Trends in Analytical Chemistry, Vol.

37, 2012 Trends

Recent advances in pharmaceutical

separations with supercritical fluid
chromatography using chiral
stationary phases
Wang Ren-Qi, Ong Teng-Teng, Tang Weihua, Ng Siu-Choon

Supercritical fluid chromatography (SFC) has developed rapidly in recent years, particularly in the area of enantioseparations.
The merits of SFC (e.g., fast analysis speed, wide polarity compatibility, lower cost of the mobile phase and higher column
efficiency) would in many cases make it a better choice than high-performance liquid chromatography in drug discovery for the
pharmaceutical industry.
Packed-column SFC meets the gradual rise in demand for simultaneous chiral and achiral separations, impurity quantitation
and direct scaling up from the pharmaceutical purifications achieved in the laboratory, especially for chiral drugs.
This review focuses mainly on the latest examples of pharmaceutical separations with chiral stationary phases in SFC for
efficient analyses and preparative-scale purifications.
ª 2012 Elsevier Ltd. All rights reserved.

Keywords: Achiral separation; Chiral separation; Chiral stationary phase (CSP); Drug discovery; Enantioseparation; Packed column; Preparative
chromatography; Screening strategy; Supercritical fluid chromatography (SFC); Tandem column

1. Introduction SFC in solving analytical problems in

Wang Ren-Qi, Ong Teng-Teng,
Ng Siu-Choon*
Division of Chemical and
Biomedical Engineering,
School of Chemical and
Biomedical Engineering,
Nanyang Technological
University,
62 Nanyang Drive,
Singapore 637459,
Singapore
Tang Weihua*
Key Laboratory of Soft
Chemistry and Functional
Materials (Ministry of Education
of China), Nanjing University of
Science and Technology,
Nanjing 210094,
PeopleÕs Republic of China
*
Corresponding authors.
Tel./Fax: +86 25 84317311
(T. Weihua),
tel.: +65 67904067;
fax: +65 67947533
(N. Siu-Choon).
E-mail: whtang@mail.njust.
edu.cn, ngsc@ntu.edu.sg
Supercritical fluid chromatography (SFC)
has developed rapidly since the 1980s,
often replacing conventional liquid-based
chromatography in both analytical scale
separations in drug discovery and pre-
parative-scale purification. The evolution
and the current developments in SFC
instrumentation were summarized in a
recently published review [1].
In recent developments, SFC is easily
coupled with mass spectrometry (MS) for
high-throughput analysis, purity assess-
ment, structure characterization, impurity
profiling and purification. The ion-source
fragmentation patterns of the separated
analytes in the MS spectra also reveal their
structural features [2].
With many SFC applications in phar-
maceutical analyses, a library of applica-
ble chiral stationary phases (CSPs) and
successful chiral resolutions of pharma-
ceutical compounds were summarized in
2008 [3]. Meanwhile, the importance of
clinical and pharmaceutical fields kept on
attracting more interest [4]. The funda-
mental challenges and opportunities for
this ‘‘green technology’’ in future devel-
opment was recently reviewed by Guio-
chon and Tarafder based on more than
500 relevant reports in the literature [5],
highlighting the state-of-the-art technique
in detail and offering readers a platform for
better understanding of SFC.
The intense research in pharmaceutical
industry makes necessary systematic
development of the strategy of SFC for
simultaneous chiral and achiral separa-
tions, impurity quantification and scaling
up from analyses of small amounts
towards preparative-scale applications.
Also, good progress in the SFC technique
requires the upgrading of both instru-
mentation and software for the industrial
applications. We therefore felt that an up-
to-date review on SFC chiral separations
would be beneficial to promote its appli-
cations in both analytical and preparative

0165-9936/$ - see front matter ª 2012 Elsevier Ltd. All rights reserved. doi:http://dx.doi.org/10.1016/j.trac.2012.02.012 83
Trends
scales. We comprehensively summarize the research
results obtained in implementing CSPs with packed-col-
umn SFC for the analysis of drugs and drug preparations.
Moreover, we provide a complete image of current
activities from the viewpoint of analytical chemists in the
field of drug development and discovery.
The rapid development of the pharmaceutical industry
demands highly efficient enrichment and purification of
active pharmaceutical ingredients and control of
impurities. Besides the successful enantioseparations of
non-polar or volatile racemates, a broad range of phar-
maceutical compounds with high polarity or thermal
instability could also be analyzed under SFC conditions
[6]. A variety of HPLC columns prepared with superfi-
cially porous particles can also be employed in SFC with
high column efficiency. Also, the mobile phase in SFC
has low viscosity, so it results in much lower pressure
drop over the column than in LC conditions. The use of
small particles (1.8 lm, 2.6 lm or 3 lm) as column
packings in SFC provides high efficiency and short
analysis time – just as they perform in ultra-high per-
formance LC but with significantly lower pressure drop
over the column [7,8].
Differing from non-polar organic solvents in normal-
phase liquid chromatography (NPLC), supercritical fluid
CO2 is miscible with both non-polar and polar organic
solvents. Furthermore, the small CO2 molecule occupy-
ing the hydrophobic cavities of chiral selectors is readily
replaced by the analytes [9]. The adsorption and the
inclusion of analytes in the cavities of chiral selectors
results in enhanced separations in many cases. However,
non-polar organic solvents usually occupy the cavities of
chiral selectors and are difficult to replace. As a conse-
quence, the separation mechanism in SFC usually differs
from NPLC with the same chiral selector [9]. Although
HPLC has been established as the main tool for the drug
development and purification, orthogonal selectivities
and some distinct advantages in preparative chroma-
tography make SFC the perfect complement to NPLC,
reversed-phase LC (RPLC) and hydrophilic interaction LC
(HILIC) in analyses and purification [10].
Similar to the screening strategy in HPLC, selectivity
in SFC can be tuned by ‘‘coarse’’ adjustments by
changing the stationary phase and ‘‘fine’’ monitoring
the mobile phases as well as setting the instrument
parameters appropriately. The solvation strength of non-
polar CO2 can be enhanced by the addition of polar or-
ganic modifiers. During the fast screening of feasible
chromatographic conditions, organic modifiers (e.g.,
methanol, ethanol and 2-propanol) are added into the
supercritical CO2 (sCO2) using an increasing gradient or
an isocratic flow approach [11]. The influence of mobile
phase on selectivity has been investigated extensively.
Acidic or basic additives (e.g., acetic acid and triethyl-
amine) are usually employed to suppress the ionization
of the analytes in the mobile phase. The additives adjust
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Trends in Analytical Chemistry, Vol. 37, 2012
the retention and the resolution of the analytes by
absorbing onto the stationary phase. Peak tailing in SFC
could also be suppressed with additives.
The density of the mobile phase correlates with the
polarity and the solvation strength of the mobile phase
influencing the selectivity of the separation. As such,
appropriate setting of the back-pressure regulator (BPR)
should be considered to optimize selectivity in SFC. It was
observed that the pressure drop over the column was not
changed with increased back-column pressure, whereas
both the pressures of mobile phase at inlet and outlet were
increased proportionally. As increased mobile phase
density might elongate or shorten the retention times of
various compounds, the selectivity could thus be modified.
Brunelli et al. demonstrated that the back pressure
exerted effect only on non-polar compounds with low
retention [11]. The non-polar analyte was eluted out
faster with the mobile phase under higher pressures but
the retention of polar analytes was rarely influenced by
the back pressure. Furthermore, the compressibility of
mobile phase decreased with the increasing ratio of the
organic modifier. Therein, the influence of pressure was
less important [11].
The fluctuation of the pressure induced by the com-
pression stokes of the CO2 pump could not be avoided in
current SFC machines; it directly correlates with the
changes in mobile-phase refraction index (RI) and results
in UV baseline noise. The pressure fluctuations and the
baseline noise could be suppressed by dual pump systems
usually stroking at different frequencies, but they could
not be eliminated if the baseline noise and sample peak
had similar frequencies (widths) [12]. The old SFC BPRs
afford a UV baseline offset of minimum 0.5 mAU, which
could not meet the requirements of a baseline noise
lower than 0.05 mAU for trace analyses.
High back pressure and/or a high content of organic
modifier in the mobile phase were recommended for
lower UV baseline noises and larger peak areas. Re-
cently, a new BPR for the Aurora SFC Fusion A5 system
was developed with an electronic filter to provide a peak-
to-peak noise lower than 0.02 mAU [13]. Moreover, the
limit of quantitation (LOQ) of impurities could be lowered
due to the higher peak efficiency in SFC [14].
Compared with the conventional HPLC protocol,
equilibration in SFC is very fast [5]. Accordingly, the
parameters (e.g., mobile-phase composition, pressure
and temperature) can be programmed swiftly in the
condition-optimization processes. After operations under
various conditions, the return to initial conditions is also
feasible and easy. In SFC analyses, traces of water in
sample solutions can also be used to substitute trifluo-
roacetic acid (TFA) as acidic modifier [1]. Also, aqueous
samples can be analyzed by packed-column SFC, which
is a good alternative to NPLC [15]. However, the
orthogonal selectivity offered by SFC is complementary
to RPLC in analyses of the impurities in raw pharma-
Trends in Analytical Chemistry, Vol. 37, 2012 Trends

Figure 1. (a) The orthogonal selectivity of SFC method vs. RP-HPLC method. SFC conditions: 100 bar, 30C, 4.0 mL/min, modifier: methanol
5–15% in 15 min, silica column. RP-HPLC conditions: 25C, 1.5 mL/min, water/acetonitrile (58:42, v/v) to water/acetonitrile (48:52, v/v) in
60 min, Ultrasphere ODS column. (b) Comparison of retention factor (kÕ) in SFC method and in RP-HPLC method [16].

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Trends
ceutical materials. As illustrated in Fig. 1, selectivity
attained on a silica column in SFC conditions is
orthogonal to those on an ODS column in RPLC condi-
tions [16]. The reversal of the elution order is important
for quantitative analyses and profiling of the minor
component, because it is usually desirable to elute the
trace-amount impurities before the major peak.
The low cost of organic modifiers in SFC makes it
attractive for preparative chromatography. There are
normally two approaches for direct scaling up from the
analytical chromatography protocol towards preparative
chromatography:
(1) first, a bench-scale preparative fractionation is
developed with micro-preparative SFC. The fast
operation of SFC affords much shorter run times
than HPLC. As a result, stacking injections of small
amount samples on single analytical column can
attain substantial throughput of purifications; and,
(2) second, larger-dimension pre-packed columns are
packed with larger amounts of CSP to improve their
productivity, since the productivity of CSP columns
is proportional to the amount of stationary phases
employed. The peak collection is monitored with a
detector (usually UV-vis).
In both operations, the collected fractions undergo
gentle decompression of CO2 to gaseous phase under the
control of electric BPR to avoid aerosol formation. A thin
layer of modifier is pushed along the capillary wall to-
wards a fraction-selective valve and the eluted com-
pounds are retained dissolved in the modifier liquid phase
[1]. The product recovered from SFC is more concen-
trated than that from HPLC and the amount of solvent
that must be evaporated is substantially reduced [17].
2. Chiral stationary phases
There are normally two modalities for chiral selector
applications in chromatography: chiral mobile-phase
additive (CMPA) and CSP.
Although CMPAs were used for SFC enantiosepara-
tions in some cases, the low solubility of chiral selectors
in SFC mobile phase restricted their applications [18]. It
is also undesirable to have chiral selectors together with
the eluted analytes, especially in drug purifications. The
employment of CSPs is therefore the first choice for SFC
enantioseparations. Almost all commercially-available
CSPs developed for LC and some newly developed CSPs
have been successfully applied in SFC chiral analytical
and preparative-scale separations.
Amongst the commercially available CSPs, cellulose-
based or amylose-based Chiralcel and Chiralpak series
developed by Chiral Technologies (Daicel) are widely used
in SFC [19]. The polysaccharide CSPs by Phenomenex
(Lux series) were also commonly used in SFC research in
recent years [8,20]. These CSPs comprise a polysaccha-
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Trends in Analytical Chemistry, Vol. 37, 2012
ride backbone, most probably adopting a helical chain.
The cavity of cellulose or amylose offers enhanced
enantioselective retention, depending on the molecular
shape of the analytes [21]. The structures of the back-
bone and the substituents of the commonly used Chiral-
cel and Chiralpak series CSPs are presented in Table 1.
In applications under the NPLC conditions, it is ob-
served that the conformation of amylose-based station-
ary phases change at approximately 30C, and is
irreversible if ethanol is applied as the organic modifier
but reversible if 2-propanol is used [22]. However, cel-
lulose-based CSPs are much more stable at working
temperatures (5-45C) [22]. Besides polysaccharide-
based CSPs, cyclodextrin-based and macrocyclic glyco-
peptide-based CSPs, together with Pirkle or brush-type
CSPs, have also been applied in SFC, and some of them
have been commercially available for a decade [19].
Protein-based columns have not been reported in SFC
applications, since these CSPs require aqueous-based
conditions (in RPLC) and their low loadability limits their
application in preparative chromatography.
The enantioseparations achieved on Chiralcel and
Chiralpak columns were compared with 500 proprietary
chiral compounds generated in drug-discovery programs
[23]. Fast, very efficient chiral SFC allows screening
throughout various conditions amongst the four most
commonly used CSPs. The enantioseparation success
rating is as follows: Chiralpak AD (60%) > Chiralcel OD
(31%) > Chiralcel OJ (8%) > Chiralpak AS (2%) [19]. In
total, 95% of the 500 proprietary racemic compounds
and 98% of 40 marketed drugs have attained resolutions
on these four CSPs in SFC. However, a different success
rate ranking was reported as Chiralpak AD > Chiralpak
AS > Chiralcel OJ > Chiralcel OD based on another li-
brary of racemic analytes [20].
It is obvious that the enantioselectivities of polysac-
charide derivatives are greatly influenced by the position
and the type of the substituent phenylcarbamate groups.
It should be noted that, in some cases, the enantiose-
lective Chiralcel OD columns are not useful for chiral
analyses of carboxylic acids (e.g., the ‘‘profen’’ series)
because they always show asymmetric peaks, interfering
with those of related compounds or impurities in the
drug when no acid additives are applied [24].
Although the most widely-employed CSPs are 3,5-
dimethylphenylcarbamoylated amylose and cellulose, it
is recognized that adding a chlorine atom to the phenyl
moieties has a positive effect on the chiral separation of
some analytes. For example, Lux Amylose-2 and Cellu-
lose-2 are able to provide good enantioseparations due to
the electron-withdrawing chlorine on the phenylcarba-
mate substituents. Therein, the acid strength of the NH
group is increased, enhancing the interactions between
analytes and the NH group, and, in many cases, this re-
sults in better enantioseparation [20]. In an attempt
to enantioseparate a synthetic ophthalmic drug
Trends in Analytical Chemistry, Vol. 37, 2012 Trends

Table 1. Commercially available polysaccharide-based chiral stationary phases

Brand (Supplier) Chiral selector backbone Substituent structure (R) Nature of CSP
Daicel series (Chiral Technology)
Chiralpak AD Physical coating
OR O CH3
C
O N
O H
n
CH3
RO
O cellulose

O CH3
C
Chiralpak IA N Chemical bonding
H
CH3
O CH3
C
Chiralpak AS N Chemical bonding
H
CH3

OR
O CH3
O C
Chiralcel OD O N Physical coating
n H
RO
OR CH3
cellulose

Chiralcel OJ CH3 Physical coating

O

Chiralcel IB CH3 Chemical bonding

O

O Cl
C
Chiralcel IC N Chemical bonding
H
Cl
Lux series (Phenomenex)

OR
O CH3
O C
Lux Cellulose-1 O N Physical coating
n H
RO CH3
OR cellulose

O Cl
C
Lux Cellulose-2 N CH3
Physical coating
H

O Cl
C
Lux Cellulose-3 N CH3
Physical coating
H

O CH3
C
Lux Cellulose-4 N Cl
Physical coating
H
OR O H3C

O C
Lux Amylose-2 O N Physical coating
n F
RO
OR amylose Cl

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Table 2. Enantioseparation of chiral drugs with commercial chiral stationary phase (CSPs) in SFC

Chiral drug Structures Separation conditions Comments Ref.

Chiral sulfoxide (asymmetric sulfoxide function)
Ricobendazole O
CSP: Chiralpak AD; Rs = 11.4 [35]
H O Column temperature: 35C
S N
H3C C CH3
N O Flow rate: 2 mL/min,
H
N BPR: 20 MPa
Mobile phase: 30% MeOH in sCO2
Oxfendazole O
Rs = 13.9 [35]
H O
S N
C CH3
N O
H
N

Omeprazole H
Rs = 9.9 [35]
H3CO H2
N
C N
S
N O
H3C CH3
OCH3

Pantoprazole H H2
Rs = 2.2 [35]
HF2CO N
C N
S
N O
H3CO
OCH3

Lansoprazole H H2
CSP: Chiralpak AD; Rs = 3.4 [35]
N
S
C N Column temperature: 35C
N O Flow rate: 2 mL/min
H3C
OCH2CF3
BPR: 20 MPa
Mobile phase: 20% MeOH in sCO2

Rabeprazole H H2
Rs = 3.3 [35]
N
C N
S
N O
H 3C
O
CH3
O

Non-steroidal anti-inflammatory drugs (NSAIDs)

Flurbiprofen CH3
CSP: Chiralpak AD; a = 1.49 [46]
OH Column temperature: 35C
O
Flow rate: 4 mL/min
BPR: 10 MPa
F
Mobile phase: Gradient Flow
(start point 7% MeOH, held for 2 min,
Ibuprofen MeOH was increased to 50% at a rate of 7%/min) a = 1.21 [46]
CH3
OH
CH3

H3C

Ketoprofen O CH3
a = 1.04 [46]
OH

Naproxen CSP: Chiralpak AD; a = 1.19 [64]

CH3
Column temperature: 30C
COOH Flow rate: 1.5 mL/min
BPR: 15 MPa
H3CO
Mobile phase: 15% MeOH in sCO2

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Table 2. (continued)

Chiral drug Structures Separation conditions Comments Ref.

a
Fenoprofen CH3
CSP: Chiralpak AD; 2.30 min, [23]
O OH
Column temperature: 30C Rs = 1.45
Flow rate: 3 mL/min
O BPR: 20 MPa,
Mobile Phase:
a, 20% 2-propanol (0.1% IPAm)
Suprofenb O CH3
8.03 min, [23]
b, 10% MeOH (0.1% IPAm) in sCO2
OH
Rs = 4.24

S O

Fungicides
Ketoconazolec CSP: Chiralpak AD; a = 1.53 [48]
N N Column temperature: 35C, Rs = 5.92
O O O
Cl
Flow rate: 2 mL/min
N N
O CH3 BPR: 20 MPa,
Mobile phase:
c, 30% EtOH
Miconazoled Cl d & e, 15% MeOH 8 min, [49]
f, 20% MeOH Rs = 3.6
g, 40% EtOH/2-Propanol 15:85 (v/v) in sCO2
Cl

O N
N
Cl Cl

Econazolee Cl
8 min, [49]
Rs = 3.4

Cl

O N
N
Cl

Sulconazolef Cl
10 min, [49]
Rs = 2.8

Cl

S N
N
Cl

Itraconazoleg 80 min, [49]

N N H3C Rs = 1.2,1.4,2.4
O
O CH3
O
N
Cl N N N
N
O

b-blockers OH H
O N CH3
Ar *

CH3
Ar =

Propranolol CSP: Chiralpak AD; k1 = 1.88 [50]

Column temperature: 40C k2 2.39
Flow rate: 2.5 mL/min a = 1.57
BPR: 10 MPa Rs = 7.50
Mobile phase: 30% EtOH (0.1% DEA) in sCO2
(continued on next page)

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Table 2. (continued)

Chiral drug Structures Separation conditions Comments Ref.

Pindolol H
CSP: Chiralcel OD; k1 = 2.12 [50]
N Column temperature: 40C k2 = 3.95
Flow rate: 2.5 mL/min a = 2.64
BPR: 10 MPa Rs = 16.41
Mobile phase: 40% MeOH (0.1% DEA) in sCO2

Acebutololh O H
CSP: Chiralcel OD; 2.94 min [23]
N CH3 Column temperature: 30C Rs = 1.48
H3C
Flow rate: 3 mL/min
O
BPR: 20 MPa,
Mobile phase:
Alprenololi h, 5% MeOH (0.1% IPAm) 4.53 min [23]
i & j, 10% MeOH (0.1% IPAm) Rs = 4.27
k, 20% MeOH (0.1% IPAm) in sCO2

Metoprololj O
9.40 min [23]
CH3 Rs = 10.23

Oxprenololk O
 3.30 min [23]
Rs = 5.94

Sympathomimetic amines
Ephedrine OH
CSP: Chiralpak AS; 5.61 min [23]
CH3
Column temperature: 30C Rs = 1.37
Flow rate: 3 mL/min
NH BPR:20 MPa
H3C
Mobile phase: 15% EtOH (0.1% IPAm) in sCO2

Epinephrine OH
CSP: Chiralpak AD; 7.99 min [23]
H Column temperature: 30C Rs = 1.58
N
CH3 Flow rate: 3 mL/min
HO
BPR: 20 MPa
OH
Mobile phase:
10% MeOH (0.1% IPAm) in sCO2

Clenbuterol OH
4.66 min [23]
H CH3 Rs = 3.59
Cl N CH3

CH3
H2N
Cl

Norephedrine OH
CSP: Chiralpak AD; k1 = 2.69 [50]
CH3
Column temperature: 40C k2 = 3.28
Flow rate: 2.5 mL/min a = 1.35
NH2 BPR: 10 MPa Rs = 4.09
Mobile phase: 20% 2-Propanol in sCO2
Diuretics
Bendroflumethiazide O O
CSP: Cationic b-cyclodextrin CSP; a = 1.84 [31]
O O
S S Column temperature: 40C Rs = 3.51
H2N NH Flow rate: 1 mL/min
F3C N BPR: 15 MPa
H Mobile phase: 10% MeOH in sCO2
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Table 2. (continued)

Chiral drug Structures Separation conditions Comments Ref.

Trichlormethiazide a = 1.71 [31]
O O O O
S S
Rs = 3.94
H2N NH

Cl N CHCl 2
H

Althiazide O O
a = 1.50 [31]
O O
S S Rs = 2.72
H2N NH
S
Cl N CH2
H

Indapamide O
a = 1.19 [31]
O O
S
Rs = 1.87
H2N NH
N
Cl
H3C

Chlorthalidone O
a = 1.29 [31]
O HO
S Rs = 2.44
H2N
HN
Cl
O

Antihistamines
1-(Chlorobenzhydryl)-piperazine Cl
CSP: Chiralpak AD; k1 = 2.46 [50]
HN Column temperature: 40C k2 = 3.23
N Flow rate: 2.5 mL/min a = 1.53
BPR: 10 MPa Rs = 7.93
Mobile phase: 40% 2-Propanol in sCO2

Promethazine S
CSP: Chiralpak AD; 2.88 min [23]
Column temperature: 30C Rs = 1.50
N CH3
Flow rate: 3 mL/min
H3C O
BPR: 20 MPa
Mobile phase: 20% 2-Propanol (0.1% IPAm) in sCO2
N
H3C CH3

Propiomazine S
CSP: Chiralcel OJ; 2.39 min [23]
Column temperature: 30C Rs = 1.49
N
Flow rate: 3 mL/min
H3C
BPR: 20 MPa
Mobile phase: 20% 2-Propanol (0.1% IPAm) in sCO2
N
H3C CH3

Maxi-K opening compound

R1
N O

R2
F 3C O
OH

Cl R3

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Table 2. (continued)

Chiral drug Structures Separation conditions Comments Ref.

1 2 3
3-Substitued-4- Compound 1: R = –H; R = –H; R = –H CSP: Chiralpak AD; tR1 = 6.20 min [51]
arylquinolinon Column temperature: 35C tR2 = 7.37 min
derivatives Flow rate: 2 mL/min a = 1.31
BPR: 15 MPa Rs = 2.93
Compound 2: R1 = –CH3; R2 = –H; R3 = –H Mobile phase: tR1 = 5.39 min [51]
10% MeOH in sCO2 tR2 = 7.40 min
a = 1.55
Rs = 3.67
Compound 3: R1 = –CN; R2 = –H; R3 = –H tR1 = 5.42 min [51]
tR2 = 7.22 min
a = 1.63
Rs = 5.00
Compound 4: R1 = –CONH2; R2 = –H; R3 = –H tR1 = 8.02 min [51]
tR2 = 9.79 min
a = 1.31
Rs = 3.54
Compound 5: R1 tR1 = 6.51 min [51]
= –CH3; R2 = –H; R3 = –CH2CHCH2 tR2 = 8.62 min
a = 1.51
Rs = 3.52
Compound 6: R1 = –CH3 tR1 = 5.50 min [51]
tR2 = 6.69 min
a = 1.39
CH3 Rs = 2.67
Si CH3
CH3

R2 = ; R3 = − CH2CHCH2

Amino acids NH2

Alaninel H3C C COOH CSP: Chirobiotic T (teicoplanin); t1 = 2.60 min [26]
H
a = 1.94
NH2 Rs = 3.1
Serinem HOH2C C COOH Column temperature: 31C t1 = 3.20 min [26]
H
Flow rate: 4 mL/min a = 1.40
O NH2 BPR: 10 MPa, Rs = 1.8
H2
Asparaginen H2N C C C COOH Mobile phase: t1 = 4.02 min [26]
H
l & v 48% MeOH, 0.1% TEA, a = 1.54
O NH2 TFA, 2% H2O, 0.3% glol Rs = 1.6
Glutamineo H2N C (CH2)2 C COOH m, 46.5% MeOH, 0.1% TEA, t1 = 4.06 min [26]
H
TFA, 3.5% H2O a = 1.36
NH2 n, o & u, 57.6% MeOH, Rs = 1.8
Methioninep H3C S (CH2)2 C COOH 0.15% TEA, t1 = 2.55 min [26]
H
TFA, 2.4% H2O a = 2.14
HN NH2
H2 p, q, r, s & v, 48% MeOH, Rs = 3.00
q C C COOH
Tryptophan H 0.1% TEA, t1 = 3.61 min [26]
TFA, 2% H2O, 0.3% glol a = 1.71
NH2 t, 67.2% MeOH, 0.15% TEA, Rs = 1.8
H2
Phenylalaniner C C COOH TFA, 2.8% H2O in sCO2 t1 = 2.72 min [26]
H
a = 1.89
OH NH2 Rs = 2.2
Threonines H3C C C COOH t1 = 2.97 min [26]
H H a = 1.52
NH2 Rs = 2.0
Lysinet H2N (CH2)4 C COOH t1 = 1.85 min [26]
H
a = 1.60
NH NH2 Rs = 2.7
Arginineu H t1 = 4.71 mina = 1.80Rs = 3.4 [26]
H 2N C N NH22)3 C COOH
(CH
H
Norvalinev H3C (CH2)3 C COOH t1 = 4.04 mina = 1.50Rs = 1.8 [26]
H
(continued on next page)

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Trends in Analytical Chemistry, Vol. 37, 2012
Table 2. (continued)
Chiral drug Structures
Other drugs
Verapamil H3C CH3
CH3
N N
CH3
O
O
H3C O O CH3
H3C
Bupivacaine
CH3
H Column temperature: 30C
N
N Flow rate: 3 mL/min
O BPR: 20 MPa
CH3
Warfarin O
OH CH3
O O
*IPAm, Isopropylamine; TEA, Triethylamine; TFA, Trifluoroacetic acid; glol, Glycerol.
{(4S-trans)-4-(N-acetamide)-5,6-dihydro-(6S)-methyl-4H-
thieno-[2,3-b]thiopyran-7,7-dioxide}, the selectivity (a)
of 1.42 and the resolution (Rs) of 6.47 were achieved on
Lux Cellulose-2 while only a 1.24 and Rs 2.73 were
afforded by Chiralcel OD-H under the same conditions
[20]. In another comparison, 3,5-dimethylphenyl
carbamates of cellulose exhibited much better enantio-
separations in SFC than with cellulose fully derivatized
with phenylcarbamate groups [25]. The two additional
methyl groups modified the secondary structure of the
polysaccharide so as to induce the favorable deformation
of the chiral cavities. The methyl groups also increased
the repulsion with the p-donor moiety in the analyte,
thus modifying the chiral selectivity.
In further developments of CSPs, the macrocyclic
glycopeptide-based CSPs (Chirobiotic series) demon-
strated versatile enantioseparation abilities towards a set
of 111 chiral compounds, which included heterocycles,
analgesics (NSAIDs), b-blockers and sulfoxides [26].
However, insight into the enantioseparation mechanism
was explored based on brush-type CSPs and cyclodex-
trin-based CSPs on account of their simpler interaction
modes. The principles derived were found applicable in
the design of molecular-imprinted polymers for enan-
tioseparations of specific families of analytes [27,28].
It is inspiring that, in LC conditions, changing the
configuration of the CSP [e.g. (R,R)-Whelk-O1 to (S,S)-
Trends
Separation conditions Comments Ref.
CSP: Chiralpak AD; 2.56 min [23]
Column temperature: 30C Rs = 1.69
Flow rate: 3 mL/min
BPR: 20 MPa
Mobile phase: 20% 2-Propanol (0.1% IPAm) in sCO2
CSP: Regis L-Leucine; Rs > 1.50 [52]
Mobile phase: 20% MeOH (0.1% IPAm) in sCO2
CSP: Regis (S,S)-DACH; Rs > 1.50 [52
Column temperature: 30C,
Flow rate: 3 mL/min
Mobile phase: 20 MPa, 20% MeOH (0.1% IPAm) in sCO2
Whelk-O1] results in the reversal of elution order, which
can be important for enantiomeric excess determination
[29]. Recently, Pirkle et al. [30] developed a new brush-
type CSP (polyWhelk-O) based on polysiloxane, which
afforded stronger enantioseparation than the former
Whelk-O series, but with lower retention. Such a CSP
was also found applicable for a wide range of tempera-
ture, mobile-phase composition and acidic/basic addi-
tives under SFC conditions. However, CSPs based on
novel structured cyclodextrins afforded stronger enan-
tioseparation capabilities and wider versatility than the
columns already commercialized (Cyclobond) [31,32].
Such novel CSPs may have great potential to be em-
ployed in a fast screening strategy for SFC separations.
3. Chromatographic conditions of chiral SFC
3.1. Organic modifiers
White et al. [33] developed a gradient screening strategy
for SFC system to choose the most suitable CSP and
optimize the mobile phase. The most frequently used
organic modifiers in SFC are methanol, ethanol and 2-
propanol, while acetonitrile, 1-propanol and some other
organic solvents are also employed in some cases to
optimize enantioselectivity. The less polar alcohols em-
ployed as mobile-phase modifiers play a role in the
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Trends
solvation of the aliphatic moieties of the analytes.
However, the polar organic solvents compete with the
solute for hydrogen-bonding donating or acceptor sites
on the stationary phases.
In the enantioseparation of (4S-trans)-4-(N-acetam-
ide)-5,6-dihydro-(6S)-methyl-4H-thieno-[2,3-b]thiopy-
ran-7,7-dioxide, the fast screening strategy was
performed with three organic modifiers using four
commercial CSPs (Chiralcel OD-H, Chiralpak AD, Lux
Cellulose-2 and Lux Amylose-2) with isocratic flow (15
vol% organic modifier in CO2). 2-Propanol offered better
enantioseparations than methanol, but higher plate
numbers were normally generated with methanol due to
the lower viscosity [20]. In another case, the mobile
phase with ethanol afforded enantioseparation of race-
mic ephedrine, while no enantioselectivity was found
using other mobile phases modified with alcohols of
higher (methanol) or lower (2-propanol) polarity [23].
The alcohols with appropriate hydrophobicity can adjust
the accessibility of the chiral cavities in polysaccharide
CSPs and result in enhanced enantioseparations [34].
Furthermore, the reversal elution order of omeprazole
enantiomers was achieved by changing the mobile phase
from ethanol to 2-propanol due to the different stability
of the enantiomer-CSP adsorption complexes in various
mobile phases [35]. However, it is worth noting that,
under LC conditions, the elution order of the enantio-
mers could be changed by changing pH, ionic strength
and organic modifier composition [36].
Although methanol does not necessarily impart the
highest enantioselectivity, it is always the preferred or-
ganic modifier in SFC. The highly efficient separations
with methanol are favorable for easy scaling up to pre-
parative separations, mainly due to its low viscosity, low
boiling point and high polarity [23]. In the modern SFC-
ESI-MS system, methanol is superior to other alcohols
due to its low surface tension, which afforded good
ionization efficiency and sensitivity in MS detection [37].
Also, it is worth noting that the addition of some non-
alcoholic organic modifiers {e.g., acetonitrile [23] or
chloroform [38]} to the mobile phase could significantly
accentuate the chiral recognition abilities of the CSPs.
3.2. Additives
In SFC, the influence of additives on enantioseparations of
analytes depends on their functionality and charges.
Highly charged chiral compounds are affected more sig-
nificantly [39]. The suppression of the ionization of ana-
lytes in the mobile phase by the additive could be the main
reason for this. However, the additives interact with the
strong adsorption sites on the stationary phase and reduce
the retention of the analytes, which also have functional
groups for competitive interactions [39]. Many chiral
pharmaceutical compounds possess one or more sites for
hydrogen-bonding or donor/acceptor functional groups to
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Trends in Analytical Chemistry, Vol. 37, 2012
interact with the stationary phase for the ultimate enan-
tioseparations. The additives can competitively adsorb onto
the stationary phase and alter the enantioselectivity by
adjusting the hydrogen bonding, or electrostatic or Cou-
lombic forces between the analytes and the CSP [31].
The effectiveness of additives could also be associated
with steric considerations [37]. For example, hindered
amines (e.g., triethylamine) are much less effective than
primary amines(e.g., isopropylamine), although they
have stronger basicity. It was considered that they were
much easier to elute than the primary amine. In
particular, Stringham et al. [40] demonstrated the
employment of a strong acidic additive, ethylsulfonic
acid (ESA), to enhance the chiral resolution of basic
racemates on Chiralpak AD in SFC. It is contrary to the
‘‘rule of thumb’’ usually recommending basic additives
for basic analytes. The acid additive forms ion pairs with
the basic analytes that could result in a different selec-
tivity when the ion-pair complex is interacting with the
stationary phase. Most recently, ammonium hydroxide
was successfully applied as basic additive for chiral sep-
arations on Chiralcel OJ and Lux Cellulose-1 columns in
SFC [41]. Such a new basic additive exhibited similar
properties with diethylamine for increasing the chiral
resolution of basic analytes. However, the easier removal
of ammonium hydroxide made it a promising replace-
ment for traditional amine additives, especially in
preparative chromatography.
3.3. Pressure and temperature
SFC parameters (e.g., backpressure and temperature)
also determine the separations. It is well known that the
stable conformation of polymer-based CSPs is always
important for successful enantioseparations. The influ-
ence of pressure on the selectivity of cellulose or amylose
CSPs was investigated in the range 80–220 bar [34].
The high pressure of the mobile phase could cause
deformation of the chiral selectors and their higher or-
dered structures. As a result, their enantioseparation
abilities could be substantially weakened. Such an effect
was observed on Chiralpak AD, but not on Chiralcel OD,
which might suggest that the skeleton of cellulose de-
rived CSP is more rigid.
The influence of column temperature on enantiosep-
arations in SFC can be evaluated by VanÕt Hoff plots
[42], wherein the selectivity is a compromise between
differences in binding enthalpy and disruptive entropic
effects. Enantioseparations with the same mechanism
afford linear plots, while non-linear plots usually suggest
a changed separation mechanism due to the changes in
the temperature [20]. Furthermore, extension of the
theory could predict the reversed elution order of the
enantiomers with the temperature beyond a point where
DDG is equal to zero and the two enantiomers co-elute
[Equation (2)] [43].
 Trends in Analytical Chemistry, Vol. 37, 2012
Figure 2. (a) Analytical chromatograms of mixture A with Chiralcel OD column. (b) Analytical chromatograms of mixture A with Chiralpak AD column. (c) Chromatograms of the Chiralpak AD
and Chiralcel OD column coupling for the separation of the four stereoisomers. Conditions: the mobile phase comprised 90% liquid CO2 and 10% ethanol, 2-propanol, or ethanol:2-propanol
(50:50 v/v). The temperature and outlet pressure were 40C and 120 bar, respectively. Total flow rate was 3.0 mL/min. [Peaks 1 and 4 are an enantiomeric pair (S,R and R,S), and Peaks 2 and 3
are an enantiomeric pair (S,S and R,R). Peak number indicates and correlates with the order of elution with the Chiralcel OD column.] [54].
http://www.elsevier.com/locate/trac

Trends
95
Trends Trends in Analytical Chemistry, Vol. 37, 2012

Figure 3. Preparative chiral SFC example of collecting by time windows. The flow rate was 70 mL/min [33].


DH  20–40C. An operating temperature beyond 45C is not
ln K ¼  DS þ lnu ð1Þ allowed, in order to avoid damaging the columns. Gen-
RT
  
DDG ¼ DDH  TDDS ð2Þ erally, because the working temperature range is below
   the temperature for enantiomers to co-elute, the
DDH DDS DDG
ln a ¼  þ ; ln a ¼  ð3Þ enantioselectivity increases at lowered temperature but
RT R RT the efficiency decreases [45].
However, under SFC conditions, the linear VanÕt Hoff
plots generally cannot be derived because the property of
CO2 changes sensitively with variations in temperature, 4. Enantioseparation of pharmaceutical
especially when the running temperature spans over the compounds
critical temperature of the mobile phase (a mixture of
CO2 and organic modifier). The status of CO2 in the The recent enantioseparation results achieved on vari-
mobile phase could have a strong influence on the ous CSPs in SFC conditions are summarized in Table 2
adsorption behaviors of the analytes [44]. According to [23,26,31,35,46–52]. Although the polysaccharide
the column manual, the polysaccharide (Daicel and CSPs are still most frequently employed in the direct
Phenomenex) and macrocyclic antibiotic CSPs (Astec) in screening strategy for enantioseparations of newly
SFC are usually employed with temperatures of developed drugs, some brush-type CSPs and cyclodex-

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Trends in Analytical Chemistry, Vol. 37, 2012 Trends

Table 3. Scale-up calculations for the enantioseparation of Naringenin based on the Loading Study on a Chiralpak AD-H column (4.6 · 250 mm,
5 lm) [58]

Column ID (mm) 4.6 10.0 41.0

Injected amount [g] (proportional to amount of CSP in column) 0.0012 0.0057 0.0549
Run time [min] (the minimal separation time for baseline separation) 16
Equilibration time [min] 1
Column length [mm] 250 250 144
Column volume [L] 0.004 0.020 0.19
Flow rate [L/min] 0.001 0.005 0.03
Linear velocity [m/min] 0.06
Injections per hour 3.53
Injections per shift (3 batches of workers per day) 28.2
Injections per day 84
Productivity per hour [g/h] 0.004 0.02 0.21
Productivity per shit [g/8 h] 0.03 0.16 1.55
Productivity per day [g/24 h] 0.10 0.48 4.65
Solvent consumption per hour [L/h] 0.06 0.28 1.80
Solvent consumption per shift [L/8 hour] 0.48 2.27 14.40
* Comparable physiochemical properties of CSPs in different dimensional columns were assumed.

trin-based CSPs for SFC enantioseparations of chiral
drugs are also briefly summarized.
The brush-type CSPs have larger applicable tem-
perature range and the choice of R- or S-configura-
tions of the selector, which have potential to supply
additional selectivity. The cyclodextrin CSPs also
demonstrated good stability and separation ability for
certain categories of chiral drugs. The newly developed
CSPs could also therefore be added to the SFC
screening strategy for pharmaceutical enantiosepara-
tions in the future.
5. Recent developments in chiral SFC
5.1. Tandem-column SFC
Due to the low viscosity of the mobile phases in SFC,
tandem columns were employed to generate higher plate
numbers without the issue of increased column pressure
[11]. In the same way, higher column efficiency could be
achieved by utilizing extended columns for SFC appli-
cations. The achiral impurities of a drug are not always
controlled by chiral SFC, particularly when a new syn-
thetic route is being explored. Thus, serial coupling of
columns for assaying both achiral and chiral compounds
in a single run is desirable. Accordingly, tandem
columns in SFC are useful in modifying selectivity and
increasing the plate number without breaking the pres-
sure-drop limitations.
In a process for purifying b-adrenergic blockers
(b-blockers) [53], serial coupling of the cyano and
Chiralcel OD columns increased the retention of the
racemic b-blockers and the plate numbers and afforded
both achiral and chiral separations in a single-method
operation. Wesley et al. [54] employed SFC for fast
screening with five mobile phases and two CSPs in order
to assay four stereoisomers of a chiral pharmaceutical
compound. Although single-column separation failed to
afford baseline resolution of all the four enantiomers, the
retention times for isomers going through the two
columns [Fig. 2 (c)] could be predicted by adding the
retention time of each isomer on each column with
various mobile phases [Fig. 2 (a) and (b)]. The mobile
phase of 90% CO2 and 10% ethanol/2-propanol (IsOH)
(50:50, v/v) was chosen to achieve baseline separation
of the four enantiomers using tandem columns [Fig. 2
(c)]. The retention times from the tandem columns are
close to the sum of retention times from each of the
coupled columns. In this way, the success of separations
on the tandem columns can be predicted. If one of the
columns provides significantly less retention than the
other columns, it would not offer any positive effect on
their separation. Therefore, the achiral/chiral column
combinations are usually selected so that each column
provides similar retentions towards the analytes [53,54].
In another case, a short Chiralpak AD column was
connected before another chiral column (Kromasil
KR100-5CHI-TBB) for evaluating enantiomeric impuri-
ties of a carboxylic-acid drug [55]. The short Chiralpak
AD column provided sufficient enantioselectivity while
the later TBB column was employed only to increase the
plate number (the enantioselectivity provided by TBB
was only 1.08).
5.2. Preparative chiral SFC
5.2.1. General considerations. SFC is advantageous over
the conventional LC separation method especially when
a large amount of mobile phase is needed [5]. Changing
the mobile phase from toxic organic solvents in prep-LC
conditions towards mainly supercritical CO2 in SFC not
only reduces the costs of manufacturing significantly but
also prevents pollution. Moreover, the loadability of

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Trends
Figure 4. (a) The apparatus of SFC-SMB. (b) Apparatus for determination of isotherms [59].
analytes and the productivity in SFC could be 5–10 times
greater than those in prep-LC. The samples can also be
easily loaded in aqueous solutions. This does not have
significant influence on the SFC separation behavior,
whereas the same is unaffordable in NPLC.
Although the prep-LC technique already established
has stifled application of SFC in the past, recent
employment of prep-SFC takes advantage of its merits of
high speed and low solvent costs. Stacked sample injec-
tions are usually adopted for small-scale purification.
However, it is envisaged that the application of
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Trends in Analytical Chemistry, Vol. 37, 2012
simulated moving bed-SFC (SMB-SFC) should have po-
tential to achieve the same success as conventional SMB-
LC if large amounts of drugs of high medical value are to
be produced.
5.2.2. Stacked sample injections. On account of the fast
screening capability of SFC, it has been accepted as an
appropriate methodology for compliance with fast new
drug-development processes, especially for chiral sepa-
rations. Because of the fast separation, the small-volume
samples can be repetitively loaded on the analytical SFC.
Trends in Analytical Chemistry, Vol. 37, 2012
Sequential injections of analytes are arranged tightly one
after another. In this way, a substantial throughput of
pure enantiomers could be accumulated in a short time.
Using CO2 as the major solvent, SFC significantly reduces
solvent usage and drug-recovery cost and increases the
drug-recovery yield, especially in the preparative scale
[56]. Currently, most of the enantioseparations scaled up
from analyses in laboratory are based on the analytical
separations with stacking injections on a large-dimen-
sion column in SFC.
Chen et al. [57] simulated the competitive Langmuir
isotherm of both enantiomers onto stationary phase of
Chiralpak AD in chiral SFC, where the effect of various
processing parameters on separation performance was
discussed. The parameters included concentration of
each enantiomer in mobile phase and CSP, the chro-
matographic linear velocity, the axial dispersion coeffi-
cient and the total porosity of the column. The
computational simulation method was practical in pre-
dicting the optimal separation conditions as well as the
maximum throughput after scaling up.
White et al. [33] integrated SFC into drug discovery
for fast screening with gradient flow. The optimum CSP
and chromatographic condition was employed with
stacked injection of small-volume racemates on a pre-
parative Chiralpak AD column (20 · 250 mm, 10 lm).
With 22 stacked injections, 72 mg of enantiomers were
collected (Fig. 3).
Similarly, Vrudhula et al. [51] separated atropisomers
of a racemic drug, known as maxi-K channel opening
3-substitued-4-arylquinolinone, on a preparative
Chiralpak AD column (30 · 250 mm, 5 lm). A total of
14 injections yielded in 202 mg and 205 mg of the
isomers, respectively.
The chromatographic conditions and injected
amounts on the large column are proportional to the
values when operated on the analytical column. Nor-
mally, the throughput of preparative SFC is calculated
based on the CSPs used in the large dimensional
columns [58], as shown in Table 3.
5.2.3. Simulated moving bed (SMB). The technique of
SMB was applied extensively in HPLC on account of its
advantage of adjustable column length. The coupling of
SFC with SMB is still the subject of intensive research.
Sustainable, environmentally-friendly production of
chemicals would prefer replacement of organic mobile
phases in LC with supercritical fluid CO2, which would
significantly reduce the cost of solvent.
Depta et al. [59] developed SFC-SMB apparatus for the
separation of stereoisomers based on the determination
of adsorption isotherms (Fig. 4). In the SMB-design
process, it is important to acquire HenryÕs constant of the
enantiomers in the operating conditions through the
measurements of the adsorption isotherm of the pure-
components. Félicie et al. [60] suggested predicting the
Trends
SMB-productivity ratio of different operating modes
when there were only linear adsorption equilibriums,
optimal operating points, and identical feed composition
considered. Based on the experimentally-derived
isotherm, one can decide the switching time for SMB.
Rajendran et al. [61] reviewed the scaling up of enan-
tioseparations from analytical-grade chromatography in
LC-SMB.
In contrast to HPLC-SMB, SFC-SMB takes the pressure
gradient of the mobile phase as an important factor to
alter the selectivity. The mean density of the mobile
phase is determined by pressure, temperature and the
ratio of the organic modifier. It has been observed that a
minimal change of the density of the mobile phases
exerts a strong influence towards their solvation ability
as well as the resolution [60]. Recently, with software
softSMB, the operation parameters on the instrument
can be easily determined [62]. The continuous operation
of SMB processes could supply higher productivity than
the batch-separation apparatus. However, the cost of the
SMB separation could be higher for purifications of small
amounts [63].
6. Conclusions
The employment of SFC for pharmaceutical analyses
and drug purification is becoming popular. Compared
with LC, SFC has merits, including providing orthog-
onal selectivity, lower pressure drop over the column
and increased efficiency. SFC is especially advanta-
geous in preparative separations due to the higher
productivity, lower cost of mobile phases and lower
pollution. The most developed applications of SFC are
for enantiomeric separations. Almost all the CSPs
originally developed for LC applications can be applied
directly in SFC. Moreover, particles for stationary
phases could be smaller than 5 lm in SFC while col-
umn pressure is not an obstacle. Polysaccharide-based
CSPs remain the CSPs applied most. However, Pirkle-
type CSPs and CSPs based on macrocyclic antibiotics
or cyclodextrins also demonstrated good, reproducible
enantioseparations.
The opposite configuration of chiral selectors could
reverse the elution order of the enantiomers, which is of
utmost importance for trace analyses and quantitative
study of impurities. Besides mobile-phase composition,
pressure and column temperature should also be con-
trolled to optimize SFC separations.
The BPR module can minimize baseline noise, making
it even more powerful for the study of trace-amount
samples.
Recent developments in SFC include choices of tandem
columns for simultaneous chiral/achiral separations,
stacked injections for rapid separations and the innova-
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Trends
tive SFC-SMB technique and instrumentation for the
development of a green pharmaceutical industry.
Acknowledgments
Financial support from the A\STAR (SERC GRANT NO.:
0921010056) and Natural Science Foundation of
Jiangsu Province (GRANT NO.: BK2010486) for this
work are gratefully acknowledged.
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