Tài liệu HPLC for Pharmaceutical Scientists 2007 (Part 22) docx

22

CHIRAL SEPARATION

Nelu Grinberg, Thomas Burakowski, and Apryll M. Stalcup

22.1 INTRODUCTION

Chirality plays a major role in biological processes, and the enantiomers of a

bioactive molecule often possess different biological effects. For example, all

pharmacological activity may reside in one enantiomer of a molecule, or enan￾tiomers may have identical qualitative and quantitative pharmacological activ￾ity. In some cases, enantiomers may have qualitatively similar pharmacological

activity, but different quantitative potencies. Since drugs that are produced by

chemical synthesis are usually a mixture of enantiomers, there is a need to

quantify the level of the isomeric impurity in the active pharmaceutical ingre￾dient. Accurate assessment of the enantiomeric purity of substances is critical

because isomeric impurities may have unwanted toxicological, pharmacologi￾cal, or other effects. Such impurities may be carried through a synthesis and

preferentially react at one or more steps and yield an undesirable level of

another impurity. The determination of a trace enantiomeric impurity in a

sample of a single enantiomer drug substance in the presence of a range of

other structurally related impurities and a large excess of the major enan￾tiomer remains challenging.

The history of enantiomeric separation starts with the work of Pasteur. In

1848 he discovered that the spontaneous resolution of racemic ammonium

sodium tartrate yielded two enantiomorphic crystals. Individual solutions of

these enantiomorphic crystals led to a levo and dextro rotation of the polar￾ized light. Because the difference of the optical rotation was observed in solu￾tion, Pasteur suggested that like the two sets of crystals, the molecules are

987

HPLC for Pharmaceutical Scientists, Edited by Yuri Kazakevich and Rosario LoBrutto

Copyright © 2007 by John Wiley & Sons, Inc.

mirror images of each other and the phenomenon is due to the molecular

asymmetry [1].

While Pasteur made the historical discovery, subsequent advances in the

resolution of enantiomers by crystallization were based on empirical results.

Several attempts to separate enantiomers using paper chromatography were

met with unsystematic results. In 1952 Dalgliesh postulated that three points

of simultaneous interaction between the enantiomeric analyte and the sta￾tionary phase are required for the separation of enantiomers [2].

Developments in the field of life sciences and in the pharmaceutical indus￾try brought enantiomeric separation to a new level. In the late 1950s/early

1960s, many of the drugs were synthesized and used in a racemic form. An

example with tragic consequences was the use of thalidomide, a sedative and

a sleeping drug used in the early 1960s which produced severe malformations

in newborn babies of women who took it in the early stage of pregnancy. Later

it was demonstrated that only the (S)-enantiomer possesses teratogenic

properties [3].

Introduction of gas chromatography gave a burst to the field of enan￾tiomeric separation. In 1966 a group from the Weizmann Institute of Science

in Israel reported the first successful separation of enantiomers using gas

chromatography.

In a letter addressed to Emanuel Gil-Av after the publication of the first

separation of enantiomers on a chiral separation of enantiomers on a chiral

gas chromatography (GC) stationary phase [4], A. J. P. Martin wrote: “As you

no doubt know, I had not expected such attempts to lead to much success,

believing that the substrate-solvent association would normally be too loose

to distinguish between the enantiomers.” At the time there were just several

reports on the separation of enantiomers using chromatographic methods.

Later developments in HPLC gave an additional boost to the field. Today,

there are over 60 types of rugged, well-characterized columns capable of

separating enantiomers. Unfortunately, there is a great deal of trial and

error in choosing a particular column for a chiral separation. Therefore this

chapter will summarize a rationale for choosing a stationary phase that is

based on the relationship that exists between the analytes and the chiral

stationary phases.

22.1.1 Enantiomers, Diastereomers, Racemates

Chirality is due to the fact that the stereogenic center, also called the chiral

center, has four different substitutions. These molecules are called asymme￾trical and have a C1 symmetry. When a chiral compound is synthesized in an

achiral environment, the compound is generated as a 50:50 equimolar mixture

of the two enantiomers and is called racemic mixture. This is because, in an

achiral environment, enantiomers are energetically degenerate and interact in

an identical way with the environment. In a similar way, enantiomers can be

differentiated from each other only in a chiral environment provided under

988 CHIRAL SEPARATION

the conditions offered by a chiral stationary/mobile phase [5]. The separation

of enantiomers using chiral stationary/mobile phases involves the formation

of transient diastereomeric complexes between the enantiomeric analytes and

the chiral moiety present in the chromatographic column.Thus, diastereomers

are chiral molecules containing two or more chiral centers with the same

chemical composition and connectivity. They differ in stereochemistry about

one or more chiral centers. If two stereoisomers are not enantiomers of one

another, they can in principle be separated in an achiral environment—that is,

using a nonchiral stationary phase [5].

22.2 SEPARATION OF ENANTIOMERS THROUGH

THE FORMATION OF DIASTEREOMERS

Formation of diastereomers for chromatographic purposes can be generated

in two ways: transient diastereomers, which occur between the enantiomers

and the chiral stationary phase (CSP) during the chromatographic process.

Such a process is also called direct separation. The second way is to generate

long-lived diastereomers that are formed by chemical reaction between the

enantiomer and a chiral derivatizing reagent prior the chromatography. Such

a process is called indirect separation. Indirect separation of enantiomers is

usually a good technique when everything in direct separation fails. However,

it requires suitable functionality in the enantiomers for reaction with a chiral

derivatizing agent. The effectiveness of this approach may also depend on a

variety of other conditions such as structural rigidity and the spatial relation￾ship between the stereogenic centers of the enantiomers and the chiral center

introduced through derivatization.

When two chiral compounds, racemic A and racemic B, react to form a cova￾lent bond between them without affecting the asymmetric center, the stereo￾chemical course of the reaction can be as follows [6]:

[(±) − A] + [(±) − B] → [+A + B] + [+A − B] + [−A + B] + [−A − B]

where the first and the last products constitute an enantiomeric pair and the

second and the third products constitute a second enantiomeric pair. In con￾trast, the first and the third products and the second and fourth products are

diastereomeric pairs. In a chiral environment, one should be able to separate

all of these four products. However, because diastereomers possess slightly dif￾ferent physicochemical properties, achiral chromatography of this mixture

should lead to two peaks (corresponding to the two diastereomers).

Indirect approaches such as chiral derivatization with chiral derivatiz￾ing reagents (CDR) offers a variety of advantages. For instance, CDRs are

cheaper than chiral columns. Separation of the product diastereomers is gen￾erally more flexible than the corresponding enantiomeric separation because

achiral columns can be used in conjunction with various mobile-phase

SEPARATION OF ENANTIOMERS THROUGH THE FORMATION 989

compositions. Depending on the functional groups on the enantiomers, there

is a variety of CDRs on the market (chiral anhydrides, acid chlorides, chloro￾formates, isocianates, isothiocianates, etc.) which can be applied, which in turn

can change the selectivity of a chromatographic system.

There are also disadvantages to the chiral derivatization approach includ￾ing extra validation. For instance, the derivatizing reagent has to be optically

pure, or the analysis can generate false-positive results. In addition, special care

needs to be taken that the chiral center of the enantiomers or derivatizing

agent is not racemized during the derivatization reaction. Furthermore,

unequal detector response of the diastereomers must be corrected via stan￾dard procedures [7]. Often, the derivatization requires a long reaction time,

which adds to the analysis time.

22.2.1 Mechanism of Separation

The separation of diastereomeric pair is due to the effect of their nonequiva￾lent shape, size, polarity, and so on, on their relative solvation and sorption

energies [8]. Their interaction with a particular stationary phase is dependent

upon their molecular structure and availability of functional groups able to

interact with the stationary phase. For instance, unsaturated bicyclic alcohols,

which are capable of internal hydrogen bonding, show shorter retention than

epimers or dihydro derivatives, which cannot undergo such types of interac￾tions [9] (Figure 22-1). The compounds of Figure 22-1 were separated by gas

chromatography on a 12-ft ×

1

/4-in. column packed with 23% by weight of Ucon

No. 50HB 2000 available from Union Carbide on Celite. As the number of

double bonds increases in the molecules, the possibility of intramolecular

hydrogen bonds between the hydroxyl groups and the double bond increases.

Simultaneously, the potential for hydrogen bond formation between the com￾pounds and the stationary phase decreases. As a consequence, the retention

time of each isomer decreases as the number of double bonds in the mole￾cules increases [10–13].

990 CHIRAL SEPARATION

Figure 22-1. Retention time of bicyclic alcohols. The numbers under each structure

represent the retention time in minutes. (Reprinted from reference 9, with permission.)

There are few differences between the separation in gas chromatography

[14–16] and the separation in liquid chromatography (LC), because it is

assumed that the differential solvation of the diastereomeric compounds

during the LC separation does not play a very important role [17]. Helmchen

et al. [18] explained the separation of diastereomeric amides using LC with a

silica gel stationary phase under normal-phase conditions. In order to explain

their separation, the authors made some assumptions:

1. Secondary amides adopt essentially the same conformation in polar solu￾tions and in the adsorbed state (on silica gel).

2. In the adsorbed state, a parallel alignment of the planar amide group

and the surface of silica gel is preferred.

3. Apolar groups (i.e., alkyl, aryl) outside the amide plane cause a distur￾bance of this preferred arrangement in proportion to their steric bulk in

a direction perpendicular to the amide plane. Such groups are classified

as large and small by indices L and S, respectively.

4. That member of a diastereomeric pair in which both faces of the amide

plane are more shielded than the least shielded face in the other member

is eluted first.

5. There is an attractive interaction between small polar groups and the

silica gel, particularly if they are hydrogen bond donors not internally

bonded to the amide group. Formally, such groups are assigned to the S

(small) class.

The actual magnitude of the interaction of a given substituent with the

adsorbent depends on the adsorbent, other substituents present, and the type

and rigidity of the backbone of the diastereomeric analytes. Although no

serious attempts at quantification have been made, repulsive interactions

toward silica and alumina can be ranked roughly as H < methyl < phenyl =

ethyl < tert-butyl < trifluoromethyl < α-naphthyl < 9-anthryl =pentafluoroethyl

< heptafluoroethyl. Size and hydrophobicity are both relevant; incorporation

of polar functionality (hydroxyl, carbalkoxy, cyano) leads to attractive rather

than repulsive interactions with silica.

22.2.2 General Concepts for Derivatization of Functional Groups

As noted previously (Section 22.2), derivatization with a chiral derivatizing

reagent (CDR) requires the presence of suitable functionality (e.g., —OH,

Ar—OH, —SH, —COOH, —CO—, —NH2

, —NRH) within the chiral analyte

to serve as a reactive site. Before addressing specific issues with regard to CDR

and analyte classes, it may be helpful to review general considerations for

achiral derivatization in chromatographic assays.

Desirable achiral derivatization reaction properties include fast, unidirec￾tional reactions with no or minimal side reactions. In addition, both the reagent

SEPARATION OF ENANTIOMERS THROUGH THE FORMATION 991

and the product should be stable. Most derivatization methods use an excess

of reagent which can present as an interfering chromatographic peak. Of

course, incorporating a derivatization step in an assay requires additional

materials, time, and effort as well as additional method validation.

In the case of chiral derivatization, there are some unique considerations

in addition to the ones noted above for achiral derivatization. Extra valida￾tion is required to establish the optical purity of the derivatizing agent. In addi￾tion, nonracemization of either the analyte or the derivatizing reagent during

the derivatization must be confirmed. Excess reagent must be used to elimi￾nate any potential chiral discrimination in the derivatization reaction. The

presence of more than one type of reactive group (e.g., amine and alcohol)

must be considered if the selected reagent has different reaction potentials for

each moiety. In some cases, chiral derivatization may be coupled with achiral

derivatization. If more than one reactive functional group is present in the

analyte, usually the derivative in which the two stereogenic centers are in

closest proximity yields the most favorable diastereomeric pair for separation

by achiral chromatography. Also, derivatives that incorporate the most struc￾tural rigidity (e.g., amides versus esters) tend to be the most amenable to sep￾arations by achiral chromatography.

22.3 MOLECULAR INTERACTIONS

Generally speaking, there are three properties involved in an intermolecular

interaction: the probability of the interaction occurring, the strength of the

interaction, and the type of interaction. These properties will be discussed in

the following sections.

22.3.1 The Probability of Molecular Interactions

Achieving enantiomeric discrimination requires understanding the interac￾tions between the selector and the selectand. In his Ph.D. thesis [19], Feibush

postulated that attaining an enantiomeric separation on a chromatographic

chiral system required that certain conditions should exist:

A necessary condition for having a difference in the standard free energy of the

two enantiomers in solution is that the solvent is chiral. The fact that the solvent is

chiral is in itself not sufficient to sustain such difference. A certain solute–solvent

correlation should exist to cause the difference in the behavior of the enantiomers.

There should be strong (solute–solvent) interactions, such as p-complexation,

coordinative bonds, [and] hydrogen bonds, to form associates between the asym￾metric solvent/solute molecules. Such association can be regarded as short-living

diastereomers. When the bonds that form these associates are in immediate prox￾imity of their asymmetric carbons, a difference in the behavior of the enantiomers

in the active phase is possible.We search for active phases and enantiomeric solutes

that can form associates through (preferably) more than one hydrogen bond, and

992 CHIRAL SEPARATION