Enantioselective potential of polysaccharide-based chiral stationary phases in supercritical fluid chromatography

Article abstract of DOI:10.1002/chir.22701

The enantioselective potential of two polysaccharide-based chiral stationary phases for analysis of chiral structurally diverse biologically active compounds was evaluated in supercritical fluid chromatography using a set of 52 analytes. The chiral selectors immobilized on 2.5?μm silica particles were tris-(3,5-dimethylphenylcarmabate) derivatives of cellulose or amylose. The influence of the polysaccharide backbone, different organic modifiers, and different mobile phase additives on retention and enantioseparation was monitored. Conditions for fast baseline enantioseparation were found for the majority of the compounds. The success rate of baseline and partial enantioseparation with cellulose-based chiral stationary phase was 51.9% and 15.4%, respectively. Using amylose-based chiral stationary phase we obtained 76.9% of baseline enantioseparations and 9.6% of partial enantioseparations of the tested compounds. The best results on cellulose-based chiral stationary phase were achieved particularly with propane-2-ol and a mixture of isopropylamine and trifluoroacetic acid as organic modifier and additive to CO₂, respectively. Methanol and basic additive isopropylamine were preferred on amylose-based chiral stationary phase. The complementary enantioselectivity of the cellulose- and amylose-based chiral stationary phases allows separation of the majority of the tested structurally different compounds. Separation systems were found to be directly applicable for analyses of biologically active compounds of interest.

Full text of DOI:10.1002/chir.22701

Received: 14 November 2016
DOI: 10.1002/chir.22701
Revised: 14 February 2017
Accepted: 21 February 2017
S P E C I A L I S S U E A R T I C L E
Enantioselective potential of polysaccharide‐based chiral
stationary phases in supercritical fluid chromatography
Gabriela Kucerova | Kveta Kalikova
| Eva Tesarova
Department of Physical and Macromolecular
Chemistry, Charles University, Prague,
Czech Republic
Abstract
The enantioselective potential of two polysaccharide‐based chiral stationary phases
for analysis of chiral structurally diverse biologically active compounds was
evaluated in supercritical fluid chromatography using a set of 52 analytes. The
chiral selectors immobilized on 2.5 μm silica particles were tris‐(3,5‐
dimethylphenylcarmabate) derivatives of cellulose or amylose. The influence of
the polysaccharide backbone, different organic modifiers, and different mobile
phase additives on retention and enantioseparation was monitored. Conditions for
fast baseline enantioseparation were found for the majority of the compounds. The
success rate of baseline and partial enantioseparation with cellulose‐based chiral
stationary phase was 51.9% and 15.4%, respectively. Using amylose‐based chiral
stationary phase we obtained 76.9% of baseline enantioseparations and 9.6% of par-
tial enantioseparations of the tested compounds. The best results on cellulose‐based
chiral stationary phase were achieved particularly with propane‐2‐ol and a mixture
of isopropylamine and trifluoroacetic acid as organic modifier and additive to
CO2, respectively. Methanol and basic additive isopropylamine were preferred on
amylose‐based chiral stationary phase. The complementary enantioselectivity of the
cellulose‐ and amylose‐based chiral stationary phases allows separation of the major-
ity of the tested structurally different compounds. Separation systems were found to
be directly applicable for analyses of biologically active compounds of interest.
Correspondence
Kveta Kalikova, Department of Physical and
Macromolecular Chemistry, Charles
University, Albertov 6, Prague 2, 12843,
Czech Republic.
Email: kveta.kalikova@natur.cuni.cz
Funding Information
Czech Science Foundation, Grant/Award
Number: 16‐05942S
KEYWORDS
amylose, biologically active compounds, cellulose, chiral separation, chiral stationary phase,
enantioselectivity, supercritical fluid chromatography
1 | INTRODUCTION
to as a “green solvent” and a further desirable MP component
thanks to its properties such as density, solvating power, or
Supercritical fluid chromatography (SFC) has become a very
successful technique for fast and efficient achiral or chiral
separations of diverse compounds in the past few years.
SFC is becoming a method of first choice in pharmaceutical
applications concerning enantioseparations and/or purifica-
tions.1-8 This is largely thanks to the commercialization of
new‐generation SFC systems, which offer enhanced sensitiv-
ity, robustness, and quantitative performance.9 Supercritical
CO2 as the main part of the mobile phase (MP) is referred
viscosity.1,10 From this point of view, SFC offers benefits
such as higher throughput or lower analysis times than the
conventional high‐performance liquid chromatography
(HPLC) technique.1,11-13
On the market, there are a number of chiral stationary
phases (CSPs) including many of HPLC CSPs, that are suit-
able for enantioseparation in SFC. Regarding HPLC as well as
SFC enantioseparations, polysaccharide‐based CSPs are clas-
sified as the versatile ones and are extensively used.5,14-24
Chirality. 2017;1–8.
wileyonlinelibrary.com/journal/chir
© 2017 Wiley Periodicals, Inc.
1

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KUCEROVA ET AL.
New‐generation SFC systems offer better compatibility
with modern stationary phases (SPs), such as those packed
with sub‐2 μm fully porous particles or sub‐3 μm
superficially porous particles.9,25 In fact, SFC columns
with 2.5 μm silica particles containing tris‐(3,5‐
dimethylphenylcarbamate) of cellulose or amylose as chiral
selectors (CSs) used in this study are recent products fully
compatible with commercial UPC2 (ultra‐performance
convergence chromatography) systems.
Organic modifiers, e.g., some alcohols, and basic or
acidic additives added to the main MP component CO2 are
used to modulate the separation ability of the SFC system.
A study has been performed on the effect of different
alcohols in MP in separation systems with immobilized poly-
saccharide‐based CSPs.26 The authors demonstrated comple-
mentary separations of pharmaceuticals in MPs containing
MEOH and propane‐2‐ol. However, in general the separation
success rate for the studied pharmaceuticals was not very
high in their work. Concerning the addition of basic
isopropylamine (IPAM) and/or acidic trifluoroacetic acid
(TFA) additives, the combination of both was reported to
reduce nonspecific interactions and so to increase
enantioselectivity. The dual addition also led to minimization
of the memory effect of SP.27 However, higher concentration
of these additives could result in undesirable precipitation of
the forming salt complexes.28
Nowadays the SFC method is used for separation of
neutral, acidic and also basic compounds.29-31 Neverthe-
less, separation of basic compounds can be hampered by
forming of ionic interactions with residual silanol‐carrier
groups.1,32,33 Among basic, acidic, bifunctional, and neu-
tral biologically active compounds (BACs) used in this
study, we classify very well‐known drugs like profens,
thiazide diuretics, flavanone derivatives, and calcium chan-
nel blockers or phenothiazines and β‐blockers.22,34,35
Moreover, newly synthesized drugs called “legal highs”
belong likewise to BACs.36 The “legal highs” used in this
work are derivatives of amphetamine or benzofuran.
Mostly, they are “abused” similarly as prohibited addictive
substances, with the difference that there is insufficient
legislation that would punish this permitted activity.
Recently, BACs based on amphetamine or benzofuran were
successfully enantioseparated using amylose‐based CSP in
SFC.1 Some enantiomers of β‐blockers used in this study
were previously separated on two different polysaccha-
ride‐based CSPs (Chiralpak IB‐3 and Chiralpak AD
columns) by SFC.37,38 Almost 20 years ago, Berger and
Wilson enantioseparated several phenothiazine substances
using packed column SFC.39
immobilized on 2.5 μm silica particles. The goal was to
show differences in the chromatographic behavior between
these two columns, as they differ in the nature of the poly-
saccharide backbone. For this purpose, a set of 52 structur-
ally different BACs was tested under diverse SFC
separation conditions, namely, different MP compositions,
to examine the enantioseparation abilities and differences
of these two columns. The other objective was to find the
best/optimal mobile phases for enantioseparation of the
tested chiral compounds.
2 | MATERIALS AND METHODS
2.1 | Chemicals and analytes
Methanol (MEOH, Chromasolv, gradient grade, ≥99.9%),
propane‐2‐ol (PROH, Chromasolv for HPLC, ≥99.8%),
isopropylamine (IPAM, ≥99.5%), trifluoroacetic acid (TFA,
99%), and tetrahydrofuran (THF, Chromasolv for HPLC)
were supplied by Sigma‐Aldrich (St. Louis, MO). Pressur-
ized liquid CO2 4.5 grade (99.995%) was purchased from
Messer (Prague, Czech Republic). Chiral analytes:
profen derivatives (PF1, ibuprofen; PF2, indoprofen;
PF3, flurbiprofen; PF4, tiaprofenic acid; PF5, carprofen,
PF6, suprofen; PF7, ketoprofen; PF8, fenoprofen),
flavanone derivatives (F1, 6‐hydroxyflavanone; F2, 7‐
hydroxyflavanone), thiazide diuretics (TD1, butizide; TD2,
mefruside; TD3, chlorthalidone; TD4, trichlormethiazide;
TD5, bendroflumethiazide), calcium channel blockers
(CB1, amlodipine; CB2, nimodipine; CB3, nitrendipine;
CB4, nicardipine; CB5, verapamil; CB6, nisoldipine),
phenothiazines (PH1, thioridazine; PH2, promethazine),
amphetamine derivatives (A1, 4‐fluoromethcathinone; A2,
4‐fluoroamphetamine; A3, 4‐bromomethcathinone; A4,
buphedrone; A5, ethylone; A6, 3‐fluoroamphetamine; A7,
2‐fluoromethcathinone; A8, methylendioxypyrovalerone),
benzofury derivatives (B1, 5‐(2‐aminopropyl)benzofuran;
B2, 6‐(2‐aminopropyl)benzofuran; B3, 5‐(2‐aminopropyl)‐
2,3‐dihydrobenzofuran;
B4,
6‐(2‐aminopropyl)‐2,3‐
dihydrobenzofuran; B5, 1‐(benzofuran‐5‐yl)‐N‐ethylpropan‐
2‐amine; B6, 1‐(benzofuran‐6‐yl)‐N‐ethylpropan‐2‐amine;
B7, 1‐(benzofuran‐5‐yl)‐N‐methylpropan‐2‐amine), β‐blockers
(BB1, propranolol; BB2, oxprenolol; BB3, metoprolol;
BB4, metipranolol; BB5, acebutolol; BB6, pindolol; BB7,
bopindolol; BB8, atenolol; BB9, alprenolol) and others
(O1, BP34; O2, BP766; O3, thalidomide; O4, tramadol;
O5, lorazepam) were purchased from Sigma‐Aldrich or
kindly donated from M.G. Schmid from Institute of
Pharmaceutical Chemistry and Pharmaceutical Technology,
Karl Franzens University, Graz, Austria. See Figures S1 in
the Supporting Information for the structures of the
compounds.
The aim of this work was to find out and compare the
enantioselective potential of new short polysaccharide‐based
columns (50 mm long), i.e., ACQUITY UPC2 Trefoil CEL1
and ACQUITY UPC2 Trefoil AMY1. The CSs are

KUCEROVA ET AL.
3
TABLE 1 MP compositions used for the enantioseparation of BACs
on CEL1 and AMY1 CSPs
2.2 | SFC instrumentation and columns
The Waters Acquity Ultra Performance Convergence Chro-
matography (UPC2) system was equipped with a binary sol-
MP compositions:
Volume ratios (v/v/v(/v)):
vent delivery pump (MP flow rates up to 4 mL min−1
,
CO2/MEOH/IPAM
CO2/PROH/IPAM
90/10/0.1
90/10/0.1
95/5/0.1
95/5/0.1
98/2/0.1
98/2/0.1
pressures up to 6000 psi), an autosampler which included a
partial loop volume injection system, a back‐pressure (BP)
regulator, a column oven, and a photodiode array detector
(Waters, Milford, MA). The Empower 3 software was used
for system control and data acquisition. Both columns:
ACQUITY UPC2 Trefoil CEL1 (CEL1) and ACQUITY
UPC2 Trefoil AMY1 (AMY1) were obtained from Waters.
The CSs immobilized on 2.5 μm silica particles were tris‐
(3,5‐dimethylphenylcarbamate) derivatives of cellulose
(CEL1) or amylose (AMY1). The dimensions of both col-
umns were 3.0 × 50 mm.
CO2/MEOH/IPAM/TFA 90/10/0.1/0.1 95/5/0.1/0.1 98/2/0.1/0.1
CO2/PROH/IPAM/TFA 90/10/0.1/0.1 95/5/0.1/0.1 98/2/0.1/0.1
CO2/MEOH/TFA
CO2/PROH/TFA
90/10/0.1
90/10/0.1
95/5/0.1
95/5/0.1
98/2/0.1
98/2/0.1
3 | RESULTS AND DISCUSSION
The columns CEL1 and AMY1 were investigated under var-
ious MP compositions in order to evaluate their
enantioselective ability for separation of acidic, basic, bifunc-
tional, and neutral compounds. Temperature and BP were
kept constant during measurements in order to monitor the
impact of the CSP type and MP compositions. The best/
optimized separation conditions were found for the majority
of the studied compounds even without the additional optimi-
zation of temperature and BP. Some of the baseline separated
compounds could be resolved under several MP composi-
tions. However, other suitable MP compositions are not
shown, particularly due to a higher duration of analysis that
is claimed to be as short as possible. Chromatographic data
collected from the measurements on the both CSPs at different
MP compositions are summarized in Table S1 in the
Supporting Information for better understanding and compar-
ison. In general, a higher retention of analytes was obtained in
MPs containing more hydrophobic PROH than those contain-
ing MEOH (comparing the same volume ratios). We did not
observe the general effect of enhanced and decreased
enantioselectivity on the cellulose‐based and amylose‐based
CSPs, respectively, caused with the dual additives in the
SFC separation systems that were reported by other authors.28
It is obvious from Table 2 and Table S1 that different groups
of tested analytes prefer different CSPs‐polysaccharide
backbone compared in this work.
2.3 | General conditions
The chromatographic measurements were performed at a
flow rate 2.5 mL min−1 based on our previous experience1
and our preliminary measurements in this work (measure-
ments were carried out in the range 1.5–3 mL min−1). Both
retention and resolution increased at lower flow rate. Thus,
the best separation conditions were considered as a compro-
mise between resolution and short analysis time. The column
temperature was 35 °C, BP of 2000 psi and UV detection at
254, 260 and 280 nm. Void volume was determined using
the solvent peak. Injection volume was in the range
0.6–1.0 μL depending on the detector response. Sample tem-
perature was 10 °C. All measurements were performed in
triplicate.
2.4 | Sample preparation
The stock solutions of profen derivatives, flavanones, thia-
zide diuretics, calcium channel blockers (except for CB6),
phenothiazines, β‐blockers, others (except for O3), and
3‐fluoroamphetamine (A7) were prepared in MEOH at a con-
centration of 1.0 mg mL−1. Amphetamine derivatives (except
for A5, A6, and A7) were dissolved in MEOH at a concentra-
tion of 0.5 mg mL−1. The stock solutions of benzofury deriv-
atives were prepared at a concentration of 0.25 mg mL−1 in
MeOH/THF 50/50 (v/v). CB6 was dissolved in MEOH/THF
80/20 (v/v) at a concentration of 1.0 mg mL−1 and O3 in
3.1 | Profen derivatives
From the set of eight profen derivatives, seven were baseline
separated under optimized conditions on the CSP with amy-
lose backbone AMY1, while just three exhibited a resolution
higher than 1.5 on CEL1 CSP. Enantiomers of ibuprofen
(PF1) could not be resolved on any of these CSPs. Despite
the general observation that better enantioseparation of
profens can be achieved on the amylose‐based column, cer-
tain separations show opposite results, as demonstrated in
Figure 1B. Comparison of the separation of flurbiprofen
MEOH/THF 75/25 (v/v) at a concentration of 1.0 mg mL−1
.
2.5 | MP compositions
MPs composed of CO2 and organic modifiers MEOH or
PROH with the addition of basic additive IPAM and/or acidic
additive TFA was prepared in various volume ratios. See
Table 1 for exact MP compositions used in this work.

4
KUCEROVA ET AL.
TABLE 2 Chromatographic data and the best MP compositions for enantioseparation of studied compounds on CEL1 and AMY1 CSPs
CEL1 CSP
AMY1 CSP
tr,1
MP composition
tr,1
MP composition
Compounds
(min)
k1
α
Rs
(v/v/v(/v))
(min)
k1
α
Rs
(v/v/v(/v))
Profen
derivatives
PF1
PF2
PF3
X
X
X
X
X
X
X
X
X
X
1.41 9.71
3.95 32.50
1.19
1.13
1.10
1.23
1.11
X
2.25 C/M/T 90/10/0.1
1.52 C/P/T 98/2/0.1
1.36 C/P/T 98/2/0.1
2.07 C/P/I/T 90/10/0.1/0.1
1.27 C/P/T 95/5/0.1
15.08 134
0.39 2.53
3.24 28.50
5.42 46.97
1.17 9.42
1.36 11.37
5.61 41.80
1.22 2.73 C/P/T 90/10/0.1
1.35 2.89 C/M/T 90/10/0.1
1.86 6.71 C/P/I/T 95/5/0.1/0.1
1.20 2.34 C/M/I/T 90/10/0.1/0.1
1.26 2.55 C/M/T 90/10/0.1
1.17 1.47 C/P/I/T 95/5/0.1/0.1
1.17 2.13 C/P/T 98/2/0.1
PF4 14.29 120
PF5
PF6
PF7
PF8
3.38 24.60
2.53 21.02
X
X
X
X
X
X
X
X
X
Flavanones
F1
F2
1.61 11.18
1.11
X
1.51 C/M/I/T 95/5/0.1/0.1
1.73 14.99
1.55 13.38
1.48 3.86 C/P/I/T 90/10/0.1/0.1
1.18 1.80 C/P/I/T 90/10/0.1/0.1
X
X
X
X
Thiazide diuretics TD1
2.69 19.36
1.52
X
1.23
1.11
1.09
4.73 C/M/I/T 90/10/0.1/0.1 15.17 133
2.63 22.86
2.53 C/M/I/T 90/10/0.1/0.1 12.18 87.91
1.24 1.81 C/P/I/T 90/10/0.1/0.1
1.70 3.85 C/M/I 90/10/0.1
1.36 2.64 C/M/I 90/10/0.1
1.13 8.68 C/P/I/T 90/10/0.1/0.1
1.26 2.03 C/M/I/T 90/10/0.1/0.1
TD2
TD3
TD4
X
X
X
X
3.40 24.83
5.75 49.41
1.51 C/M/T 90/10/0.1
1.05 C/P/I 90/10/0.1
18.74 165
3.36 28.69
TD5 17.39 130
CB1 7.16 52.75
CB2 10.72 89.83
Calcium channel
blockers
1.24
1.09
X
X
1.28
1.09
1.70 C/P/I/T 95/5/0.1/0.1
X
X
X
X
X
1.05 C/P/T 98/2/0.1
0.72 5.43
3.37 4.23 C/P/T 90/10/0.1
CB3
CB4
CB5
CB6
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
0.73 4.50
8.00 58.84
1.87 C/M/I/T 90/10/0.1/0.1
0.85 6.11
1.14 0.84 C/P/I 90/10/0.1
1.23 C/P/I 98/2/0.1
X
X
X
X
X
Phenothiazines
PH1
PH2
X
X
X
1.10
X
X
2.85 22.73
1.95 12.73
1.24 2.04 C/P/I 90/10/0.1
1.26 1.56 C/P/I 98/2/0.1
1.41 9.73
0.93 C/P/I 98/2/0.1
Amphetamine
derivatives
A1
A2
A3
A4
A5
A6
A7
A8
X
X
X
X
X
X
X
X
X
1.16
X
X
X
X
X
X
X
X
1.65 12.85
10.53 88.23
0.43 2.80
3.05 25.07
1.75 14.92
3.06 25.14
3.06 25.50
0.56 3.80
1.30 1.20 C/P/T 95/5/0.1
1.15 1.83 C/P/I 98/2/0.1
1.51 1.78 C/P/I/T 90/10/0.1/0.1
1.35 2.68 C/P/I 95/5/0.1
1.66 2.54 C/P/I/T 95/5/0.1/0.1
1.27 2.15 C/P/I 95/5/0.1
1.26 2.66 C/P/I 95/5/0.1
1.24 1.84 C/P/I 95/5/0.1
2.25 18.72
1.43 C/M/T 98/2/0.1
X
X
X
X
X
X
X
X
X
X
X
X
X
1.27
1.34 9.12
2.25 C/P/I/T 95/5/0.1/0.1
Benzofury
derivatives
B1
B2
B3
B4
B5
B6
B7
1.45 9.99
1.45 9.93
2.71 19.70
0.54 3.05
1.24
1.23
1.92
1.50
X
1.80 C/P/I/T 95/5/0.1/0.1
1.68 C/P/I/T 95/5/0.1/0.1
7.33 C/M/I 98/2/0.1
7.25 60.97
7.28 61.18
7.58 63.79
0.80 5.94
1.99 16.04
2.08 16.81
2.92 23.96
1.31 3.72 C/P/I 95/5/0.1
1.32 3.48 C/P/I 95/5/0.1
1.11 1.37 C/P/I 95/5/0.1
1.35 1.97 C/M/I/T 95/5/0.1/0.1
1.24 2.38 C/P/I 95/5/0.1
1.13 1.42 C/P/I 95/5/0.1
1.19 2.07 C/P/I 95/5/0.1
2.42 C/P/I/T 90/10/0.1/0.1
X
X
X
X
X
X
X
X
X
1.12
3.27 23.78
1.01 C/P/I 98/2/0.1
β‐blockers
BB1
BB2
BB3
BB4
BB5
BB6
BB7
BB8
BB9
0.80 5.09
0.31 1.35
0.28 1.13
0.60 3.54
5.43 40.10
2.01 14.25
1.76 12.25
1.55 10.61
0.23 0.76
2.00
2.44
4.02
1.30
1.15
6.36 C/M/I/T 90/10/0.1/0.1
4.14 C/M/I/T 90/10/0.1/0.1
5.00 C/M/I/T 90/10/0.1/0.1
2.26 C/M/I/T 95/5/0.1/0.1
1.71 C/M/I/T 95/5/0.1/0.1
2.48 21.37
1.25 10.25
1.83 15.46
13.58 115
15.69 140
1.50 2.92 C/P/I 90/10/0.1
1.28 1.63 C/P/I 90/10/0.1
1.34 1.94 C/P/I 90/10/0.1
1.20 2.92 C/P/I 95/5/0.1
1.26 1.62 C/P/I 90/10/0.1
4.46 14.2
C/M/I/T 90/10/0.1/0.1
X
X
X
X
X
1.72
2.31
1.79
5.66 C/M/I/T 90/10/0.1/0.1
8.62 C/M/I/T 90/10/0.1/0.1
2.05 C/M/I/T 90/10/0.1/0.1
5.56 47.4
1.86 5.52 C/M/I/ 95/5/0.1
X
X
X
X
X
0.93 7.35
1.41 2.07 C/P/I 90/10/0.1
Others
O1
O2
2.70 19.41
1.41
X
1.92 C/P/I/T 95/5/0.1/0.1
3.46 27.83
2.02 15.97
1.40 1.68 C/P/I 90/10/0.1
1.31 1.58 C/P/T 95/5/0.1
X
X
X
X
(Continues)

KUCEROVA ET AL.
5
TABLE 2 (Continued)
CEL1 CSP
AMY1 CSP
tr,1
MP composition
tr,1
MP composition
Compounds
(min)
k1
α
Rs
(v/v/v(/v))
(min)
k1
α
Rs
(v/v/v(/v))
O3
O4
O5
2.43 20.29
0.53 2.99
1.46 10.07
1.13
1.18
1.27
1.70 C/M/T 95/5/0.1
1.56 C/M/I/ 95/5/0.1
2.72 C/M/I/T 90/10/0.1/0.1
3.40 29.11
0.38 2.13
1.96 13.77
1.30 2.34 C/P/I/T 90/10/0.1/0.1
1.29 1.58 C/P/I 90/10/0.1
1.30 2.36 C/M/I/T 90/10/0.1/0.1
In the case of baseline separation, the optimized chromatographic data are reported with respect to the shortest analysis time; resolution value in bold indicates baseline
separation; MP composition in bold indicates the best chromatographic conditions (including CSP) for enantioseparation, C: CO2, M: MEOH, P: PROH, I: IPAM, T:
TFA; tR,1: retention time of the first eluted enantiomer, k1: retention factor of the first eluted enantiomer, α: selectivity, RS: resolution of the two enantiomers, X: no indi-
cation of enantioseparation.
in their molecule) were successfully enantioseparated using
AMY1 CSP and MP composed of CO2/PROH/IPAM/TFA
90/10/0.1/0.1 (v/v/v/v) (Table 2). The combination of both
MP additives (IPAM and TFA) was very supportive for fast
enantioseparation of both analytes on AMY1 CSP. On the
other hand, they were not baseline enantioresolved on
CEL1 CSP under the above‐mentioned MP. However,
enantioresolution RS = 1.5 for F1 enantiomers was achieved
on CEL1 CSP in MP with higher CO2 content. The represen-
tative chromatograms of analysis of 7‐hydroxyflavanone on
both columns in the same MP composition are depicted in
Figure 2.
3.3 | Thiazide diuretics
All the enantiomers of analytes from the group of thiazide
diuretics could be baseline separated under optimized separa-
FIGURE 1 Analyses of flurbiprofen A, and indoprofen B, on the
polysaccharide‐based CSPs. MP composition: CO2/MEOH/TFA 90/
tion conditions on AMY1 CSP. However, their retention and
thus analysis time was too long for practical purposes. On the
other hand, butizide (TD1), chlorthalidone (TD3), and
trichlormethiazide (TD4) enantiomers could be baseline sep-
arated on CEL1 CSP in a significantly shorter analysis time.
Concerning the MPs, the most suitable composition varies by
CSP used and analyte of interest (Table 2).
10/0.1 (v/v/v)
and indoprofen in the same MP composed of CO2/MEOH/
TFA 90/10/0.1 (v/v/v) supports this statement, i.e., better
result of indoprofen enantioseparation obtained on CEL1
CSP. Moreover, indoprofen (PF2) was baseline
enantioseparated in all the MPs tested using CEL1 CSP
(Table S1 in the Supporting Information). MPs with basic
IPAM additive were not suitable for enantioseparation of
profen derivatives. The basic additive increases “dissocia-
tion” of acidic profens, while compounds to be separated on
the polysaccharide‐based CSPs are nondissociated. Thus,
addition of TFA or a combination of IPAM and TFA resulted
in improved enantioseparation. Certain complementarity of
the enantioseparation ability of the compared CSPs is clearly
seen from the obtained results (Figure 1).
3.2 | Flavanones
FIGURE 2 Analyses of 7‐hydroxyflavanone on the polysaccharide‐
based CSPs. MP composition: CO2/PROH/IPAM/TFA 90/10/0.1/0.1
(v/v/v/v)
The two flavanone derivatives with very similar structures
(the difference lies only in the position of the hydroxy group

6
KUCEROVA ET AL.
MPs tested on CEL1 CSP, and just partial enantioseparation
was achieved for B7 with this CSP. The use of MP composed
of PROH and both basic IPAM and acidic TFA additives
seemed to be advantageous. On the other hand, MP consisted
of CO2, PROH and just the basic additive, mostly in the vol-
ume ratio CO2/PROH/IPAM 95/5/0.1 (v/v/v), was a better
choice if AMY1 CSP was used. A special result was
observed with compound B3 (Table 2). The highest resolu-
tion value (RS = 7.33) of all the compounds of this group
was achieved with cellulose‐based CSP, while on an amy-
lose‐based column the lowest resolution value (RS = 1.37)
of B3 was obtained under the “best” MP compositions for a
given system.
3.4 | Calcium channel blockers
Neither AMY1 nor CEL1 CSPs were suitable for
enantioseparation of this group of analytes. Despite the fact
that calcium channel blockers possess in their molecules
structurally similar motifs, we observed rather different
results for diverse derivatives. Whereas amlodipine, verapa-
mil, and nisoldipine enantiomers were baseline separated on
CEL1 CSP, nimodipine could be enantioseparated on
AMY1 CSP. No CSP could be preferred; moreover, the best
MP composition for analysis of the calcium channel blockers
differed in the type of organic modifier as well as in MP
additive.
3.5 | Phenothiazines
3.8 | β‐blockers
AMY1 CSP better suited for separation of phenothiazine
enantiomers than CEL1 CSP. Thioridazine (PH1) and
promethazine (PH2) were both baseline resolved in MP con-
taining CO2, PROH, and IPAM (Table 2). A better resolution
value was achieved for PH1 than for PH2 with amylose‐based
column, while the opposite result was observed on cellulose‐
based CSP. On the latter, partial enantioseparation was
obtained for PH2, while no enantioseparation was achieved
for PH1.
It is obvious from Table 2 as well as from Table S1 that for
separation of enantiomers of β‐blockers cellulose‐based
CSP should be considered the column of first choice. All
β–blockers were baseline enantioseparated with very high
resolution values in very short retention times (except for
BB5) using CEL1 CSP (Table 2). The best MP compositions
for the enantioseparation of β‐blockers on the CEL1 CSP
were: CO2/MEOH/IPAM/TFA 90/10/0.1/0.1 or 95/5/0.1/0.1
(v/v/v/v). MPs suited for enantioseparation of β–blockers on
AMY1 CSP contained PROH as organic modifier (except
for BB7). The presence of the less polar alcohol PROH
instead of MEOH in MP caused longer analyses on AMY1
than on CEL1 CSP (Table S1). Illustrative chromatograms
of enantioseparation of metoprolol (BB3) are shown in
Figure 3.
3.6 | Amphetamine derivatives
All amphetamine derivatives were baseline enantioseparated,
except of 4‐F‐methcathinone (A1) enantiomers, which were
partially separated using AMY1 CSP. PROH was a better
organic modifier for separation of all the analytes from this
group and also the best MP composition was mostly the
same, i.e., CO2/PROH/TFA 95/5/0.1 (v/v/v) (Table 2). Using
CEL1 CSP, all these analytes eluted in very short retention
times and no enantioseparation was observed (except for A4
and A8) (Table 2 and Table S1 in the Supporting Informa-
tion). Only one amphetamine derivative, A8, was baseline
enantioresolved using CEL1 CSP. Nevertheless, faster base-
line separation of A8 was observed using AMY1 CSP. Com-
paring the observed results AMY1 CSP is definitely the
better choice for the enantioseparation of amphetamine deriv-
atives in SFC.
3.9 | Others
The last tested analytes are structurally less similar com-
pounds than those in the other groups. Thus, any general dis-
cussion cannot be performed. However, we show these
results that can be helpful to those who have to carry out sep-
arations of these enantiomers because these are compounds
of interest (thalidomide, tramadol, lorazepam) for analyses
3.7 | Benzofury derivatives
The measurements of the group of benzofury derivatives
brought interesting results. No general trends could be related
to the structure of these analytes, to the structure of the poly-
saccharide backbone of the CSP, or to MP composition. On
the one hand, enantiomers of B1, B2, B3, and B4 were base-
line enantioseparated using CEL1 CSP. On the other hand, no
enantioseparation of B5 and B6 was observed in any of the
FIGURE 3 Analyses of metoprolol on the polysaccharide‐based
CSPs. MP composition: CO2/MEOH/IPAM/TFA 90/10/0.1/0.1 (v/v/v/v)

KUCEROVA ET AL.
7
4. Wang RQ, Ong TT, Tang WH, Siu‐Choon N. Recent advances
in pharmaceutical separations with supercritical fluid chromatog-
raphy using chiral stationary phases. Trac‐Trend Anal Chem.
2012;37:83‐100.
in clinical or pharmaceutical laboratories. As can be seen
from Table 2, both columns are applicable for fast
enantioseparation of thalidomide, tramadol, as well as loraz-
epam enantiomers.
5. Lee J, Lee JT, Watts WL, et al. On the method development of
immobilized polysaccharide chiral stationary phases in supercritical
fluid chromatography using an extended range of modifiers.
J Chromatogr A. 2014;1374:238‐246.
4 | CONCLUSION
A set of 52 structurally different chiral BACs were used to
reveal the enantioselective potential of two polysaccharide‐
based CSs immobilized on 2.5 μm silica particles, i.e.,
CEL1 and AMY1 CSPs in SFC. We monitored the influence
of the type of CS backbone, the type and amount of organic
modifier, as well as MP additives on enantioresolution of
the studied compounds. MPs were composed of CO2, organic
modifier, i.e., MEOH or PROH and MP additive, i.e., IPAM
and/or TFA. The results showed that the tris‐(3,5‐
dimethylphenylcarbamate) derivatives of amylose and cellu-
lose show very broad and complementary enantiorecognition
abilities. In general, tris‐(3,5‐dimethylphenylcarbamate) of
amylose was more suitable for enantioseparation of the stud-
ied compounds than tris‐(3,5‐dimethylphenylcarbamate) of
cellulose. However, certain enantiomers could be better
resolved using the CSP with cellulose backbone. We
obtained baseline and partial enantioseparations of 45 and 4
tested compounds, respectively, even without further BP
and temperature optimization. Three compounds were not
enantioseparated under any conditions used.
6. Lemasson E, Bertin S, West C. Use and practice of achiral and chiral
supercritical fluid chromatography in pharmaceutical analysis and
purification. J Sep Sci. 2016;39:212‐233.
7. West C, Cieslikiewicz‐Bouet M, Lewinski K, Gillaizeau I. Enantio-
meric separation of original heterocyclic organophosphorus
compounds in supercritical fluid chromatography. Chirality.
2013;25:230‐237.
8. Nováková L, Douša M. General screening and optimization strategy
for fast chiral separations in modern supercritical fluid chromatogra-
phy. Anal Chim Acta. 2017;950:199‐210.
9. Guillarme D. SFC star. Anal Sci. 2015;09:27
10. De Klerck K, Parewyck G, Mangelings D, Vander HY.
Enantioselectivity of polysaccharide‐based chiral stationary phases
in supercritical fluid chromatography using methanolcontaining car-
bon dioxide mobile phases. J Chromatogr A. 2012;1269:336‐345.
11. Kalikova K, Slechtova T, Vozka J, Tesarova E. Supercritical fluid
chromatography as a tool for enantioselective separation; a review.
Anal Chim Acta. 2014;821:1‐33.
12. West C. Enantioselective separations with supercritical fluids —
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In summary, 27 analytes were baseline enatioresolved on
CEL1 CSP, whereas 40 on AMY1 CSP. Furthermore, eight
and five analytes were partially enantioseparated on CEL1
and AMY1 CSPs, respectively. We were not able to achieve
enantioseparation of 17 analytes on CEL1 CSP, while seven
on AMY1 CSP. Some complementary behavior of the CSPs
was observed. Thus, the combination of these two CSPs
offers a powerful tool for enantioseparation of different types
of BACs in SFC.
13. Novakova L, Desfontaine V, Ponzetto F, et al. Fast and sensitive
supercritical fluid chromatography — Tandem mass spectrometry
multi‐class screening method for the determination of doping agents
in urine. Anal Chim Acta. 2016;915:102‐110.
14. Toribio L, del Nozal MJ, Bernal JL, Bernal J, Martin MT. Study of
the enantiomeric separation of an acetamide intermediate by using
supercritical fluid chromatography and several polysaccharide based
chiral stationary phases. J Chromatogr A. 2011;1218:4886‐4891.
15. Franco P, Zhang T. Common approaches for efficient method devel-
opment with immobilised polysaccharide‐derived chiral stationary
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ACKNOWLEDGEMENTS
16. Vozka J, Kalikova K, Tesarova E. Rapid supercritical fluid chroma-
tography method for separation of chlorthalidone enantiomers. Anal
Lett. 2013;46:2860‐2869.
The authors gratefully acknowledge the financial support of
the Czech Science Foundation, Grant No. 16‐05942S.
17. Geryk R, Kalikova K, Vozka J, Plecita D, Schmid MG, Tesarova E.
Enantioselective potential of chiral stationary phases based on
immobilized polysaccharides in reversed phase mode. J Chromatogr
A. 2014;1363:155‐161.
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SUPPORTING INFORMATION
Additional supporting information may be found online in
the supporting information tab for this article.
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