Introduction to Modern Liquid Chromatography, Third Edition part 72 ppt

Among the available tools, direct separation with a chiral stationary phase CSP; an enantioselective or ‘‘chiral’’ column has become the pre-dominant and most accepted procedure but not

14.7.2 Thermodynamics of Direct Chromatographic Enantiomer

Separation, 716

14.7.3 Site-Selective Thermodynamics, 717

Previous chapters have described HPLC procedures of wide applicability; that is, separations that can be used for many different kinds of samples Usually method development can be started with any of various columns or conditions, and resolution

is then systematically improved by varying separation conditions Consequently there are often many different ways of successfully separating a particular sample Such an approach cannot be used for enantiomers, however, which require highly specialized techniques and separation materials Furthermore, in most cases, the selection of the column is the critical step; unless a suitable column is selected, subsequent changes in other conditions are unlikely to be successful Nevertheless, enantiomer separations are today performed routinely in many research and routine laboratories, and are of great importance in the pharmaceutical industries Various technologies and tools have been developed that provide a rich toolbox to separate

virtually any sample Among the available tools, direct separation with a chiral

stationary phase (CSP; an enantioselective or ‘‘chiral’’ column) has become the

pre-dominant and most accepted procedure (but not the only procedure) However, the

identification of the most suitable CSP/mobile-phase combination for a particular analyte can be challenging Due to the specificity of molecular-recognition for each

combination of CSP (chiral selector) and analyte—and its sensitivity to minor

struc-tural variations in either the solute or CSP—reliable predictions of an appropriate column from analyte molecular structure are as yet hardly possible

Databases such as ChirBase (http://chirbase.u-3mrs.fr) can provide help in the selection of column and starting mobile phase, based on analyte structure [1, 2]

A database approach will be of greatest value when the enantiomers of interest are included in the database This approach can also be useful for enantiomers

of related structures—where presumably similar separation conditions will be successful, but it must fail for completely new structures Alternatively, automated screening procedures are used in large pharmaceutical companies to solve this problem efficiently [3, 4] This way the most promising CSP can be found quickly, followed by optimization of the mobile phase Overall, direct HPLC enantiomer separation based on CSPs has become an extraordinarily powerful technology, and this will be the primary focus of the present chapter

Non-enantiomeric separations can often be developed from only a general descrip-tion of the sample components, for example, acidic, basic, or neutral solutes In

some cases the class of compounds within a sample (e.g., peptides, proteins, car-bohydrates) suggest a range of suitable conditions, including column type (but not requiring a specific column) Consequently further details of solute molecular structure are unnecessary for non-enantioselective method development This is not the case for enantiomeric separations, where solute molecular structure and related physicochemical properties play a critical role in method development For this reason a basic understanding of the behavior of enantiomers is essential for their efficient separation

14.2.1 Isomerism and Chirality

Isomers are molecules that possess identical atomic composition, yet are not superimposible upon each other [5, 6] A classification of isomeric structures is given in Figure 14.1 [6] As distinguished from the case of molecules which are actually identical (‘‘homomers’’), a structural isomer can be defined in various ways Compounds whose atom-to-atom connections are different (e.g., 1-butanol,

2-butanol) are defined as constitutional isomers Stereoisomers, by contrast, have

identical atom-to-atom connections, but distinct orientation of atoms or groups

in three-dimensional space The latter can be further divided into enantiomers and diastereomers (or ‘‘diastereoisomers’’) While the former are always chiral, the latter may also include nonchiral stereoisomers such as cis/trans isomers (e.g., cis/trans-1,2-dichloroethylene).

Chirality, the synonym for ‘‘handedness’’ (from the Greek word for hand),

refers to the geometric property of an object which is nonsuperimposable on its mirror image (e.g., the left and right hand) Such an object has no symmetry elements of the second kind, such as a plane of symmetry, a center of inversion, or

a rotation-reflection axis Chirality may arise from various distinct chiral elements

(Fig 14.2): centers of chirality (stereogenic centers; Fig 14.2a), chiral axes (axial

Constitutional isomers

Isomers (structural isomers) Homomers

(identical)

Molecules with identical atomic composition

yes

no

superimposible

Same constitution

Enantiomers

(R S)

(1:1 mixture = racemate)

Diastereomers

(SR SS)

Stereoisomers

Criteria for distinction:

Property Non-enantiomers Enantiomers

Symmetry No mirror images Non-superimposible

mirror images Energy Distinct Identical

Figure14.1 Classification of isomeric structures Adapted from [6]

chirality; Fig 14.2b), chiral planes (planar chirality; Fig 14.2c), chiral helices (helical chirality; Fig 14.2d), and topologically chiral elements (topological chirality) The

most well known example is a center of chirality, where typically a carbon atom within the molecule is substituted by four different entities (see the example of

carbons 1 and 2 in Figure 14.3a.

Enantiomers and diastereomers can be differentiated by either of two

proper-ties: symmetry or energy content (i.e., the free energy of the molecule) Enantiomers

are molecules that are nonsuperimposable mirror images of each other having identical energy content (because of exactly identical atomic distances, angles, and torsions, as well as interatomic interactions) They are indistinguishable in

an achiral environment and therefore cannot be separated by achiral chromato-graphic methods such as conventional reversed-phase chromatography (RPC) For

example, (1S, 2R)-ephedrine and (1R, 2S)-ephedrine (Fig 14.3a) are enantiomers Moreover (1R, 2R)- and (1S, 2S)-pseudoephedrines are also enantiomeric to each other (Fig 14.3b) Note that the corresponding enantiomers always exhibit opposite

configurations at the two stereogenic centers (as in Fig 14.3) An exactly equimolar

mixture of enantiomers is called a racemate All other mixtures of enantiomers with

a composition deviating from 1:1 are defined as nonracemic mixtures.

Enantiomers are provided with stereochemical descriptors (e.g., R and S, or

L and D, or + and −) that distinguish them (with the R and S system being

(a) Centers of chirality

X = C, Si, Ge, Sn, N +, P+, As+

a X

d a X b c d

tetra-coordinate

X = N, S +, P tri-coordinate

X c b

a X c b a

(b) Axial chirality

C C C a b

c d

c d

a b

c d

a b a

b d

c

N a

b

c d

C C C H HOOC

COOH

H COOH

HOOC H

CH3

COOH

H3C

HOOC

H3C

HOOC

CH3 COOH

Figure14.2 Structural features that contribute to chirality

(c ) Planar chirality

CH2

CH2

H2C

H2C

a

CH2

CH2

H2C

H2C

COOH

H2C

H2C

CH2

CH2 HOOC

P (plus)

M (minus)

(d ) Helical chirality

Figure14.2 (Continued)

highly preferred) R and S refer to configurations in which the substituents at the

stereogenic center are in clockwise and counterclockwise arrangement regarding their Cahn–Ingold– Prelog priorities when the substituent with the lowest priority

is oriented away from the observer.+ and − refer to the optical rotation properties

of a chiral molecule; that is, its ability to rotate the plane of linear polarized light to the right or left (dextrorotatory and levorotatory) L and D refer to the Fisher designations for amino acids and sugars that specify relative configurations chemically derived fromD-(+)-glyceraldehyde [6]

Diastereomers are not mirror images of each other, are characterized by distinct

physical and chemical properties, and can be separated by achiral chromatography

In the example of Figure 14.3, each stereoisomer of Figure 14.3a is a diastereomer of any of the stereoisomers in Figure 14.3b because they differ solely in the configuration

of one stereogenic center rather than both (as for enantiomers) Thus ephedrines and pseudoephedrines are diastereomers because they are not mirror images; they can

also be termed epimers, since only one of several stereogenic centers is inverted This

distinction between enantiomers and diastereomers is the fundamental basis for all chiral differentiation processes including all enantiomer separation concepts

14.2.2 Chiral Recognition and Enantiomer Separation

Enantiomers have identical physicochemical properties, so their separation requires

their conversion to either (1) diastereomers (the indirect method) or (2) diastere-omeric complexes (the direct method) [7, 8] Today the use of the indirect approach

(1S, 2R)−(+) (1R, 2S)−(−)

erythro

ephedrine

(1R, 2R)−(−) (1S, 2S)−(+)

threo

pseudo-ephedrine

C6H5

C C

CH3

OH H

NHCH3 H

C6H5 C

C

CH3

1

2

C6H5

C

C

CH3

H HO

NHCH3 H

C6H5

C

C

CH3

(a)

(b)

Figure14.3 Structures of ephedrines and pseudoephedrines

is decreasing because of certain problems discussed below [8] Nevertheless, the indirect method is the procedure of choice for some applications, and its discussion

in following Section 14.3 will cover issues that are also relevant to the later treatment

of the direct method in Section 14.4 When planning an enantioselective separation

by the direct method, different chromatographic modes can be used, as in the case

of achiral chromatography In this connection we will distinguish enantioselective separations by designating them as reversed phase (RP) or normal phase (NP); this contrasts with the previously used abbreviations RPC and NPC for achiral separations

The indirect method involves the formation of diastereomers by reaction of an analyte (X) in the R or S configuration with an enantiomerically pure compound (hereafter with R-configuration), which we will refer to as a chiral derivatizing

reagent (CDR):

The reactions above must go to completion, and the diastereomeric products must

be stable (chemically and configurationally) The diastereomers (R, R)-X-CDR and (S, R)-X-CDR can then be separated by achiral chromatography (usually RPC).

One advantage of the indirect method is its use of conventional HPLC columns, which offer higher plate numbers compared to the enantioselective (‘‘chiral’’) columns of Section 14.4 A higher column efficiency can be especially important for the measurement of impurities at the<0.1% level in complex mixtures, as required

in pharmaceutial products Even more important is the possible use of CDRs for enhanced detection by UV, fluorescent, electrochemical, or mass spectromet-ric means, for example, fluorescent tags for the sensitive detection of otherwise difficult-to-detect amino acids The indirect method is also relatively economical,

in contrast to the direct method with its requirement of a battery of different (and generally expensive) enantioselective columns

A large number of CDRs have been developed that provide adequate diastereo-selectivity and in some cases enhanced detection [8] Chiral derivatizing reagents must be both chemically and stereochemically stable, and should be commercially

available in both enantiomeric forms (e.g., R and S) The choice of R or S CDRs

Commonly Employed Chiral Derivatizing Reagents (CDR) and Their Application

1 (R) or

(S)−α-methoxy-α-trifluoromethyl

phenylacetic acid and corresponding

acid chloride (Mosher’s reagent)

Alcohols, amines [9]

2 O,O-dibenzoyl tartaric acid

anhydride (DBTAAN)

Primary and secondary amines, alcohols, aminoalcohols

[10]

3 (R)- or (S)−1-(9-fluorenyl)ethyl

chloroformate (FLEC)

Primary and secondary amines, amino acids

[11]

4 ortho-phthaldialdehyde (OPA) in

combination with chiral thiols such

as (S)-or (R)-enantiomers of

N-acetyl-cysteine, N-t-Boc-cysteine,

N-acetyl-penicillamine,

1-thio-β-glucose

Primary amines, primary amino acids

[12, 13]

5

1-fluoro-2,4-dinitrophenyl-5-(S)-alanine amide (FDAA)

(Marfey’s reagent)

Primary and secondary amines, amino acids, thiols

[14, 15]

6

2,3,4,6-tetra-O-acetyl-β- D -glucopyranosyl isothiocyanate

(GITC)

Primary and secondary amines, amino acids, thiols

[16]

enables a reversal of elution order for two enantiomers, which can be used to position

a minor peak in front of a major peak, when one enantiomer is in considerable excess (see Section 2.4.2 and compare Fig 2.17e, f ) Some popular examples for

CDRs are given in Table 14.1

A still popular, indirect method for the analysis of the enantiomer composition

of amino acids is the use of the o-phthaldialdehyde (OPA) reagent with a chiral thiol such as N-acetyl-cysteine (CDR 4 in Table 14.1) The reaction scheme is shown in Figure 14.4 OPA derivatization is fast and amenable to automation, so

derivative instability can be overcome by reacting just before injection (an excess

of the non-fluorescent reagent will not interfere with detection) The chromatogram

in Figure 14.5 shows the analysis of amino acids in a bacitracin sample, after its hydrolysis and oxidation of Cys to cysteic acid (Cya) by the OPA method [13] This example illustrates the ability of the indirect approach to resolve several enantiomer pairs in a single separation, which is much less likely with a direct method (unless MS detection is used, which introduces additional chemoselectivity so that co-elution

of species with distinct MS properties does not matter) Nevertheless, despite its apparent simplicity, the development of indirect enantiomer separation methods is far from a trivial task [8]

A crucial requirement of the indirect method is an analyte that possesses

a selectively derivatizable functional group such as hydroxyl, amino, carboxylic, carbonyl, or thiol Another important requirement is a derivatizing reagent that is chemically and (especially) enantiomerically pure, that is, an enantiomeric excess

>99.9% If the CDR contains a significant amount of enantiomeric impurity, erroneous quantification data will result For example, the (S)-enantiomer impurity in (R)-CDR gives upon derivatization, besides the main diastereomeric pair of products,

the formation of a second pair of diastereomers yielding all four stereoisomers (see Fig 14.6; note that the impurity and its diastereomeric products are distinguished

CHO

CHO +

analyte

H2N

R1

R2

HS

COOH N H

CH3 O

R

S

R1

R2

COOH N

H

CH3 O

R

R

+

N

S

R1

R2

COOH N

H

CH3 O

R

S

fluorescent derivatives

frequently employed reagents:

• OPA / N-acetyl-cysteine

• OPA / Boc-cysteine

• OPA / Isobutyryl-cysteine

• OPA / N-acetyl-O-penicillamine

• OPA / 1-thio- β-glucose

• OPA / 1-thio- β-mannose

Figure14.4 OPA-chiral thiol derivatization for the stereoselective analysis of primary amines and amino acids (indirect enantiomer separation)

Asp Glu

L-His L-Val

L-Ile

D-Phe L-Leu

D-Orn L-Lys D-Lys

D-His L

D

L

D D

L

(min)

Figure14.5 Enantiomeric separation of amino acids obtained from the hydrolysis of baci-tracin A, followed by OPA-chiral thiol derivatization (indirect separation with fluorescence detection; Cya= cysteic acid) Adapted from [13]

pair of enantiomeric

analytes

(R )-X

kR

kS

chiral derivatizing

agent (bold, R )

with enantiomeric

impurity (plain, S)

two pairs of diastereomers (4 stereoisomers, two pairs of enantiomers)

(S )-X

+ (R)-CDR

+ (S)-CDR

k ′ R

k ′ S

(R, R )-X-CDR

(S, R )-X-CDR

(R, S )-X-CDR

(S, S )-X-CDR

e e d

d

d d

d diastereomeric to each other e enantiomeric to each other

Figure14.6 Reaction scheme for indirect HPLC enantiomer separation (in the presence of

an S-CDR impurity in the chiral derivatization reagent R-CDR) All four stereoisomers are formed and two pairs of enantiomers, respectively (d, diastereomeric to each other; e,

enan-tiomeric to each other)

by unbolded type) Achiral chromatography (e.g., RPC) will be unable to resolve the products that are enantiomeric to each other Hence the stereoisomers arising from the enantiomeric contamination of the CDR will co-elute with the peaks of the opposite enantiomers (i.e., opposite configurations), and only two peaks will

be observed Needless to say, these co-elutions will prohibit accurate quantitation Corrections are possible for CDR contamination, if the enantiomeric impurity level

in the CDR is known; however, this adds considerable complexity to both method development and validation

kR> kS

Time (h)

100

50

0

R

S

Peak area ratio:

(R)/(S)= 55:45

Kinetic resolution

Peak area ratio:

(R)/(S)= 50:50

No kinetic resolution

Figure14.7 Illustrative kinetic profiles for the reaction of a sample with a chiral derivatizing reagent The two enantiomers are assumed to have different rate constants and an identical detector response for the resulting diastereomers

Two additional complications exist for the indirect method First, for a

given derivatization procedure, stereochemical integrity must be fully preserved;

no racemization is allowed of the derivatizing reagent, analytes, or diastereomeric products [8] The absence of racemization can be easily checked by derivatizing and

analyzing a sample of known enantiomer composition Second, it must be verified that the derivatization has reached completion, in order to avoid potential kinetic resolution problems (Fig 14.7) [8] Specifically, the reaction rates for derivatization

of the R- and S-enantiomers of the analyte may differ; if the reaction is stopped

before completion, this can give rise to a stereoisomer ratio that deviates from the actual ratio of enantiomers in the sample For example, if the sample is a racemate

and k R > k S (with k being the rate constants of the derivatization reaction), the

analyzed enantiomer composition after 2 minutes in Figure 14.7 would significantly deviate from the 1:1 ratio that is expected for a racemate Systematic error due

to kinetic resolution can be easily avoided by driving the derivatization reaction

to completion This is generally achieved by increasing the reaction temperature and time, and by the use of a large excess of the corresponding CDR (the CDR is typically employed in 10-fold molar excess relative to the enantiomers)

Another drawback of the indirect method is that enantiomeric ratios cannot

be directly calculated from peak-area ratios measured in the final separation (as is possible with the direct method), since the two diastereomeric products can differ considerably in their detector response [8] This holds true for most detection modes, including UV, fluorescence, and mass spectrometry, with response factors varying typically by a factor of 1.1 to 1.5 Consequently a correction for this difference in response factors will be necessary External calibration with individual standards of

the diastereomers circumvents this problem, but individual standards are not always available

It might appear that these limitations make indirect methods questionable, which is certainly not the case However, users should carefully check for all of the complications above, most important, the enantiomeric purity of the CDR [17] (it

is unacceptable to rely only on specifications given by the supplier) Moreover the numerous method-validation issues that need to be addressed during the development

of an indirect assay render this approach time-consuming, labor intensive, and more prone to systematic errors Nevertheless, some enantioselective analysis assays are still carried out by indirect methods

The direct approach relies on the reversible formation of (transient) diastereomeric complexes between the two enantiomers [(R) − X and (S) − X] and a chiral com-plexing agent termed chiral selector (CS) As in the case of the indirect method,

the selector must be enantiomerically pure, but this requirement is less stringent for the direct method If the difference in complex stabilities is sufficiently large for the diastereomeric associates, a less-pure selector can still be used in a direct method:

and

The direct method circumvents the laborious derivatization with a CDR Two

distinct experimental modes for direct enantiomer separation exist: The chiral mobile-phase-additive (CMPA) mode (or simply, ‘‘additive mode’’) and the chiral stationary-phase (CSP) mode.

14.4.1 Chiral Mobile-Phase-Additive Mode (CMPA)

The additive mode makes use of an achiral stationary phase (e.g., a reversed-phase

or normal-phase column) with a mobile phase that contains the chiral

mobile-phase-additive (CMPA) at an appropriate concentration The CMPA (selector, CS)

may be present in the mobile phase and/or retained by the stationary phase as

described by its distribution coefficient K d,CS Upon injection of the sample, various

equilibria will be established that involve both the analyte X and the selector CS (Fig 14.8; note that subscripts m and s refer to species in the mobile and stationary phases, respectively, and the R-form of the selector is assumed) These equilibria

include:

• complex formation between selector (R)-CS and enantiomers (R)-X and (S)-X in the mobile phase, with association constants Ka,(R)-X and Ka,(S)-X

• distribution of (R)-X and (S)-X between the mobile and stationary phases with distribution constants K d,(R)-X and K d,(S)-X