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Chiral Separation of Pharmaceuticals by High Performance Liquid

Chromatography

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114 Current Pharmaceutical Analysis, 2010, 6, 114-130

Chiral Separation of Pharmaceuticals by High Performance Liquid

Chromatography

Yiwen Zhang, Shun Yao, Hao Zeng and Hang Song*

Department of Pharmaceutical and Biological Engineering, Sichuan University, Chengdu 610065, P. R. China

Abstract: The increasing demand for enantiopure drugs has led to the development of a variety of stereoselective separa-
tion technologies. Among them, high performance liquid chromatography (HPLC) is well recognized as a powerful, fast
and efficient technique, which has been successfully employed for analysis and preparation of enantiomers of drugs.
Nowadays, liquid chromatographic techniques are the focus of intensive research, which lead to the rational design and
production of highly selective and efficient chromatographic materials. This review focuses on various HPLC methods
and related hyphenated techniques (including LC-MS, LC-OR detector, LC-CD detector) for chiral separation of pharma-
ceuticals, many new developments and applications are introduced in chiral HPLC separations in recent years. In this pa-
per, the usage of new materials as chiral stationary phases (CSPs) in liquid chromatography for enantiomeric discrimina-
tion is investigated in detail. Moreover, hyphenated techniques are very useful for chiral separation of HPLC, especially
for separation of new pharmaceuticals.

Keywords: High performance liquid chromatography, Chiral separation, Enantiomeric purity, Hyphenated techniques.

1. INTRODUCTION Nowadays, chiral separation covers an important area of

Molecular chirality is a fundamental consideration in
drug discovery, it is very necessary to understand and de-
scribe biological targets as well as to design effective phar-
maceutical agents (2005) [1]. A visual context was ever pro-
vided to position chiral technology in the highly complex
and evolving drug discovery environment with its numerous
challenges, hurdles, and tools (see Fig. (1)) (2007) [2].
Fig. (1). Positioning Chiral Technology in a highly complex and
evolving drug discovery environment [2].
*
Address correspondence to this author at the School of Chemical Engi-
neering, Sichuan University, Chengdu 610065, P. R. China;
Tel: +86-28-8540-5221; Fax: +86-28-8540-5221;
E-mail: hangsong@vip.sina.com
analytical chemistry of relevance to a wide variety of scien-
tific professionals. Many enantiomeric forms of drugs have
been recognized to have different physiological and thera-
peutic effects. Very often, only one form is pharmacologi-
cally active in an enantiomeric pair. It is, thus, hardly sur-
prising that the pharmaceutical industry needs effective
methods to determine enantiomeric purity and obtain effec-
tive and safe drugs with single steroconfiguration (2006) [3].
Ward et al. (2006, 2008) [4, 5] ever reviewed various devel-
opments and applications in chiral separations. A large
number of approaches have been used to isolate enantiomers
(such as gas chromatography, thin-layer liquid chromatog-
raphy, capillary electrophoresis, liquid chromatography, su-
percritical fluid chromatography, electrokinetic chromatog-
raphy, etc.), among the approaches enantioselective liquid
chromatography has become the most popular technique for
quickly obtaining limited quantities (from mg to
multi-grams) of pure single enantiomers, especially in drug
discovery [1]. Moreover, HPLC is well recognized as a
powerful, fast, highly efficient and selective technique, suc-
cessfully employed for preparation and analysis of enanti-
omers of drugs (2007) [6]. Okamoto et al. (2008) [7] re-
ported about chiral HPLC for efficient resolution of enanti-
omers and mentioned that more than half of the determina-
tions have been carried out by chiral HPLC in all the relative
reports. In recent years, some new HPLC methods have been
developed for the analytical and semipreparative resolution
of new drug enantiomers (2005) [8]. Although HPLC may be
considered as a mature technology, many advances have
been made to improve the separation capability and the effi-
ciency of method development and sample analysis con-
tinuously. Ever increasing demands for chiral analysis and
for higher laboratory efficiency have driven the development

1573-4129/10 $55.00+.00 © 2010 Bentham Science Publishers Ltd.

Chiral Separation of Pharmaceuticals
Table 1. The Characterization of Three Chromatographic Methods
Method Advantages
Chiral (1) Less expensive, i.e., convention nal chromatographic
deriva- columns can be used;
tization (2) Flexible, because various achiral columns and mobile
reagents phase conditions, as in HPLC, can be used;
(CDR) (3) Numerous types of derivatization chemistry are
available and the cost of each reagent may be less expen-
sive than for a chiral column;
(4) Different selectivities can be achieved.
Chiral (1) A large number of commercially available chiral
stationary columns can be selected;
phases (2) Convenience, specifically in preparative chromatog-
raphy;
(CSPs)
(3) The mode of operation is easy;
Chiral (1) Less expensive conventional LC columns can be used;
mobile (2) A wide variety of possible additives are available;
phase (3) Different selectivities from the chiral phases can be
additives obtained.
(CMPA)
of various options for high-speed analysis with the desire to
improve chiral resolution (2006) [9].
HPLC can be used either indirectly with chiral derivati-
zation reagents (CDR) or directly with chiral stationary
phases (CSPs) or chiral mobile phase additives (CMPA) to
separate chiral compounds. The characteristics of these three
chromatographic methods are summarized in Table 1. At
present, large kinds of new materials have been developed as
chiral stationary phases (CSPs) in liquid chromatography.
Also, some new chiral derivatization reagents and new chiral
mobile phase additives have been used for enantiomeric
separation successfully. Moreover, large amounts of applica-
tions of several hyphenated techniques (LC-MS, LC-CD and
LC-OR, etc.) were reported succesively in chiral analysis
(2000-2007) [10-14]. For example, when an enantiomer ex-
presses exciton coupling that is observed in the CD spec-
trum, the absolute stereoconfiguration can be assessed in
combination with molecular modeling studies of the lowest
energy conformers (2005-2008) [15-22].
This review focuses on the current developments in col-
umn technology and method development approaches, then a
brief discussion of advances is given in hyphenated tech-
niques. Related methods of rapid analysis include mi-
cro-columns, small particle columns and multidimensional
chiral high performance liquid chromatography.
2. NOVEL CHIRAL STATIONARY PHASES (CSPS)
As is well known, chiral separation by HPLC with CSPs
has become a powerful tool in the development of chiral
drugs (2009) [23]. The HPLC-CSPs demonstrated great suc-
cess for performing chiral separation in pharmaceutical in-
dustry, because the HPLC can be used both for analytical or
preparative purpose in a very efficient and flexible way
(2005, 2005) [8, 24]. So far, there are numerous commer-
Current Pharmaceutical Analysis, 2010, Vol. 6, No. 2 115
Disadvantages
(1) Long analysis time that include sample preparation and verification of
the derivatization chemistry;
(2) Inconvenience, specifically in preparative chromatography, when re-
versal of derivatization is needed to recover the pure enantiomers;
(3) Yhe need to synthesize noncommercially available pure derivatizing
reagent;
(4) Biased results for enantiomeric composition due to partial racemization
of derivatizing agent or unequal reaction rates.
(1) Many commercially available chiral columns are expensive;
(2) One specific chiral column sometimes, can only separate very few
kinds of enantiomers;
(3) It is difficult to select CSPs for efficient resolution of specific enanti-
omers
(1) Many chiral additives are costly, and sometimes, have to be synthe-
sized;
(2) The mode of operation is complex,
(3) Incovenient for preparative applications because the chiral additive
must be removed from the enantiomeric solutes.
cially available chiral selectors or CSPs for the resolution of
racemic drugs. Several new selectors for HPLC applications
have been developed in recent years
such as cyclodextrin,
polysaccharide, protein, macrocyclic antibiotics, crown
ethers, etc. Table 2 presents various typical kinds of chiral
selectors, including their characterizations, structures and
possible separation analytes.
2.1. Brush-type CSP
Brush-type CSPs, also called ‘Pirkle CSPs’, were in-
vented by Pirkle’s group since the early 1980s (1980, 1991)
[25, 26]. The chemical structural characteristics of this type
of CSPs are based on the presence of -donor groups,
-acceptors group or the groups that could form multiple
hydrogen bonds, and these groups can give a good fit to the
“three point interaction principle” theory. This type of CSPs
(such as DNB-Leu, DNB-PG, Whelk-O 1, Whelk-O 2,
ULMO,
-Burke, -Gem 1, all from Regis Technologies,
Inc. USA) have been extensively used for chiral analysis
(2003-2008) [27-34]. Some structural improvement of exist-
ing brush-type CSP have been carried out for improving lim-
ited separating capability of the CSPs. These novel CSPs
show strong separating capability for many specific chiral
compounds. Landek et al. (2006) [35] adopted brush type
chiral stationary phases derived from L-leucine for enanti-
omeric separation of 1,1’-binaphthyl-2,2’-diol enantiomers.
A novel chiral packing material for HPLC was prepared by
connecting (R)-1-phenyl-2-(4-methylphenyl) ethylamine
(PTE) amide derivative of (S)-isoleucine to aminopropyl
silica gel through 2-amino-3, 5-dinitro-1-carboxamido-ben-
zene unit (2007) [36]. A Pirkle-type chiral stationary phase
for column chromatography based on N-p-Amino ben-
zoyl-L-glutamic acid has been synthesized for resolution of
-methylphenylethylamine (2010) [37].
116 Current Pharmaceutical Analysis, 2010, Vol. 6, No. 2 Zhang et al.

Table 2. Most Popular Commercially Available LC CSPs

CSP’s Symbolic Separation

Characteristics Structures of the CSP
Type Column Analytes

Brush- Based on “three point Whelk-O 1 Acids, amines,

type interaction principle” for alcohols,
CH3
chiral discrimination amides, esters,
Si O
sulfoxides, thiols,
H N CH3
amino alcohols,
O
alkaloids, cyclic
O2N ketones,
alkaloids,
NO2

OH
Cyclo- Cyclodextrin cavity con- CYCLOBOND
Amino acids,

dextrin tains hydroxyls for 2000AC HO O O amines, alcohols,

O
OH RO
H-bonding with polar OH
amides, crown
O
groups of analyte; hydro- OR ether, metallo-
HO RO
O
phobic portion of analyte OH cenes, polycyclic
O O
fits into non-polar cavity aromatic hydro-
OH
RO O
(inclusion complexes) carbons, lactones,
OH
OH
O
OR
OH
O OH O
OH OR O
O OH
HO
O
OH

R=CH3CO-
Immobilised on silica gel

Poly- Enantiomers form CHIRALPAK IC H Acids, amino

N Cl
O
saccharide H-bonds with carbamate acids, amines,
O
links between side chains alcohols, amides,
O Cl
and polysaccharide back- esters, sulfoxides,
O
bone Cl lactams, lactones,
O n
O cyclic ketones,
O
NSAIDS,
Cl N HN
H O

Cl

Cl

Immobilised on silica gel

Protein- Natural proteins bonded CHIRAL-AGP
1-acid glycoprotein Acids, amines,

based to a silica matrix; proteins Immobilised on silica gel alcohols, amides,
contain large numbers of sulfoxides, cyclic
chiral centers and interact drugs, aromatic
strongly with small chiral drugs,NSAIDS,
analytes through hydro- peptides
phobic and electrostatic
interactions, H-bonding
Chiral Separation of Pharmaceuticals Current Pharmaceutical Analysis, 2010, Vol. 6, No. 2 117

Table 2. contd….

CSP’s Symbolic Separation

Characteristics Structures of the CSP
Type Column Analytes

Macro- Contain a large number of CHIROBIOTIC R OH HO Acids, amino

cyclic antibi- chiral centers together CH3 acids, amines,
O
O OH CH2OH
otics with cavities for analytes H2N OH HO
amides, esters,
OH
to enter and interact sulfoxides, amino
HO
O NH HN alcohols,
NH
O
HO O O COOCH3
HN
HN
H
N O
O O
H3C O
O O O
HO
O O

HO CH2OH CH3
O O
NH2
HO OH O
HO OH
O OH

O OH

OH
OH

Immobilised on silica gel

Crown ethers Crown ethers are macro- CROWNPAK CR Amino acids,

cyclic polyethers that can amines, alcohols,
form host-guest com- crown ether,
O
plexes metallocenes,
O O
polycyclic aro-

O O matic hydrocar-

O bons,

CR+=(S)-18-crown-6-ether

Immobilised on silica gel

2.2. Cyclodextrin CSP and neutral analytes by chromatographic techniques. These

The first CSP containing cyclodextrin (CD) bonded to
silica gel was developed by Armstrong and DeMond in 1984
(1984) [38]. Recently, both the native CD CSPs and the
modified CD CSPs with improved enantioseparation per-
formances have been used in many cases (2008) [39].
Berkecz et al. (2007) [40] employed a kind of -CD based
chiral stationary phase for enantioseparation of 1-(
-amino-
benzyl)-2-naphthol and 2-(
-aminobenzyl)-1-naphthol
analogs. Despite the numerous CD derivatives were
developed as chiral selectors in the past decades for the
purpose of chiral separation, a great need still exists for
further effort to synthesize novel CD-CSPs with improved
chiral recognition ability towards a large pool of racemic
analytes with shorter time and greater accuracy. Tang et al.
(2008) [41] provided a meaningful overview of recent work
carried out in their laboratory on the synthesis of four series
of mono-substituted positively charged CDs which were
applied as chiral selectors in the separation of derivatized
amino acids, anionic pharmaceutics and neutral analytes by
novel cyclodextrin CSPs showed good chiral recognition
ability in actual separation.
2.3. Polysaccharide CSP
Polysaccharide type CSPs have proven to be one of the
most useful CSPs because of their versatility, durability, and
loadability (2001) [42]. In recent years, a broad range of
commercially available polysaccharide phases have been
produced by Daicel group (Osaka, Japan), based on cellu-
lose, amylose esters or carbamates. The cellulose-based
columns (e.g. Chiralcel OD) and the amylose-based columns
(e.g. Chiralcel AD) are frequently used for the enantiosepa-
ration (2001-2009) [42-46]. Immobilised polysaccha-
ride-based CSPs are a new generation of chiral chroma-
tographic materials combining the remarkable enantioselec-
tive performance of the polysaccharide derivatives with sol-
vent versatility in enantiomeric resolution. Since 2004 three
immobilised polysaccharide-derived CSPs have become
commercially available: Chiralpak IA, Chiralpak IB and
118 Current Pharmaceutical Analysis, 2010, Vol. 6, No. 2 Zhang et al.

Fig. (2). Enantioseparations of (a) D/L-tryptophan, (b) R/S-warfarin and (c) R/S-ibuprofen on HSA columns prepared by the SMCC and SIA
methods. The HPLC conditions were as follows: sample concentration, 20μM tryptophan, ibuprofen or warfarin; sample volume, 20μL; mo-
bile phase for tryptophan, pH 7.4, 0.067M potassium phosphate buffer; mobile phase for ibuprofen, pH 7.0, 0.067M potassium phosphate
buffer containing 8% 2-propanol and 1mM octanoic acid; mobile phase forwarfarin, pH 7.0, 0.067M potassium phosphate buffer containing
5% 2-propanol and 1mM octanoic acid; flow rate for the SMCC HSA column, 1.5 mL/min for tryptophan, 1.0 mL/min for ibuprofen and
warfarin; flow rate for the SIA HSA column, 0.3 mL/min for tryptophan and warfarin, 0.5 mL/min for ibuprofen; column size, 5 cm
4.6mm
i.d.; temperature, 25
[58].

Chiralpak IC by Daicel Chemical Industries, Ltd. (Tokyo,
Japan) (2008) [47]. Zhang et al. (2008) [48] employed
Chiralpak IC for separation of 70 chiral molecules. The re-
sults indicated that Chiralpak IC exhibited high enantioselec-
tivity for a large number of enantiomers. In addition, the
versatility of mobile phase is an important factor for chiral
separation on Chiralpak column. The chromatographic per-
formances of Chiralpak IA under various mobile phases
were investigated (2005) [49]. It was found that four solvents
(methyl tert-butyl ether, dichlormethane, tetrahydrofuran and
ethyl acetate) were particularly versatile in mobile phase. So
it was highly recommended to screen these solvents first for
enantioselectivity.
Jin et al. compared several polysaccharide-derived chiral
stationary phases for the enantiomeric separation of
N-fluorenylmethoxycarbonyl -amino acids (2007) [50] and
N-protected -amino acids (2009) [51]. The result indicated
that all the CSPs resulted in the greatest separation factor.
However, Chiralpak AD and Chiralcel OD of the typical
coated-type CSPs are not compatible with all solvents of the
mobile phase (normal phase mode is mainly used), because
the chiral selectors of polysaccharide derivatives for these
CSPs are coated on a silica matrix. Moreover, owing to the
compatibility with a wide range of solvents of the covalently
bonded CSPs, it is expected some new application of
Chiralpak IA and IB in enantiomer separation, especially for
preparative purpose. Some literatures reported the technique
for immobilizing the polysaccharide derivatives onto the
silica gel formerly (2008, 2007) [52, 53].
2.4. Protein-based CSP
The advantages of protein-based CSP include enantiose-
lectivity to a wide range of compounds because all proteins
have certain ability to discriminate chiral molecules. CSPs
based on bovine serum albumin (BSA), human serum albu-
min (HAS), 1-acid glycoprotein (AGP), ovomucoid from
chicken egg whites (OMCHI), avidin (AVI), cellobiohydro-
lase I (CBH I) and pepsin are now commercially available
(2001) [54]. So far, many protein-based CSPs have been
developed, and some studies (2000-2003) [55–57] were pub-
lished on the mechanisms and applications of enantiosepara-
tion of chiral drugs.
In recent years, serum albumin of other species, penicillin
G-acylase, antibodies, fatty acid binding protein and strep-
tavidin were newly introduced as the chiral protein-based
selectors in CSPs (2008, 2008) [58, 59]. Fig. (2) shows good
enantioseparation of tryptophan, warfarin and ibuprofen on
Chiral Separation of Pharmaceuticals
HSA columns prepared by N-(4-carboxycyclohexylmethyl)
maleimide (SMCC) and succinimidyl iodoacetate (SIA)
methods [58]. The chiral recognition mechanisms of these
proteins were persumed by molecular modeling, ligand
docking, and X-ray crystallography, which can effectively
promote the rapid development in designing new pro-
tein-based CSPs with specific group proteins used for chiral
selectors.
2.5. Macrocyclic Antibiotics CSP
Macrocyclic antibiotics used as chiral selectors were first
introduced by Armstrong’s group (1994) [60] and represent a
relatively new class of chiral selectors in separation science
(2001, 2006) [61, 62]. The selectors have a large number of
stereogenic centers and functional groups, which could real-
ize multiple interactions with chiral molecules. So far, sev-
eral macrocyclic antibiotics (including rifamycins B and SV,
avoparcin, teicoplanin, ristocetin A and vancomycin) have
been used as chiral selectors widely (2009) [63]. A vanco-
mycin degradation product-based CSP has been adopted for
chiral separation of racemic compounds (2007, 2008) [64,
65]. Honetschlagerova-Vadinska et al. (2009) [66] made
satisfying evaluation of the enantioselective performance of
two teicoplanin-based CSPs Chirobiotic T and Chirobiotic
T2, and then the authors discussed the possible interaction
mechanisms on the basis of the separation results and some
theories. Pataj et al. (2009) [67] compared three teico-
planin-derived CSPs (Chirobiotic T, T2 and TAG) for the
enantioseparation of
2-homoamino acids. The result indi-
cated that Chirobiotic TAG, whose structure contain no
sugar units, may promote the interactions of the enantiomers
of
2-homoamino acids with branched alkyl or aryl
side-chains, whereas
2-homoamino acids with alkyl
side-chains Chirobiotic T and T2 seem to be more favorable.
Vancomycin-CSP was used for determinating thalidomide
enantiomers in biological samples (2006) [68].
2.6. Other CSPs
Chiral synthetic polymers, crown ethers, chiral imprinted
polymers, ionic compounds, chiral ionic liquid, and some
natural products have also been used as CSPs for the enanti-
oseparation of pharmaceuticals and their unique structure
always result in uncontemplated separation ability for chiral
drugs.
A comprehensive review on the synthesis and application
of chiral synthetic polymers was reported by Nakano (2001)
[69]. Two new synthetic polymeric CSPs based on trans-(1S,
2S)-cyclohexanedicarboxylic acid bis-4-vinylphenylamide
and trans-N,N-(1R,2R)-cyclohexanediyl-bis-4-ethenylben-
zamide monomers were prepared and evaluated by HPLC in
normal phase system (2008) [70]. Further, a new synthetic
polymeric CSP for liquid chromatography was prepared via
freeradical-initiated polymerization of trans-9,10-dihydro-9,
10-ethano-anthracene- (11S,12S)-11,12-dicarboxylic acid
bis-4-vinylphenylamide. This new polymeric CSP showed
special enantioselectivity for many chiral compounds in
multiple mobile phases (2007) [71].
Crown ethers are macrocyclic polyethers that can form
host-guest complexes. The CSPs of crown ethers have an
Current Pharmaceutical Analysis, 2010, Vol. 6, No. 2 119
excellent chiral recognition ability and are easily bound co-
valently to the support. Berkecz et al. (2008) [72] employed
a chiral stationary phase containing (+)-(18-crown-6)-2,3,11,
12-tetracarboxylic acid for chiral separation of several

-amino acids. A novel CSP prepared by immobilizing a
chiral pseudo-18-crown-6-type host onto 3-aminopropyl
silica gel was used for the enantioseparation of 18 common
natural amino acids efficiently (2005) [73]. In addition, a
CSP based on (-)-(18-crown-6)-2,3,11,12-tetracarboxylic
acid was evaluated for the direct resolution of the enanti-
omers of dipeptides and tripeptides (2005) [74].
Molecularly imprinted polymers (MIP) have been ex-
ploited as CSPs since the pioneering work of Wulff in the
1970s (2005) [75]. Several excellent reviews were given in
past years on the use of molecularly imprinted stationary
phases in HPLC (1999-2001) [76-78]. Molecularly imprinted
polymers CSPs can be extremely successful in most chiral
for many certain compounds.
CSPs with ionizable groups have been developed in past
few years. Hoffmann et al. (2008) [79] investigated the syn-
ergistic effects on enantioselectivity of zwitterionic CSPs for
the separation of chiral acids, bases, and amino acids. For
example, a novel strong cation-exchange-type of chiral sta-
tionary phase has been used for separation of cinchona alka-
loids (2009) [80].
Ionic liquids (ILs) are a group of organic salts that can
keep liquid-state at room temperature. They have unique
chemical and physical properties, including air- and mois-
ture-stable, a high dissolving capacity and virtually no vapor
pressure. Because of these properties, they can serve as
“green” recyclable alternatives to the volatile organic com-
pounds which are traditionally used as industrial solvents
(1999-2003) [81-84]. Advances in ILs have made the syn-
thesis of chiral ILs become a subject of intense study in re-
cent years (1999-2006) [85-96]. The popularity stems from
the fact that it is possible to use chiral ILs as a chiral station-
ary phase in chromatography for the determination of phar-
maceutical compounds (2005) [97]. Tran et al. (2006) [3]
successfully synthesized both enantiomers of a novel chiral
ionic liquid, (R)- or (S)-[(3-chloro-2-hydroxypropyl)
trimethylammonium] [bis((trifluoromethyl)-sulfonyl) amide]
((R)- and (S)-[CHTA]+[Tf2N]-) in optically pure form which
are commercially available by simple ion exchange reaction
from corresponding chloride salts, and they successfully
developed a novel method based on the near-infrared tech-
nique with this chiral IL serving both as solvent and as a
chiral selector for the determination of enantiomeric purity.
Yu et al. (2008) [98] also synthesized a series of structurally
novel chiral ionic liquids with an either chiral cation or chiral
anion, or both. Structurally novel and strong intra- and in-
termolecular chiral recognition ability of these chiral ILs
showed that they could be used in a variety of applications
including as chiral solvents for asymmetric synthesis and as
CSPs for chromatographic separation.
In addition, a large number of natural products have been
used as novel chiral stationary phases. A novel chiral sta-
tionary phase based on the L-RNA aptamer was a kind of
highly biostable stationary phase due to the intrinsic insensi-
tivity of L-RNA to the RNase degradation. Such an approach
120 Current Pharmaceutical Analysis, 2010, Vol. 6, No. 2
allowed one to reverse the elution order of enantiomer rela-
tive to that obtained with the corresponding D-RNA CSP
(2005) [99]. Wang et al. (2008) [100] adopted glucose, cel-
lobiose, lactose and raffinose as chiral stationary phases in
HPLC, and the novel chiral stationary phases also possess
high enantioseparation selectivity, which may be used in
normal-phase and reverse-phase mode, and there is a great
chiral discriminating complementary to present study.
3. CHIRAL DERIVATIZATION REAGENTS AND
CHIRAL MOBILE PHASE ADDITIVES
3.1. Chiral Derivatization Reagents (CDRs)
The indirect method involving the derivatization process
with some suitable chiral tagging reagent is an efficient way
for the separation of many enantiomers, especially when
suitable CSPs were not available. The mechanism of indirect
separation is based on the use of chiral derivatization rea-
gents to form a complex of diastereomers, the diastereomers
differing in their physicochemical properties can hence gen-
erally be separated by conventional chromatographic col-
umns. So far, enormous chiral derivatization agents are
available. A review described the application of chiral de-
rivatization agents in the HPLC separation of amino acid
enantiomers (2008) [101]. A new chiral derivatization agent,
2,3,4,6-tetra-O-acetyl--D-galacto-pyranosyl isothiocyanate
(GATC) was adopted for chiral separation of several
-blockers (2006) [102]. Ten novel CDRs have now been
used for HPLC enantioresolution of some -amino alcohols.
The method was also found successful for the separation of
20 diastereomers from a mixture (2009) [103]. Kurosu et al.
(2009) [104] reported unprecedented chiral derivatizing
agents that allowed for the determination of the absolute
configurations of a wide range of amino acids by only ana-
lyzing the carbamate nitrogen protons in 1H NMR spectra.
Bhushan et al. (2008) [105] synthesized chiral hydrazine
reagents for enantioseparation of carbonyl compounds. An-
other new chiral derivatizing reagent synthesized from
s-triazine chloride was employed for enantioseparation of

-amino acids by reversed-phase liquid chromatography
(2008) [106]. With more and more new chiral derivatizing
agents appear for selection, the methodology of chiral de-
rivatizing agents will play a more important role in chiral
separation.
3.2. Chrial Mobile Phase Additives (CMPAs)
It is possible to effect an enantiomeric separation using
conventional HPLC stationary phases by adding a chiral se-
lector in the mobile phase. In this method, enantiomeric
separation is accomplished by the formation of a pair of
transient diastereomeric complexes between racemic analyte
and the chiral mobile phase additive. The advantages of this
technique are so many, the most obvious of them are sim-
plicity and flexibility; moreover there is no need for chiral
derivatization. The frequently used CMPAs are ion-pairing
reagents, ligand-exchanging reagents, protein-affinity rea-
gents and cyclodextrin inclusion reagents. Among them,
cyclodextrin was used for chrial mobile phase additives gen-
erally (1997-2001) [107-111]. Ma et al. (2009) [112] em-
ployed sulfated -cyclodextrin (S--CD) as CMPA for enan-
Zhang et al.
tiomeric separation of chiral amine and explored the chiral
recognition. The results showed that S--CD could undergo
a conformational change with increasing temperature. Par-
ticularly, the aromatic part of the molecule was included
inside the cyclodextrin through hydrophobic interactions
with the interior of the macrocycle, which could reflect the
interactions between the CMPA and analytes. Moreover,
norvancomycin and vancomycin were adopted as CMPAs in
chiral separation and gained expected affect for several cer-
tain analytes (2006, 2000) [113, 114].
4. HYPHENATED TECHNIQUES
4.1. LC-MS
During the last decade, separation techniques coupled
with mass spectrometry became an essential and powerful
tool in molecular biochemistry, especially in the analysis of
chiral compounds of pharmaceutical, clinical, environmental
and/or agro-chemical interests. In order to evaluate the
pharmacokinetics of a single enantiomeror of enantiomer
mixture, manufacturers should develop quantitative assay for
the individual enantiomers in vivo samples early in the drug
development process. Currently, there is a trend toward con-
verting chiral HPLC methodologies into more sensitive mass
spectrometry-based approaches without losing the enanti-
oselectivity of the assay (2004) [115]. The methodology of
chiral LC-MS is becoming increasingly important for the
routine analysis of chiral drug and their metabolites in bio-
logical matrices (2005-2009) [116-121], also for toxicology
research (2005) [122].
The recent developments in chiral liquid chromatography
coupled with atmospheric pressure ionization tandem mass
spectrometry (LC-API-MS/MS) for the analysis of pharma-
ceuticals were reviewed by Chen et al. (2005) [123]. Various
new ion sources including electrospray ionization (ESI), at-
mospheric pressure chemical ionization (APCI) and atmos-
pheric photoionization (APPI) have been employed for chiral
separation of pharmaceuticals. Among these soft ionization
techniques, only APPI source is compatible with both re-
versed phase and normal phase conditions without the safety
concerns of a potential explosion. The schematic of APPI
with a chiral column for the pharmaceuticals is shown in Fig.
(3). D’Orazio et al. (2008) [124] employed a novel chiral
nano-LC-MS for analysis amino acids in orange juice profil-
ing. The complete set-up used for nano-LC-MS analysis of
amino acid enantiomers is shown in Fig. (4). Luo et al.
(2010) [125] adopted chiral liquid chromatography and tan-
dem mass spectrometry for simultaneous analysis of bam-
buterol and its active metabolite terbutaline enantiomers in
rat plasma, the typical chromatograms are shown in Fig. (5).
4.2. LC-CD
It is always required to determine the enantiomeric purity
in the quality control of chiral drugs production. At present,
the importance of the circular dichroism (CD) spectroscopy
is increasing importance in pharmaceutical analysis because
of the continuous appearance of commercially available CD
detector in liquid chromatography (2007) [126]. Fig. (6)
shows the basic components of the CD detector (2007)
[127].
Chiral Separation of Pharmaceuticals Current Pharmaceutical Analysis, 2010, Vol. 6, No. 2 121

Fig. (3). Schematic diagram of an atmospheric pressure photoionization (APPI) source [123].

Fig. (4). Schematic representation of the column-switching system coupled with MS [124].

Fig. (5). Typical MRM chromatograms of R-bambuterol, S-bambuterol, R-terbutaline, S-terbutaline, and S-propranolol (I.S.) in Wistar rat
plasma. The retention times were approximately 19.5, 21.6, 15.1, 16.9, and 20.1 min, respectively. (A) Blank plasma sample; (B) plasma
spiked with 1 ng/mL of bambuterol and terbutaline enantiomers and 1.5μg/mL S-propranolol; (C) plasma sample 4 h after intravenous infu-
sion of 5mg/kg rac-bambuterol. Panels marked are (1) for bambuterol (m/z 368
294); (2) for terbutaline (226
152); and (3) for I.S. (260

183) [125].

HPLC with CD detector analysis (LC-CD) can establish
a correlation between the absolute configuration of separated
enantiomers and their characteristic CD transitions. So,
LC-CD hyphenated technique is playing a gradual key role
for determination of absolute configuration of chiral drug,
especially in drug discovery. There are lasting LC-CD ap-
plications reported about determination of absolute configu-
ration for enantiomers (2001-2009) [127-134]. Iwasa et al.
(2009) [130] employed on-line HPLC-CD in combination
with HPLC-MS for analysis of benzylisoquinoline alkaloids,
122 Current Pharmaceutical Analysis, 2010, Vol. 6, No. 2 Zhang et al.

Fig. (6). Optical components of a CD detector [127].

Fig. (7). LC-CD data and stop-flow LC-CD of (+)-reticuline and (±)-reticuline. CD detector: Jasco CD-2095Plus; pump: Jasco
PU-2SOOPlus; column: chiralcel OJ-H (4.6
250 mm); fluent: N-hexane/ethanol/diethylamine: 50/50/0.1; flow rate: 0.5 mL/min. LC/CD
detection: 236 nm; sample injection: 5 μg [130].

the chromatogram is presented in Fig. (7). A novel chiral
detector, a circular-dichroism thermal lens microscope
(CD-TLM), was developed to realize sensitive and selective
detection of small volume chiral samples on a microchip
(2006) [135]. Thermal lens spectrometry (TLS) is a kind of
photothermal spectrometry which holds promise for over-
coming the low sensitivity of absorption-based detection
methods, and the principle of CD-TLM is illustrated in
Fig. (8). This novel chiral detector is more than 250 times
higher sensitivity versus that in a CD spectrophotometer for
pure enantiomer detection.
Ranjbar et al. (2009) [136] introduced circular dichroism
techniques for biomolecular and nanostructural analyses. The
result indicated circular dichroism is a powerful technique
for evaluating the conformation of polypeptides and proteins.
In a word, the development tendency of CD detector is aim-
ing to improve its sensitivity to detect smaller amounts of
enantiomers.
4.3. LC-OR
Some applications for OR detectors have received the
most attention to date. The most fundamental is to verify the
optical activity of the compounds of interest and to assign
either a positive or negative rotation of the structure. LC-OR
can also be adopted for the identification and characteriza-
tion of impurities. However, this application has received
little attention to date, primarily due to the relatively low
sensitivity of most OR detectors and the need for optically
pure standards to facilitate identification (2000) [137].
Kott et al. (2007) [127] made a meaningful evaluation of
four commercial HPLC chiral detectors (three polarimeters
and a circular dichroism detector). Moreover, LC-OR hy-
Chiral Separation of Pharmaceuticals Current Pharmaceutical Analysis, 2010, Vol. 6, No. 2 123

Fig. (8). Principle of CD-TLM [126].

Fig. (9). Enantioseparation of a mixture containing 5 rac-
-blockers after derivatization with AITC reagent. Column: Acquity CB18B BEH
(50 3 2.1 mm I.D., 1.7 μm); Mobile phase: acetate buffer 20 mM pH 5-MeCN, gradient from 23.5 to 56.5% MeCN in 2.34 min. (slope of
14.1%/min); F=1000 μl/min; Injection: 2 μl; Temperature: 30
. UV detection: 254 nm. Each rac-
-blocker was between 100 and 500 μg/ml
before derivatization [143].

phenated technique has been developed for determining the
change of enantiomeric excess in the chiral synthesis in order
to confirm the reaction termination (2006, 2008) [138,139].
5. SOME NEW DEVELOPMENTS AND APPLICA-
TIONS IN HPLC CHIRAL SEPARATIONS
Today, rapid analysis for chiral pharmaceuticals has be-
come an inevitable trend. Fmoc- and Z-derivatives of natural
and unnatural sulfur containing amino acids were separated
by micro-HPLC with very short time (2004) [140]. The
separation was carried out in microbore columns packed
with a new material based on Ristocetin A bonded to 3.5 μm
silica gel. The major advantage of micro-HPLC is much less
consumption of expensive packing material, solvent and
sample. Furthermore, the small particle size leads to higher
efficiency and ideal resolution with shorter retention times.
Currently, particle size can be decreased even more, and
the use of columns packed with sub-2 μm particles working
under ultra-high pressure have been developed over the last 5
year (2005, 2006) [141, 142]. So, the use of columns packed
with sub-2 μm particles in liquid chromatography with very
high pressure conditions (known as Ultra High Pressure Liq-
uid Chromatography, UHPLC) was investigated for the fast
enantioseparation of drugs. Guillarme et al. (2010) [143]
employed two alternative approaches for evaluation for rapid
chiral analysis by UHPLC using amphetamine derivatives
and
-blockers as model compounds. Using this strategy, it
was possible to achieve the robust separation presented in
Fig. (9). As a result 10 enantiomers from 5 different

-blockers were well separated in less than 3 min, which
demonstrated that the developed method could be applied for
the establishment of optical purity of pure compounds at low
levels in very short analysis times.
124 Current Pharmaceutical Analysis, 2010, Vol. 6, No. 2
Fig. (10). Simulated moving bed chromatographic unit placed on the UNICAMP Bioseparation Laboratory. (A) Detectors controller module,
(B) raffinate pump, (C) feed pump, (D) UV detector, (E) extract pump, (F) inlet desorbent pump, (G) polarimeter, (H) valves system, (I)
columns, (J) valves controller system, (K) sampling valve [146].
CSPs associated with simulated moving bed (SMB)
technology have also shown excellent performance in the
separation of racemates. This technology has been mainly
used in the area of chiral purification (2007, 1997)
[144,145], where the resolution of enantiomers is usually a
binary separation task with low selectivity and high opera-
tional cost (2007) [144]. Therefore, SMB is not only the
method to prepare few grams of the chiral drug required by
preliminary clinical tests but even more so most suitable way
for the large-scale production. A laboratory-scale SMB unit
made in home was adopted in an eight-column configuration
(two column per section), as shown in Fig. (10) (2008) [146].
Rajendran et al. (2009) [147] reviewed simulated moving
bed chromatography using for the separation of enantiomers.
After realizing its potential for enantiomer separations, now
the SMB technology is an important component of the sepa-
ration toolbox in the pharmaceutical industry, particularly to
manufacture single pure enantiomers of racemic drugs.
Nowadays, multidimensional high performance liquid
chromatography technique is a powerful tool for analysis of
complicated samples especially biological fluids. It is a
powerful tool for chiral analysis too, which can be used to
determine not only drugs’ metabolites in plasma but also
value of enantiomeric excess (e.e.%). Mullett et al. (2007)
[148] provided an overview of the existing literatures with
emphasis on advances in automated sample preparation
methods for liquid chromatography. The column configura-
tions including multi-dimensional-column mode can provide
additional dimensions of selectivity. Albendazole metabo-
lites were analyzed by direct injection of bovine plasma on
multidimensional high performance liquid chromatography
Zhang et al.
(2008) [149], and the column-switching system employed is
illustrated in Fig. (11).
Shi et al. (2009) [150] reviewed a recently developed
technique that couples HPLC with on-line, post-column
(bio)chemical assays and parallel chemical analysis to screen
and identify bioactive compounds from complex mixtures
without the need for cumbersome purification and subse-
quent screening. Those strategies have proved to be very
useful for rapid profiling and identification of individual
active components in mixtures, hence to provide a powerful
method for natural product-based drug discovery. This sys-
tem coupled with chiral columns can be employed for analy-
sis of complicated samples with the high selectivity and sen-
sitivity of detection. The on-line assays can always be
adopted in a qualitative way and known active compounds
can be screened and identified rapidly, while novel active
compounds could be further investigated after target purifi-
cation.
6. MECHANISM OF CHIRAL RECOGNITION
Although there are so many commercially available
chiral columns used for chiral analysis, it is difficult to select
suitable CSPs for efficient resolution of specific enantiomers
rapidly. Therefore, it is very important for us to understand
the mechanism of enantioseparation. Today, the intermo-
lecular interactions between chiral selector and enantiomers
can be calculated with quantum mechanics, molecular me-
chanics and molecular dynamics. The various molecular
modelings have been established by X-ray crystallography,
NMR and computer simulation in order to study chiral dis-
crimination mechanism (2004) [151]. Among them, molecu-
Chiral Separation of Pharmaceuticals
Fig. (11). Schematic diagram of the column-switching system [149].
lar modeling is considered as a practical tool for evaluating
the complex interactions taking place as analytes migrate
through a chromatographic column, which could provide
detailed information at the atomic level about how chroma-
tographic process undergoes. Several molecular simulation
theories have been employed to simulate the liquid chroma-
tography process in enantioseparation. Chemometric analysis
of enantiospecific retention data was first described by
Francotte and co-workers (1988, 1991) [152, 153]. These
authors applied multiple regression analysis to relate enanti-
oselective separation on cellulose triacetate and cellulose
tribenzoate columns to structural descriptors of analytes
(2007) [154]. In order to study the retention and chiral rec-
ognition mechanism, the method of quantitative struc-
ture-enantioselectivity retention relationships (QSERR) was
investigated from the quantitative equations established be-
tween the chromatographic retention of enantiomers and
their molecular descriptors of physico chemical properties.
QSERR was first introduced as a special term in a report on
studies of HPLC data determined on human serum albumin
(HSA) column (1992) [155]. Then, QSERR has been exten-
sively studied since decades (1996-2001) [156-159]. Heber-
get (2007) [160] reviewed Quantitative structure (chroma-
tographic) retention relationships (including QSERR) from
1996 to 2006. QSERR models can be used for successful
classification of chiral drugs of various compound classes
and/or chromatographic columns (systems), which is very
useful for HPLC practitioners. Consequently, in QSERR
studies one can address either variation in the
non-enantiospecific strength of solute-CSP interactions due
to differences in the chemical structure and properties (such
as polarizability, number of hydrogen atoms, etc.) which
increases the overall retention of both isomers or the forces
which lead to chiral discrimination (2009) [161]. Moreover,
Lipkowitz (2001) [162] introduced the atomistic modeling of
enantioselection in chromatography, and the mechanism of
chiral recognition for five types of CSPs has been studied
and direct interactions are calculated between CSPs and
analytes. The chromatographic process can be well analyzed
using molecular dynamic model theories. The determination
of the adsorption sojourn times and the number of adsorp-
tion–desorption or mass-transfer events can give a practical
view of the molecular process of separation (2008) [163].
The studies of various kinds of molecular modeling for chiral
Current Pharmaceutical Analysis, 2010, Vol. 6, No. 2 125
separation by LC have been investigated extensively
(2004-2009) [164-172]. With researching the chiral recogni-
tion mechanisms at the molecular level, we can not only ex-
plain the experiment phenomena but also predict the results.
It can guide us to adjust chromatographic conditions in order
to improve the resolution of enantioseparation. What is more
important, the new types of CSPs can be designed to realize
the expected separation.
7. CONCLUSION
A major trend in LC for the separation of drug enanti-
omers is clearly heading toward faster and more efficient
separation with comparable or improved separation capabil-
ity. The development of liquid chromatographic procedures
focuses on usage of efficient novel materials in column
chromatography and hyphenated techniques with highly se-
lectivity.
After the rapid development in many years, enantioselec-
tive liquid chromatography, particularly HPLC on CSPs, has
become an indispensable part of drug discovery not only in
daily chiral analysis but also in the fast preparation of drug
molecules. As more chiral selectors are being developed and
commercialized as CSPs, a systematic and deeply under-
standing of the chiral recognition mechanisms at the mo-
lecular level is essential for the future, so that researchers can
rapidly select or even create suitable CSPs for efficient reso-
lution of specific enantiomers in one day. Moreover, by us-
ing hyphenated techniques (LC-MS, LC-CD, LC-OR), we
can know more information about molecule structure, phar-
macokinetics, spatial configuration, eluting order for drug
enantiomers. With the advances and promotion of related
techniques, recent developments in HPLC enantiomeric
separation, including micro-columns, small particle columns,
simulated bed moving chromatography and multidimen-
sional chiral high performance liquid chromatography, have
been reviewed in this paper. Collectively, these advances
will lead to persistent improvement of chiral separation ca-
pability.
ABBREVIATIONS
API = Atmospheric pressure ionization
APPI = Aatmospheric pressure photoionization
126 Current Pharmaceutical Analysis, 2010, Vol. 6, No. 2 Zhang et al.

AVI = Avidin high-performance liquid chromatography with optical rotation and

mass spectrometric detection. J. Chromatogr. A, 2000, 878, 35-43.
AGP =
1-acid glycoprotein [11] Roussel, C.; Rio, A.D.; Pierrot-Sanders, J.; Piras, P.; Vanthuyne, N.
Chiral liquid chromatography contribution to the determination of
BSA = Bovine serum albumin the absolute configuration of enantiomers. J. Chromatogr. A, 2004,
1037, 311-328.
CBH I = Cellobiohydrolase I [12] Borovkov, V.V.; Muranaka, A.; Hembury, G.A.; Origane, Y.;
CD = Circular dichroism Ponomarev, G.V.; Kobayashi, N.; Inoue, Y. Chiral bis-chlorin:
enantiomer resolution and absolute configuration determination.
CD = Cyclodextrin Org. Lett., 2005, 7(6), 1015-1018.
[13] Stephens, P. J.; Devlin, F. J. Determination of the absolute con-
CDR = Chiral derivatization reagents figuration of a chiral oxadiazol-3-one calcium channel blocker, re-
solved using chiral chromatography, via concerted density func-
CD-TLM = Circular-dichroism thermal lens micro- tional theory calculations of its vibrational circular dichroism, elec-
scope tronic circular dichroism, and optical rotation. J. Org. Chem., 2007,
72, 4707-4715.
CMPA = Chiral mobile phase additives [14] Lee, J.; Huang, B.X.; Yuan, Z.; Kim, H.Y. Simultaneous determi-
CSPs = Chiral stationary phases nation of salsolinol enantiomers and dopamine in human plasma
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HAS = Human serum albumin liquid chromatography/electrospray ionization-tandem mass spec-
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HPLC = High performance liquid chromatogra- [15] Mcconnell, O.; He, Y.; Nogle, L.; Sarkahian, A. Application of
phy chiral technology in a pharmaceutical company. enantiomeric
separation and spectroscopic studies of key asymmetric intermedi-
ILs = Ionic liquids ates using a combination of techniques. phenylglycidols. Chirality,
2007, 19, 716-730.
MIP = Molecularly imprinted polymers [16] Bui, T.T.T.; Coles, S.J.; Davies, D.B.; Drake, A.F.; Eaton, R.J.;
Hursthouse, M.B.; Kilic, A.; Shaw, R.A.; Yesilot, S. Chiral separa-
MS = Mass spectra tion and cd characterisation of enantiomeric cyclotriphosphazene
OMCHI = Ovomucoid from chicken egg whites derivatives. Chirality, 2005, 17, 438-443.
[17] Villan, C.; Laleu, B.; Mobian, P.; Lacour, J. Effective HPLC reso-
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Accepted: 03 March, 2010