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.
Trang 1Contents lists available at ScienceDirect
journal homepage: www.elsevier.com/locate/chroma
electrochromatography
Giovanni D’Orazioa, Chiara Fanalib, Salvatore Fanalic, ∗, Alessandra Gentilid,
Marina Karchkhadzee, Bezhan Chankvetadzee
a Istituto per i Sistemi Biologici (ISB), CNR- Consiglio Nazionale delle Ricerche, Via Salaria Km 29,300 – 00015 Monterotondo (Rome), Italy
b 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
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 ana- lytes was investigated on the chiral stationary phase (CSP) made by covalent immobilization of amy- lose tris(3-chloro-5-methylphenylcarbamate) onto aminopropylsilanized (APS) silica in nano-liquid chro- matography (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 re- ported 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 sep-
aration techniques and among these also in nano liquid chro-
matography (nano-LC) and capillary electrochromatography (CEC)
[1-5] As it has been shown in many studies the chiral recogni-
tion 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: salvatore.fanali@gmail.com (S Fanali)
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 sub- stituents [ 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 chromato- graphic 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 fam- ily, namely of amylose tris(3-chloro-5-methyphenylcarbamate) was described in 1997 [14] However, the packing materials and chiral https://doi.org/10.1016/j.chroma.2020.461213
0021-9673/© 2020 Elsevier B.V All rights reserved
Trang 2columns 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 stud-
ied 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-chloro-
5-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-chloro-
5-methyphenylcarbamate) was covalently immobilized on APS sil-
ica and its applicability was studied in nano-LC separation of enan-
tiomers of basic, neutral and acidic chiral analytes In addition, the
attempt was made to use the same material for separation of enan-
tiomers 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 (NH 4HCO 3 ≥ 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 (Interna-
tional 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, hexo-
barbital, temazepam, carbinoxamine, warfarin, and Trưger’s base
were obtained from Sigma-Aldrich Diclofop, fenoxaprop, dichlor-
prop, 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 Hặn (Seelze, Germany) The non-
steroidal 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 Crystallogra-
phy, CNR, Monterotondo, Roma, Italy) Ketoprofen, ketorolac, and
ibuprofen were purchased from Sigma-Aldrich Racemic thalido-
mide 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 fluoxe-
tine 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 H 2O/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; am- monium hydrogen carbonate was weighed and dissolved in ultra- pure 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
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 solu- tions 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 homoge- neous packing bed and stable stationary phase-slurry during pack- ing process
A Stereozoom 4 optical microscope (Cambridge Instruments, Vi- enna, Austria) with illuminator was used to inspect the status of the capillary columns and checking the fused silica capillary dur- ing 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 Technologies TM, 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- ica (nominal particle size, 5 μm; nominal pore size, 10 0 0 ˚A) This material was provided by Enantiosep GmbH (Münster, Germany) Amylose tris(3-chloro-5-methylphenylcarbamate) (Fig S1) was syn- thesized as described earlier [14] The product was isolated by pre- cipitation 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 photo- chemical technology
2.3 Packing of the capillary columns
The capillary columns were prepared in our laboratory follow- ing 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 proce- dure previously described by our group [30] was adjusted as fol- lowing: 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 LC- pump 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 mechan- ical LC-frit The maximum pressure during packing procedure was
in the range 30–35 MPa (300–350 bar) For CEC experiments, at
Trang 3the inlet side of the capillary, a 5 cm sector was packed with
Kromasil Si-NH 2 (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
CEC experiments were carried out with Agilent 3DCE sys-
tem, (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 and 20 Hz while the col-
umn 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 Af-
ter 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 sub-
merged into the vials containing MeOH/water 90:10 (v/v) In order
to avoid bubble formation, CEC experiments were performed ap-
plying to both vials a pressure of 10 bar The sample was hydro-
dynamically 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
Nano-LC experiments were performed using a laboratory-
assembled instrumentation as previously described [28] Briefly, for
this purpose an Agilent 1100 series LC (G1376A) (Agilent Tech-
nologies, 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 or-
der 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 cap-
illary column (after the splitting system) was estimated by con-
necting 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
NH 4FA pH 2.5 in 90/10, ACN/H 2O (v/v) for acid and neutral com-
pounds, while 50 mM NH 4HCO 3 pH 11 in 90/10, MeOH/H 2O (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 Chem-
station software (Rev.A.09.01, Agilent Technologies,) while the UV
Fig 1 Enantiomeric separation of basic compounds in nano-LC Separation con-
ditions: capillary column, 100 μm I.D x 25.0 cm (packed length), L eff = 26.5 cm,
L tot = 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); mo- bile phase, 50 mM NH 4 HCO 3 pH 11 in 90/10, MeOH/H 2 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 col- umn 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 Corpo- ration ( https://www.curveexpert.net/download/)
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 men- tioned 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 differ- ent 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 NH 4FA pH 2.5 in 90/10, ACN/H 2O (v/v) ( Table1) 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, fe- namiphos and profenofos The enantiomers of the most of these analytes (except dialifos and profenofos) were well separated
Trang 4Table 1
Chromatographic data of the enantioseparation of some selected racemic basic compounds by nano-LC For experimental conditions see
text
t 0 (min)- flow rate
50 mM NH 4 HCO 3 pH 11 in
90/10, MeOH/H 2 O (v/v)
Alprenolol
6.643 - 200
0.43 0.60 1.40 1.7 14943 14017
15 mM NH 4 FA pH 2.5 in
90/10, ACN/H 2 O (v/v)
Lorazepam
3.73 - 355
Table 2
Chromatographic data of some selected neutral chiral analytes obtained using nano-LC Mobile phase, 15
mM NH 4 FA pH 2.5 in 90/10, ACN/H 2 O (v/v) For other experimental conditions, see text
Compounds
t 0 (min)- flow rate
Dialifos
3.73 - 355
( Table 2) Since among twelve studied analytes eight were fla-
vanone and its derivatives some correlations could be drawn be-
tween 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 re-
solved 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 consid-
ering that this substituent has an electron-donating effect increas-
ing the electron density on the conjugated rings of analytes and
thus, the interaction with the chiral selector through π-π mecha-
nism
The introduction of a hydroxyl group, although with electron-
donating properties, caused a lower enantioseparation than the
methoxy one However, 6-OH-Fla exhibited higher enantioresolu-
tion ( Rs=7.7) than flavanone Although good resolution of enan-
tiomers was obtained for the other hydroxy-derivative, their enan-
tioresolution factors were lower than the one of unsubstituted fla-
vanone 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 phospho- ric acid esters used as pesticides have been also studied Interest- ingly, two of studied three compounds, in particular, fenamiphos and profenofos owe their chirality to the asymmetrically substi- tuted phosphor atom in their structure Of this set of chiral an- alytes 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-chloro- 5-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 proto- nated aminopropyl moieties on the surface of silica may cause un- desirable peak tailing The mobile phase containing 5 mM am- monium formate pH 2.5 in 90:10, v/v ACN/H 2O was applied for the enantioseparation of selected acidic compounds such as nons- teroidal anti-inflammatory drugs (carprofen, cicloprofen, flurbipro- fen, ketoprofen, ketorolac, ibuprofen, indoprofen, naproxen, supro-
Trang 5Fig, 2 Enantiomeric separation of studied flavanone derivatives in nano-LC Exper-
imental conditions: 15 mM NH 4 FA pH 2.5 in 90/10, ACN/H 2 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 hexo-
barbital These chiral analytes belong to different structural
groups such as arylpropionic acid derivatives, coumarins and
barbiturates
Table3 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 an-
alytes and silica surface One of the possible reasons of this could
be low apparent pH of the mobile phase suppressing the nega-
tive charge on the analytes Based on literature [18]amylose tris(3-
chloro-5-methyphenylcarbamate) shows very high success rate for
separation of enantiomers of weak chiral acids, among them also
Fig 3 Nano-LC chiral separation of hexobarbital, suprofen, carprofen, and ketoro-
lac For experimental conditions see Fig 2 and text
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
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 chro- matographic performance of this material in nano-LC and CEC
As some selected chromatograms ( Fig 4), as well as plate num- bers ( 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
NH 4 FA pH 2.5 in 90/10, ACN/H 2 O (v/v) For other experimental conditions, see text
Compounds t (nL/min) 0 (min)- flow rate k’ 1 k’ 2 α Rs N 1 /m N 2 /m
Carprofen
3.73 - 355
0.35 0.50 1.43 2.4 34963 31909
Trang 6Fig 4 Comparative separation of enantiomers on CSP, i-amylose tris(3-chloro-5-
methylphenylcarbamate) (20%, w/w), (A) native silica (B) APS silica in nano-LC sys-
tem Experimental conditions: as reported in Fig 2 and text
tage of CSP based on APS silica over the CSP based on native sil- ica in the present study is also supported with van Deemter de- pendences shown for flurbiprofen, hexobarbital and flavanone on Fig.6
3.5 Preliminary attempts of enantioseparations in CEC
As mentioned above APS silica-based CSP showed some ad- vantages over the CSP based on native silica for nano-LC appli- cations under this study On the next step we tried to apply this CSP in CEC and were surprised with the absence of the electroos- motic flow (EOF) in these capillaries In many of our earlier stud- ies 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 for- mate pH 2.5 in 90/10, ACN/H 2O (v/v) a significant EOF appeared there but it was not stable and thus not suitable for providing ade- quate run to run repeatability Our detailed experiments for under- standing 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 ob- served 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
Trang 7Fig 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
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-NH 2 (5 μm) and, 20 cm CSP- i-amylose tris(3-chloro-
5-methylphenylcarbamate) (20%, w/w), APS silica (5 μm)), L eff = 26.5 cm, L tot = 34.9
cm; mobile phase, 5 mM NH 4 FA pH 2.5 in 90/10, ACN/H 2 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
application of this CSP made on the basis of APS silica in CEC re- quires further studies
5 Conclusions
As the results of this study indicate amylose tris(3-chloro-5- methylphenylcarbamate) is very useful chiral selector also in com- bination 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 car- bon but also nitrogen and phosphor The chiral selector immobi- lized 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 How- ever, further studies are required for optimizing CEC experiments and realizing potential advantages of CEC over nano-LC
Declaration of Competing Interest
The authors declare that they have no known competing finan- cial interests or personal relationships that could have appeared to influence the work reported in this paper
Trang 8Supplementary 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, Visual-
ization, Writing review & editing Alessandra Gentili: Writing
review & editing Marina Karchkhadze: Formal analysis Bezhan
Chankvetadze: Supervision, Methodology, Resources, Writing re-
view & editing
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