Analytica Chimica Acta 950 (2017) 199e210

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Analytica Chimica Acta

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General screening and optimization strategy for fast chiral separations

in modern supercritical fluid chromatography
 a, *, Michal Dousa b
kova
Lucie Nova
a
Department of Analytical Chemistry, Faculty of Pharmacy, Charles University in Prague, Heyrovsk lov
eho 1203, 500 05 Hradec Kra e, Czechia
b
Zentiva, k. s. A Sanofi Company, Praha, U Kabelovny 130, 102 37 Praha 10, Czechia

h i g h l i g h t s g r a p h i c a l a b s t r a c t

General screening approach for fast

SFC chiral separation was developed.

The best enantioselectivity in SFC
was obtained with tris(3,5-
dimethylphenylcarbamate)
substituted stationary phases based
on both amylose and cellulose.

Combined additive composed of 0.1%
DEA and 0.1% TFA provided the best
enantioselectivity for generic
screening.

Volatile buffers were found to be a
viable option for SFC chiral screening.

Tetrahydrofuran was the most
convenient injection solvent for chi-
ral SFC analyses.

a r t i c l e i n f o a b s t r a c t

Article history: High throughput general chiral screening method using supercritical fluid chromatography was devel-
Received 2 September 2016 oped. This method takes an advantage of very fast gradient screening (3 min þ 1 min isocratic hold) and
Received in revised form generic enantioselectivity of the combined additive formed by 0.1% trifluoroacetic (TFA) acid and 0.1%
22 October 2016
diethylamine (DEA). The TFA/DEA combined additive was systematically added to organic modifiers
Accepted 2 November 2016
Available online 21 November 2016
methanol and isopropanol. Among five tested polysaccharide-based chiral stationary phases, amylose
tris(3,5-dimethylphenylcarbamate) and cellulose tris(3,5-dimethylphenylcarbamate) provided the best
enantioseparation success rate. Therefore, the proposed initial first-line screening includes four exper-
Keywords:
Supercritical fluid chromatography
iments using these two stationary phases and the above mentioned two combinations: CO2/methanol
SFC and CO2/isopropanol þ the combined additive. If these stationary phases fail in the screening step,
Chiral stationary phases cellulose tris(3-chloro-4-methylphenylcarbamate) and cellulose tris(3,5-dichlorophenylcarbamate) can
Organic modifier be proposed for the screening in the second line.
Combined additive For further optimization in case of insufficient resolution obtained in the screening phase fine tuning
Enantioselectivity of temperature, BPR pressure and gradient slope was tested with unsuccessful results. An improvement
Pharmaceuticals of enantioselectivity was obtained only when gradient elution was replaced by isocratic elution with
substantially lower amount of organic modifier, when changing the concentration of the additive or
when using combined organic modifier, such as methanol/acetonitrile (1:1). Finally, to enable the MS
compatibility, also volatile additives including ammonium formate and ammonium acetate were tested.

* Corresponding author.
E-mail address: nol@email.cz (L. Nov
akov
a).

http://dx.doi.org/10.1016/j.aca.2016.11.002
0003-2670/© 2016 Elsevier B.V. All rights reserved.
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L. Nova , M. Dousa / Analytica Chimica Acta 950 (2017) 199e210
The results were more encouraging than expected. Volatile buffers thus make an interesting option in
chiral SFC screening methods, however, at the cost of somewhat lower enantioselectivity.
1. Introduction
Supercritical fluid chromatography (SFC) has already become
quite well-established in separation of chiral compounds [1e10].
However, it is not yet considered a first-choice option for routine
quality control (QC) in pharmaceutical industry, although it meets
the criteria of high efficiency and fast separation, which are
required due to the huge number of samples to be analyzed. The
reasons for a lower popularity of SFC in routine QC originates in the
past drawbacks of the technique, such as much lower method
robustness, repeatability and sensitivity of available instrumenta-
tion. Therefore, HPLC in both reversed phase and normal phase
modes are still considered to be the methods of the first choice
[11e13] with the current trends of ultra-fast enantioseparations
using superficially porous particles [14e16] or teicoplanin sub-2
mm totally porous silica particles [17] stationary phases providing
separations within seconds [18]. Recently, new SFC platforms and
new stationary phases have been commercially introduced in order
to extend application potential and reliability of SFC method and to
compensate for the above stated drawbacks [19,20].
The generic screening should consist of limited number of fast
chromatographic experiments, which can be applied to various
racemates and be helpful in quick finding of conditions for suc-
cessful separation. The state of the art in screening chiral SFC
strategies employs several polysaccharide based chiral stationary
phases and CO2/methanol mobile phase with the addition of tri-
fluoroacetic acid (TFA) for the analysis of acidic compounds and
isopropylamine (IPA) or another amine for basic and neutral com-
pounds [1,3,21e23]. Isocratic elution using 10 and/or 20% of alco-
holic organic modifiers (typically methanol and/or isopropanol) has
been still preferred even in several quite recent modern methods
for chiral screening despite very long retention times for some
compounds and difficulty of prediction and set-up of analysis times
[21,23e25]. In this regard the method development remains sub-
stantially less effective and still time-consuming. Modern screening
approaches request for very fast analyses, even in chiral analysis.
Therefore, a quick gradient elution is much more convenient for
fast screening purposes. Similarly to isocratic methods, gradient
screening approaches were using alcoholic modifiers and various
additives, such as TFA, IPA, diethylamine (DEA) or thiethylamine
(TEA) for the improvement of the peak shapes [22,26e28]. How-
ever, in many cases these were still very time consuming requiring
about 15e25 min analysis times [22,26,27]. Until recently, gradient
approaches were less commonly used, probably due to instru-
mentation constraints that did not enable sufficient method
reproducibility. Few rapid methods using 2.5 min gradient method
with CO2/methanol mobile phases and 0.1% TEA [28], methanol,
ethanol or isopropanol with the addition of NH4OH [29] and CO2/
methanol with 25 mM isobutylamine [3] as the additives have been
published. The latter employed a combination of 3 min isocratic
and 6 min gradient elution. However, considering the basic nature
of additives, these approaches may be considered less generic. The
chiral screening method speed and efficiency may be further
increased also using multi-column parallel approaches [30,31].
Currently available commercial SFC systems enable using up to 8
columns in parallel.
An important advance in the screening approaches in SFC
© 2016 Elsevier B.V. All rights reserved.
included the application of a combined additive, i. e. utilization of
IPA and TFA simultaneously in one mobile phase [32e36]. Due to
this approach there was no need for compound classification any
more. Moreover, an improved separation efficiency and enatiose-
lectivity has been reported, especially for zwitterionic compounds.
However, a combined additive (0.5% TFA þ 0.5% IPA) present in
methanol/CO2 mobile phases resulted in problems with the system
stability in this study due to the salt complex formation [33].
Alternatively, also a combination of TFA/TEA (0.2e0.3%) was re-
ported to provide excellent enantioselectivity [37]. Unfortunately,
use of isocratic elution significantly decreased the throughput of
these reported approaches [24,25,32e34].
The aim of this work was to develop a quick general screening
and optimization strategy for fast SFC separation of chiral phar-
maceuticals using modern approaches to SFC chiral screening in
one design. SFC method development was performed using poly-
saccharide based chiral columns packed with 3 mm particles and
new ultra-high performance supercritical fluid chromatography
(UHPSFC) instrumentation platform. A procedure for fast and
straightforward method development was designed for separation
of chiral basic, neutral and acidic compounds from the group of APIs
(active pharmaceutical ingredients) and their intermediates using
combined acidic/basic additive added to the organic modifiers. The
influence of individual variables on enantioresolution, analysis time
and peak asymmetry was evaluated. Finally, the feasibility of use of
volatile MS friendly additives in chiral screening and the influence
of injection solvent were evaluated as well.
2. Experimental
2.1. Chemicals and reagents
Reference standards of pharmaceuticals including neutrals and
bases (alaptide, aprepitant intermediate, atomoxetine, boc-
saxagliptin, cinacalcet, darifenacin, epinephrine, ezetimibe, feso-
terodine, fesoterodine intermediate, indacaterol, maraviroc, mar-
aviroc intermediate ester, tamsulosin, tolterodine, zolmitriptan)
and acids (ambrisentan, cetirizin, ibuprofen, ketoprofen) were
provided by Zentiva a.s., (Prague, Czech Republic) and Sigma
Aldrich (Prague, Czech Republic), respectively. Except for aprepi-
tant, epinephrine and indacaterol which were at the disposal only
in a racemate form, all pharmaceuticals were available in both R
and S configuration, for boc-saxagliptin in S,S,S,S and R,R,R,R and
for ezetimibe in R,S,S and S,R,R respectively.
Methanol, ethanol, isopropanol, acetonitrile, heptane HPLC
gradient grade and tetrahydrofuran HPLC grade were provided by
Sigma Aldrich (Prague, Czech Republic). Mobile phase additives
including diethylamine (99.5%), trifluoroacetic acid (99%), ammo-
nium acetate (>99.99%) and ammonium formate (>98%) were
purchased from Sigma Aldrich (Prague, Czech Republic).
2.2. Ultra-high performance supercritical fluid chromatography
The supercritical fluid chromatography system Acquity UPC2
(Waters, Prague, Czech Republic) consisted of Acquity UPC2 binary
solvent manager, Acquity UPC2-FL sample manager, Acquity UPC2
convergence manager, Acquity column manager and Acquity UPC2
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L. Nova , M. Dousa / Analytica Chimica Acta 950 (2017) 199e210 201

PDA detector.
All injected sample solutions were stored in the autosampler at
4  C. The partial loop with needle overfill mode was set up to inject
10 mL. Methanol was used as a needle wash solvent. Gradient
elution was performed using CO2 (>99.995%, LindeGas, Hradec
Kralove, Czech Republic) and various combinations of modifiers
(methanol, ethanol, isopropanol and acetonitrile) and additives
(trifluoroacetic acid, diethylamine, ammonium acetate, ammonium
formate) at a flow-rate of 3.0 mL/min. Gradient program started at
5% of organic modifier and was linearly increased up to 30% in
3 min. An isocratic step was kept at 30% of organic modifier for
1 min followed by column equilibration (2 min). The influence of
temperature was evaluated in the range of 10e60  C. The BPR
(back-pressure regulator) pressure was set to 1500 psi and its
variations were evaluated in the range of 1500e2500 psi. UV
detection by means of PDA detector was performed at extracted
wavelength corresponding to the absorption maximum of indi-
vidual tested compounds.
The separation was performed using polysaccharide based chi-
ral stationary phases including: Lux 3u Amylose-2: coated amylose
tris(5-chloro-2-methylphenylcarbamate), provided by Phenom-
enex (Chromservis, Prague, Czech Republic), Kromasil 3-Amycoat:
coated amylose tris(3,5-dimethylphenylcarbamate), provided by
Kromasil (Chromservis, Prague, Czech Republic), Lux 3u Cellulose-
1: coated cellulose tris(3,5-dimethylphenylcarbamate), provided
by Phenomenex (Chromservis, Prague, Czech Republic), Lux 3u
Cellulose-2: coated cellulose tris(3-chloro-4-
methylphenylcarbamate), provided by Phenomenex (Chromservis,
Prague, Czech Republic) and Chiralpak IC-3: immobilized cellulose
tris(3,5-dichlorophenylcarbamate), provided by Daicel (Illkirch
Cedex, France). All columns in this study had dimensions of
150  4.6 mm and 3 mm particles.
2.3. Standard solutions
The stock standard solutions of the pharmaceuticals were pre-
pared in tetrahydrofuran due to its good compatibility with SFC
mobile phases and overall good solubility for the selected set of
compounds. Less soluble compounds including alaptide, atom-
oxetine, darifenacin, epinephrine, indacaterol and cetirizine were
dissolved in a mixture of tetrahydrofuran/methanol (9:1). Tamsu-
losin was not soluble at all in tetrahydrofuran, therefore the stock
solution was prepared in methanol. The concentrations of the stock
solutions were 1 mg/mL. Stock solutions were further diluted with
tetrahydrofuran in order to obtain a mixture of both enantiomers at
a concentration of 0.1 mg/mL, which was used for the injection into
SFC system for SST measurements. For screening purposes, a con-
centration of 0.3 mg/mL was used for an active isomer for faster and
easier identification of the isomers. In case of doubts individual in-
jections of each isomer were performed to confirm the peak identity.
3. Results and discussion
3.1. Selection of stationary and mobile phases for fast SFC screening
Selection of correct stationary phase for chiral separation still
remains challenging both in LC and SFC techniques. Based on
research previously performed in SFC and LC screening approaches
[11e13,21e25,38] and based on practical experience with chiral
separations, five the most promising stationary phases were
selected for the initial screening and subsequent comparison. These
included cellulose tris(3,5-dimethylphenylcarbamate), amylose
tris(3,5-dimethylphenylcarbamate) and chlorinated phases
including cellulose tris(3-chloro-4-methylphenylcarbamate),
amylose tris(5-chloro-2-methylphenylcarbamate) and finally cel-
lulose tris(3,5-dichlorophenylcarbamate). The group of analytes
was selected in order to reflect the current state at the drug market
including acidic, basic and neutral analytes and being both APIs and
pharmaceutical intermediates that have to be subjected to chiral
purity control. Table 1 shows basic physico-chemical properties of
the tested chiral compounds. Log P values reveal, that the range of
polarity was quite large (0.06e6.19). Most of the tested com-
pounds had basic character which also corresponds to the typical
situation at the drug market.
Following the state-of-the art of SFC separations and in agree-
ment with the previous findings, the first comparison was made
using CO2/methanol þ0.1% TFA for the analysis of acidic compounds
and CO2/methanol þ0.1% DEA for basic and neutral compounds.
Two comparative mobile phases were made by a combination of
0.1% DEA and 0.1% TFA added simultaneously first to methanol/CO2
and secondly to isopropanol/CO2. In contrast to previously
described technical problems with the system stability described in
Ref. [33] when using a combination of 0.5% TFA and 0.5% IPA in
methanol/CO2, similar issues have never been observed through

Table 1
Physico-chemical properties of chiral pharmaceuticals used in this study.

Compound Molecular formula Molecular weight (g/mol) log P pKa acidic pKa basic Detection wavelength (nm) Acid-base characteristic Active enantiomer

Alaptide C9H14N2O2 182.22 0.06 e e 210 N S

Ambrisentan C22H22N2O4 378.42 2.82 0.97 2.84 220 A S
Aprepitant int C21H19F6O3N 447.37 5.05 e e 220 N R
Atomoxetin C17H21NO 255.35 3.36 e 10.15 220 B R
Boc-saxagliptin C19H26N2O2 314.42 1.12 14.74 7.58 215 B S,S,S,S
Cetirizine C21H25ClN2O3 388.89 0.86 3.60 7.79 230 Z R
Cinacalcet C22H22F3N 357.41 6.19 e 9.19 220 B R
Darifenacin C28H30N2O2 426.55 3.79 15.70 9.32 220 B S
Epinephrine C9H13NO3 183.20 0.43 9.69 8.91 220 B R
Ezetimibe C24H21F2NO3 409.43 3.96 9.72 0.20 230 N R,S,S
Fesoterodine C26H37NO3 411.58 4.25 14.27 10.60 220 B R
Fesoterodine int C22H31NO2 341.49 3.43 9.58 10.82 220 B R
Ibuprofen C13H18O2 206.29 3.84 4.85 e 220 A R,S
Indacaterol C24H28N2O3 392.49 4.09 8.68 0.90 260 B R
Ketoprofen C16H14O3 254.28 3.61 3.88 e 250 A R,S
Maraviroc C29H41F2N5O 513.67 5.30 14.80 10.24 215 B S
Maraviroc int ester C9H11NO2 165.18 0.93 e 6.70 215 N S
Tamsulosin C20H28N2O5S 408.51 2.14 10.08 8.78 220 B R
Tolterodine C22H31NO 325.49 5.23 10.13 10.68 220 B R
Zolmitriptan C16H21N3O2 287.36 0.46 12.57 9.52 220 B S

Note: Acid-base characteristic: A eacid, B e base, N eneutral, Z - zwitterionic.

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the course of this study despite using also higher concentrations of was the different enantioselectivity provided by the two organic
the additives later in the method optimization phase. This may be modifiers with the combined TFA/DEA additive, which is shown in
attributed to a better solubility of TFA-DEA salt complexes Fig. 2 for the two columns providing the best results. Thus, putting
compared to TFA-IPA salt. TFA-DEA salt is formed in the ratio 1:1, the two conditions together provided very good success separation
while in case of TFA-IPA it may be 2:1 leading to worse salt solu- rate (18 enantiomeric pairs out of 20 at least partially resolved
bility in CO2/methanol mobile phases. using these four conditions). To complete this set of experiments,
All the screening experiments were performed in quick gradient further analyses were performed also with CO2/ethanol þ0.1% DEA
elution (3 min þ 1 min isocratic hold) described in Section 2.2, and 0.1% TFA on Amycoat and Cellulose 1 stationary phases to
which substantially increased the throughput of the screening verify, if there was not some interesting enantioselectivity missed.
phase compared to the isocratic elution. Longer separation time The results showed, that no additional enantioselectivity was
(6 min þ 1 min isocratic hold) was also tested in the preliminary observed compared to CO2/methanol and CO2/isopropanol with the
experiments to verify if the enantioresolution was not compro- combined TFA/DEA additive. The selectivity of this CO2/ethanol-
mised due to the short analysis times. The impact of the time on
enantioseparation was negligible, thus short gradient time of 3 min
was selected.
The comparison of the success rate of the enantioseparation
using single and combined additives on all five tested stationary
phases is shown in Fig. 1. In this presentation completely separated
peaks as well as partially separated peaks are taken as “successful
separation”, because method optimization is considered as
following step, if necessary. Generally, combined TFA/DEA additive
in methanol/CO2 (blue column in Fig. 1) provided always at least the
same, but generally better results compared to single additives in
methanol/CO2 (black column). This improvement could be both
improvement of partial separation to complete baseline separation
or non-resolved enantiomeric pair obtained in case of single ad-
ditives while the separation was obtained with the combined TFA/
DEA additive. The combined TFA/DEA additive added to iso-
propanol/CO2 demonstrated overall lower success rate in the
enantioseparation. However, its benefit was different and often
complementary enantioselectivity compared to methanol with the
combined TFA/DEA additive, especially on Amycoat stationary
phase. Overall, the best enantioselectivity was obtained on Amy-
coat and on Cellulose 1 columns when using CO2/methanol þ0.1%
DEA and 0.1% TFA leading to at least partial separation of 60% of
enantiomeric pairs in both cases (12 out of 20). The second best Fig. 2. Comparison of the success-rate of enantioseparation of individual compounds
enantionselectivity was observed on Amycoat column when using for Cellulose 1 (blue) and Amycoat (black/gray) stationary phases using CO2/methanol
CO2/isopropanol þ0.1% DEA and 0.1% TFA providing separation for and CO2/isopropanol with the combined TFA/DEA additive. Successful separation (full
55% of enantiomeric pairs. Interestingly, both stationary phases separations and partial separations) is represented by the columns above the x-axis
(þ), while non-successful separation is represented by the columns bellow the x-axis
contained 3,5-dimethylphenylcarbamoyl functional groups, either
(). (For interpretation of the references to colour in this figure legend, the reader is
on amylose or on cellulose. As already mentioned, a great benefit referred to the web version of this article.)

Fig. 1. Comparison of the success-rate of enantioseparation of the 20 tested compounds on the five tested polysaccharide-based stationary phases in gradient chiral screening. CO2/
methanol with the addition of 0.1%DEA was used for the analysis of basic and neutral compounds and 0.1% TFA for the analysis of acidic compounds. Comparison was made with
CO2/methanol and CO2/isopropanol with a combined additive composed of 0.1% TFA and 0.1% DEA.
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L. Nova , M. Dousa / Analytica Chimica Acta 950 (2017) 199e210 203

based mobile phase was mostly similar to that of CO2/methanol enantioselectivity.

with the combined TFA/DEA additive.
Graphical presentation in Fig. 2 reveals straightforward and
challenging separations. There are several compounds (e.g. alap-
tide, boc-saxagliptin, cinacalcet and few others), for which 3e4
different conditions out of the 4 provided at least partial separation.
On the other hand, challenging analytes (e.g. epinephrine, feso-
terodine int. and cetirizine) have shown only one successful sepa-
ration providing less flexibility for further method optimization.
There were only two compounds which did not provide any sep-
aration at all in the initial screening phase at selected first-line
screening conditions e tolterodine (no enantioresolution at all)
and ambrisentan (resolved on Cellulose 2). In the light of these
results the recommendation for the initial chiral screening in
UHPSFC would be a combination of cellulose tris(3,5-
dimethylphenylcarbamate) and amylose tris(3,5-
dimethylphenylcarbamate) using CO2/methanol and CO2/iso-
propanol with the combined additive composed of 0.1% DEA and
0.1% TFA. The third ranked stationary phase was Cellulose 2 (cel-
lulose tris(3-chloro-4-methylphenylcarbamate)) using CO2/iso-
propanol with the combined TFA/DEA additive and Chiralpak IC3
(cellulose tris(3,5-dichlorophenylcarbamate)) using CO2/methanol
with the combined TFA/DEA additive providing the same success
rate of separation (45%, 9 pairs out of 20) in the screening phase.
These stationary phases are further options for fine-tuning
3.2. Detailed optimization of SFC chiral methods
Effective screening approach enabled by gradient elution and
selection of a good combination of stationary and mobile phases
enabled to obtain full enantioseparation for some compounds
already in the screening phase. In this study strict criteria were
defined for the separation of the two enantiomers with the respect
to the real-life situation. When the enantiopurity control is per-
formed, typical limit for a chiral impurity is 0.15% relative to API. In
the real-life situation this results in very wide peak of API often
leading to strong tailing due to the column overload. Therefore, the
criteria for resolution (Rs) was set-up to 2.0 rather than commonly
accepted 1.5, which would not be sufficient in the above described
case if the impurity is eluted right after the API. If the criteria of
Rs  2.0 was met, the system suitability test (SST) measurement
was performed in order to verify method repeatability (Table 2). If
the resolution was lower and also in case of partial separations,
where the resolution could not have been calculated, further
optimization followed.
Complete enantioseparation was obtained for several com-
pounds already during the screening step up to a different extent
on the five tested stationary phases. This success rate corresponded
to the overall success rate of the columns shown in Fig. 1. Thus, for

Table 2
Final analytical conditions (stationary phase, mobile phase, gradient elution (G) or isocratic elution (ISO)) for both screening phase optimal separations and finely optimized
separations of all compounds. Retention times, repeatability and resolution are also shown.

tR1 tR2 RSD tR1 [%] RSD tR2 [%] RSD A1 [%] RSD A2 [%] RS Column Elution Mobile phase

Alaptide 3.224 (R) 3.529 (S*) 0.04 0.04 0.86 0.45 3.59 Amycoat G MeOH þ 0.1% TFA þ 0.1% DEA
3.008 (R) 3.697 (S*) 0.04 0.04 0.33 0.49 3.55 Amycoat ISO 20% MeOH þ 0.1% TFA þ 0.1% DEA
3.529 (S*) 4.538 (R) 0.06 0.05 0.82 0.40 8.97 Cellulose 2 G MeOH þ 0.1% TFA þ 0.1% DEA
2.212 (S*) 3.474 (R) 0.04 0.03 0.29 0.33 8.20 Cellulose 2 ISO 25% MeOH þ 0.1% TFA þ 0.1% DEA
Aprepitant int. 1.184 1.623 0.08 0.05 0.77 0.32 3.77 Cellulose 2 ISO 15% 2-Pr-OH þ 0.1% TFA þ 0.1% DEA
1.918 2.269 0.03 0.02 0.95 0.27 3.86 Cellulose 2 G 2-Pr-OH þ DEA 0.1%
Atomoxetine 2.375 (S) 2.924 (R*) 0.02 0.01 0.15 0.23 8.55 Cellulose 1 G MeOH þ 0.1% TFA þ 0.1% DEA
1.204 (S) 1.826 (R*) 0.06 0.05 0.22 0.21 4.40 Cellulose 1 ISO 20% MeOH þ 0.1% TFA þ 0.1% DEA
3.928 (S) 4.138 (R*) 0.04 0.03 0.31 0.48 2.14 Cellulose 2 G 2-Pr-OH þ 0.1% TFA þ 0.1% DEA
3.638 (S) 4.796 (R*) 0.03 0.02 0.32 0.05 2.75 Cellulose 2 ISO 15% 2-Pr-OH þ 0.1% TFA þ 0.1% DEA
Boc-saxagliptin 2.583 (S*SSS) 3.236 (RRRR) 0.02 0.04 0.38 0.14 4.01 Amycoat G MeOH þ 0.1% TFA þ 0.1% DEA
0.911 (S*SSS) 1.312 (RRRR) 0.08 0.04 0.28 0.46 2.22 Amycoat ISO 30% MeOH þ 0.1% TFA þ 0.1% DEA
2.408 (S*SSS) 2.597 (RRRR) 0.02 0.02 0.22 0.16 2.50 Cellulose 1 G MeOH þ 0.1% TFA þ 0.1% DEA
1.772 (S*SSS) 2.180 (RRRR) 0.04 0.05 0.24 0.79 2.24 Cellulose 1 ISO 15% MeOH þ 0.1% TFA þ 0.1% DEA
Cinacalcet 2.147 (S) 2.682 (R*) 0.06 0.05 0.16 0.20 7.60 Amycoat G 2-Pr-OH þ 0.1% TFA þ 0.1% DEA
1.639 (S) 2.806 (R*) 0.05 0.06 0.24 0.36 6.49 Amycoat ISO 13% 2-Pr-OH þ 0.1% TFA þ 0.1% DEA
2.385 (R*) 2.530 (S) 0.02 0.02 0.10 0.12 2.00 Cellulose 1 G MeOH þ 0.1% TFA þ 0.1% DEA
3.080 (R*) 3.604 (S) 0.04 0.05 0.20 0.18 2.75 Cellulose 1 ISO 10% MeOH þ 0.1% TFA þ 0.1% DEA
Darifenacin 4.716 (R) 5.840 (S*) 0.03 0.05 0.41 0.44 2.46 Cellulose 1 ISO 25% 2-Pr-OH þ 0.1% TFA þ 0.1% DEA
2.637 (S*) 3.368 (R) 0.10 0.09 0.21 0.17 2.95 Amycoat ISO 25% 2-Pr-OH þ 0.1% TFA þ 0.1% DEA
3.149 (R) 5.030 (S*) 0.05 0.08 0.96 0.79 6.55 Amycoat ISO 35% 2-Pr-OH þ 0.1% TFA þ 0.5% DEA
Epinephrine 4.867 5.960 0.08 0.09 0.23 0.31 2.82 Cellulose 2 ISO 13% MeOH þ 0.1% TFA þ 0.1% DEA
Ezetimibe 3.577 (RSS*) 4.098 (SRR) 0.09 0.12 0.22 0.43 4.51 Amycoat G MeOH þ 0.1% TFA þ 0.1% DEA
2.261 (RSS*) 3.218 (SRR) 0.04 0.03 0.24 0.34 3.90 Amycoat ISO 23% MeOH þ 0.1% TFA þ 0.1% DEA
Fesoterodine 5.875 (R*) 6.792 (S) 0.08 0.07 0.42 0.46 2.27 Cellulose 1 ISO 15% 2-Pr-OH þ 0.1% TFA þ 0.1% DEA
Fesoterodine int 6.949 (R*) 8.206 (S) 0.07 0.11 0.27 0.45 2.13 Amycoat ISO 17% 2-Pr-OH þ 0.1% TFA þ 0.1% DEA
Indacaterol 3.459 5.010 0.12 0.18 0.49 0.57 2.86 Amycoat ISO 25% MeOH þ 0.1% TFA þ 0.5% DEA
Maraviroc 5.346 (S*) 6.405 (R) 0.07 0.07 0.37 0.37 2.69 Cellulose 1 ISO 15% MeOH þ 0.1% TFA þ 0.1% DEA
Maraviroc int ester 2.128 (R) 2.476 (S*) 0.05 0.03 0.37 0.29 5.82 Chiralpak IC3 G MeOH þ 0.1% TFA þ 0.1% DEA
1.916 (R) 2.035 (S*) 0.04 0.02 0.50 0.28 2.36 Cellulose 1 G MeOH þ 0.1% TFA þ 0.1% DEA
2.647 (R) 2.815 (S*) 0.03 0.02 0.59 0.53 1.45 Cellulose 2 G MeOH þ 0.1% DEA
2.506 (S*) 2.698 (R) 0.03 0.04 0.39 0.65 2.84 Cellulose 2 G MeOH þ 0.1% TFA þ 0.1% DEA
Tamsulosin 8.508 (R*) 10.094 (S) 0.06 0.10 0.34 0.53 2.33 Amycoat ISO 20% 2-Pr-OH þ 0.1% TFA þ 0.1% DEA
Tolterodine 11.109 (S) 12.365 (R) 0.31 0.29 0.56 0.85 1.25 Amycoat ISO 5% MeOH þ DEA 0.5%
Zolmitriptan 3.940 (S*) 4.765 (R) 0.03 0.03 0.33 0.52 2.49 Cellulose 2 ISO 27% MeOH þ 0.1% TFA þ 0.1% DEA
3.717 (R) 4.668 (S*) 0.02 0.04 0.10 0.10 2.51 Amycoat ISO 17% MeOH þ 0.1% TFA þ 0.1% DEA
Ambrisentan 2.615 (S,S*) 3.201 (R,R) 0.05 0.06 0.32 0.40 3.07 Cellulose 2 ISO 12% MeOH þ 0.1% TFA þ 0.1% DEA
Cetirizine 3.836 (S) 4.038 (R*) 0.01 0.01 0.10 0.31 2.22 Cellulose 1 G MeOH þ 0.1% TFA þ 0.1% DEA
4.132 (S) 4.762 (R*) 0.04 0.04 0.19 0.13 2.30 Cellulose 1 ISO 20% MeOH þ 0.1% TFA þ 0.1% DEA
Ibuprofen 5.460 (S*) 5.940 (R) 0.06 0.24 0.30 0.53 2.08 Chiralpak IC3 ISO 2.5% MeOH:ACN (1:1) þ 0.1% TFA þ 0.1% DEA
Ketoprofen 8.375 (S*) 9.109 (R) 0.12 0.11 0.45 0.43 2.25 Chiralpak IC3 ISO 8% MeOH:ACN (1:1) þ 0.1% TFA þ 0.1% DEA
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Cellulose 1 and Amycoat columns 5 compounds and for Cellulose 2
and Chiralpak IC3 columns 3 compounds provided complete
baseline separation with Rs  2 during the screening step. Some
examples are shown in Fig. 3 for boc-saxagliptin, cinacalcet and
maraviroc intermediate ester showing two different gradient con-
ditions providing successful enantioseparations. In total, 8 com-
pounds could be completely resolved in the screening phase using
some of the 5 stationary phases, see Table 2. Among these 8 com-
pounds 7 could be completely resolved on the two stationary
phases proposed for the first-line screening. These compounds
belonged among less challenging compounds based on the
graphical representation in Fig. 2, except for cetirizine. It is partic-
ularly interesting to mention the cases, where the change of elution
order of the two enantiomers can be observed between the two
stationary phases, such as in case of cinacalcet and maraviroc in-
termediate ester. In enantiomeric purity control when the minor
enantiomeric impurity has to be analyzed in the presence of the
major enantiomer, it is more desirable to elute the minor compo-
nent before the major one. This usually allows for more sensitive
and reproducible quantification of the relevant enantiomeric im-
purity [39,40].
The separations of enantiomeric pairs with Rs < 2 required
further method optimization. Fine tuning parameters, such as
temperature, BPR pressure, gradient slope and change in additive
concentration were considered on the stationary phases, which
provided at least partial separation. The influence of the tempera-
ture was tested in the range from 10 to 60  C. Increasing the tem-
perature reduces the mobile phase density, which results in higher
retention factors. This implies that the resolution of chiral separa-
tions may improve at lower temperatures. However, the tempera-
ture effect is not always so straightforward, since it can also affect
the analyte affinity for the stationary phase hereby influencing the
enantioselectivity [41e43]. In agreement with some previous re-
ports, the influence of the temperature on the enantioseparation in
SFC was very limited also in this study. As a result, in the tested set
of 20 analytes the change in temperature did not improve any
enantioseparation in the tested set of compounds, thus all the an-
alyses were further performed at 40  C. Similarly, fine tuning of BPR
pressure (1500e2500 psi) and change in gradient slope did not lead
to any improvement of the enantioselectivity for any of tested
enantiomeric pairs. The former conclusion is also in agreement
with the previously published report [44], which observed the in-
fluence of the pressure on the enantioresolution to be up to 6%. It
clearly indicates, that this parameter can't be successfully used as
an important parameter in method optimization.
Further testing included the use of partially successful gradient
screening conditions (typically Amycoat or Cellulose 1 stationary
phase with CO2, both tested modifiers and combined TFA/DEA
additive) in the isocratic mode. However, substantially lower
amount of organic modifier was applied compared to the gradient
elution. This way a complete baseline separation with the Rs  2 for
aprepitant intermediate, darifenacin, epinephrine, fesoterodine
and its intermediate, maraviroc, tamsulosin, zolmitriptan and
ambrisentan were obtained. Detailed results are shown in Table 2
and selected chromatograms in Fig. 4A. Due to the success of this
approach, isocratic conditions were additionally applied also for
compounds, which were successfully separated already in the
screening phase to show the differences and to see the potential for
further possibility to decrease the analysis time and eliminate the
equilibration step in final QC method. Some examples are shown in
Fig. 4B. The same compounds were selected to show the difference
between gradient elution (Fig. 3) and isocratic elution. Indeed, very
fast separations, sometimes between 2 and 4 min were obtained for
some enantiomeric pairs, see Table 2. However, for some com-
pounds, such as maraviroc intermediate ester, longer analysis time
was needed in isocratic mode, thus the gradient method would be
still the most convenient one also in the final QC method. Separa-
tions of more challenging compounds took typically 5e10 min after

Fig. 3. The chromatograms of the gradient SFC screening experiments for selected analytes e boc-saxagliptin, cinacalcet and maraviroc intermediate-ester and the structures of the
analytes. Right: Amycoat column - boc-saxagliptin and cinacalcet, Chiralpak IC3 - maraviroc intermediate. Left: Cellulose 1 for boc-saxagliptin and cinacalcet, Cellulose 2 for
maraviroc intermediate. The active isomers is depicted with asterisk. Detailed conditions are given in Table 2.
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L. Nova , M. Dousa / Analytica Chimica Acta 950 (2017) 199e210 205

Fig. 4. Chromatograms of SFC enantioseparations fine-tuned using isocratic elution: (A) maraviroc on Cellulose 1 using CO2/methanol and TFA/DEA combined additive, darifenacin
on Cellulose 1 and tamsulosin on Amycoat column using CO2/isopropanol and TFA/DEA combined additive. (B) boc-saxagliptin and cinacalcet on Cellulose 1 using CO2/methanol and
TFA/DEA combined additive and cinacalcet on Amycoat column using CO2/isopropanol and TFA/DEA combined additive. The active isomer is depicted with asterisk. Detailed
conditions are given in Table 2.

the fine method tuning (Table 2). approach has an important influence only for some analytes, while
Another option to tune the enantioselectivity is the change in in other cases the resolution is influenced only slightly as it is
the additive concentration. It is important to note, that this shown for zolmitriptan separation on Amycoat column in Fig. 5.

Fig. 5. Effect of the amount of organic modifier and the concentration of DEA on the enantioseparation of zolmitriptan on Amycoat column. (A) CO2/methanol with 0.1% TFA/DEA
combined additive using 25, 20 and 15% or organic modifier. (B) CO2/methanol with increased concentration of DEA (0.5%) in the combined TFA/DEA additive using 25, 20 and 15% of
organic modifier. The active isomer is depicted with asterisk.
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Fig. 6. Effect of the amount of organic modifier and the concentration of DEA on the enantioseparation of darifenacin on Amycoat column. (A) CO2/methanol and (C) CO2/iso-
propanol with 0.1% TFA/DEA combined additive showing the separation at 25 and 20% of organic modifier. (B) CO2/methanol and (D) CO2/isopropanol with increased concentration
of DEA (0.5%) in the combined TFA/DEA additive showing the separation at 25, 20 and 35% of organic modifier, respectively. The active isomer is depicted with asterisk.

Due to basic properties of this molecule combined TFA/DEA addi-
tive in CO2/methanol at 0.1% concentration and the combined TFA/
DEA additive with increased concentration of DEA (0.5%) were
compared. An increase in DEA concentration lead to substantially
higher retention of both isomers and to slightly increased enan-
tioresolution (Fig. 5). However, easier way to manipulate the
enantioresolution was changing the amount of the organic modi-
fier. Nevertheless, even slight change might sometimes be impor-
tant to meet the strict requirements of regulations. Similar
approach, i. e. increase in DEA concentration to 0.5% in a combined
additive, was successfully applied for the analysis of indacaterol on
Amycoat column (data not shown) enabling to obtain complete
enantioresolution. Absolutely different behavior was observed in
case of darifenacin separation on Amycoat column (Fig. 6). When
increasing the concentration of DEA (0.5%) in the combined TFA/
DEA additive in CO2/methanol, the enantioresolution was
completely lost (Fig. 6B), while it was around 1.6 at the concen-
tration 0.1% of combined TFA/DEA additive (Fig. 6A). Even more
interesting behavior was observed when CO2/methanol was
replaced with CO2/isopropanol. The complete baseline enantior-
esolution with Rs  2 was obtained for both combined TFA/DEA
0.1% and 0.5% concentrations in CO2/isopropanol (Fig. 6C and D).
However, the elution order of the enantiomers reversed, which was
quite surprising considering quite a small concentration change.
This kind of behavior was not typical and was not found to be
equally effective in other cases.
The last three remaining compounds, tolterodine, ibuprofen and
ketoprofen belong among the most challenging compounds in this
set of analytes. Only in case of tolterodine, which was also the
compound showing no separation in the screening phase, the strict
requirement for Rs  2 was not met. Nevertheless, baseline sepa-
ration was finally obtained when using CO2/methanol with 0.5%
DEA. This option was considered in the light of the above described
experiments and a broadening not yet splitting peak shape
observed on Amycoat column providing the clue that the two iso-
mers may separate. Ketoprofen and ibuprofen demonstrated partial
separation when using CO2/methanol with TFA or combined TFA/
DEA additive on Amycoat column and on Chiralpak IC3. This sep-
aration seemed to be quite straightforward. However, fine tuning
through isocratic elution or change in additive concentration, both
using TFA and combined TFA/DEA additive, was not successful in
this case. Another option to change the selectivity is the use CO2/
combined organic modifiers, such as mixtures of alcohols and
acetonitrile, different alcohols or others. Both separations were
finally enabled using isocratic elution with CO2/methanol:acetoni-
trile (1:1) and the combined TFA/DEA additive. All results are
shown in Table 2.
3.3. MS compatibility of the chiral SFC screening method
Coupling of separation techniques with mass spectrometry (MS)
is important for the enhancement of specificity, sensitivity and
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L. Nova , M. Dousa / Analytica Chimica Acta 950 (2017) 199e210 207

Fig. 7. A comparison of enantioselectivity of CO2/methanol mobile phases with volatile additives ammonium formate (AmF), ammonium acetate (AmAc) and the combined TFA/
DEA additive on Cellulose 1 (A) and Amycoat (B) column. Successful separation (full enantioresolution or partial separations) is represented by the columns above the x-axis (þ),
while non-successful separation is represented by the columns bellow the x-axis ().

facilitation of compound identification. While it is already quite ammonium formate or ammonium acetate, in chiral SFC separa-
common in achiral SFC separations, there is limited information tions [45] and they have never been employed for chiral screening.
published so far on the use of MS friendly buffer additives, such as Therefore, MS compatible additives ammonium acetate and

Fig. 8. Changes observed in the enantioresolution when the combined 0.1% TFA/DEA additive was replaced with volatile additives in CO2/methanol mobile phases. Separation of
cinacalcet was performed on Cellulose 1 column using CO2/methanol with the addition (A) combined 0.1% TFA/DEA additive, (B) 20 mM ammonium formate and (C) 20 mM
ammonium acetate. Separation of maraviroc was performed on Amycoat column using CO2/methanol with the addition (D) combined TFA/DEA additive, (E) 20 mM ammonium
formate and (F) 20 mM ammonium acetate.
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Fig. 9. Effect of the injection solvent on the peak shape of atomoxetine (elution order S,R*) in gradient (A) and isocratic (B) elution using following injection solvents: heptane/
isopropanol (9:1, black, a reference in all presented chromatograms), acetonitrile (green), tetrahydrofuran (dark blue), methanol (red), ethanol (light blue) and isopropanol (violet).
Injection volume was 10 ml on the Cellulose 1 column using mobile phase composed of CO2 and methanol with the combined 0.1% TFA/DEA additive in the gradient mode (5e30% in
3 min) and in the isocratic mode (20% of organic modifier). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this
article.)

ammonium formate at a concentration 20 mM added to CO2/
methanol were further tested on Amycoat and Cellulose 1 sta-
tionary phases to verify the suitability of these additives for the
enantioseparation. The results in Fig. 7A demonstrate that the
change in enantioselectivity was very small on Cellulose 1 column.
Only the enantioresolution of cinacalcet (Fig. 8) and tamsulosine
was lost using whatever of the two buffers, while maraviroc in-
termediate ester was not separated only when using ammonium
acetate. The additive comparison results were different on Amycoat
column (Fig. 7B). The loss of enantioresolution was found for
darifenacin, epinephrine and indacaterol using both volatile
buffers. On the other hand, on this stationary phase also some
enantioresolution improvements were observed when replacing
the combined TFA/DEA additive in CO2/methanol mobile phase
with the volatile ones. This improvement was observed for cetir-
izine and maraviroc (Fig. 8) with both buffers. These experiments
reveal that volatile additives can also be successfully employed in
chiral screening, but generally at the cost of loss of enantionse-
lectivity for some compounds. As the results are quite encouraging
and to the best of our knowledge, no reports using volatile
ammonium acetate and ammonium formate in SFC chiral screening
have been published so far, further research is currently being
performed on this topic in our laboratory.
3.4. Injection solvent for SFC chiral methods
mode. Tested solvents included methanol, isopropanol, ethanol,
acetonitrile, tetrahydrofuran and a mixture of heptane/isopropanol
(9:1), which is recommended as the best solvent for SFC due to the
close properties to SFC mobile phase. However, this dilution solvent
is non-polar and many pharmaceutical compounds are not well
soluble in such solvent. Therefore, some compromise has to be
found. The choice of the solvent for the injection was found to be
more critical in the isocratic mode than in the gradient elution, as it
is shown in Fig. 9 for atomoxetine. As expected, alcohol solvents
compromised the peak shape with the worst tendency for meth-
anol. In the gradient elution a partial peak splitting was observed
for the first eluted enantiomer, while only band broadening was
observed for the second enantiomer and for both enantiomers in
the isocratic elution. Tetrahydrofuran and acetonitrile were found
to provide the best peak shapes in the gradient mode, where the
results were comparable to the mixture of heptane/isopropanol
(9:1), Fig. 9A. In isocratic elution some band broadening was
observed even when using these two solvents and this phenome-
non was more important for acetonitrile than for tetrahydrofuran
(Fig. 9B). When tetrahydrofuran was combined with alcohols, such
as methanol or ethanol, some peak broadening was still observed in
both isocratic and gradient modes (data not shown). Based on these
results, tetrahydrofuran was chosen as a good compromise in this
study and provided overall good peak shapes for all 20 tested
analytes.

The importance of injection solvent in SFC have already been 4. Conclusion

widely discussed in the previously published works [19,20,46,47].
For this reason a set of experiments was performed also on this Fast and efficient SFC general screening separation approach
topic. Several solvents typical for SFC were tested using two probe enabled by quick gradient elution was developed. The screening
analytes, atomoxetine and cinacalcet in both isocratic and gradient analysis took only 4 min followed with 2 min of column
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L. Nova , M. Dousa / Analytica Chimica Acta 950 (2017) 199e210
equilibration. Higher enantioselectivity compared to the conven-
tional screening approaches using single acidic and basic additives
was obtained when using the combined additive composed of 0.1%
TFA and 0.1% DEA. Another advantage of the combined additive was
no need for compound classification. This additive was systemati-
cally added to CO2/methanol or CO2/isopropanol mobile phases.
Using the combination of these two mobile phases, 18 enantiomeric
pairs out of 20 including all, acidic, basic and neutral structures,
were at least partially resolved already in the screening phase using
two stationary phases, Cellulose 1 and Amycoat. Both stationary
phases contained the same 3,5-dimethylphenylcarbamoyl func-
tional groups. The first-line screening step thus included testing of
two stationary phases and two mobile phases, which fully meets
the criteria of limited number of experiments needed for fast
method development. If these separations were not successful,
further screening should be performed using cellulose tris(3-
chloro-4-methylphenylcarbamate) and subsequently cellulose
tris(3,5-dichlorophenylcarbamate) with the same mobile phases.
While temperature, BPR pressure and gradient slope changes were
found ineffective in the fine method optimization step, change in
the concentration of additive, combined organic modifiers, such as
methanol/acetonitrile (1:1) or use of isocratic elution at lower
concentration of organic modifier were effective tools to achieve
requested enantioresolution. SST results for these optimized
methods demonstrated very good repeatability for both retention
times and peak area. When testing volatile buffers, ammonium
formate and ammonium acetate, at the place of the combined TFA/
DEA additive, quite encouraging results were obtained showing
that only some enantioselectivity was lost during the screening
phase.
Acknowledgement
The authors gratefully acknowledge research projects of Charles
University in Prague UNCE 204026/2012.
Appendix A. Supplementary data
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.aca.2016.11.002.
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