This manuscript is based on the following old article:
S. Levin and S. Abu-Lafi, "The Role of Enantioselective Liquid Chromatographic Separations Using Chiral Stationary Phases in Pharmaceutical Analysis", in Advances in Chromatography. E. Grushka and P. R. Brown, Eds., Marcel Dekker Inc.: NY, Vol. 33, 1993: 233-266.
(There are plenty of new developments since this review was written!)
The biological activity of chiral substances often depends
stereochemistry, since the living body is a highly chiral environment.
A large percentage of commercial and investigational pharmaceutical
are enantiomers, and many of them show significant enantioselective
in their pharmacokinetics and pharmacodynamics. The importance of
of drugs has been increasingly recognized, and the consequences of
them as racemates or as enantiomers has been frequently discussed in
pharmaceutical literature during recent years. With increasing
of problems related to stereoselectivity in drug action,
analysis by chromatographic methods has become the focus of intensive
of separation scientists. Most of the pharmaceutical and
studies of stereoselectivity of chiral drugs before the mid eighties
pre-column derivatization of the enantiomers with chiral reagents,
diastereomers. The diastereomers were subsequently separated in the
or reversed phase mode of chromatography.
Great efforts have been devoted to the development of better
for enantioselective chromatography during the past decade, and have
in new chiral stationary phases, pioneered by Pirkle [1 and references
therein]. Chiral agents were derivatized and immobilized on the surface
of the support (silica gel mostly), and served as the in situ chiral
during the chromatographic process. The preference of chiral stationary
phases lies in the inherent advantages of any chromatographic
such as the speed of the analysis, the possibility to analyze or purify
the enantiomers in complex mixtures, the reproducibility of the
and its flexibility. Moreover, analytical chromatographic systems can
adapted to preparative separations, in which pure enantiomers can be
A. Chiral affinity by proteins (serum albumin, a1-acid
ovomucoid and chymotrypsin).
Most of the analytical methods for pharmaceutical compounds in
samples use types A-D of the aforementioned stationary phases, and
the discussion will be focused on them. The parameters of importance in
chiral recognition by the chromatographic stationary phases will be
in each section. It may be generalized that in most cases the
in steric fit, anchored by hydrogen bonding of the solutes into the
environment in the specific discriminating sites, is responsible for
A. PROTEIN IMMOBILIZED ON SILICA GEL
One of the most appealing types of chiral stationary phases for pharmaceutical analysis involves the use of protein immobilized to the surface of silica gel, or other support, as the chiral discriminator. Many small chiral biomolecules have shown stereoselective affinity to serum albumin and to a1-acid glycoprotein, and consequently, the two proteins have been chosen as chiral selectors for the analysis of these molecules. Naturally, the mobile phases are mostly aqueous buffers containing a limited percentage of organic modifiers. When the protein stationary phases are efficient, even very small differences in binding affinity of the enantiomers to the protein give rise to resolution between them. Further more, if the immobilized protein maintains its native binding ability and the mobile phase composition does not affect its chiral binding properties, valuable information of drug-protein interaction can be deduced from chromatographic parameters.
a. Serum Albumin
The most abundant protein in the blood is serum albumin, which
as a non-specific binder and carrier. The bioavailability of
protein-bound molecules exceeds that of the free molecules, since they
have temporary protection and slower metabolism and excretion.
enantioselective binding of drugs by blood proteins is a vital function
in their action, and can be explored clinically and pharmacologically
using enantioselective analysis using immobilized proteins.
b. a1-acid glycoprotein (AGP)
The concentration of AGP in blood is much lower than HSA, and
its binding capacity is lower. Nevertheless, basic drugs have
affinity to this protein, and the stereoselectivity of their binding
considerably affect their pharmacokinetic and pharmacodynamic
Not all the properties of the binding sites in AGP are known. It
is anticipated that binding studies using chromatographic parameters
shed light on the mechanism of chiral recognition by the protein.
An efficient analytical column can be constructed with the AGP
phase, for an increasing number of applications in pharmaceutical
The mechanism of the stereoselective affinity of this protein cannot be
easily deduced, until the structural features are fully established.
c. Ovomucoid and a-chymotrypsin
Another type of chiral affinity stationary phase is ovomucoid, immobilized on silica gel, which has also proven effective in chiral discrimination of various pharmaceutical compounds. Also in use is immobilized a-chymotrypsin, which has a known recognition site for specific chiral substrates. The a-chymotrypsin stationary phase was developed mainly by Wainer and co-workers. They were able to resolve a number of the enantiomeric D,D- and L,L-dipeptides as well as the diastereomeric D,D-/L,L- and L,D-/D,L-dipeptides. Another attempt to explore the binding of dipeptides to immobilized a-chymotrypsin indicated that the observed enantioselectivity of the stationary phase to the particular dipeptides is a measure of the difference in the binding affinities at two sites rather than differential affinities at a single site.
B. POLYSACCHARIDE DERIVATIVES
Polysaccharides such as cellulose and amylose consist of D
linked by 1-4 glucosidic bonds, forming the natural polymers with a
ordered helical structure. The three hydroxyls on each glucose
can be derivatized to form strands around the chiral glucose. The
derivatized glucose unit can in principle act as a chiral site
between enantiomers that interact differently with the strands.
can sometimes be achieved with unsupported natural cellulose, but the
version has proven far better. The acetate ester, bezoate ester,
or phenylcarbamate derivatives of glucose, have shown better
Mobile phases are usually organic, normal phase type solvents, however,
aqueous solvents can also be used in many versions of the stationary
The structure provides the possibilities of p-p interaction of aromatic groups with the aromatic amide at the chiral site with the anchoring effect of hydrogen bonding with the amide groups. Wainer studied aromatic alcohols on cellulose tribenzoate and suggested that both insertion of the aromatic group and hydrogen bonding stabilize the enantiomers inside the chiral cavity. The discrimination is affected by the steric fit in the cavity.
An example of the separation of enantiomers of cannabidiol,
one of the
substances in Marijuana is shown in the following Figure.
C. CHIRAL CAVITY
Another general strategy for chiral discrimination on a stationary phase is creation of chiral cavities, in which stereoselective guest-host interactions govern the resolution. The first important consideration for retention and chiral recognition in such stationary phases is the proper fit of the molecule to the chiral cavity in terms of size and shape. This category of stationary phases includes crown ethers, imprinted polymers and cyclodextrins. A majority of pharmaceutical applications were accomplished using cyclodextrins, and therefore, the discussion is concentrated on them.
The monomers are arranged so that a shape of a hollow truncated cone is obtained. A relatively hydrophobic chiral cavity is formed, comprised of essentially methylene and 1,4 glucoside bonds, with which the intercalated solute interacts. In contrast to the interior, the exterior surface is hydrophilic, surrounded by hydroxyls. Mobile phases are usually aqueous solutions mixed with organic solvents, however, normal phase type solvents can also be used. When cyclodextrin stationary phases are used with aqueous mobile phases, the mechanism of retention is based on inclusion complexation. This mechanism represents the attraction of the apolar molecular segment to the apolar cavity. When an aromatic group is present, the orientation in the cavity will be stereoselective due to the interactions with the glucoside oxygens. Linear or acyclic hydrocarbons can occupy positions in the cavity in a random fashion. It is therefore essential that the solute has at least one aromatic ring, if a chiral separation is attempted in the reversed phase mode. The high density of secondary hydroxyls at the larger opening of the torroid is responsible for the preferential hydrogen bonding. Amines and carboxyl groups react strongly with these hydroxyl groups, as a function of the pK of the solute and pH of the aqueous mobile phase.
D. p-DONOR p-ACCEPTOR - PIRKLE TYPE
Historically, this type of chiral stationary phase preceded all the others described here . The pioneering work of Pirkle had such an impact on the field that the whole category of donor-acceptor type stationary phases was named after him. The structure of these type of stationary phases is based on single strands of chiral selectors, connected via amidic linkage onto aminopropyl silica as shown in Figure 4.
 Pirkle W.H. and Pochapsky T.C., "Advances in Chromatography" eds. Giddings J.C., Grushka E. and Brown P.R., Marcel Dekker Inc. NY, vol 27, 73-127, 1987.
 D. R. Taylor and K. Maher, "Chiral Separations by High-Performance Liquid Chromatography", J. Chromatogr. Sci., 30, 67-85 (1992).
 G. Gubitz, "Separation of Drug Enantiomers by HPLC Using Chiral Stationary Phases- A Selective Review", Chromatographia, 30, 555-564 (1990). W. H. Pirkle, D. W. House and J. M. Finn, "Broad spectrum resolution of optical isomers using chiral HPLC bonded phases", J. Chromatogr. , 192, 143-158 (1980).
Copyright (C)1997 Dr. Shulamit Levin
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