them have been used i n racemic or diastereomeric forms (12). The differences i n the physiologic properties between enantiomers of these racemic drugs have not yet been examined i n many cases, probably because of d i f f i c u l t i e s of obtaining both enantiomers i n o p t i c a l l y pure forms. Some enantiomers may e x h i b i t p o t e n t i a l l y d i f f e r e n t pharmacologic a c t i v i t i e s , and the patient may be taking a useless, or even undesirable, enantiomer when ingesting a racemic

In Chiral Separations by Liquid Chromatography; Ahuja, S.; ACS Symposium Series; American Chemical Society: Washington, DC, 1991.

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Chiral Separations: Overview

Table 1.1

A c t i v i t i e s of Some Stereoisomers (6)

Compound

Stereoisomers and A c t i v i t i e s

Amphetamine

d-Isomer i s a potent CNS stimulant, while £isomer has l i t t l e , i f any, e f f e c t trans-Isomer i s much more estrogenic than c i s -

Diethylstilbestrol (DES)


Ascorbic acid Propoxyphene Epinephrine Synephrine Quinine/Quinidine Bermethrin Propranolol Warfarin

(+) Isomer i s a good antiascorbutic, while (-) has no such properties a-£ i s an active a n t i t u s s i v e , a-d i s a potent analgesic, but p-d and p-£ are s u b s t a n t i a l l y inactive (-) Isomer i s more than 10 times more active a vasoconstrictor than (+) (-) Isomer has 60 times the pressor a c t i v i t y than (+) Quinidine i s (+) enantiomer, w i t h cardiac suppressant e f f e c t s ; quinine i s (-) enantiomer with other medicinal uses d-Isomers of t h i s i n s e c t i c i d e are much more t o x i c than Si Racemic propranolol i s administered but only S-(-)isomer has desired p-adrenergic blocking activity Racemic warfarin i s administered but S-(-) isomer i s 5 times more potent as a blood a n t i coagulant than R-(+) isomer


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mixture. To ensure the safety and e f f e c t of currently used and newly developing drugs, i t i s important to i s o l a t e and examine both enantiomers separately. Furthermore, i t i s necessary, i n at l e a s t three s i t u a t i o n s , to measure and control the stereochemical composition of drugs. Each s i t u a t i o n presents a s p e c i f i c t e c h n i c a l problem: (a) during manufacture, where problems of p h y s i c a l , preparative scale separations may be involved; (b) during q u a l i t y control (or regulatory a n a l y s i s ) , where a n a l y t i c a l questions of p u r i t y and s t a b i l i t y predominate; (c) during pharmacologic studies of plasma d i s p o s i t i o n and drug e f f i c a c y , where u l t r a t r a c e methods may be required (4). 1.1

Chromatographic Methods

Optical r e s o l u t i o n of racemates i s one way to obtain pure isomers. Since Pasteur reported the f i r s t example of o p t i c a l r e s o l u t i o n i n 1848, more than 7000 compounds have been resolved, mainly by f r a c t i o n a l c r y s t a l l i z a t i o n of diastereomeric s a l t s . Resolution by entrainment or with enzyme or b a c t e r i a has also been applied to a large-scale separation of some racemates, f o r example, amino acids. Chromatographic methods are considered most u s e f u l f o r o p t i c a l resol u t i o n . A h i s t o r i c a l account of c h i r a l separations by chromatography i s given i n Table 1.2. D e r i v a t i z a t i o n of a given enantiomeric mixture with a c h i r a l reagent, leading to a p a i r of diastereomers, ( i n d i r e c t method) allows separation of samples by chromatography. On the other hand, using the c h i r a l stationary or mobile-phase systems i n chromatography ( d i r e c t method) i s an a l t e r n a t i v e procedure that has recently come into use. This approach has been examined rather extensively by many research s c i e n t i s t s . E a r l y successful r e s u l t s d i d not a t t r a c t much i n t e r e s t ; the technique remained r e l a t i v e l y dormant and l i t t l e was done to develop t h i s approach into a generally applicable method. Less than 20 years ago, systematic research was i n i t i a t e d f o r the design of c h i r a l stationary phases functioning to separate enantiomers by gas chromatography. Molecular design and preparation of the c h i r a l phase systems f o r l i q u i d chromatography have been examined since then. More recently, e f f o r t s have been directed to f i n d i n g new types of c h i r a l stationary and mobile phases on the basis of the stereochemical viewpoint and the t e c h n i c a l evolution of modern l i q u i d chromatography. Since HPLC i s now one of the most powerful separation techniques, r e s o l u t i o n of enantiomers by HPLC i s expected to move r a p i d l y i f an e f f i c i e n t c h i r a l stationary phase (CSP) i s a v a i l a b l e . Large-scale, preparative l i q u i d chromatography systems have already been put on the market as process u n i t s f o r i s o l a t i n g and p u r i f y i n g chemicals and n a t u r a l products. C h i r a l HPLC i s i d e a l l y suited f o r large-scale preparation of o p t i c a l isomers. I t i s now recognized that chromatographic methods (TLC, GLC, and HPLC) o f f e r d i s t i n c t advantages over c l a s s i c techniques i n the separation and analysis of stereoisomers, p a r t i c u l a r l y f o r the more d i f f i c u l t c l a s s , enantiomers (2,3,13-16). Most of the discussion i n t h i s book i s on HPLC, as i t o f f e r s the greatest promise. Chromatographic methods show promise f o r moderate-scale separations of syn-


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Table 1.2

Chiral Separations: Overview H i s t o r i c a l Account of C h i r a l Separations by Chromatography

1939

Henderson and Rule: r e s o l u t i o n of a racemic camphor d e r i v a t i v e by chromatography on lactose

1952

D a l g l i e s h : p o s t u l a t i o n of the three-point r u l e i n the paper chromatography of amino acids

1966

Gil-Av et a l . : d i r e c t r e s o l u t i o n of enantiomers by GC

1971

Davankov and Rogozhin: Introduction of c h i r a l ligand exchange chromatography

1972

Wulff and Sarhan: preparation of enzyme analogue polymers f o r c h i r a l LC

1973

Hesse and Hagel: preparation of c e l l u l o s e t r i a c e t a t e f o r chiral resolution

1973

Stewart and Doherty: use of agarose-bonded bovine serum a l bumin (BSA) f o r c h i r a l r e s o l u t i o n .

1974

Blaschke: synthesis of c h i r a l polymers from o p t i c a l l y a c t i v e monomers, f o r c h i r a l LC

1975

Cram and co-workers: development of host-guest chromatography using c h i r a l crown ethers

1979

P i r k l e and House: synthesis of f i r s t silica-bonded CSP and a p p l i c a t i o n i n c h i r a l LC

1979

Okamoto et a l . : synthesis of h e l i c a l polymers f o r c h i r a l LC

1982

Allenmark et a l . : use of agarose-bonded BSA i n c h i r a l LC

1983

Hermansson: use of silica-bonded ax-acid glycoprotein f o r chiral resolution

1984

Armstrong and DeMond: preparation of silica-bonded cyclodextrins


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t h e t i c intermediates as w e l l as f o r f i n a l products. For large-scale separations and i n consideration of the cost of plant-scale resolut i o n processes, the sorption methods o f f e r s u b s t a n t i a l increases i n e f f i c i e n c y over r e c r y s t a l l i z a t i o n techniques. Of various stereoisomers, diastereomers s p e c i f i c a l l y are inherently easier to separate because they already possess differences i n phys i c a l properties. In recent years many s i g n i f i c a n t advances have occurred which allow the chromatographic r e s o l u t i o n of enantiomers. There are b a s i c a l l y two approaches to the separation of an enantiomeric p a i r by chromatography. In the i n d i r e c t approach, the enantiomers may be converted into covalent, diastereomeric compounds by a reaction with a c h i r a l reagent, and these diastereomers are t y p i c a l l y separated on a routine, a c h i r a l stationary phase. In the d i r e c t approach, several v a r i a t i o n s can be t r i e d : (a) the enantiomers or t h e i r d e r i v a t i v e s are passed through a column containing a c h i r a l stationary phase, or (b) the d e r i v a t i v e s are passed through an a c h i r a l column using a c h i r a l solvent or, more commonly, a mobile phase that contains a c h i r a l a d d i t i v e . In e i t h e r v a r i a t i o n of the second case, one depends on d i f f e r e n t i a l , t r a n s i e n t diastereomer formations between the solutes and the s e l e c t o r to e f f e c t the observed separation. 1.2 Modes of Separation The chromatographic separation of enantiomers can be achieved by various methods; however, i t i s always necessary to use some kind of c h i r a l discriminator or s e l e c t o r (7,17). Two d i f f e r e n t types of selectors can be distinguished: a c h i r a l a d d i t i v e i n the mobile phase (see Section 1.2.2) or a c h i r a l stationary phase (Section 1.2.3). Another p o s s i b i l i t y i s precolumn d e r i v a t i z a t i o n (Section 1.2.1) of the sample with c h i r a l reagents to produce diastereomeric molecules which can be separated by non-chiral chromatographic methods. The mechanism of separation i s dependent on the mode of separation used. Some discussion on the mechanism of separation i s provided f o r each mode of separation; however, i t should be recognized that det a i l e d mechanisms f o r c h i r a l separations have not been worked out. The proposals made by c e r t a i n s c i e n t i s t s appear a t t r a c t i v e ; however, vigorous differences p r e v a i l , so an attempt has been made not to h i g h l i g h t a s i n g l e proposal. Various types of columns used f o r c h i r a l separations are given i n Table 1.3. Detailed below are various approaches that can be used f o r c h i r a l separations. 1.2.1

Chromatography of Diastereomeric Derivatives

This i s the oldest and most widely used chromatographic approach to the r e s o l u t i o n of enantiomers (8). The precolumn d e r i v a t i z a t i o n of an o p t i c a l l y active solute with another o p t i c a l l y a c t i v e molecule depends on the a b i l i t y to d e r i v a t i z e the target molecule. A large number of f u n c t i o n a l groups and d e r i v a t i v e s have been investigated including amino groups ( d e r i v a t i z e d t o amides, carbamates, ureas, thioureas, and sulfonamides), hydroxyl groups (esters, carbonates,


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7

and carbamates), carboxy groups (esters and amides), epoxides ( i s o thiocyanate), o l e f i n s ( c h i r a l platinum complexes), and t h i o l s ( t h i o esters).


This method has been used with a wide v a r i e t y of HPLC columns and mobile phases, including normal- and reversed-phase approaches. At present there i s no d e f i n i t i v e way of determining which chromatographic approach w i l l work. The advantages of d e r i v a t i z a t i o n are: 1.

The methodology has been extensively studied, making the a p p l i cation r e l a t i v e l y easy and accessible.

2.

I t i s possible to use r e a d i l y a v a i l a b l e , standard HPLC supports and mobile phases.

3.

D e t e c t a b i l i t y can be improved by appropriate s e l e c t i o n of a d e r i v a t i z i n g agent with a strong chromophore or fluorophore.

The main l i m i t a t i o n s are as follows: 1.

The synthesis of the diastereomeric derivatives requires the i n i t i a l i s o l a t i o n of the compounds of i n t e r e s t p r i o r to t h e i r derivatization. This hinders the development of an automated procedure f o r large numbers of samples.

2.

The a p p l i c a t i o n to routine assays often i s l i m i t e d by enantiomeric contamination of the d e r i v a t i z i n g agent, which can lead to inaccurate determinations. The problem of enantiomeric contamination of the d e r i v a t i z i n g agent has been encountered i n a number of studies. S i l b e r and Riegelman (18), f o r example, used ( - ) - N - t r i f l u o r o a c e t y l - l - p r o l y l chloride (TPC) i n the determinat i o n of the enantiomeric composition of propranolol i n b i o l o g i c a l samples. They found that commercial TPC was contaminated with 4 to 15% of the (+)-enantiomer and that the reagent r a p i d l y racemized during storage.

3.

Enantiomers can have d i f f e r e n t rates of reaction and/or e q u i l i brium constants when they react with another c h i r a l molecule. As a r e s u l t , two diastereomeric products may be generated i n proportions d i f f e r e n t from the s t a r t i n g enantiomeric composition (19).

1.2.2

Enantiomeric Resolution Using C h i r a l Mobile-phase Additives

The resolution of enantiomeric compounds has been accomplished through the formation of diastereomeric complexes with a c h i r a l molecule (s) added to the mobile phase. The c h i r a l r e s o l u t i o n i s due to differences i n the s t a b i l i t i e s of the diastereomeric complexes, s o l v a t i o n i n the mobile phase, or binding of the complexes to the s o l i d support. A general overview of t h i s method has been published by Lindner and Pettersson (20). There are three major approaches to the formation of diastereomeric complexes: t r a n s i t i o n metal i o n complexes (ligand exchange), ion



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Table 1.3 Commercial C h i r a l Columns

Name

Chemical Name

Apex p r e p s i l L-ualylphenylurea

(S)-valyl-phenylurea

Bakerbond c h i r a l covalent DNBLeu

(S)-DNB-Leucin (covalent)

Bakerbond c h i r a l covalent DNBPG

(R)-DNB-Phenylglycine

(covalent)

Bakerbond c h i r a l i o n i c DNBPG


(ionic)

Cellulose CEL-AC-40 XF

Cellulose-triacetate

Chiral-AGP

Ofi-Acid-glycoprotein

C h i r a l c e l CA-1


ChiraIce1 OA


C h i r a l c e l OB

Cellulose-tribenzoate

C h i r a l c e l OC

Cellulose-trisphenylcarbamate

C h i r a l c e l 00

Cellulose-tris-3,5-dimethylphenylcarbamate

C h i r a l c e l OF

Cellulose-tris-4-chlorphenylcarbamate

C h i r a l c e l OG

Cellulose-tris-4-toluylcarbamate

C h i r a l c e l OJ

Cellulose-tris-4-toluylate

C h i r a l c e l OK

Cellulose-tricinnamate

C h i r a l c e l WE

N-(2-Hydroxy-1,2-diphenylethyl)glycine-copper

Chiralpak 0T(+)

Poly(triphenylmethy1-methacry1ate)

Chiralpak 0P(+)

Poly(2-pyridyl-diphenylmethyl-methacrylate)

Chiralpak WH

P r o l i n e copper

Chiralpak UM

Amino acid copper

C h i r a l D-DL=Daltosil 100

(R)-DNB-Leucine (covalent)

C h i r a l L-DL=Daltosil 100

(S)-DNB-Leucine (covalent)

C h i r a l D-DPG=SilOO


(covalent)

C h i r a l L-DPG=Daltosil 100

(S)-DNB-Phenylglycine

(covalent)

C h i r a l hypra-Cu=Daltosil 100

Hydroxyproline copper

C h i r a l proCu=SilOO

Proline copper

C h i r a l valCu=SilOO

Valine copper

Chiral protein 1

Beef serum albumin

C h i r a l protein 2

Human serum albumin

ChiraSpher

Poly-n-acryloyl-(S)-phenylalaninethylester

ChiRSil I

(R)-ONB-Phenylglycine

(ionic)

Chi-RoSil


(ionic)

Covalent L-leucine

(S)-DNB-Leucin (covalent)

Covalent

D-naphthylalanine

(R)-Naphthylalanine

Covalent

L-naphthylalanine

(S)-Naphthylalanine

Covalent

D,L-naphthylalanine

(R,S)-Naphthylalanine

Covalent D-phenyl glycine

(R)-DMB-Phenylglycine (covalent)

Covalent L-phenyl glycine

(S)-DMB-Phenylglycine

Covalent D,L-phenyl glycine

(R,S)-DNB-Phenylglycine

(covalent) (covalent)


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for HPLC*

Type

Particle Size

Source

Brush

8

Jones

Brush

5

Baker

Brush

3,5,10

Baker

Brush

5

Baker

Helix

7

Macherey-Nagel ChromTech

Protein

5

Helix

10

Daicel

Helix

10

Daicel

Helix

10

Daicel

Helix

10

Daicel

Helix

10

Daicel

Helix

10

Daicel

Helix

10

Daicel

Helix

10

Daicel

Helix

10

Daicel

Ligand exchange

10

Daicel

Helix

10

Daicel

Helix

10

Daicel

Ligand exchange

10

Daicel

Ligand exchange

10

Daicel

Brush

4

Serva

Brush

4

Serva

Brush

3,5

Serva

Brush

4

Serva

Ligand exchange

4

Serva

Ligand exchange

5

Serva

Ligand exchange

5

Serva

Protein

SFCC

Protein

SFCC

Helix

5

Brush

5,10

Merck RSL

Brush

5

RSL

Brush

5

Regis, A l l t e c h

Brush

5

" 11

11

Brush

5

Brush

5

"

Brush

5,10

Brush

5,10

"

"

Brush

5

"

"

" 11

" "

Continued on


10



Table 1.3

Commercial C h i r a l Columns f o r HPLC*

Name

Chemical Name

Crownpak CR

Crown ether

Cyclobond I

p-Cyclodextrin

Cyclobond I I

Y-Cyclodextrin

Cyclobond I I I

a-Cyclodextrin

Cyclobond I-acetylated

Acetylated p-cyclodextrin

Cyclobond I I I - a c e t y l a t e d

Acetylated-cyclodextrin

Of-Cyclodextrin=Daltosil 100

a-Cyclodextrin

p-Cyclodextrin=Daltosil 100

p-Cyclodextrin

EnantioPac

Ofi-Acid-glycoprotein

ES D-DMB-LEU

(R)-DNB-Leucine

(covalent

ES L-DNB-LEU

(S)-DNB-Leucine

(covalent)

ES D-DNB-PHGLY

(R)-DNB-Phenylglycine (covalent)

ES L-DNB-PHGLY

(S)-DNB-Phenylglycine (covalent)

ES R-PU

(R)-Phenylethylurea

ES S-PU

(S)-Phenylethylurea

Grom-chiral-(R)-DNBPG-C

(R)-DNB-Phenylglycine (covalent)

Grom-chiral-(R)-DNBPG-I

(R)-DNB-Phenylglycine ( i o n i c )

Grom-chiral-(S)-DNBPG-C

(S)-DNB-Phenylglycine (covalent)

Grom-chiral-(S)-DNBPG-I

(S)-DNB-Phenylglycine ( i o n i c )

Grom-chiral-(R)-DNBL-C

(R)-DNB-Leucine

Grom-chiral-(R)-DNBL-I

(R)-DMB-Leucine ( i o n i c )

(covalent)

Grom-chiral-(S)-DNBL-C

(S)-DNB-Leucine

Grom-chiral-(S)-DNBL-I

(S)-DNB-Leucine ( i o n i c )

(covalent)

Grom-chiral-beta-CD

p-Cyclodextrin

Grom-chiral-HP


Grom-chiral-P

Prolinamide

Grom-chiral-PC


Grom-chiral-PC


Grom-chiral-U

(R)-N-a-Phenylethylurea

Grom-chiral-UC

U a l i n copper

Ionic L-leucine

(S)-DMB-leucine ( i o n i c )

Ionic D-phenyl glycine

(R)-DMB-phenylglycine ( i o n i c )

MCI g e l CRSI0W

C - S i l i c a g e l with N,N-dioctyl-(S)-alanine

Nucleosil chiral-1


1 8

Nucleosil chiral-2 Optimer PI

Aromatic amide

Optimer LI

A l i p h a t i c amino acid copper

Optimer L2

Aromatic amino acid copper

Resolvosil

Beef serum albumin


1.


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11

(continued) Particle Size

Source

Cavity

10

Daicel

Cavity

5

Astec

Cavity

5

Astec


Type

Cavity

5

Astec

Cavity

5

Astec

Cavity

5

Astec

Cavity

4

Serva

Cavity

4

Serva

Protein

10

Pharmacia

Brush

5

ES

Brush

5

ES

Brush

5

ES

Brush

5

ES

Brush

5

ES

Brush

5

ES

Brush

5

Grom

Brush

5

Grom

Brush

5

Grom

Brush

5

Grom

Brush

5

Grom

Brush

5

Grom

Brush

5

Grom

Brush

5

Grom

Cavity

5

Grom

Ligand exchange

5

Grom

Ligand exchange

5

Grom

Ligand exchange

5

Grom

Ligand exchange

5

Grom

Brush

5

Grom

Ligand exchange

5

Grom

Brush

5


Brush

5


Ligand exchange

Mitsubishi

Ligand exchange

5

Macherey-Nagel

Brush

5

Macherey-Nagel

Brush

4

Toyo Soda

Ligand exchange

5

Toyo Soda

Ligand exchange

5

Toyo Soda

Protein

7

Macherey-Nagel

Continued on next page


12



Table 1.3 Commercial C h i r a l Columns f o r HPLC*

Name

Chemical Name

Spherisorb c h i r a l 1

(R)-N-ct-Phenylethylurea

Spherisorb c h i r a l 2

(R)-Naphthylethylurea

Sumichiral OA-1000, OA

a-Naphthylethylamide

Sumichiral OA-2000

(R)-DMB-Phenylglycine ( i o n i c )

Sumichiral OA-200OA


Sumichiral OA-2100

Chlorphenyl-isovaleroyl-phenylglycine

Sumichiral 0A-2200

Chrys anthemoy1-phenylglycine

Sumichiral OA-3000

t e r t , Butylaminocarbonyl-valine

Sumichiral OA-4000

(S),(S)-a-Naphthylethyl-aminocarbonyl-valine

Sumichiral OA-4100

(R),(R)-a-Naphthylethyl-aminocarbonyl-valine

Supelcosil

LC-(R)-naphthylurea

(R)-Naphthylethylurea

Supelcosil

LC-(R)-urea

Triacetylcellulose

(covalent)

(R)-Phenylethylurea Cellulose-triacetate

Trichsep-100

Cellulase

TSKgel Enantio L i

A l i p h a t i c amino acid copper

TSKgel Enatio L2

Aromatic amino acid copper

TSKgel Enantio PI

(S)-Aromatic amide

Ultron OVM

Ovomucoid

VMC-Pak K

Polymer with

(R)-naphthylethylamine

*Adapted from V. Meyer


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(continued)

Type

Particle Size

Source

Brush

5

PhaseSep

Brush

5

PhaseSep

Brush

5,10

Sumika

Brush

5,10

Sumika

Brush

5,10

Sumika

Brush

5,10

Sumika

Brush

5,10

Sumika

Brush

5,10

Sumika

Brush

5,10

Sumika

Brush

5,10

Sumika

Brush

5

Supelco

Brush

5

Supelco

Helix

10

Merck

Protein

10

Sonsep

Ligand exchange

5

TosoHaas

Ligand exchange

5

TosoHaas

Brush

4

TosoHaas

5

YMC

Shinwa

Protein Brush


13

14


p a i r s , and i n c l u s i o n complexes. Each method i s based on the format i o n of r e v e r s i b l e complexes and uses an a c h i r a l chromatographic packing. A.

Ligand Exchange


C h i r a l ligand exchange i s an excellent method for the r e s o l u t i o n of amino acids and amino-acid-like compounds. The molecules need not be d e r i v a t i z e d , and the aqueous mobile phases are compatible with automated column-switching techniques. A number of c h i r a l molecules have been resolved by ligand-exchange chromatography. However, the r e s o l u t i o n i s possible f o r only those molecules that are able to form coordination complexes with t r a n s i t i o n metal ions. This method i s most often u t i l i z e d with free and d e r i v a t i z e d amino acids and s i m i l a r compounds. There has been some success with other classes of compounds including carboxylic acids, amino alcohols (as S c h i f f bases), barbiturates, hydantoins, and succinimides (20). The mobile phases employed with c h i r a l ligand exchange are aqueous, with the metal ions and selector ligands added as modifiers. C h i r a l ligand-exchange chromatography i s based on the formation of diastereomeric complexes i n v o l v i n g a t r a n s i t i o n metal ion (M), a single enantiomer of a c h i r a l molecule ( L ) , and the racemic solute (d and 1). The diastereomeric mixed chelate complexes formed i n t h i s system are represented by the following formulas: L-M-d and L-M-l. The most common t r a n s i t i o n metal ion used i n these separations i s C u , and the selector ligands are usual ly amino acids such as Lproline. The chromatography i s most often c a r r i e d out using an a c h i r a l HPLC packing (such as C-18) with these compounds added to the mobile phase. +2

The e f f i c i e n c y and s e l e c t i v i t y of a c h i r a l ligand-exchange system can be improved by binding the selector ligand to the stationary phase. Some examples of t h i s approach are the L-proline-containing stationary phase, an L-(+)-tartaric-acid-modified s i l i c a reported by K i c i n s k i and Kettrup, and a c h i r a l phase composed of a C-18 column dynamically coated with (R,R)-tartaric acid mono-n-octylamide (7). The major disadvantage of c h i r a l ligand exchange i s the small number of compounds that can be resolved by t h i s approach. Many of the c a t i o n i c and anionic molecules of pharmacologic i n t e r e s t have not been resolved by t h i s method. B.

Ion P a i r i n g

Ion-pair chromatography i s a l i q u i d chromatographic method commonly used with charged solutes. The method i s based on the formation of a "neutral complex (ion p a i r , SC) between a charged solute (S ) and a counterion of opposite charge (C~). 11

+

When both the solute and the counterion are o p t i c a l l y a c t i v e , d i a stereoisomeric ion p a i r s are formed. These ion p a i r s often can be


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15

separated by differences i n t h e i r s o l v a t i o n i n the mobile phase or i n t h e i r binding to the stationary phase. A number of d i f f e r e n t counterions have been employed i n t h i s approach, including (+)-10-camphorsulfonic a c i d , quinine, quinidine, cinchonidine, (+)-di-n-butylt a r t r a t e , and the protein albumin. This method has been reviewed recently by Pettersson and S c h i l l (21).


The solutes resolved by c h i r a l ion-pairing chromatography have i n cluded amino alcohols such as a l p r e n o l o l , carboxylic acids such as t r o p i c acid and naproxen, and amino acids such as tryptophan. The composition of the mobile phase depends on the c h i r a l agent used. When a c h i r a l counterion i s used, a mobile phase of low p o l a r i t y such as methylene chloride i s used to promote a high degree of ion-pair formation. The retention of the solute can be decreased by increasing the concentration of the counterion or by the addition of a polar modifier such as 1-pentanol. The l a t t e r approach usually r e s u l t s i n a decrease i n the s t e r e o s e l e c t i v i t y . The water content of the mobile phase also appears to be important, and a water content of 80 to 90 ppm has been recommended. With serum albumin as the c h i r a l agent, aqueous mobile phases cont a i n i n g phosphate buffers are used. The retention and s t e r e o s e l e c t i v i t y can be altered by changing the pH. Both aqueous and nonaqueous mobile phases can be used when (+)-di-n-butyltartrate i s the c h i r a l modifier. In some cases i t appears that the modifier i s retained by the stationary phase when the column i s e q u i l i b r a t e d with an aqueous mobile phase. The system then can be used with an organic mobile phase (21). The c h i r a l ion-pairing systems are not stable. The chromatography can be affected by the water content of the mobile phase, temperature, pH, and a number of other v a r i a b l e s . This makes the routine applications d i f f i c u l t . In a d d i t i o n , the counterions often absorb i n the UV region, reducing the s e n s i t i v i t y of the system; i n d i r e c t photometric detection (22) or other detection methods must be used. C.

Inclusion

Cyclodextrins are c y c l i c oligosaccharides composed of d-a-glucose units linked through the 1,4 p o s i t i o n . The three most common forms of t h i s molecule are a-,p-, and y-cyclodextrin, which contain 6, 7, and 8 glucose u n i t s , respectively. Because of the d-a-glucose u n i t s , cyclodextrin has a s t e r e o s p e c i f i c , doughnut-shaped structure. The i n t e r i o r cavity i s r e l a t i v e l y hydrophobic and a v a r i e t y of watersoluble and insoluble compounds can f i t into i t , forming i n c l u s i o n complexes. I f these compounds are c h i r a l , diastereoisomeric i n c l u sion complexes are formed. p-Cyclodextrin has been used by Sybilska et a l (23) as a c h i r a l mobile-phase additive i n the resolution of mephenytoin, methylphenob a r b i t a l , and hexobarbital. They a t t r i b u t e the observed r e s o l u t i o n to two d i f f e r e n t mechanisms. The r e s o l u t i o n of mephenytoin i s due to a difference i n the absorption of the diastereoisomeric complexes on


16


the a c h i r a l C-18 support. For methylphenobarbital and hexobarbital, the r e l a t i v e s t a b i l i t i e s of the diastereoisomeric complexes are responsible for the r e s o l u t i o n of these compounds.


In addition to the compounds l i s t e d above, mobile phases modified with p-cyclodextrin can resolve mandelic acid and some of i t s derivat i v e s (24,25). Aqueous mobile phases modified with a buffer such as sodium acetate are commonly u t i l i z e d . A l c o h o l i c modifiers such as ethanol can be added to the mobile phase to reduce retention. Automation i s possible f o r the d i r e c t measurement of b i o l o g i c a l samples. Sybilska et a l (23) have used i t f o r preparative separations of mephenytoin, methylphenobarbital, and hexobarbital. The applications of t h i s approach seem l i m i t e d . For example, unlike hexobarbital and methylphenobarbital, the c h i r a l barbiturates secob a r b i t a l , pentobarbital, and thiopental are not resolved when chromatographed with a p-cyclodextrin-containing mobile phase (21). Other s t r u c t u r a l l i m i t a t i o n s of r e s o l u t i o n i n v o l v i n g cyclodextrin i n c l u s i o n complexes are discussed i n Section 1.2.3. 1.2.3

Enantiomeric Resolution Using C h i r a l Stationary Phases

Enantiomers can be resolved by the formation of diastereomeric complexes between the solute and a c h i r a l molecule that i s bound to the stationary phase. The stationary phase i s c a l l e d a CSP, and the use of these phases i s the fastest-growing area of c h i r a l separations. The f i r s t commercially a v a i l a b l e HPLC-CSP was introduced by P i r k l e i n 1981 (26). Currently a large number of c h i r a l phases are commerc i a l l y available. The separation of enantiomeric compounds on CSP i s due to differences i n energy between temporary diastereomeric complexes formed between the solute isomers and the CSP; the larger the difference, the greater the separation. The observed retention and e f f i c i e n c y of a CSP i s the t o t a l of a l l the i n t e r a c t i o n s between the solutes and the CSP, including a c h i r a l i n t e r a c t i o n s . Since there are so many HPLC-CSPs a v a i l a b l e to the chromatographer, i t i s d i f f i c u l t to determine which i s most s u i t a b l e to solve a p a r t i cular problem. This d i f f i c u l t y can be p a r t i a l l y overcome by grouping the CSPs for c h i r a l separations according to a common c h a r a c t e r i s t i c . The f i r s t step, that i s , the formation of the solute-CSP complexes, i s more r e a d i l y adaptable to the development of a c l a s s i f i c a t i o n system. Using t h i s as a c r i t e r i o n f o r the d i v i s i o n of CSPs i n t o groups, the current commercially a v a i l a b l e CSPs can be divided i n t o f i v e categories (7). Type 1 - The solute-CSP complexes are formed by a t t r a c t i v e i n t e r a c t i o n s , hydrogen bonding, 71-71 i n t e r a c t i o n s , dipole stacking, e t c . , between the solute and CSP. Type 2 - The primary mechanism for the formation of the solute-CSP complex i s through a t t r a c t i v e i n t e r a c t i o n s but where i n c l u s i o n complexes also play an important r o l e .


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17

Type 3 - The solute enters i n t o c h i r a l c a v i t i e s w i t h i n the CSP form i n c l u s i o n complexes. Type 4 - The solute i s part of a diastereoisomeric ( c h i r a l ligand-exchange chromatography).

to

metal complex


Type 5 - The CSP i s a protein and the solute-CSP complexes are based on combinations of hydrophobic and polar i n t e r a c t i o n s . HPLC resolutions of enantiomers induced by molecular associations, i n which the main d r i v i n g force i s the action of weak hydrogen bonds, have been described (27-28). The hydrogen-bond association p o t e n t i a l was f i r s t demonstrated through the o p t i c a l r e s o l u t i o n of racemic Nacylated amino acid esters using a c h i r a l stationary phase (N-acyl-Lvalylamino) propyl s i l i c a gel. Following t h i s preliminary study, a p p l i c a t i o n was made of the c h i r a l mobile phase additive (CMPA1) on which the fundamental structure of the c h i r a l g r a f t of CSP i s reproduced, to resolve the above enantiomers i n l i q u i d - s o l i d chromatography. The addition of N-acetyl-L-valine tert-butylamide to the nonaqueous mobile phase solvent of a s i l i c a gel column successfully brought about t h i s o p t i c a l r e s o l u t i o n . Two types of c h i r a l additives derived from a c h i r a l skeleton (R,R)-tartaric acid were found capable of resolving various kinds of enantiomers, such as d i a l k y l t a r t r a t e and d i a l k y l tartramide. Of these two, the l a t t e r having an isopropyl substituent (CMPA2), led to a wide range of r e s o l u t i o n of enantiomers of the following categories: a- and p-hydroxycarboxylic a c i d , p-hydroxy ketone, p-amino alcohol, Of-amino a c i d , Of-hydroxy ketoxime d e r i v a t i v e s , and bi-p-naphthol. This occurred when the enantiomers, except p-hydroxy ketones, a-hydroxy ketoximes, 1,2-diols, and b i p-naphthol, were derivatized to respond to the hydrogen bonding s i t e s of the additive molecules. Cyclodextrin c h i r a l phases have been shown to be widely applicable for the separation of enantiomers, diastereoisomers, s t r u c t u r a l i s o mers, and routine compounds (Table 1.4). The e f f i c i e n c y and select i v i t y of the p-cyclodextrin column have been improved. In addition, the mechanism of separation on cyclodextrin bonded media, solvent e f f e c t s , temperature e f f e c t s , and s t r u c t u r a l e f f e c t s on c h i r a l separations have been investigated (29). I t i s widely believed that an i n c l u s i o n complex should be formed for c h i r a l recognition to be possible (30). This has been v e r i f i e d by performing a normal-phase separation, f o r example, using a hexanol: 2-propanol mobile phase, on a p-cyclodextrin column. The hydrophobic solvent occupies the cyclodextrin cavity and the enantiomeric solute i s r e s t r i c t e d to the outside surface of the cyclodextrin cavity. No enantiomeric resolutions have been achieved i n t h i s mode as yet, although excellent routine separations are common. Apparently, the i n c l u s i o n complex formed should be r e l a t i v e l y " t i g h t f i t " f o r the hydrophobic species i n the cyclodextrin cavity (29,31). For example, p-cyclodextrin seems to e x h i b i t better e n a n t i o s e l e c t i v i t y f o r molecules the s i z e of biphenyl or naphthalene than i t does f o r smaller molecules (29). Smaller molecules are not t i g h t l y held and appear to move i n a manner where they f e e l the same average environment. I t


18


Table 1.4

A B r i e f Summary of Stereoisomeric Separations of D i f f e r e n t Classes of Compounds. One S p e c i f i c Example i s Given f o r Each Class (29)

a Enantiomeric Compounds

k'

Of

R _s

Mobile Phase

b


Dansyl amino acids (l)-norleucine (d)-norleucine p-Naphthyl amino acid derivatives 1-alanine p-naphthylamide d-alanine p-naphthylamide Barbiturates (-)mephobarbital (-)mephol (+)mephobarbital

c

Metallocenes (-)s-(l-ferrocenylethyl)thiophenol (+)s-(l-ferrocenylethyl)thiophenol Carboxylic acids a-methoxy-a-trifluoromethylof-methoxy-Ofphenyl acetic acid"" Miscellaneous (-)DIOP (+)DI0P

1.90 2.40

1.26

2.30

50:50

5.1 6.1

1.20

2.00

50:50

14.8 16.9

1.14

1.6

20:80

3.1

1.39

2.27

90:10

7.5 9.8

1.31

0.6

50:50

10.56 11.84

1.12

1.2

48:52

4.3

A 10 cm p-cyclodextrin column was used. Numbers represent the volume percent of methanol to water. flow rate was 1.0 mL/min.

The

C

25 cm p-cyclodextrin i n column.

d

2,3,0-isopropylidene-2,3-dihydroxy-1,4-bis(diphenylphosphino)butane.


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also appears that the c h i r a l center must be near the c a v i t y entrance or have a substituent oriented i n a s p e c i f i c p o s i t i o n so that i t would be able to form at l e a s t one strong i n t e r a c t i o n with the groups present at the c a v i t y entrance. When an enantiomer i s able to f u l f i l l the above conditions, the p o s s i b i l i t y f o r c h i r a l recognition i s good. E n a n t i o s e l e c t i v i t y appears to be due to a combination of c y c l o d e x t r i n s gross geometry, which allows i n c l u s i o n complex format i o n and the c h i r a l i t y of the number 2 and 3 glucose carbons at the entrance of the cavity.


1

I t i s necessary for a solute to i n t e r a c t with the mouth of the cyclod e x t r i n c a v i t y i n order to observe e n a n t i o s e l e c t i v i t y (30,32-35). Extensive use of CD-bonded phases makes i t apparent that small changes i n the structure of e i t h e r the cyclodextrin or the c h i r a l solute, can i n some cases cause large differences i n e n a n t i o s e l e c t i v i t y . Norgestrel i s an example where the c h i r a l center (the number 17 carbon) and i t s substituents are s p a t i a l l y too f a r from the mouth of the CD c a v i t y to i n t e r a c t with the 2-hydroxyl groups. D e r i v a t i z ing the hydroxyl group e f f e c t i v e l y changes the e n a n t i o s e l e c t i v i t y of the stationary phase and enhances c h i r a l recognition. Conversely, i f the c h i r a l center of the solute i s hidden between large bulky s u b s t i tuents, one can a l t e r the structure of the solute to enhance c h i r a l recognition. This has been demonstrated with a series of metallocene compounds (30). Resolution of (±) a-ferrocenylbenzylalcohol i s not possible since the hydroxyl substituent attached to the c h i r a l carbon i s apparently hidden between the bulky ferrocene and phenyl groups. By replacing the hydroxyl group with thioethanol, the length of the hydroxy-substituent on the c h i r a l carbon was extended beyond the bulky groups and good r e s o l u t i o n was observed. Another example of how small changes i n a solute's structure can e f f e c t s e l e c t i v i t y i s the compound binaphthyl crown-5 (36). This crown ether was baselineseparated on a 25-cm p-cyclodextrin column. But when one of the crown oxygen atoms was replaced with a nitrogen atom (binaphthyl mono-azo-crown-5), r e s o l u t i o n was no longer observed. I t i s apparent that the a b i l i t y to make small changes i n e i t h e r the cyclodextrin or enantiomer structure provides an a d d i t i o n a l powerful t o o l to resolve enantiomeric mixtures. A wide range of solvents can be used with cyclodextrin-bonded phases depending on the p a r t i c u l a r a p p l i c a t i o n . By using mobile-phase mixtures such as hexanol:2-propanol, the cyclodextrin stationary phase i s made to function as a normal phase. Separations tend to be analogous to those of a d i o l column because solutes adsorb to the hydroxyls on the outside of the cyclodextrin while the hydrophobic solvent occupies the c a v i t y . Inclusion complexes u s u a l l y are formed only i n the presence of water and c e r t a i n organic modifiers such as dimethyl s u l f o x i d e , dimethyl formamide, a c e t o n i t r i l e , and alcohols (29,32). Since the i n t e r a c t i o n of solutes with cyclodextrin i s greatest i n water, retention can be increased by increasing the water concentration i n the mobile phase. While broad peaks and t a i l i n g are a r t i f a c t s often associated with long retention times, they can be minimized by the use of buffers. Buffers such as 0.1 to 1% t r i e t h y l ammonium acetate (TEAA), pH = 4.1, sharpen e l u t i n g peaks and increase r e s o l u t i o n and e f f i c i e n c y . For the derivatives of amino acids and


20



peptides, use of the buffer TEAA (1%, pH = 4.1) instead of water, has produced up to f o u r f o l d increases i n e f f i c i e n c y . Other buffers such as ammonium acetate and phosphate buffers that are compatible with cyclodextrin bonded phases, may also be used. I t should be noted that the e l u t i o n order of most compounds i n the reversed^phase mode on p-cyclodextrin media can be d i f f e r e n t from that using t r a d i t i o n a l reversed-phase columns. This i s i n d i c a t i v e of the f a c t that the retention mechanisms are not the same. For examp l e , at a l l solvent compositions measured, the arene t r i c a r b o n y l chromium complex of benzene i s retained much longer on p-CD than on C-18 while the benzene free-ligand i s retained much longer on ODS than p-CD (37). Mobile-phase composition a f f e c t s enantiomeric separations. For example, as the methanol concentration i s decreased, r e s o l u t i o n and retention time increase. Therefore, the higher the concentration of the organic modifier, the easier i t i s f o r a solute to be displaced from the cyclodextrin c a v i t y . A c e t o n i t r i l e and ethanol e x h i b i t a greater a f f i n i t y f o r the cyclodextrin c a v i t y than methanol, consequently much lower concentrations of these modifiers are needed to obtain comparable retention times. Furthermore, s e l e c t i v i t i e s of some compounds are very d i f f e r e n t i n MeOH/HaO. While most compounds studied have exhibited a higher degree of s e l e c t i v i t y i n methanol/ water, a few compounds give better separations i n a c e t o n i t r i l e / w a t e r mobile phases (29). Further studies are currently i n progress to understand f u l l y the mechanisms involved. Temperature changes have a greater e f f e c t on the retention of solutes on cyclodextrin bonded phases than on comparable reversed-phase columns. This i s because the binding constant of a solute to the cyclodextrin i s s i g n i f i c a n t l y affected by temperature. As temperature i s increased, the binding of the solute to cyclodextrin decreases r a p i d l y . In f a c t , K approaches zero between 60°C and 80°C f o r most compounds (29). S

Chromatographic techniques have been widely used f o r the separation of various metal complexes and have been recognized by coordination chemists as indispensable f o r the separation and p u r i f i c a t i o n of various kinds of isomers (geometric, diastereomeric, and enantiomeric) of a wide v a r i e t y of coordination compounds. A vast number of studies have been c a r r i e d out i n t h i s area. Most of them, however, had as t h e i r aim the i s o l a t i o n of pure isomers, with a discussion of the c o r r e l a t i o n between properties and structures, so that t h e i r e f f o r t s were concentrated mainly on the search f o r appropriate separation conditions. From the chromatographic viewpoint, the data are diverse and non-systematic. The addition of c h i r a l N-acetyl-L-valine-tert-butylamide to the mobile phase using a s i l i c a gel column has resulted i n the o p t i c a l r e s o l u t i o n of d- and 1-amino acid derivatives of N-acetyl-O-tert.b u t y l esters. The e n a n t i o s e l e c t i v i t y generated by the diastereoisomeric c h e l a t e - l i k e solvates i s based on i n t e m o l e c u l a r hydrogen bonds between the c h i r a l additive and amino acid enantiomers. The degree of enantioselection was found to depend markedly on the composition


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of the chloroform-n-hexane mobile phase containing the c h i r a l addit i v e . The chromatographic process responsible f o r the recognition of the enantiomers i s related to the equilibrium r e l a t i o n s h i p i n the column, involving the c h i r a l additive and amino acid derivatives to be resolved (38). D i r e c t o p t i c a l resolution by HPLC based on the enantioselective prope r t i e s of a p r o t e i n , p a r t i c u l a r l y bovine serum albumin (BSA), has been shown to be a very v e r s a t i l e method with many useful a n a l y t i c a l applications. Although the mechanism of c h i r a l recognition by the p r o t e i n i s l a r g e l y unknown, some e m p i r i c a l l y found correlations between retention behavior and mobile phase composition gave a gene r a l idea of the main types of solute-protein i n t e r a c t i o n s involved (39). A n a l y t i c a l - s c a l e o p t i c a l r e s o l u t i o n of a series of N-(2,4-dinitrophenyl)-and dansyl-d,l-amino acids has been affected by the use of a bovine serum albumin (BSA) s i l i c a column ( R e s o l v o s i l ) . Decreasing retention was found f o r both types of amino acid with increasing pH or 1-propanol content of the mobile phase. In the dinitrophenyl s e r i e s , the aspartic acid derivatives showed very large enantiomeric separation factors compared with the glutamic acid homologue, and an analogous, but less pronounced, e f f e c t was found f o r the phenylglycine-phenylalanine pair. F l u o r i m e t r i c studies of dansyl-alanine showed that by a simple postcolumn addition of 1-propanol, the f l u o rescence y i e l d can be increased by a factor of over 20, giving a very low detection l i m i t . The a n a l y t i c a l technique i s useful f o r the determination of the b a c t e r i a l marker compounds, D-alanine and D-glutamic a c i d , present i n c e l l w a l l hydrolysates (40). A commercially available c h i r a l stationary phase containing a-x-acid glycoprotein on s i l i c a (EnantioPac, LKB) has been applied to the r e s o l u t i o n of a number of pharmacologically important enantiomeric ammonium compounds. The optimization of retention and s e l e c t i v i t y by c a t i o n i c , anionic, and neutral modifiers i n the mobile phase was studied. The r e s u l t s suggest that the solutes are retained according to an ion-pair d i s t r i b u t i o n model. Compounds of widely d i f f e r e n t structures were studied, and high separation factors were achieved for a majority (41). Some examples covering d i f f e r e n t kinds of hydrogen bonding (HB) groups are given i n Table 1.5. The r e s u l t s suggest that the magnitude of the enantiomeric r e s o l u t i o n i s affected by the strength of the HB substituent. Previous studies on separation of enantiomeric ions by binding to the HB group and the charged s i t e of a c h i r a l reagent indicate that the distance between these binding s i t e s i s of v i t a l importance. These relationships could not be confirmed i n t h i s study. No c h i r a l resol u t i o n i s obtained when the asymmetric carbon atom i s i n a betap o s i t i o n r e l a t i v e to the amide. Differences i n s t e r e o s e l e c t i v i t y also appear between diastereoisomers. The R,S;S,R enantiomers of some a,p-amino alcohols, f o r example, l a b e t a l o l A and ephedrine, d i s play a higher s e l e c t i v i t y than the corresponding R,R;S,S enantiomers, l a b e t a l o l B and pseudoephedrine. Nadolol displays the same tendency when chromatographed with 0.001M tetrabutylammonium bromide as the


22

CHIRAL SEPARATIONS BY LIQUID CHROMATOGRAPHY

Table 1.5

Highest Separation Factors with a Selection of Mobile Phases (41)

Mobile Phase:

Modifier i n 0.02M Phosphate Buffer


Solute

a

Atropine Bromodiphenhydramine B rompheniramine Bupivicaine^ Butorphanol Carbinoxamine Chlorpheniramine Clidinium Cocaine Cyclopentolate Dimethindene Diperodone Disopyramide Doxylamine Ephedrine Ephedrine, pseudoHomatropine Labetalol A Labetalol B Mepensolate

1.64 1.17 1.50 1.41 1.99 1.33 2.26 1.21 1.46 3.86 1.53 1.47 2.70 1.37 1.83 1.34 1.63 2.10 1.36 1.32

M o d i f i e r Solute 3

8 5 14 3 2 16 14 1 15 11 6 17 6 13 7 7 8 14 7 5

Mepivicaine Methadone Methorphan Methylatropine Methylhomatropige Methylphenidate Metoprolol Nadolol A Nadolol B Oxyphencyclimine Oxprenolol ^ Phenmetrazine Phenoxybenzamine Promethazine Pronethalol ^ Propoxyphene Propranolol Terbutaline Tocainide Tridihexethyl

or

Modifier

1.25 1.59 2.54 1.27 4.2 1.70 1.64 3.98 3.03 1.42 1.25 1.57 1.37 1.25 1.26 2.3 1.13 1.22 1.44 1.64

3 6 12 9 11 14 4 12 12 6 5 8 16 5 16 6 17 11 10 17

M o d i f i e r s : (H P0 or NaOH added to give the indicated pH) 1 = 0.33M 2-propanol, pH 7.0; 2 = 0.67M 2-propanol, pH 7.0; 3 = 1.33M 2-propanol, pH 7.0; 4 = 0.1M NaCl, pH 7.0; 5 = 0.1M NaCl + 1.74M ethanol, pH 7.0; 6 = 0.1M Nacl + 1.33M 2-propanol, pH 7.0; 7 = 0.05M b u t y r i c a c i d , pH 7.0; 8 = 0.01M octanoic a c i d , pH 7.0; 9 = 0.25M cyclohexylsulfamic a c i d , pH 7.0; 10 = 0.001M tetrapropylammonium bromide (TPrABr), pH 6.0; 11 = 0.003M TPrABr, pH 7.0; 12 = 0.001M tetrabutylammonium bromide (TBuABr), pH 6.0; 13 = 0.003M TBuABr, pH 6.0; 14 = 0.003M TBuABr, pH 7.0; 15 = 0.001M dimethyloctylamine (DM0A), pH 7.0; 16 = 0.001M DM0A + 0.17M 2-propanol, pH 7.0; 17 = 0.002M DM0A + 0.33M 2-propanol, pH 7.0; 3

b

4

0ne of the diastereoisomers ( f o r which there are no generally accepted names).


3

1.

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modifier. However, when 0.025 M b u t y r i c acid i s used as the modif i e r , the R,R;S,S enantiomeric p a i r , nadolol B, has a higher select i v i t y , that i s , 2.30, compared to 2.16 f o r nadolol A. A c h i r a l Of-x-acid glycoprotein column (EnantioPac )has been used f o r the separation of the enantiomers of some a c i d i c drugs (ibuprofen, ketoprofen, naproxen, 2-phenoxypropionic a c i d , bendroflumethiazide, ethotoin, and hexobarbital) and basic drugs, such as disopyramide. The column was prepared by immobilization of the human plasma p r o t e i n O f - i - a c i d glycoprotein on s i l i c a p a r t i c l e s . The retention and the e n a n t i o s e l e c t i v i t y of the solutes were e a s i l y regulated by the addit i o n of the t e r t i a r y amine N,N,-dimethyloctylamine (DMOA) to the mobile phase. DMOA decreased the retention and the enantioselect i v i t y of the weaker acids, whereas the retention and the enantios e l e c t i v i t y of the stronger acids increased d r a s t i c a l l y with increasing DMOA concentration. The influence of column temperatures between 25°C and 80°C on the separation f a c t o r , separation e f f i c i e n c y , and the r e s o l u t i o n has also been evaluated. S t a b i l i t y ^ t u d i e s indicated that the O f - i - a c i d glycoprotein column (EnantioPac ) i s very stable. I t can be used a t e l e vated temperatures and with 2-propanol, and loses

Chiral Separations - ACS Symposium Series (ACS Publications)

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