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

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.

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 May 2020 Accepted May 2020 Available online 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 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: salvatore.fanali@gmail.com (S Fanali) https://doi.org/10.1016/j.chroma.2020.461213 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 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 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 M) to the pH 2.5; ammonium hydrogen carbonate was weighed and dissolved in ultrapure water and titrated with ammonia solution (approx 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 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, μm; nominal pore size, 10 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 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 the inlet side of the capillary, a 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 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 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 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 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 (https://www.curveexpert.net/download/) 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 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 G D’Orazio, C Fanali and S Fanali et al / Journal of Chromatography A 1623 (2020) 461213 Table 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 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 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 Fig Nano-LC chiral separation of hexobarbital, suprofen, carprofen, and ketorolac For experimental conditions see Fig and text Fig, 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 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 reports the chromatographic data on the enantiomeric separation of the studied compounds Analytes were eluted in less than seven 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 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 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 3.5 Preliminary attempts of enantioseparations in CEC Fig 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 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 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 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 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 and text G D’Orazio, C Fanali and S Fanali et al / Journal of Chromatography A 1623 (2020) 461213 Fig 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 and text application of this CSP made on the basis of APS silica in CEC requires further studies 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 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, 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, mM NH4 FA pH 2.5 in 90/10, ACN/H2 O (v/v); Inj: 10 bar x 0.3 ; 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 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Comparative study on enantiomer resolving ability of amylose tris(3-chloro-5methylphenylcarbamate) covalently immobilized onto silica in nano-liquid chromatography and capillary electrochromatography,... 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... 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