Further study on enantiomer resolving ability of amylose tris(3-chloro-5-methylphenylcarbamate) covalently immobilized onto silica in nano-liquid chromatography and capillary

Journal of Chromatography A 1623 (2020) 461213

Contents lists available at ScienceDirect

Journal of Chromatography A
journal homepage: www.elsevier.com/locate/chroma

Further study on enantiomer resolving ability of amylose
tris(3-chloro-5-methylphenylcarbamate) covalently immobilized onto
silica in nano-liquid chromatography and capillary
electrochromatography
Giovanni D’Orazio a, Chiara Fanali b, Salvatore Fanali c,∗, Alessandra Gentili d,
Marina Karchkhadze e, Bezhan Chankvetadze e
a

Istituto per i Sistemi Biologici (ISB), CNR- Consiglio Nazionale delle Ricerche, Via Salaria Km 29,300 – 00015 Monterotondo (Rome), Italy
Department of Science and Technology for Humans and the Environment, University Campus Bio-Medico of Rome, Via Alvaro del Portillo 21, 00128 Rome,
Italy
c
Teaching Committee of Ph.D. School in Natural Science and Engineering, University of Verona, Strada Le Grazie, 15 – 37129 Verona, Italy
d
Department of Chemistry “Sapienza” University of Rome, P.le Aldo Moro 5, 00185, Rome, Italy
e
Institute of Physical and Analytical Chemistry, School of Exact and Natural Sciences, Iv. Javakhishvili Tbilisi State University, Chavchavadze Ave 3, 0179
Tbilisi, Georgia
b

a r t i c l e

i n f o

Article history:
Received 26 March 2020
Revised 2 May 2020
Accepted 5 May 2020
Available online 8 May 2020
Keywords:
Amylose
tris(3-chloro-5-methylphenylcarbamate)
Capillary electrochromatography
Covalently immobilized
polysaccharide-based chiral stationary
phase
Enantioseparations
nano-Liquid Chromatography

a b s t r a c t
In the present study separation of enantiomers of some chiral neutral, basic and weakly acidic analytes was investigated on the chiral stationary phase (CSP) made by covalent immobilization of amylose tris(3-chloro-5-methylphenylcarbamate) onto aminopropylsilanized (APS) silica in nano-liquid chromatography (nano-LC) in aqueous methanol or acetonitrile mixtures. It has been shown that similar to
high-performance liquid chromatography (HPLC) and supercritical fluid chromatography (SFC) this chiral
selector is useful for separation of enantiomers of neutral, basic and acidic analytes also in nano-LC. In
comparison to our previous research, in which the chiral selector (CS) was bonded on native silica, in this
study, the CS was immobilized on APS silica in order to improve chromatographic performance towards
basic analytes. In fact, some improvement was observed and surprisingly not only for basic but also for
neutral and acidic analytes. Again, quite unexpectedly almost no electroosmotic flow (EOF) was observed
in capillaries packed with ca. 20% (w/w) amylose tris(3-chloro-5-methylphenylcarbamate) immobilized
onto APS silica although the same APS silica before attachment of chiral selector exhibited significant
EOF. In order to generate EOF in the capillaries with the CSP and enable capillary electrochromatographic
(CEC) experiment on it, the short segment of the capillary column was packed with APS silica without
chiral selector. The EOF in such capillary enabled CEC experiment and some preliminary results are reported here.
© 2020 Elsevier B.V. All rights reserved.

1. Introduction
Polysaccharide phenylcarbamates and esters are widely used
chiral selectors for separation of enantiomers in liquid phase separation techniques and among these also in nano liquid chromatography (nano-LC) and capillary electrochromatography (CEC)
[1-5]. As it has been shown in many studies the chiral recognition ability of polysaccharide derivatives strongly depends not only
on the type of polysaccharide but also on the pendant groups and
on the substituents on these pendant groups [6-10]. The phenyl
∗

Corresponding author.
E-mail address: (S. Fanali).

/>0021-9673/© 2020 Elsevier B.V. All rights reserved.

moiety on cellulose and amylose phenylcarbamates introduced by
Okamoto and co-workers in early 1980s was unsubstituted or
contained either electron-donating or electron withdrawing substituents [9,10]. In early 1990s Chankvetadze and co-authors have
introduced polysaccharide phenylcarbamate derivatives containing
both, electron-donating and electron-withdrawing substituents on
the phenyl moiety [11-14]. Some spectroscopic and chromatographic studies of these materials indicated their extended chiral
recognition ability and many of these derivatives became the part
of commercially available chiral packing materials and columns.
The synthesis of one of the powerful chiral selectors in this family, namely of amylose tris(3-chloro-5-methyphenylcarbamate) was
described in 1997 [14]. However, the packing materials and chiral


2

G. D’Orazio, C. Fanali and S. Fanali et al. / Journal of Chromatography A 1623 (2020) 461213

columns on its base became commercially available just in last few

years. In spite of short availability period the columns based on
amylose tris(3-chloro-5-methyphenylcarbamate) have been studied by several groups and its usefulness has been shown in
HPLC in combination with various mobile phases [15-26], as well
as in supercritical fluid chromatography (SFC) [27]. Recently we
have published a paper on applicability of amylose tris(3-chloro5-methyphenylcarbamate) covalently immobilized on native silica
for separation of enantiomers of neutral and acidic chiral analytes
in nano-LC and CEC [28]. In the present work, in order to improve
the peak shape of basic chiral analytes, the amylose tris(3-chloro5-methyphenylcarbamate) was covalently immobilized on APS silica and its applicability was studied in nano-LC separation of enantiomers of basic, neutral and acidic chiral analytes. In addition, the
attempt was made to use the same material for separation of enantiomers in CEC.
2. Experimental
2.1. Chemicals and materials
Methanol (MeOH), 2-propanol (2-PrOH), ammonia solution
(30%, w/w), glacial acetic acid (99.0%, w/w) and formic acid (99.0%,
w/w) (FA) were purchased from Carlo Erba (Rodano, Milan, Italy),
while ammonium hydrogen carbonate (NH4 HCO3 ≥ 99.0%, w/w)
was obtained from Sigma-Aldrich (St. Louis, MO, USA). Acetonitrile
of HPLC grade (ACN) and HPLC ultrapure water (filtered through
0.2 μm and packaged under nitrogen) were from VWR (International PBI S.r.l. Milan, Italy).
Racemic mixtures of flavanone (Fla), 4-methoxyflavanone
´
(4-MeO-Fla),
6-methoxyflavanone
(6-MeO-Fla),
7´
methoxyflavanone (7-MeO-Fla), 2-hydroxyflavanone
(2-OH-Fla),
´
´
4-hydroxyflavanone
(4-OH-Fla),

6-hydroxyflavanone (6-OH-Fla),
´
´
7-hydroxyflavanone (7-OH-Fla) and lorazepam, oxazepam, hexobarbital, temazepam, carbinoxamine, warfarin, and Tröger’s base
were obtained from Sigma-Aldrich. Diclofop, fenoxaprop, dichlorprop, haloxyfop, fluazifop (herbicides in the free acidic form) were
purchased from Dr. Ehrenstorfer GmbH (Augsburg, Germany).
Profenofos, dialifos, fenamiphos (organophosphorus pesticides)
were purchased from Riedel-de Haën (Seelze, Germany). The nonsteroidal anti-inflammatory drugs (NSAIDs) racemic indoprofen,
naproxen, carprofen, cicloprofen, flurbiprofen, suprofen, and the
single enantiomers of S(+)-flurbiprofen and S(+)-suprofen were
kindly provided by Dr. Cecilia Bartolucci (Institute of Crystallography, CNR, Monterotondo, Roma, Italy). Ketoprofen, ketorolac, and
ibuprofen were purchased from Sigma-Aldrich. Racemic thalidomide and its (-)-enantiomer were kindly provided by the Institute
of Pharmaceutical and Medicinal Chemistry, University of Münster,
Münster, Germany. Racemic standard basic compounds, namely
alprenolol, ambucetamide, bupivacaine, clenbuterol, metoprolol,
mianserin, nadolol, oxprenolol, pindolol, propranolol, tolperisone,
were obtained from Sigma Aldrich. Racemic venlafaxine was kindly
supplied by Prof. J.-L. Veuthey (Laboratoire de Chimie Analytique
Pharmaceutique, University of Geneva, Switzerland), while fluoxetine and citalopram were kind gift from Lilly Research Laboratories
(Eli Lilly and Company, Indianapolis, IN, USA) and by H. Lundbeck
A/S (Copenhagen, Denmark), respectively.
The stock of racemic mixtures and pure enantiomer standard
solutions (1 mg/mL) were prepared by dissolving the appropriate
weighted powder of each analyte in MeOH or ACN and stored at
−18 °C. The working solutions were prepared by diluting the stock
solution at 100 μg/mL with H2 O/2-PrOH/MeOH (80:10:10, v/v/v)
for acid and neutral compounds and MeOH/water or ACN/water for
basic compounds. 50 mL (500 mM) of stock buffer solutions were
prepared every week as below: ammonium formate was obtained
by diluting the appropriate volume of FA with ultrapure water and

titrated with ammonia solution (approx. 5 M) to the pH 2.5; ammonium hydrogen carbonate was weighed and dissolved in ultrapure water and titrated with ammonia solution (approx. 5 M) to
the pH 11. All solutions were stored at +4 ◦C.
10 mL of polar organic mobile phases were daily prepared by
dissolving the appropriate amount of buffer solution in ACN/water
or MeOH/water mixture.
2.2. Instrumentation
Measurements of pH during titration of buffer solution were
performed with a Crison Basic pH 20 (Crison Instruments SA,
Barcelona, Spain), with a combined electrode and a temperature
sensor. The accurate measurement of pH was performed by a
three-point calibration with the appropriate certified buffer solutions at pH 4.01, 7.00 and 9.21.
An ultrasonic bath model FS 100b Decon (Hove, UK) was used
to sonicate mobile phases, to dissolve analytes, to have homogeneous packing bed and stable stationary phase-slurry during packing process.
A Stereozoom 4 optical microscope (Cambridge Instruments, Vienna, Austria) with illuminator was used to inspect the status of
the capillary columns and checking the fused silica capillary during the capillary column packing procedure.
An HPLC pump (Perkin Elmer Series 10, Palo Alto, CA, USA) was
used for packing and equilibration the capillary columns.
An outside polyimide-coated fused silica capillary (Polymicro
TechnologiesTM , Silsden, UK), with 375 μm O.D. and 100 μm I.D.
was used for preparation capillary columns for both, nano-LC and
CEC.
The polysaccharide-based CSP used in this experimental work
was 20% (w/w) amylose tris(3-chloro-5-methylphenylcarbamate) as
chiral selector covalently immobilized on APS silica or native sil˚ This
ica (nominal particle size, 5 μm; nominal pore size, 10 0 0 A).
material was provided by Enantiosep GmbH (Münster, Germany).
Amylose tris(3-chloro-5-methylphenylcarbamate) (Fig. S1) was synthesized as described earlier [14]. The product was isolated by precipitation in MeOH, filtrated, washed with excess of methanol and
dried in the vacuum oven at 70°C for 12 hrs. The carbamate was
dissolved and coated on native or APS silica (Daiso, Osaka, Japan).

The coated material was immobilized using a proprietary photochemical technology.
2.3. Packing of the capillary columns
The capillary columns were prepared in our laboratory following a packing procedure previously published by our group based
on the slurry packing method [29,30].
Considering our previous experience regarding packing
polysaccharide-based CSPs into capillary columns, the good
homogenous slurry suspension of packing material was obtained
with about 50 mg/mL in ACN.
Due to inability making semi-permeable frits on this CSP the
frits were made by using LiChrosorb® 10 μm RP-18 100 A˚ from
Merck KGaA (Darmstadt, Germany). The modified packing procedure previously described by our group [30] was adjusted as following: ACN and ACN/distilled water, 80/20 (v/v), as slurry and
flushing solvents during frit preparation, were used, respectively.
The slurry of packing materials were sonicated for 2 min and
quickly transferred into an HPLC pre-column 50 × 4.1 mm I.D.
(Valco, Houston, TX, USA) connected at the inlet end to the LCpump while the outlet end was connected to the silica capillary
(40 cm length). MeOH was the LC-pumping solvent that delivered
the packing material into fused silica capillary towards a mechanical LC-frit. The maximum pressure during packing procedure was
in the range 30–35 MPa (300–350 bar). For CEC experiments, at


G. D’Orazio, C. Fanali and S. Fanali et al. / Journal of Chromatography A 1623 (2020) 461213

3

the inlet side of the capillary, a 5 cm sector was packed with
Kromasil Si-NH2 (5 μm) phase (Sigma-Aldrich) followed by 20 cm
of CSP. Afterwards LiChrosorb® 10 μm particles were packed (4-5
cm). The column was flushed with ACN/distilled water, 80/20 (v/v),
for about 15 min and the frits prepared (at about 650 °C x 10 s)
close to the end of CSP packing segment. The rest of LiChrosorb®

10 μm particles were flushed out of the capillary.
The detection window was prepared at 1.5 cm from the outlet
frit empty side by removing the polyimide coating by means of a
razor blade.
2.4. Capillary electrochromatography
CEC experiments were carried out with Agilent 3D CE system, (Agilent Technologies, Waldbronn, Germany), equipped with a
diode-array UV detector and an autosampler device. Detection was
performed at 205 nm, rise time: 0.5 s and 20 Hz. while the column temperature (20 °C) was controlled by an air thermostating
system.
The capillary column packed with APS-silica (5 cm) plus CSP
(20 cm) was firstly equilibrated with the mobile phase with the
HPLC pump at 10 MPa and then placed into the CE instrument. After the typical conditioning step (applying a voltage ramp from -5
to -20 kV) for 30 min, the capillary was ready for CEC experiments.
At the end of the working day, both ends of the capillary were submerged into the vials containing MeOH/water 90:10 (v/v). In order
to avoid bubble formation, CEC experiments were performed applying to both vials a pressure of 10 bar. The sample was hydrodynamically injected applying 10 bar pressure for 0.3 min at the
cathodic end. The separation voltage was -15 kV.
A Chemstation software (Rev. A.09.01, Agilent Technologies) was
used for managing the instrument and collecting and reprocessing
the obtained data.
2.5. Nano-liquid chromatography
Nano-LC experiments were performed using a laboratoryassembled instrumentation as previously described [28]. Briefly, for
this purpose an Agilent 1100 series LC (G1376A) (Agilent Technologies, Waldbronn, Germany) micro-pump was used in isocratic
mode delivering MeOH. It was connected to a three port steel
union (Vici Valco, Houston, TX, USA) as passive split system in order to reduce the flow rate to nL/min range. A nanoliter injection
was obtained by using a modified LC injector valve (Enantiosep,
Münster, Germany) where its external configuration included a 40
μL external loop allowing both sample loading, as well as its use
as a mobile phase reservoir during the chiral separation. The nano
volume injection was obtained by using the pressure-pulse driven
stopped-flow injection time method [31]. The flow rate in the capillary column (after the splitting system) was estimated by connecting a 10 μL syringe (Hamilton, Reno, NV, USA) to the outlet

column through a Teflon® tube (TF-350, LC-Packing, CA, USA) and
measuring the mobile phase volume for approximately 5 min. The
flow rate was changed in the range 70-1440 nL/min. In order to
reduce dead volume and the band broadening effect, the column
inlet was directly connected to the modified valve. Samples were
eluted in isocratic mode with a mobile phase consisting 15 mM
NH4 FA pH 2.5 in 90/10, ACN/H2 O (v/v) for acid and neutral compounds, while 50 mM NH4 HCO3 pH 11 in 90/10, MeOH/H2 O (v/v)
was used for basic compounds except chiral diazepine derivatives
which were eluted with the former mobile phase. A Spectra 100
UV instrument (Thermo Separation Products, San Jose, CA, USA),
was employed for the on-capillary UV detection. The detector was
set at 205 nm; data acquisition and rise time were adjusted at 20
Hz and 0.5 s, respectively. The LC pump was controlled by Chemstation software (Rev.A.09.01, Agilent Technologies,) while the UV

Fig. 1. Enantiomeric separation of basic compounds in nano-LC. Separation conditions: capillary column, 100 μm I.D. x 25.0 cm (packed length), Leff = 26.5 cm,
Ltot = 34.9 cm. CSP, i-amylose tris(3-chloro-5-methylphenylcarbamate) (20%, w/w),
APS silica (5 μm); sample, 100 μg/mL in 80/10/10 water/2-PrOH/MeOH (v/v/v); mobile phase, 50 mM NH4 HCO3 pH 11 in 90/10, MeOH/H2 O (v/v); flow rate: about 200
nL/min, inj. volume, 60 nL; UV detection, 205 nm; room temperature.

detector data were acquired and processed with the ChromQuest
version 3.0 software (Thermo-Finnigan, San Jose, CA, USA). The column temperature was controlled by continuous conditioning room
(about 25°C).
The A, B, and C coefficients part of the van Deemter equation
were estimated by using Curve expert 1.40 from Microsoft Corporation ( />3. Results and Discussion
3.1. Enantioseparations of basic analytes
Since separation of enantiomers of basic chiral analytes has
not included in our previous study on the application of amylose
tris(3-chloro-5-methylphenylcarbamate) in nano-LC and CEC [28],
this was the important goal of the present study. As already mentioned above the major difference between the CSPs used in the
previous and the present studies is that in the previous study the

chiral selector was immobilized on native silica with free silanol
groups while in the present study it was immobilized on APS silica.
This should enable improved peak shape and higher resolution for
basic analytes in nano-LC and CEC and in addition, the anodic EOF
in the latter technique. Basic chiral analytes belonging to different structural groups were used as chiral test compounds (some of
these analytes are well known chiral drugs). The new CSP showed
good results for separation of enantiomers of basic chiral analytes
in methanol containing 10% ammonium bicarbonate buffer at pH
11.0 (v/v). The enantiomers of some chiral diazepine derivatives
were separated in 15 mM NH4 FA pH 2.5 in 90/10, ACN/H2 O (v/v)
(Table 1). Some representative chromatograms are shown in Fig. 1.
3.2. Enantioseparations of neutral analytes
The group of studied neutral chiral analytes together with
structurally similar flavanone derivatives included also chiral drugs
such as thalidomide, as well as chiral agrochemicals dialifos, fenamiphos and profenofos. The enantiomers of the most of these
analytes (except dialifos and profenofos) were well separated


4

G. D’Orazio, C. Fanali and S. Fanali et al. / Journal of Chromatography A 1623 (2020) 461213
Table 1
Chromatographic data of the enantioseparation of some selected racemic basic compounds by nano-LC. For experimental conditions see
text.

Mobile phase

Compounds

50 mM NH4 HCO3 pH 11 in

90/10, MeOH/H2 O (v/v)

Alprenolol
Ambucetamide
Bupivacaine
Carbinoxamine
Citalopram
Clenbuterol
Fluoxetine
Metoprolol
Mianserine
Nadolol
Oxprenolol
Pindolol
Promethazine
Propranolol
Tolperisone
Tröger’s base
Venlafaxine

15 mM NH4 FA pH 2.5 in
90/10, ACN/H2 O (v/v)

Lorazepam
Oxazepam
Temazepam

t0 (min)- flow rate
(nL/min)

k’1

k’2

α

Rs

N1 /m

N2 /m

6.643 - 200

0.43
0.56
0.52
0.79
0.93
0.29
0.36
1.12
0.88
0.29
0.52
0.30
0.96
0.56
1.44
1.87

1.60

0.60
0.86
0.60
0.95
1.03
0.37
0.36
1.45
1.17
0.55
0.82
0.38
1.12
0.72
1.70
5.73
2.28

1.40
1.54
1.16
1.20
1.11
1.26
1.00
1.29
1.34
1.88

1.58
1.30
1.17
1.28
1.18
3.06
1.42

1.7
2.5
0.6
1.3
1.1
0.8
<0.3
1.9
2.1
0.35; 1.38
2.6
1.0
1.0
1.3
1.7
9.2
1.7

14943
14807
15187
11071

13498
14700
16548
10960
12654
14613
10933
4374

14017
11504
12138
10471
12021
11301
14858
10261
10939
12769
7651
3231

3.73 - 355

0.40
0.53
0.78

0.40
0.60

1.14

1.13
1.47

0.7
3.3

23868

17626

Table 2
Chromatographic data of some selected neutral chiral analytes obtained using nano-LC. Mobile phase, 15
mM NH4 FA pH 2.5 in 90/10, ACN/H2 O (v/v). For other experimental conditions, see text.
Compounds
Dialifos
Flavanone
4 -MeO-Fla
6-MeO-Fla
7-MeO-Fla
2 -OH-Fla
4 -OH-Fla
6-OH-Fla
7-OH-Fla
Fenamiphos
Profenofos
Thalidomide

t0 (min)- flow rate

(nL/min)

3.73 - 355

k’1

k’2

α

Rs

N1 /m

N2 /m

0.23
0.50
0.74
0.90
0.84
0.27
0.35
0.62
0.45
0.43
0.56
0.83

0.23

0.99
1.80
2.14
1.88
0.50
0.77
1.35
0.87
0.49
0.56
1.38

1.97
2.42
2.37
2.24
1.88
2.17
2.18
1.93
1.14
1.67

6.8
9.7
9.8
9.4
4.2
6.2
7.7

5.4
1.0
5.8

41683
38761
32210
33220
42836
40484
34608
35362
35358
27923

35181
22796
22681
26360
35548
31912
24091
26167
34592
34850

(Table 2). Since among twelve studied analytes eight were flavanone and its derivatives some correlations could be drawn between structure of analytes on the one hand, and the retention
and separation factors on the other one (Fig. 2). All three methoxy
derivatives of flavanone were longer retained on this CSP compared
to unsubstituted flavanone, while most of all hydroxy derivatives

retained less than unsubstituted flavanone.
The enantiomers of all flavanone derivatives were baseline resolved with resolution factors in the range 4.2-9.8. The highest Rs
values were recorded for the methoxy derivatives (6-MeO- > 4’MeO- >7-MeO-Fla). The introduction of a methoxy group on one of
the two aromatic rings resulted in an increase of enantioresolution
factor compared to flavanone (Rs=6.8). This together with longer
retention of these derivatives can most likely be explained considering that this substituent has an electron-donating effect increasing the electron density on the conjugated rings of analytes and
thus, the interaction with the chiral selector through π -π mechanism.
The introduction of a hydroxyl group, although with electrondonating properties, caused a lower enantioseparation than the
methoxy one. However, 6-OH-Fla exhibited higher enantioresolution (Rs=7.7) than flavanone. Although good resolution of enantiomers was obtained for the other hydroxy-derivative, their enantioresolution factors were lower than the one of unsubstituted flavanone. This trend was quite similar to that observed earlier in

HPLC on amylose tris(3,5-dimethylphenylcarbamate)-based chiral
column in methanol as a mobile phase [32].
Together with above mentioned neutral analytes few phosphoric acid esters used as pesticides have been also studied. Interestingly, two of studied three compounds, in particular, fenamiphos
and profenofos owe their chirality to the asymmetrically substituted phosphor atom in their structure. Of this set of chiral analytes the enantiomers of fenamiphos were partially separated
(Rs= 1.0) under the experimental conditions of this study (Fig.
S2). Exceptional chiral recognition ability of amylose tris(3-chloro5-methylphenylcarbamate)-based columns towards enantiomers of
chiral pesticides belonging to various chemical groups has been
also shown in the references [20-24].
3.3. Enantioseparations of acidic analytes
In theory the CSP prepared on the basis of APS silica may not
be ideal for separation of acidic analytes. In fact, some kind of
electrostatic interaction between the anionic analytes and protonated aminopropyl moieties on the surface of silica may cause undesirable peak tailing. The mobile phase containing 5 mM ammonium formate pH 2.5 in 90:10, v/v ACN/H2 O was applied for
the enantioseparation of selected acidic compounds such as nonsteroidal anti-inflammatory drugs (carprofen, cicloprofen, flurbiprofen, ketoprofen, ketorolac, ibuprofen, indoprofen, naproxen, supro-


G. D’Orazio, C. Fanali and S. Fanali et al. / Journal of Chromatography A 1623 (2020) 461213

5

Fig. 3. Nano-LC chiral separation of hexobarbital, suprofen, carprofen, and ketorolac. For experimental conditions see Fig. 2 and text.

Fig, 2. Enantiomeric separation of studied flavanone derivatives in nano-LC. Experimental conditions: 15 mM NH4 FA pH 2.5 in 90/10, ACN/H2 O (v/v), flow rate, 355
nL/min; inj. volume, 60 nL. For additional experimental conditions see Fig. 1 and
text.

fen), anticoagulant drug warfarin, herbicides (diclofop, fenoxaprop,
fluazyfop, and haloxyfop) and hypnotic and sedative drug hexobarbital. These chiral analytes belong to different structural
groups such as arylpropionic acid derivatives, coumarins and
barbiturates.
Table 3 reports the chromatographic data on the enantiomeric
separation of the studied compounds. Analytes were eluted in less
than seven min. As can be observed, among these compounds,
phenoxaprop was the most retained analyte (k’2 = 1.56). Good
baseline resolution was obtained for the enantiomers of several
racemic analytes (Fig. 3). There was no measurable negative effect
on the peak shape due to electrostatic interaction between the analytes and silica surface. One of the possible reasons of this could
be low apparent pH of the mobile phase suppressing the negative charge on the analytes. Based on literature [18] amylose tris(3chloro-5-methyphenylcarbamate) shows very high success rate for
separation of enantiomers of weak chiral acids, among them also

included in this project with n-hexane/alcohol type mobile phases.
In addition, our unpublished results also show that in polar organic
solvents such as MeOH, and especially ACN, the success rate is also
high. Thus, rather low enantiomer resolving ability of this material
towards the enantiomers of weakly acidic chiral analytes observed
in the present study may relate to poor quality of capillary column
packing or unoptimized mobile phase.
3.4. Comparative results between Amylose
tris(3-chloro-5-methylphenylcarbamate) immobilized on native and
on aminopropylsilanized silica
As mentioned above we have already studied application of

amylose tris(3-chloro methylphenylcarbamate) as chiral selector
in nano-LC and CEC [28]. Our goal in the present study was to
extend the applicability of this CSP also to basic analytes and
observe the effect of surface chemistry of silica on the chromatographic performance of this material in nano-LC and CEC.
As some selected chromatograms (Fig. 4), as well as plate numbers (Fig. 5a) and resolutions (Fig. 5b) show the CSP based on
APS silica performed slightly better (with very few exceptions)
for all type of analytes (basic, neutral and acidic). Some advan-

Table 3
Chromatographic data obtained in the separation of chiral acidic analytes by nano-LC. Mobile phase, 15 mM
NH4 FA pH 2.5 in 90/10, ACN/H2 O (v/v). For other experimental conditions, see text.
Compounds
Carprofen
Cicloprofen
Diclofop
Fenoxaprop
Fluazifop
Flurbiprofen
Haloxyfop
Hexobarbital
Ibuprofen
Indoprofen
Ketoprofen
Ketorolac
Naproxen
Suprofen
Warfarin

t0 (min)- flow rate
(nL/min)

3.73 - 355

k’1

k’2

α

Rs

N1 /m

N2 /m

0.35
0.32
1.16
1.45
0.81
0.31
0.75
0.31
0.23
0.81
0.30
0.64
0.27
0.37
0.39

0.50
0.40
1.16
1.56
0.87
0.43
0.75
0.87
0.23
0.87
0.30
0.87
0.31
0.47
0.47

1.43
1.25
1.08
1.08
1.41
2.78
1.07
1.36
1.16
1.26
1.21

2.4

1.4
0.7
0.6
2.3
8.1
0.4
2.6
0.7
1.6
1.6

34963
41802
39274
42948
27680
38555
65180

31909
32901
38554
29318
22360
34860
40049


6

G. D’Orazio, C. Fanali and S. Fanali et al. / Journal of Chromatography A 1623 (2020) 461213

tage of CSP based on APS silica over the CSP based on native silica in the present study is also supported with van Deemter dependences shown for flurbiprofen, hexobarbital and flavanone on
Fig. 6.

3.5. Preliminary attempts of enantioseparations in CEC

Fig. 4. Comparative separation of enantiomers on CSP, i-amylose tris(3-chloro-5methylphenylcarbamate) (20%, w/w), (A) native silica (B) APS silica in nano-LC system. Experimental conditions: as reported in Fig. 2 and text.

As mentioned above APS silica-based CSP showed some advantages over the CSP based on native silica for nano-LC applications under this study. On the next step we tried to apply this
CSP in CEC and were surprised with the absence of the electroosmotic flow (EOF) in these capillaries. In many of our earlier studies we have observed quite strong anodic EOF in the capillaries
packed with APS silica-based polysaccharide-type CSPs [1,29,33].
After overnight flushing the capillaries with 5 mM ammonium formate pH 2.5 in 90/10, ACN/H2 O (v/v) a significant EOF appeared
there but it was not stable and thus not suitable for providing adequate run to run repeatability. Our detailed experiments for understanding the reasons of the initial EOF absence, its appearance and
fluctuations did not lead to a conclusive answer. The preparation of
this CSP involved new proprietary technology for immobilization
of a chiral selector onto the APS silica. However, it is less likely
that this technique could be a reason for the absence of the EOF
in these capillary columns. In order to perform some preliminary
tests of these capillaries under CEC conditions a 5 cm long segment
of the capillary column was packed with APS silica not containing
a chiral selector while another 20 cm was packed with CSP used
in nano-LC experiments. In these capillaries the EOF was sufficient
for performing CEC experiments (Fig. 7) but the plate numbers observed in these separations were not high enough. Thus, successful

Fig. 5. The comparative results of enantioseparation of acid and neutral compounds on native silica and APS silica CSP-polysaccharide based by nano-LC: A) enantioresolution,
B) number of theoretical plates. For experimental conditions see Fig. 2 and text.


G. D’Orazio, C. Fanali and S. Fanali et al. / Journal of Chromatography A 1623 (2020) 461213

7

Fig. 6. van Deemter dependences for the first peaks of flurbiprofen, hexobarbital and flavanone in nano-LC modes. Experimental conditions: flow rates, 70-1440 nL/min. For
other conditions, see Fig. 2 and text.

application of this CSP made on the basis of APS silica in CEC requires further studies.

5. Conclusions
As the results of this study indicate amylose tris(3-chloro-5methylphenylcarbamate) is very useful chiral selector also in combination with aqueous methanol or acetonitrile as a mobile phase.
The set of basic, neutral and acidic analytes in the present study
included as a chiral center not only asymmetrically substituted carbon but also nitrogen and phosphor. The chiral selector immobilized on APS silica showed some advantages over its counterpart
immobilized onto a native silica in nano-LC, however, it failed in
CEC due to EOF generation and stability problems. Using a short
segment of APS silica without chiral selector together with this
APS silica-based CSP enabled to perform CEC experiments. However, further studies are required for optimizing CEC experiments
and realizing potential advantages of CEC over nano-LC.
Fig. 7. Enantiomeric separation of some selected neutral and acidic compounds by
CEC. Experimental conditions: capillary column, 100 μm I.D. x 25.0 cm (packed
length, 5 cm with Kromasil Si-NH2 (5 μm) and, 20 cm CSP- i-amylose tris(3-chloro5-methylphenylcarbamate) (20%, w/w), APS silica (5 μm)), Leff = 26.5 cm, Ltot = 34.9
cm; mobile phase, 5 mM NH4 FA pH 2.5 in 90/10, ACN/H2 O (v/v); Inj: 10 bar x 0.3
min ; applied Voltage: -15 kV; I= -0.9 μA; Detection, 205 nm. 10 bar on both vials.

Declaration of Competing Interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to
influence the work reported in this paper.


8

G. D’Orazio, C. Fanali and S. Fanali et al. / Journal of Chromatography A 1623 (2020) 461213

Supplementary materials
Supplementary material associated with this article can be
found, in the online version, at doi:10.1016/j.chroma.2020.461213.
CRediT authorship contribution statement
Giovanni D’Orazio: Investigation, Validation, Formal analysis,
Writing - original draft. Chiara Fanali: Conceptualization, Writing
- original draft. Salvatore Fanali: Project administration, Visualization, Writing - review & editing. Alessandra Gentili: Writing review & editing. Marina Karchkhadze: Formal analysis. Bezhan
Chankvetadze: Supervision, Methodology, Resources, Writing - review & editing.
References
[1] S. Fanali, B. Chankvetadze, P. Catarcini, G. Blaschke, Enantioseparations by
capillary electrochromatography, Electrophoresis 22 (2001) 3131–3151, doi:10.
1002/1522-2683(200109)22:15 3131::AID-ELPS3131 3.0.CO;2-S.
[2] G. D’ Orazio, M. Asensio-Ramos, C. Fanali, Enantiomers separation by capillary
electrochromatography using polysaccharide-based stationary phases, J. Sep.
Sci. 42 (2019) 360–384, doi:10.10 02/jssc.20180 0798.
[3] B. Chankvetadze, T. Kubota, T. Ikai, C. Yamamoto, N. Tanaka, K. Nakanishi, Y. Okamoto, High-performance liquid chromatographic enantioseparations
on capillary columns containing crosslinked polysaccharide phenylcarbamate
derivatives attached to monolithic silica, J. Sep. Sci. 29 (2006) 1988–1995,
doi:10.10 02/jssc.20 050 0388.
[4] S. Fanali, Nano-liquid chromatography applied to enantiomers separation, J.
Chromatogr. A 1486 (2017) 20–34, doi:10.1016/j.chroma.2016.10.028.
[5] S. Rocchi, S. Fanali, T. Farkas, B. Chankvetadze, Effect of content of chiral selector and pore size of core-shell type silica support on the performance
of amylose tris(3,5-dimethylphenylcarbamate)-based chiral stationary phases
in nano-liquid chromatography and capillary electrochromatography, J. Chromatogr. A 1363 (2014) 363–371, doi:10.1016/j.chroma.2014.05.029.
[6] J. Shen, Y. Okamoto, Efficient separation of enantiomers using stereoregular
chiral polymers, Chem. Rev. 116 (2016) 1094–1138, doi:10.1021/acs.chemrev.
5b00317.
[7] B. Chankvetadze, Recent developments on polysaccharide-based chiral stationary phases for liquid-phase separation of enantiomers, J. Chromatogr. A 1269

(2012) 26–51, doi:10.1016/j.chroma.2012.10.033.
[8] B. Chankvetadze, Recent trends in preparation, investigation and application of
polysaccharide-based chiral stationary phases for separation of enantiomers in
high-performance liquid chromatography, TrAC-Trend Anal. Chem. 122 (2020)
115709, doi:10.1016/j.trac.2019.115709.
[9] Y. Okamoto, M. Kawashima, K. Hatada, Useful chiral packing materials for highperformance liquid chromatographic resolution of enantiomers: Phenylcarbamates of polysaccharides coated on silica gel, J. Am. Chem. Soc. 106 (1984)
5357–5359, doi:10.1021/ja00330a057.
[10] Y. Okamoto, M. Kawashima, K. Hatada, Chromatographic resolution: XI. Controlled chiral recognition of cellulose triphenylcarbamate derivatives supported
on silica gel, J. Chromatogr. 363 (1986) 173–186, doi:10.1016/S0021-9673(01)
83736-5.
[11] B. Chankvetadze, E. Yashima, Y. Okamoto, Tris(chloro- and methyldisubstituted phenylcarbamate)s of cellulose as chiral stationary phases
for chromatographic enantioseparation, Chem. Lett. 22 (1993) 617–620,
doi:10.1246/cl.1993.617.
[12] B. Chankvetadze, E. Yashima, Y. Okamoto, Chloro-methyl-phenylcarbamate
derivatives of cellulose as chiral stationary phases for high performance liquid chromatography, J. Chromatogr. A. 670 (1994) 39–49, doi:10.1016/j.chroma.
2019.460572.
[13] B. Chankvetadze, E. Yashima, Y. Okamoto, Dimethyl-, dichloro- and
chloromethyl-phenylcarbamate derivatives of amylose as chiral stationary
phases for high performance liquid chromatography, J. Chromatogr. A 694
(1995) 101–109, doi:10.1016/0 021-9673(94)0 0729-S.
[14] B. Chankvetadze, L. Chankvetadze, Sh. Sidamonidze, E. Kasashima, E. Yashima,
Y. Okamoto, 3-Fluoro-, 3-bromo-, and 3-chloro-5-methylphenylcarbamates of
cellulose and amylose as chiral stationary phases for HPLC enantioseparation,
J. Chromatog. A 787 (1997) 67–77, doi:10.1016/S0 021-9673(97)0 0648-1.
[15] R. Cirilli, S. Carradori, A. Casulli, M. Pierini, A chromatographic study on the retention behavior of the amylose tris(3-chloro-5-methylphenylcarbamate) chiral
stationary phase under aqueous conditions, J. Sep. Sci. 41 (2018) 4014–4021,
doi:10.10 02/jssc.20180 0696.

[16] A. Ghanem, C. Wang, Enantioselective separation of racemates using Chiralpak
IG amylose-based chiral stationary phase under normal standard, non-standard

and reversed phase high performance liquid chromatography, J. Chromatogr. A
1532 (2018) 89–97, doi:10.1016/j.chroma.2017.11.049.
[17] R. Ferretti, L. Zanitti, A. Casulli, R. Cirilli, Unusual retention behavior of
omeprazole and its chiral impurities B and E on the amylose tris (3-chloro5-methylphenylcarbamate) chiral stationary phase in polar organic mode, J.
Pharm. Anal. 8 (2018) 234–239, doi:10.1016/j.jpha.2018.04.001.
[18] M. Maisuradze, Sheklashvili G, A. Chokheli, I. Matarashvili, T. Gogatishvili,
T. Farkas, B. Chankvetadze, Chromatographic and thermodynamic comparison
of amylose tris(3-chloro-5-methylphenylcarbamate) coated or covalently immobilized on silica in high-performance liquid chromatographic separation of
the enantiomers of selected chiral weak acids, J. Chromatogr. A 1602 (2019)
228–236, doi:10.1016/j.chroma.2019.05.026.
[19] W. Bia, F. Wang, J. Han, B. Liu, L. Zhang, J. Shen, Y. Okamoto, Influence of
the substituents on phenyl groups on enantioseparation property of amylose
phenylcarbamates, Carbohydr. Polym. (2020) in press.
[20] M.E. Díaz Merino, R.N. Echevarría, E. Lubomirsky, J.M. Padró, C.B. Castells,
Enantioseparation of the racemates of a number of pesticides on a silica-based
column with immobilized amylose tris(3-chloro-5-methylphenylcarbamate),
Microchem. J. 149 (2019) 103970, doi:10.1016/j.microc.2019.103970.
[21] M.E. Díaz Merino, C. Lancioni, J.M. Padró, C.B. Castells, Chiral separation of several pesticides on an immobilized amylosetris(3-chloro-5-methylphenylcarbamate) column under polar-organic conditions. Influence of mobile phase and temperature on enantioselectivity, J.
Chromatogr. A (2020) submitted.
[22] P. Zhao, Z. Wang, K. Li, X. Guo, L. Zhao, Multi-residue enantiomeric analysis
of 18 chiral pesticides in water, soil and river sediment using magnetic solidphase extraction based on amino modified multiwalled carbon nanotubes and
chiral liquid chromatography coupled with tandem mass spectrometry, J. Chromatogr. A. 1568 (2018) 8–21, doi:10.1016/j.chroma.2018.07.022.
[23] P. Zhao, Z. Wang, X. Gao, X. Guo, L. Zhao, Simultaneous enantioselective determination of 22 chiral pesticides in fruits and vegetables using chiral liquid chromatography coupled with tandem mass spectrometry, Food Chem 277
(2019) 298–306, doi:10.1016/j.foodchem.2018.10.128.
[24] X. Yuan, X. Li, P. Guo, Z. Xiong, L. Zhao, Simultaneous enantiomeric analysis
of chiral non-steroidal anti-inflammatory drugs in water, river sediment, and
sludge using chiral liquid chromatography-tandem mass spectrometry, Anal.
Methods. 10 (2018) 4404–4413, doi:10.1039/c8ay01417e.
[25] C. Panella, R. Ferretti, A. Casulli, R. Cirilli, Temperature and eluent composition effects on enantiomer separation of carvedilol by high-performance liquid chromatography on immobilized amylose-based chiral stationary phases, J.
Pharm. Anal. 9 (2019) 324–331, doi:10.1016/j.jpha.2019.04.002.

[26] P. Zhao, S. Li, X. Chen, X. Guo, L. Zhao, Simultaneous enantiomeric analysis of
six chiral pesticides in functional foods using magnetic solid-phase extraction
based on carbon nanospheres as adsorbent and chiral liquid chromatography
coupled with tandem mass spectrometry, J. Pharm. Biomed. Anal. 175 (2019)
112784, doi:10.1016/j.jpba.2019.112784.
[27] A.-E. Dascalu, A. Ghinet, B. Chankvetadze, E. Lipka, Comparison of dimethylated and methylchlorinated amylose stationary phases, coated and covalently immobilized on silica, for the separation of some chiral compounds in supercritical fluid chromatography, J. Chromatogr. A, in press.
10.1016/j.chroma.2020.461053.
[28] G. D’ Orazio, C. Fanali, A. Gentili, S. Fanali, B. Chankvetadze, Comparative study on enantiomer resolving ability of amylose tris(3-chloro-5methylphenylcarbamate) covalently immobilized onto silica in nano-liquid
chromatography and capillary electrochromatography, J. Chromatogr. A 1606
(2019) 460425, doi:10.1016/j.chroma.2019.460425.
[29] S. Fanali, G. D’Orazio, K. Lomsadze, B. Chankvetadze, Enantioseparations with
cellulose(3-chloro-4-methylphenylcarbamate) in nano liquid chromatography
and capillary electrochromatography, J. Chromatogr. B 875 (2008) 296–303.
[30] G. D’Orazio, Z. Aturki, M. Cristalli, M.G. Quaglia, S. Fanali, Use of vancomycin
chiral stationary phase for the enantiomeric resolution of basic and acidic
compounds by nano-liquid chromatography, J. Chromatogr. A 1081 (2005) 105–
113, doi:10.1016/j.chroma.2005.02.025.
[31] J.P.C Vissers, H.A. Claessens, C.A. Cramers, Microcolumn liquid chromatography:
instrumentation, detection and applications, J. Chromatogr. A 779 (1997) 1–28,
doi:10.1016/S0 021-9673(97)0 0422-6.
[32] C. Fanali, S. Fanali, B. Chankvetadze, HPLC Separation of enantiomers of
some flavanone derivatives using polysaccharide-based chiral selectors covalently immobilized on silica, Chromatographia 79 (2016) 119–124, doi:10.1007/
s10337-015-3014-8.
[33] M. Girod, B. Chankvetadze, G. Blaschke, Enantioseparations in nonaqueous capillary electrochromatography using polysaccharide type chiral stationary phase,
J. Chromatogr. A 887 (20 0 0) 439–455.