Enantiomers separation by nano-liquid chromatography: Use of a novel sub-2 μm vancomycin silica hydride stationary phase

Accepted Manuscript Title: Enantiomers separation by nano-liquid chromatography: use of a novel sub-2 ␮m vancomycin silica hydride stationary phase Author: Silvia Rocchi Anna Rocco Joseph J. Pesek Maria T. Matyska Donatella Capitani Salvatore Fanali PII: DOI: Reference:

S0021-9673(15)00061-8 http://dx.doi.org/doi:10.1016/j.chroma.2015.01.015 CHROMA 356176

To appear in:

Journal of Chromatography A

Received date: Revised date: Accepted date:

11-11-2014 8-1-2015 9-1-2015

Please cite this article as: S. Rocchi, A. Rocco, J.J. Pesek, M.T. Matyska, D. Capitani, S. Fanali, Enantiomers separation by nano-liquid chromatography: use of a novel sub¨ ¨/>rmmum vancomycin silica hydride stationary phase, Journal of 2
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Highlights

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A novel sub-2 m vancomycin modified silica hydride particles was synthesized.

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The CSP material was packed into capillaries and used in nano-LC.

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Experiments were carried out studying both acidic and basic compounds.

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The novel CSP offered high enantioresolution for acidic compounds.

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Short analysis time were recorded using a conventional pump.

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Enantiomers separation by nano-liquid chromatography: use of a novel sub-2 m vancomycin silica hydride stationary phase.

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Silvia Rocchi1,2, Anna Rocco1, Joseph J. Pesek3, Maria T. Matyska3, Donatella Capitani1 and Salvatore Fanali1*

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Institute of Chemical Methodologies, Italian National Research Council (C.N.R.), 00015 Monterotondo (Rome) Italy Department of Physical and Chemical Sciences, University of L’Aquila, 67100 Coppito (L’Aquila) Italy

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Department of Chemistry, San José State University, San José, CA 95112, United States

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*Corresponding author: Dr. Salvatore Fanali, 1Institute of Chemical Methodologies, Italian National Research Council (C.N.R.), Via Salaria km 29,300 - 00015 Monterotondo (Rome) Italy

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Tel: +390690672256: e-mail: [email protected]

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Abstract

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A novel sub-2 µm chiral stationary phase (CSP) was prepared immobilizing vancomycin onto 1.8

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µm diol hydride-based silica particles. The CSP was packed into fused silica capillaries of 75 µm

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I.D. with a length of 11 cm and evaluated by means of nano-liquid chromatography (nano-LC)

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using model compounds of both pharmaceutical and environmental interest (some non-steroidal

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anti-inflammatory drugs, β-blockers and herbicides). The study of the effect of the linear velocity of

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the mobile phase on chromatographic efficiency showed good enantioresolutions up to a value of

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5.11 at the optimal linear velocity with efficiencies in terms of number of plates per meter in the

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range 51,650-68,330. The results were compared with the ones obtained employing 5 µm

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vancomycin modified diol-silica particles packed in capillaries of the same I.D. For the acidic

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analytes the sub-2 µm CSP showed better performances, the baseline chiral separation of several

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studied compounds occurred in an analysis time of less than 3 minutes. Column-to-column packing

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reproducibility (n=3) expressed as relative standard deviation was in the range 2.2-5.8% and 0.5-

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7.7% for retention times and peak areas, respectively.

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Keywords: nano-liquid chromatography, capillary columns, enantiomers, vancomycin stationary phase, sub-2 m silica hydride particles, herbicides, non-steroidal anti-inflammatory drugs, blockers

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1. Introduction

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Recently, there has been a growing interest in speeding up analysis considering the high number of

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samples to be analysed in applications such as pharmaceutical, food, and environmental. The

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pharmaceutical industry especially requires the development of faster analytical methods in order to

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enhance throughput and reduce costs required in many critical operational areas: drug discovery,

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assessment of purity and quality control of drug formulations, pharmacokinetic and drug

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metabolism studies, as well as enantiomeric separations [1].

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Several different approaches in liquid chromatography, that in several areas remains the separation

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technique of choice, have been developed to satisfy the demand for fast analysis without

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compromising separation efficiency and resolution. The main efforts have been focused on

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performing separations at high temperature (high-temperature liquid chromatography) [2] and

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developing column technology. With regard to the latter aspect, numerous investigations were

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undertaken to produce new stationary phases, such as monolithic continuous separation media [3]

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sub-3 µm superficially porous particles [4], and to reduce the particle size of the packing material

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up to sub-2 µm working under ultra-high pressure conditions (ultra-high pressure liquid

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chromatography, UHPLC) [5-8].

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In particular, the use of short columns packed with sub-2 µm porous silica-based particles, recently

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provided by different manufacturers, by means of UHPLC systems at high flow rates has shown a

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remarkable success in obtaining fast achiral separations in various application areas such as food

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chemistry, metabolic identification, pesticide residue analysis, environmental water analysis and

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pharmaceutical determinations [5, 9-11].

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The use of sub-2 µm particles can offer some advantages over the 3-5 µm materials, e.g., increased

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resolution and the possibility to reduce the analysis time working at flow rates higher than the

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optimum conditions (minimum plate height) without significant loss of efficiency. This is

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demonstrated observing the van Deemter equation. In fact the A- and C-terms are directly

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proportional to the particle size and to the square of the particle size, respectively. Therefore the

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use of smaller particles provides a decrease of the plate height together with a flatter profile of the

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right branch of the van Deemter curve [12, 13].

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Even though, as reported above, the sub-2 µm silica-based stationary phases have established

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themselves as an effective analytical tool in achiral applications, but in the field of chiral

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separations the technology related to the development of sub-2 µm chiral stationary phases (CSPs)

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is still under study. Chiral UHPLC columns are not yet commercially available. However some

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recent research articles, still limited in number, clearly reveal the potential of sub-2 µm silica-based

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CSPs in liquid chromatography. In this respect, some investigations report the preparation of brush-

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type CSPs, and their successfully application for UHPLC analysis, employing the Whelk-O1 and π-

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acidic bis-(3,5-dinitrobenzoyl)-derivative of trans-1,2-diaminocyclohexane as chiral selectors

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chemically immobilized onto sub-2 µm porous silica particles [14-16]. Other studies of UHPLC

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describe the use of sub-2 µm porous silica particles and sub-1 µm mesoporous silica particles

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functionalized with α-cyclodextrin and perphenylcarbamoylated-β-cyclodextrin, respectively [17,

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18]. The potential of mesoporous silica particles for HPLC as well as UHPLC chiral applications

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has been recently reviewed [19]. It is worth mentioning that interesting results in enantioselective

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UHPLC separations were obtained by using achiral sub-2 µm stationary phases with chiral additives

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added in the mobile phase or after precolumn derivatization of the compounds with chiral reagents

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[20-27] Also nano-liquid chromatography (nano-LC) has shown promising results in fast

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enantiomeric analysis utilizing two types of crown ether-capped β-cyclodextrin bonded stationary

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phases prepared by derivatizing non-porous 1.5 µm silica particles [28, 29]. Very short analysis

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times have been reported utilizing polysaccharides based chiral selectors coated either on

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monolithic or core-shell silica particles [30-32]. In general, the miniaturized versions of liquid

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chromatography, including nano-LC, offer several advantages arising from the low flow rate of the

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mobile phase (µL-nL/min) in column. Among them there are the consumption of minute volumes of

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mobile phase with a reduction of waste solvents and costs, the need of small sample volumes and an

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easier coupling with mass spectrometry. In addition, the requirement of small amounts of stationary

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phase is of particular interest while using expensive packing materials such as CSPs [ 33].

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In the present work for the first time, to the best of our knowledge, the well-known chiral selector

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vancomycin was immobilized onto sub-2 µm hydride-based silica particles and the

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chromatographic properties evaluated by nano-LC.

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The fundamental feature that distinguishes the type of silica used in this study from the ordinary

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material is that in the latter silanol groups (Si–OH) are present on the surface, while Si–H moieties

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dominate the surface of the hydride silica [ 34, 35]. The synthesized CSP was packed into 75 m

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I.D. capillaries and tested for enantiomeric separations using model compounds of both

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pharmaceutical and environmental interest and studying the effect of the linear velocity of the

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mobile phase on the chromatographic efficiency and enantioresolution factor (Rs). The results, in

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terms of enantioresolution, retention factor (k) and selectivity (α) were compared with those

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achieved employing 75 m I.D. columns packed with 5 µm ordinary silica diol particles modified

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with vancomycin in order to also investigate the influence of the silica support on the chiral

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recognition of acidic and basic compounds. In order to characterize the CSPs, NMR analysis on

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solid-state of the two stationary phases was also carried out.

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2. Material and methods

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2.1. Chemicals and samples

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All chemicals were of analytical reagent grade and used as received. Acetonitrile (ACN), methanol

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(MeOH), acetone, ammonium hydroxide solution (30% w/w ), sodium hydroxide, acetic acid,

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orthophosphoric acid (85% w/v) were provided by Carlo Erba (Milan, Italy). Formic acid, sodium

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metaperiodate, sodium cyanoborohydride were from Merck (Darmstadt, Germany). Sodium

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phosphate monobasic monohydrate and vancomycin hydrochloride were purchased from Sigma6

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Aldrich (St. Louis, MO, USA). A Milli-Q system (Millipore Waters, Milford, MA, USA) was

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employed to purify water. The buffers used in the synthesis of vancomycin-based CSPs were

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prepared by diluting the proper amounts of sodium phosphate monobasic and orthophosphoric acid

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in water and adjusting the pH to the desired values with sodium hydroxide solution. Ammonium

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acetate and formate buffers (500 mM), utilized for the chromatographic runs, were obtained by

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titrating the appropriate volumes of acetic or formic acid with concentrated ammonia solution to pH

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4.5 and 3.5, respectively. Mobile phases employed for the nano-LC experiments were prepared

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daily by mixing suitable volumes of buffer solutions, water and organic solvents (ACN or MeOH).

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The following herbicides in the free acid form, dichlorprop, diclofop, haloxyfop, fenoprop,

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fluazifop, mecoprop (all racemic mixtures) were purchased from Dr. Ehrenstorfer GmbH

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(Augsburg, Germany). Racemic standard compounds of non-steroidal anti-inflammatory drugs

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(NSAIDs) selected for this study were purchased from Sigma-Aldrich (St. Louis, MO, USA),

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namely, ibuprofen, indoprofen, ketoprofen, naproxen. Racemic carprofen, cicloprofen, flurbiprofen,

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suprofen and single enantiomers of R(-) and S(+) of flurbiprofen, naproxen, suprofen and S(+)-

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ibuprofen were kindly provided by Dr. Cecilia Bartolucci (Institute of Crystallography, CNR,

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Monterotondo,

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(DF1738Y) were kindly supplied by Dompé (L’Aquila, Italy). The standard compounds of the

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following β-blockers were bought from Sigma-Aldrich (St. Louis, MO, USA): alprenolol,

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metoprolol, oxprenolol, pindolol, propranolol (all racemic mixtures), (-)-alprenolol, (-)-atenolol,

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(+)-atenolol, (-)-propranolol.

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Stock standard solutions of NSAIDs (1 mg/mL) were prepared in ACN, while all other studied

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analytes were dissolveld in MeOH. Individual working solutions at concentrations of 50-100 µg/mL

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were obtained by dilution of the stock solutions in ACN, while for the β-blockers MeOH was used

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as the sample solvent. All solutions were stored at -18°C when not in use. Figures 1a, b and c shows

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the chemical structure of the studied racemic compounds.

Italy).

Racemic

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2-[(5’-benzoyl-2’-hydroxy)phenyl]propionic

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2.2. Instrumentation

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A BASIC 20 pH meter (Crison, Barcelona, Spain) was employed for accurate pH measurements in

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aqueous buffer solutions. A Decon model FS 100b (Hove, UK) ultrasonic bath and an R-200

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Rotavapor (BÜCHI Labortechnik AG, Flawil, Switzerland) were used during the derivatization

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procedure of silica-based stationary phases. A Series 10 LC HPLC pump (Perkin Elmer, Palo Alto,

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CA, USA) and a Stereozoom 4 optical microscope (Cambridge Instruments, Vienna, Austria) were

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employed for the capillary packing process.

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To perform nano-LC experiments a laboratory-made instrument was assembled utilizing an HPLC

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pump (Perkin Elmer series 10 LC, Palo Alto, CA, USA), a modified six-port injection valve

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(Enantiosep GmbH Münster, Germany), and an UV/VIS on-column detector (Spectra Focus

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PC1000, Thermo Separation Products, San Jose, CA, USA). The wavelength for UV detection was

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set at 200 nm. Data were collected using ClarityTM Advanced Chromatography Software (DataApex

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Ltd., Prague, Czech Republic). The HPLC pump, delivering continuously MeOH isocraticly, and

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the injector were connected so as to obtain a passive split-flow system needed to reduce the flow

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rate at nL/min levels, as follows. Both pump and injection valve were joined to a stainless steel T

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piece (Vici, Valco, Houston, TX, USA) by means of 500 m I.D. stainless steel tubes with lengths

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of 70 and 5 cm, respectively. The third entrance of the tee was connected to the waste, consisting of

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the MeOH reservoir of the pump, through a fused silica capillary (50 m I.D. x 20 cm) achieving a

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continuous recycling of the organic solvent. The capillary column was directly inserted into the

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modified injector equipped with a loop of about 11 L.

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Both sample and mobile phase were introduced in the chromatographic system through the injection

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valve. Injections were carried out by filling the loop with the sample solutions, switching the valve

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for the suitable time and then flushing the loop with the mobile phase. Change of the time switching

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allowed control of the injected sample volume.

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The flow rate of the mobile phase was calculated by measuring the volume of mobile phase eluting

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from the capillary column after a known time by means of a 10 L syringe (Hamilton, Reno, NV,

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USA) connected to the column by a Teflon tube (TF-350; LC-Packing, CA, USA).

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2.3. Preparation of vancomycin-based chiral stationary phases and capillary columns

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The CSPs used in this work were prepared by chemically bonding of vancomycin to diol hydride-

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based silica particles with a 1.8 µm particle size, donated by MicroSolv Technology Corp,

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Eatontown NJ, USA ), and to LiChrospher® 100 DIOL silica particles with a 5 µm particle size

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from Merck (Darmstadt, Germany), following a previously reported procedure [ 36]. Both types of

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silica particles had a nominal pore size of 100 Å.

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Fused silica capillaries (75 m I.D. x 375 m O.D.), purchased from CM Scientific (Silsden, West

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Yorkshire, UK), were packed with the synthesized CSPs employing a slurry packing method

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already described in our previous works [ 37, 38]. The packed length was 11 cm .

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2.4. Chromatographic conditions

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For each class of compounds the experimental conditions were chosen on the basis of our previous

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results achieved employing capillary columns packed with 5 µm vancomycin modified diol-silica

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particles [ 38, 39]. A mobile phase composed of 500 mM ammonium acetate buffer pH

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4.5/H2O/MeOH (5:10:85, v/v/v was used for herbicides and 500 mM ammonium acetate buffer pH

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4.5/H2O/ACN (1:9:90, v/v/v ) for NSAIDs. In the case of enantiomeric separation of DF 1738Y

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and β-blockers mobile phases consisting of 500 mM ammonium formate buffer pH 3.5/H2O/ACN

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(1:9:90, v/v/v) and 500 mM ammonium acetate buffer pH 4.5/H2O/MeOH (1:4:95, v/v/v ) were

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employed, respectively. Flow rates of mobile phase were in the range 10-1700 nL/min, while

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injected sample volumes in the range 40-90 nL. All experiments were carried out with isocratic

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elution at ambient temperature (25 °C) controlled with a continuous room air conditioning system.

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2.5. Calculation of chromatographic parameters

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For the evaluation of chromatographic efficiency, the number of theoretical plates (N) was 9

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calculated using the following equation :  t N  5.54 R  w1 / 2

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where tR and w1/2 are the retention time and the peak width at half height, respectively.

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The resolution factor, Rs, was calculated according to the following formula:

(1)

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Rs  2 

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t R 2  t R1 w1  w 2

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where tR1 and tR2 are the retention times of the enantiomers and w1 and w2 are the peak widths at

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baseline.

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The van Deemter equation reported below was employed to fit nano-LC data by Curve expert 1.40

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(http://www.curveexpert.net):

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H = A + B/u + Cu

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(3)

where H (H=L/N, with L the packed length) is the height equivalent to the theoretical plate (HETP)

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in m and u is the linear velocity of the mobile phase in µm/ms. The system peaks in the baseline

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were used as marker of dead time.

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2.6. Solid-state NMR analysis for the characterization of CSPs

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Samples were packed into 4-mm zirconia rotors, and sealed with Kel-F caps.

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Bruker Avance III spectrometer operating at the proton frequency of 400.13 MHz. The spinning

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rate was set at 8 kHz. 29Si CPMAS spectra were obtained with a contact time of 3 ms and a recycle

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delay of 3 s. 13C CPMAS spectra were obtained with a contact time of 1.5 ms and a recycle delay

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of 3 s. In both cases the cross-polarization was carried out by applying the variable spin-lock

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sequence RAMP–CPMAS [ 40], the RAMP was applied on the 1H channel, and during the contact

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time the amplitude of the RAMP was increased from 50 to 100% of the maximum value.

Si and

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C solid state NMR spectra were recorded at 79.49 and 100.63 MHz respectively on a

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Si MAS spectra were obtained with a pulse length of 3 s and a

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Quantitative proton decoupled

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recycle delay optimized at 150 s.

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Spectra were recorded with a time domain of 1024 data points, zero filled and Fourier transformed

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with a size of 4096 data points.

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exponential multiplication with a line broadening of 16 Hz whereas

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apodized applying an exponential multiplication with a line broadening of 32 Hz.

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chemical shifts were referenced to tetramethylsilane used as an external reference.

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The deconvolution of

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[41]. Each resonance was modelled by a gaussian lineshape, and characterized by amplitude,

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chemical shift and width at half height.

C CPMAS were apodized applying an 29

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Si MAS spectra were 29

Si and

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Si MAS spectra was carried out by using the dm2006 software package

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3. Results and discussion

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Diol hydride-based silica particles of 1.8 µm diameter chemically modified with vancomycin were

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packed into fused silica capillaries of 75 µm I.D. and evaluated by means of nano-LC for the chiral

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separation of standard racemic mixtures of some non-steroidal anti-inflammatory drugs (NSAIDs),

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herbicides and β-blockers. As described in Section 2.4., for this work the experimental conditions in

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terms of mobile phase were chosen on the basis of our previous studies and used for both 1.8 µm

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and 5 µm vancomycin-based CSPs in order to compare their chromatographic performances in

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capillary columns with the same I.D.

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To perform fast liquid chromatographic separations with sub-2 µm particles requires the use of

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dedicated pumps capable of operating at the high backpressures generated, although columns

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packed for only a few centimeters are employed. In this study, as already reported in our previous

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work with capillary columns packed with sub-2 µm C18 particles [12, 42, 43], the assembly of a

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laboratory-made nano-LC instrument by means of a passive split-flow system allowed the use of a

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conventional HPLC pump for all the experiments carried out on 75 m I.D. capillary columns

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packed for 11 cm with 1.8 µm vancomycin modified diol-silica particles. The highest backpressure,

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equal to 360 bar, measured in the pump (before splitting) were recorded using the mobile phase

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selected for DF 1738Y (500 mM ammonium formate buffer pH 3.5/H2O/ACN 1:9:90, v/v/v) at a

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flow rate of 1.7 µL/min (the flow rate of the MeOH from the pump was 2.6 mL/min).

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Although in LC the composition of mobile phase as well as separation selectivity are important

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parameters to be controlled for achieving optimal separations affecting both peak shape and width,

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and consequently the number of theoretical plates, this is not the only factor to be considered.

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Among others the sample solvent plays an important role in the chromatographic performance

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because, if appropriate, band broadening can be reduced . For the above mentioned reasons first, in

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this work, preliminary studies were carried out to find the proper sample solvent for the selected

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acidic analytes, herbicides, NSAIDs and DF1738Y, employed as model compounds to evaluate the

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vancomycin-based CSPs performances. For this purpose, the mobile phase and solvents as water,

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ACN and MeOH in presence and absence of the buffer (same type and concentration of the mobile

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phase) were tested. For all substances pure ACN, as sample solvent, provided the best results in

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terms of resolution, peak symmetry and efficiency. Therefore this solvent was chosen for further

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experiments.

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3.1. Chiral separations with sub-2 µm vancomycin-based stationary phase

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Tables 1 and 2 report the chromatographic parameters, namely, retention time (tR), retention factor

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(k), selectivity (α) and resolution factor (Rs) for the selected herbicides and NSAIDs analyzed on a

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75 µm I.D. capillary packed with 1.8 µm diol hydride-based silica particles derivatized with

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vancomycin at a flow rate of 230 and 360 nL/min, respectively, corresponding in both cases to a

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linear velocity of mobile phase of 1.2 mm/s. Figure 2 shows the nano-LC separation of the studied

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enantiomers. As can be seen, good resolution was achieved although the flow rates of the mobile

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phase was not optimal with retention times less than 8 minutes. From a first comparison with the

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data of our previous works done with 5 µm vancomycin modified diol silica particles packed in

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columns having the same I.D. [ 38, 39] the novel CSP produced better results, in terms of resolution

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factors. In fact, in the previous studies (CSP with 5 µm silica), using a longer packed column (23

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cm), resolution values between 1.3 and 1.9 were obtained for the herbicides (dichlorprop, fenoprop

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and mecoprop) in a longer analysis time (about 24 minutes). Furthermore for the studied NSAIDs

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(ketoprofen and indoprofen) Rs < 1 were obtained.

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In order to better investigate the potential of sub-2 µm vancomycin modified silica hydride

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particles, their chromatographic performance was evaluated studying the effect of the linear

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velocity of the mobile phase on plate height by means of the van Deemter equation for the retention

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of flurbiprofen, chosen as test compound for the NSAIDs (flow rates in the range 10-600 nL/min),

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and fenoprop selected for the herbicides (flow rates in the range 15-440 nL/min). The plots obtained

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for these analytes are shown in Figure 3. As can be observed, for both enantiomers an analogous

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curve trend was obtained, with an evident increase of column performance with decreasing the

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linear velocity. As a consequence, the highest efficiency occurred at rather low flow rate. However,

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at the optimum linear velocity good results were obtained with an Rs value of 5.11 and 67,000 and

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51,650 N/m for S-(+)-flurbiprofen and R-(-)-flurbiprofen, respectively. A similar trend was

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observed for both enantiomers of fenoprop (data not shown) in terms of the profile of the van

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Deemter curves, optimal linear velocity and best performances (Rs = 5.07, 68,330 and 57,460 N/m

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for the first and the second eluting enantiomer, respectively).

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3.2. Chromatographic comparison between sub-2 µm and 5 µm CSPs

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With the aim of carrying out a direct comparison between 1.8 µm and 5 µm diol silica particles

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modified with vancomycin, some acidic and basic model substances of pharmaceutical interest,

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namely NSAIDs and DF1738Y (related compound of ketoprofen), basic drugs (alprenolol, atenolol,

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metoprolol, oxprenolol, pindolol and propranolol) and some herbicides were analyzed using the

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same chromatographic conditions on capillary columns packed with the CSP of 5 µm silica (the

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same length and capillary I.D.). Table 3 reports the chromatographic data obtained analyzing the

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studied acidic compounds using the CSP synthesized with conventional silica.

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As can be seen poor enantioresolution was obtained for all analyzed acidic compounds with Rs

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values in the range 0.00-0.64 and 0.45-1.09 for NSAIDs and herbicides, respectively. These values

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were much lower than the ones obtained with the novel CSP 1.27-2.85 and 2.26-3.36; retention

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factors and  were also lower. Figure 4 shows the nano-LC chromatograms of the enantioseparation

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of the studied acidic compounds.

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The comparison of the nano-LC enantioseparation of the basic compounds under study utilizing the

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two CSPs revealed opposite results than the ones obtained for acidic analytes as reported in Table 4.

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Both columns exhibited quite similar enantioselectivity but different retention times, retention

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factors and enantioresolutions. Higher values of these parameters were recorded demonstrating a

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higher affinity for the studied compounds towards the 5 µm CSP 5 particles. Figure 5 shows the

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nano-LC chromatograms of the separation of the basic compounds under study utilizing the two

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different CSPs.

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Based on the above data it can be concluded that the two CSPs exhibited different behavior when

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analyzing basic or acidic compounds especially concerning retention factor and resolution, while

329

comparable enantioselectivity was obtained. The different behavior can be explained considering

330

the different type of silica used. The presence of silanol groups in the 5µm silica CSP influences

331

non-specific interactions between analytes and the surface for positively charged compounds due to

332

the more hydrophilic properties of the surface. The surface of the 1.8 µm silica does not contain

333

silanols and therefore is more hydrophobic than the other CSP interacting more with acidic

334

compounds.

335

These conclusion are supported by the NMR analysis of the two CSPs. Figure 6 (a) and (b) shows

336

the

337

elements of bare silica consisting of Q2, Q3, and Q4 species are observed. Specifically, signals of

338

bulk siloxane Q4 units are observed at -109.8 ppm, whereas Q3 units due to isolated silanol groups

339

and Q2 units due to geminal silanol groups, are observed at -100.6 and -90.7 ppm, respectively, see

340

Figure 6 . As is well known [ 44], resonances of trifunctional silanes are found between -45 and -

341

70 ppm. In the 5 m CSP resonances at -65.5 and -56.2 ppm indicate the presence of T3 and T2

342

units (see Figure 6 panel e), whereas in the 1.8 m CSP , besides resonances due to T2 and T3 units

343

there is also a resonance centred at -84.5 ppm due to C unit, see Figure 6 panel (e).

344

Whereas 29Si CPMAS spectra are not quantitative, 29Si MAS spectra collected with a long, suitable

345

recycle delay may be used for quantitative analysis.

346

an

M

Si CPMAS NMR spectra of the 5 m (a) and 1.8 m (b) CSPs. In both spectra structural

Ac ce p

te

d

29

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cr

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325

29

Si MAS spectra of 5 and 1.8 m CSPs are reported in Figure 6 c and d respectively. In the figure

347

the deconvoluted spectra are superimposed to the experimental ones. The deconvolution procedure

348

allowed the obtainment of the integral of each resonance to be used for the quantitative analysis, see

15

Page 15 of 43

Table 5. In 5 m CSP the amount of bare silica was found to be 87.5% (Q4 68.4%, Q3 14.5%, and

350

Q2 4.6 %), and units T3 and T2 accounted for 6.6 and 5.9 % respectively.

351

In 1.8 m CSP the amount of bare silica was found to be 93.7% (Q4 72.0%, Q3 19.6%, and Q2

352

2.1%), T3 and T2 units accounted for 2.0 and 1.0 % respectively, and C units for 3.3 %.

353

Figure 7 shows the

354

m (b) and 1.8 m (d) diol particles. In the same figure the assignment of peaks of carbon atoms of

355

diol is reported. In the spectrum of 5 m diol particles methylene carbons 1a and 2a are observed at

356

8.7 and 22.9 ppm respectively, methylene carbon 6a resonates at 63.7 ppm whereas other carbons

357

resonate at 71.6 ppm. In the spectrum of 1.8 m diol particles methylene carbon 1b is observed at

358

13.3 ppm, methylene carbons 2b,3b, and 4b resonate between 26 and 33 ppm, methylene carbon 6b

359

is observed at 67.6 ppm, and methine carbon 5b at 72.7 ppm. All resonances of the carbon spectrum

360

of diol particles are also observed in the corresponding spectra of CSPs, see figure 7a and c. All

361

other peaks in these spectra belong to vancomycin.

362

3.3. Column-to-column and synthesis reproducibility

363

Column-to-column reproducibility was evaluated packing three capillary columns with the 1.8 µm

364

CSP following the procedure reported in Section 2.3. and analysing the studied NSAIDs at the same

365

linear velocity of the mobile phase. The linear velocity selected is the same as the data reported in

366

Table 2 (1.2 mm/s). Column-to-column reproducibility, expressed as relative standard deviation

367

(RSD), was calculated using the mean values obtained from the packed columns performing three

368

replicates for each analysis injecting 70 nL of the sample solution. For the retention times and peak

369

areas RSD% values are in the range of 2.2-5.8 and 0.5-7.7%, while for the selectivity the values

370

were between 0.8 and 5.4%. Concerning the chromatographic efficiency in terms of height

371

equivalent to the theoretical plate, RSD% values ranging from 5.2 to 13.9% were achieved.

372

The reproducibility of the synthetic procedure for the chemical immobilization of vancomycin on

373

the 1.8 µm diol hydride-based silica particles was also evaluated. The derivatization process

C CPMAS NMR spectra of 5 m (a) and 1.8 m (c) CSPs, and spectra of 5

Ac ce p

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13

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349

16

Page 16 of 43

(Section 2.3.) was repeated by another co-worker in our laboratory employing the sub-2 µm

375

particles and a 75 µm I.D. capillary was packed with this new CSP following the established

376

packing procedure. The reliability of the synthesis for this type of silica support and the results

377

obtained in terms of chiral recognition ability were confirmed, as shown in Figure 8.

ip t

374

378

3.4. Improving enantioresolution and analysis time changing the mobile phase flow rate

380

It is known that changes of flow rate can influence the resolution and analysis time. Therefore, in

381

order to verify this principle, the flow rate was decreased at 50 nL/min for the separation of the

382

selected compounds exhibiting poor enantioresolution utilizing the column containing 5 µm

383

particles.

384

Figure 9 shows the chiral separation of ketoprofen and flurbiprofen at 380 nL/min and at 50

385

nL/min. As can be observed the two compounds were poorly resolved at the highest flow rate, while

386

the enantiomers were almost baseline separated at the lowest. Although better results were obtained,

387

analysis time increased (from 3.0 and 3.5 min to 22 and 27 min for flurbiprofen and ketoprofen,

388

respectively). To verify the possibility to speed up the analysis DF1738Y was analyzed with the

389

CSP 1.8 m particles at mobile phase flow rate of 330 nL/min and 1.7 µL/min. As can be seen in

390

Figure 10 analysis time was reduced to 1 min while having a high Rs value of 2.12. From the

391

reported example it is evident that the novel CSP, due to its good enantioselectivity, can be also

392

advantageously used for fast chiral analysis. Finally in order to reduce analysis time of NSAIDs

393

compounds were analyzed by nano-LC at a relatively high flow rate (900 nL/min) achieving Rs

394

values in the range 1.05-3.12 and with retention times of 1-3 min.

Ac ce p

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cr

379

395 396

4. Concluding remarks

397

A novel CSP prepared by chemical reaction between 1.8 µm diameter diol-silica hydride and

398

vancomycin was packed into capillaries having a 75 m I.D. with a length of 11 cm and studied for

399

enantiomer separation of basic and acidic racemic compounds. The CSP was effective in the 17

Page 17 of 43

resolution of chiral acidic compounds, showing better chromatographic performance compared to

401

that obtained employing 5 µm ordinary silica diol particles modified with vancomycin. However,

402

basic compounds were better resolved with CSPs synthesized utilizing ordinary silica. Selected

403

NSAIDs, DF1738Y and diclofop were enantiomerically resolved in a short time (less than 3 min)

404

with very high resolution factors (Rs = 1.05-3.12) at high flow rates on the silica hydride-based

405

column. Considering the good results achieved, further studies will be carried out derivatizating the

406

sub-2 µm diol-silica hydride particles with other glycopeptide antibiotics and evaluating their

407

potential for chiral nano-LC separations.

us

cr

ip t

400

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408

18

Page 18 of 43

5. References

410

[1] S. Fekete, I. Kohler, S. Rudaz, D. Guillarme, Importance of instrumentation for fast liquid

411

chromatography in pharmaceutical analysis, J. Pharm. Biomed. Anal. 87 (2014) 105-119.

412

[2] S. Heinisch, J.-L. Rocca, Sense and nonsense of high-temperature liquid chromatography, J.

413

Chromatogr. A 1216 (2009) 642-658.

414

[3] P. Jandera, Advances in the development of organic polymer monolithic columns and their

415

applications in food analysis-A review, J. Chromatogr. A 1313 (2013) 37-53.

416

[4] G. Guiochon, F. Gritti, Shell particles, trials, tribulations and triumphs, J. Chromatogr. A 1218

417

(2011) 1915-1938.

418

[5] Y. Wang, F. Ai, S.-C. Ng, T.T.Y. Tan, Sub-2 µm porous silica materials for enhanced separation

419

performance in liquid chromatography, J. Chromatogr. A 1228 (2012) 99-109.

420

[6] F. Badoud, E. Grata, J. Boccard, D. Guillarme, J.-L. Veuthey, S. Rudaz, M. Saugy,

421

Quantification of glucuronidated and sulfated steroids in human urine by ultra-high pressure liquid

422

chromatography quadrupole time-of-flight mass spectrometry, Anal. Bioanal. Chem. 400 (2011)

423

503-516.

424

[7] J.W. Jorgenson, Capillary Liquid Chromatography at Ultrahigh Pressures, Annu. Rev. Anal.

425

Chem. 3 (2010) 129-150.

426

[8] M.M. Aguilera-Luiz, J.L. Martínez Vidal, R. Romero-González, A. Garrido Frenich, Multi-

427

residue determination of veterinary drugs in milk by ultra-high-pressure liquid chromatography–

428

tandem mass spectrometry, J. Chromatogr. A 1205 (2008) 10-16.

429

[9] L. Bijlsma, J.V. Sancho, E. Pitarch, M. Ibáňez, F. Hernández, Simultaneous ultra-high-pressure

430

liquid

431

amphetamine-like stimulants, cocaine and its metabolites, and a cannabis metabolite in surface

432

water and urban wastewater, J. Chromatogr. A 1216 (2009) 3078-3089.

Ac ce p

te

d

M

an

us

cr

ip t

408 409

chromatography–tandem

mass

spectrometry

determination

of

amphetamine

and

19

Page 19 of 43

[10] A. Garrido Frenich, J.L. Martínez Vidal, R. Romero-González, M. del Mar Aguilera-Luiz,

434

Simple and high-throughput method for the multimycotoxin analysis in cereals and related foods by

435

ultra-high performance liquid chromatography/tandem mass spectrometry, Food Chem. 117 (2009)

436

705-712.

437

[11] E. Grata, J. Boccard, D. Guillarme, G. Glauser, P.-A. Carrupt, E.E. Farmer, J.-L. Wolfender, S.

438

Rudaz, UPLC–TOF-MS for plant metabolomics: A sequential approach for wound marker analysis

439

in Arabidopsis thaliana, J. Chromatogr. B 871 (2008) 261-270.

440

[12] G. D’Orazio, A. Rocco, S. Fanali, Fast-liquid chromatography using columns of different

441

internal diameters packed with sub-2 µm silica particles J. Chromatogr. A 1228 (2012) 213-220.

442

[13] J.J. van Deemter, F.J. Zuiderweg, A. Klinkenberg, Longitudinal diffusion and resistance to

443

mass transfer as causes of nonideality in chromatography, Chem. Eng. Sci. 5 (1956) 271-289.

444

[14] D. Kotoni, A. Ciogli, C. Molinaro, I. D’Acquarica, J. Kocergin, T. Szczerba, H. Ritchie, C.

445

Villani, F. Gasparrini, Introducing Enantioselective Ultrahigh-Pressure Liquid Chromatography

446

(eUHPLC): Theoretical Inspections and Ultrafast Separations on a New Sub-2-μm Whelk-O1

447

Stationary Phase, Anal. Chem. 84 (2012) 6805-6813.

448

[15] D. Kotoni, A. Ciogli, I. D’Acquarica, J. Kocergin, T. Szczerba, H. Ritchie, C. Villani, F.

449

Gasparrini, Enantioselective ultra-high and high performance liquid chromatography: A

450

comparative study of columns based on the Whelk-O1 selector, J. Chromatogr. A 1269 (2012) 226-

451

241.

452

[16] G. Cancelliere, A. Ciogli, I. D’Acquarica, F. Gasparrini, J. Kocergin, D. Misiti, M. Pierini, H.

453

Ritchie, P. Simone, C. Villani, Transition from enantioselective high performance to ultra-high

454

performance liquid chromatography: A case study of a brush-type chiral stationary phase based on

455

sub-5-micron to sub-2-micron silica particles, J. Chromatogr. A 1217 (2010) 990-999.

456

[17] F. Ai, Y. Wang, H. Chen, Y. Yang, T.T.Y. Tan, S.-C. Ng, Enantioselective separation of

457

dansyl-DL-amino acids and some racemates on “click” functionalized native α-cyclodextrin based

458

sub-2 µm columns, Analyst 138 (2013) 2289-2294.

Ac ce p

te

d

M

an

us

cr

ip t

433

20

Page 20 of 43

[18] F. Ai, L. Li, S.-C. Ng, T.T.Y. Tan, Sub-1-micron mesoporous silica particles functionalized

460

with cyclodextrin derivative for rapid enantioseparations on ultra-high pressure liquid

461

chromatography, J. Chromatogr. A 1217 (2010) 7502-7506.

462

[19] I. Sierra, D. Pérez-Quintanilla, S. Morante, J. Gaňán, Novel supports in chiral stationary phase

463

development for liquid chromatography. Preparation, characterization and application of ordered

464

mesoporous silica particles, J. Chromatogr. A 1363 (2014) 27–40.

465

[20] C. Müller, J.R. Fonseca, T.M. Rock, S. Krauss-Etschmann, P. Schmitt-Kopplin,

466

Enantioseparation and selective detection of D-amino acids by ultra-high-performance liquid

467

chromatography/mass spectrometry in analysis of complex biological samples, J. Chromatogr. A

468

1324 (2014) 109-114.

469

[21] M.L. Reyes-Reyes, G. Roa-Morales, R. Melgar-Fernández, H. Reyes-Pérez, P. Balderas-

470

Hernández, UHPLC Determination of Enantiomeric Purity of Sertraline in the Presence of its

471

Production Impurities, Chromatrographia 77 (2014) 1315–1321.

472

[22] H. Tsutsui, T. Mochizuki, T. Maeda, I. Noge, Y. Kitagawa, J.Z. Min, K. Todoroki, K. Inoue, T.

473

Toyo’oka, Simultaneous determination of DL-lactic acid and DL-3-hydroxybutyric acid

474

enantiomers in saliva of diabetes mellitus patients by high-throughput LC–ESI-MS/MS, Anal.

475

Bioanal. Chem. 404 (2012) 1925-1934.

476

[23] M.T. Furlong, B. He, W. Mylott, S. Zhao, T. Mariannino, J. Shen, B. Stouffer, A validated

477

enantioselective LC–MS/MS assay for the simultaneous determination of carvedilol and its

478

pharmacologically active 4’-hydroxyphenyl metabolite in human plasma: Application to a clinical

479

pharmacokinetic study, J. Pharm. Biomed. Anal. 70 (2012) 574-579.

480

[24] Y.-H. Wang, B. Avula, X. Fu, M. Wang, I.A. Khan, Simultaneous Determination of the

481

Absolute Configuration of Twelve Monosaccharide Enantiomers from Natural Products in a Single

482

Injection by a UPLC-UV/MS Method, Planta Med. 78 (2012) 834-837 .

Ac ce p

te

d

M

an

us

cr

ip t

459

21

Page 21 of 43

[25] Y. Xiao, T.T.Y. Tan, S.-C.Ng, Enantioseparation of dansyl amino acids by ultra-high pressure

484

liquid chromatography using cationic β-cyclodextrins as chiral additives, Analyst 136 (2011) 1433-

485

1439.

486

[26] Guillarme, G. Bonvin, F. Badoud, J. Schappler, S. Rudaz, J.-L. Veuthey, Fast Chiral

487

Separation of Drugs Using Columns Packed with Sub-2 µm Particles and Ultra-High Pressure,

488

Chirality 22 (2010) 320-330.

489

[27] J. Yang, Y. Wang, L. Pan, N. L., X. Lu, J. Guan, M. Cheng, F. L., Enantioselective

490

determination of trantinterol in rat plasma by ultra performance liquid chromatography–electrospray

491

ionization mass spectrometry after derivatization, Talanta 79 (2009) 1204-1208.

492

[28] Y. Gong, Y. Xiang , B. Yue , G. Xue , J.S. Bradshaw, H.K. Lee, M.L. Lee, A pplication of

493

diaza-18-crown-6-capped β-cyclodextrin bonded silica particles as chiral stationary phases for

494

ultrahigh pressure capillary liquid chromatography, J. Chromatogr. A 1002 (2003) 63-70.

495

[29] Y. Gong, G. Xue, J.S. Bradshaw, M.L. Lee, H.K. Lee, Synthesis of Crown Ether-capped 3-(2-

496

O-β-Cyclodextrin)-2-hydroxypropylsilyl Silica Particles for Use as Chiral Stationary Phases in

497

Chromatography, J. Heterocycl. Chem. 38 (2001) 1317-1321.

498

[30] B. Chankvetadze, C. Yamamoto, Y. Okamoto, Very Fast Enantioseparation in High-

499

performance Liquid Chromatography Using Cellulose Tris(3,5-dimethylphenylcarbamate) Coated

500

on Monolithic Silica Support, Chem. Lett. 32 (2003) 850-851.

501

[31] B. Chankvetadze, C. Yamamoto, M. Kamigaito, N. Tanaka, K. Nakanishi, Y. Okamoto, High-

502

performance liquid chromatographic enantioseparations on capillary columns containing monolithic

503

silica modified with amylose tris(3,5-dimethylphenylcarbamate), J. Chromatogr. A 1110 (2006) 46-

504

52.

505

[32] K. Lomsadze, G. Jibuti, T. Farkas, B. Chankvetadze, Comparative high-performance liquid

506

chromatography enantioseparations on polysaccharide based chiral stationary phases prepared by

507

coating totally porous and core–shell silica particles, J. Chromatogr. A 1234 (2012) 50-55.

Ac ce p

te

d

M

an

us

cr

ip t

483

508 22

Page 22 of 43

[ 33] J. Hernández-Borges, Z. Aturki, A. Rocco, S. Fanali, Recent applications in nanoliquid

510

chromatography, J. Sep. Sci. 30 (2007) 1589-1610.

511

[ 34] J.J. Pesek, M.T. Matyska, Our favorite materials: Silica hydride stationary phases, J. Sep. Sci.

512

32 (2009) 3999-4011.

513

[ 35] J.J. Pesek, M.T. Matyska, Silica Hydride Surfaces: Versatile Separation Media for

514

Chromatographic and Electrophoretic Analyses, J. Liq. Chromatogr. Related Technol. 29 (2006)

515

1105-1124.

516

[ 36] C. Desiderio, Z. Aturki, S. Fanali, Use of vancomycin silica stationary phase in packed

517

capillary electrochromatography. I. Enantiomer separation of basic compounds, Electrophoresis 22

518

(2001) 535-543.

519

[ 37] S. Fanali, Z. Aturki, V. Kašicka, M.A. Raggi, G. D’Orazio, Enantiomeric separation of

520

mirtazapine and its metabolites by nano-liquid chromatography with UV-absorption and mass

521

spectrometric detection, J. Sep. Sci. 28 (2005) 1719-1728.

522

[ 38] G. D’Orazio, Z. Aturki, M. Cristalli, M.G. Quaglia, S. Fanali, Use of vancomycin chiral

523

stationary phase for the enantiomeric resolution of basic and acidic compounds by nano-liquid

524

chromatography, J. Chromatogr. A 1081 (2005) 105-113.

525

[ 39] N. Rosales-Conrado, M.E. León-González, G. D’Orazio, S. Fanali, Enantiomeric separation of

526

chlorophenoxy acid herbicides by nano liquid chromatography-UV detection on a vancomycin-

527

based chiral stationary phase, J. Sep. Sci. 27 (2004) 1303-1308.

528

[ 40] G. Metz, X.L. Wu, S.O. Smith, Ramped-Amplitude Cross Polarization in Magic-Angle-

529

Spinning NMR, J. Magn. Reson., Ser A 110 (1994) 219-227.

530

[41] D. Massiot, F. Fayon, M. Capron, I. King, S. Le Calvé, B. Alonso, J.O. Durand, B. Bujoli, Z.

531

Gan, G. Hoatson, Modelling one- and two-dimensional solid-state NMR spectra, Magn. Reson.

532

Chem. 40 (2002) 70-76.

Ac ce p

te

d

M

an

us

cr

ip t

509

23

Page 23 of 43

[ 42] A. Rocco, C. Fanali, L. Dugo, L. Mondello, A nano-LC/UV method for the analysis of

534

principal phenolic compounds in commercial citrus juices and evaluation of antioxidant potential,

535

Electrophoresis 35 (2014) 1701-1708.

536

[ 43] A. Rocco, S. Fanali, Analysis of phytosterols in extra-virgin olive oil by nano-liquid

537

chromatography, J. Chromatogr. A 1216 (2009) 7173-7178.

538

[ 44] K. Albert, NMR investigations of stationary phases, J. Sep. Sci. 26 (2003) 215-224.

Ac ce p

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d

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an

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cr

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533

24

Page 24 of 43

539

Legends to the figures:

540

Fig. 1 (a), (b) and (c) Chemical structures of the studied compounds.

541

Fig. 2 Enantioseparations of selected herbicides (a) and NSAIDs (b) analyzed by nano-LC

543

employing a capillary column packed with 1.8 µm diol hydride-based silica particles derivatized

544

with vancomycin (75 µm I.D., Lpack: 11 cm, Leff: 13 cm). For the experimental conditions see

545

Tables 1 and 2.

cr

ip t

542

us

546

Fig. 3 Van Deemter curves for flurbiprofen enantiomers separated by nano-LC on 75 µm I.D.

548

capillary column packed having a length of 11 cm (Leff: 13 cm) with the synthesized 1.8 µm

549

vancomycin-based CSP; mobile phase, 500 mM ammonium acetate buffer pH 4.5/H2O/ACN

550

(1:9:90, v/v/v), elution in isocratic mode, flow rates 10-600 nL/min; sample, flurbiprofen 50 µg/mL

551

in ACN, ~70 nL injected.

d

M

an

547

te

552

Fig. 4 Enantioseparations of acidic compounds on a capillary column packed with 5 µm CSP. For

554

the experimental conditions see Table 3.

555

Ac ce p

553

556

Fig. 5 Chiral separations of basic compounds on capillary columns packed with 5 µm and 1.8 µm

557

CSPs. For the experimental conditions see Table 4.

558 559

Fig. 6 29Si CPMAS NMR spectra of 5 m (a) and 1.8 m (b) CSPs. 29Si MAS NMR spectra of 5

560

m (c) and 1.8 m (d) CSPs. Sketch of units found in 5 m and 1.8 m CSPs (e).

561 562

Fig. 7 13C CPMAS NMR spectra of 5 m (a) and 1.8 m (c) CSPs, and spectra of 5 m (b) and 1.8

563

m (d) diol particles.

564 25

Page 25 of 43

Fig. 8 Reproducibility of derivatization process for the immobilization of vancomycin onto 1.8 µm

566

diol hydride-based silica particles. Chiral separations of flurbiprofen performed on two columns (75

567

µm I.D., Lpack: 11 cm, Leff: 13 cm ) packed with two 1.8 µm CSPs prepared by the authors of this

568

study (a) and another co-worker (b) following the same procedure. Mobile phase, 500 mM

569

ammonium acetate buffer pH 4.5/H2O/ACN (1:9:90, v/v/v), elution in isocratic mode, flow rate 340

570

nL/min; sample, flurbiprofen 50 µg/mL in ACN, ~70 nL injected.

cr

ip t

565

571

Fig. 9 Enantioseparations of flurbiprofen and ketoprofen with the capillary column packed with 5

573

µm CSP (75 µm I.D., Lpack: 11 cm, Leff: 13 cm) decreasing the flow rate of mobile phase (500

574

mM ammonium acetate buffer pH 4.5/H2O/ACN 1:9:90, v/v/v, elution in isocratic mode). Samples

575

50 µg/mL in ACN, ~70 nL injected. a and c, b and d flow rate at 380 and 50 nL/min, respectively.

M

an

us

572

576

Fig. 10 Nano-LC chiral separations of DF1738Y with the synthesized 1.8 µm vancomycin-based

578

CSP increasing the flow rate of the mobile phase (500 mM ammonium formiate buffer pH

579

3.5/H2O/ACN 1:9:90, v/v/v, elution in isocratic mode). Capillary column, 75 µm I.D., Lpack: 11

580

cm, Leff: 13 cm; sample: solution 100 µg/mL diluted in ACN, ~ 60 nL injected for 330 nL/min

581

flow rate and solution 50 µg/mL diluted in ACN, ~ 90 nL injected for 1.7 µL/min flow rate.

583 584 585

te

Ac ce p

582

d

577

586

26

Page 26 of 43

586 Table 1 Separation characteristics of the enantiomers of selected herbicides on capillary column packed with 587 1.8 µm diol hydride-based silica particles derivatized with vancomycin. 588 Experimental conditions: capillary column, 75 µm I.D., Lpack: 11 cm, Leff: 13 cm; mobile phase, 500 mM 589 ammonium acetate buffer pH 4.5/H2O/MeOH (5:10:85, v/v/v ), elution in isocratic mode, flow rate 230 590 nL/min (to ~ 1.8 min); samples, solutions 50 µg/mL diluted in ACN, ~ 40 nL injected.

k1

k2

α

Rs

2.77

3.70

0.59 1.12 1.91 2.95

Diclofop

2.96

3.81

0.72 1.22 1.69 2.26

Fenoprop

2.91

3.70

0.61 1.04 1.72 2.60

Fluazifop

2.23

2.90

0.28 0.67 2.38 2.81

Haloxyfop

2.29

3.24

0.32 0.87 2.69 3.36

Mecoprop

2.63

3.35

0.49 0.89 1.83 2.50

M

an

Dichlorprop

cr

tR2 (min)

us

Compounds tR1 (min)

ip t

591

592

Ac ce p

te

d

593

27

Page 27 of 43

593 Table 2 Separation characteristics of the enantiomers of selected NSAIDs on capillary column packed with 1.8 594 µm diol hydride-based silica particles derivatized with vancomycin. 595 Experimental conditions: capillary column, 75 µm I.D., Lpack: 11 cm, Leff: 13 cm; mobile phase, 500 mM 596 ammonium acetate buffer pH 4.5/H2O/ACN (1:9:90, v/v/v ), elution in isocratic mode, flow rate 360 nL/min (to 597 ~ 1.8 min); samples, solutions 50 µg/mL in ACN, ~70 nL injected.

ip t

598 599 α

Rs

4.76

5.63

1.70 2.19 1.29 1.72

Cicloprofen

3.31

4.10

0.88 1.33 1.50 2.38

Flurbiprofen

3.09

3.97

0.75 1.24 1.66 2.85

Ibuprofen

2.31

2.62

0.33 0.52 1.54 1.72

Indoprofen

6.38

7.30

2.62 3.14 1.20 1.27

Ketoprofen

3.60

4.45

1.16 1.66 1.44 2.29

Naproxen

3.22

4.01

0.80 1.25 1.55 2.52

Suprofen

4.24

5.16

1.45 1.98 1.37 2.19

d

M

an

Carprofen

cr

k2

te

601

k1

Ac ce p

600

tR1 (min) tR2 (min)

us

Compounds

28

Page 28 of 43

601 602 603 604 605

Table 3 Chromatographic data for acidic compounds analyzed by nano-LC employing 5 µm conventional diol silica particles derivatized with vancomycin. Experimental conditions: capillary column, 75 µm I.D., Lpack: 11 cm, Leff: 13 cm; flow rates 380 nL/min and 260 nL/min for the analysis of NSAIDs and herbicides, respectively (to ~ 1.8 min). For the other conditions see Tables 1 and 2.

Compounds

tR1 (min) tR2 (min)

k1

k2

α

ip t

606 Rs

2.90

0.58

0.00 0.00

Cicloprofen

2.44

0.37

0.00 0.00

2.32

Ibuprofen

2.47 1.78

0.31 0.40 1.27 0.61

an

Flurbiprofen

0.00

0.00 0.00

4.77

4.98

1.68 1.79 1.07 0.49

Ketoprofen

2.93

3.06

0.69 0.76 1.11 0.46

Naproxen

2.56

2.65

0.42 0.47 1.11 0.39

Suprofen

3.66

3.95

1.01 1.17 1.16 0.64

2.23

2.51

d

te

0.26 0.42 1.61 1.09

Ac ce p

Dichlorprop

M

Indoprofen

Herbicides

us

Carprofen

cr

NSAIDs

Diclofop

2.09

2.22

0.18 0.26 1.40 0.58

Fenoprop

2.27

2.38

0.26 0.32 1.24 0.45

Fluazifop

1.88

2.00

0.08 0.15 1.83 0.57

Haloxyfop

1.84

2.00

0.06 0.15 2.43 0.66

Mecoprop

2.18

2.30

0.23 0.30 1.31 0.59

607 608

29

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608 609 610 611 612

Table 4 Chromatographic data for the studied basic compounds analyzed by nano-LC employing 5 µm conventional diol silica particles and 1.8 µm diol hydride-based silica particles derivatized with vancomycin. Experimental conditions: capillary columns, 75 µm I.D., Lpack: 11 cm, Leff: 13 cm; mobile phase, 500 mM ammonium acetate buffer pH 4.5/H2O/MeOH (1:4:95, v/v/v ), elution in isocratic mode, flow rate 150 nL/min for 5 µm CSP and 135 nL/min for 1.8 µm CSP (to ~ 3.3 min); samples, 100 µg/mL in MeOH, ~ 40 nL injected.

5 µm CSP tR2 (min)

k1

k2

618 619 620 621

tR1 (min)

tR2 (min)

k1

k2

α

Rs

14.22

2.91 3.29 1.13 1.46

8.44

9.08

1.54 1.73 1.13 1.01

Atenolol

23.20

25.67

5.85 6.58 1.12 1.48

11.98

12.82

2.57 2.82 1.10 0.70

Metoprolol

13.55

14.83

3.15 3.54 1.12 1.38

8.24

8.76

1.48 1.64 1.10 0.83

Oxprenolol

12.59

13.54

2.78 3.06 1.10 1.17

8.09

8.49

1.43 1.55 1.09 0.61

Pindolol

14.86

16.31

3.51 3.95 1.13 1.34

9.96

10.76

1.96 2.20 1.12 0.99

Propranolol

15.89

17.63

3.80 4.33 1.14 1.45

11.32

12.31

2.39 2.69 1.12 1.07

M

an

us

12.97

d te

617

Rs

Ac ce p

616

α

Alprenolol

614 615

1.8 µm CSP

cr

Compounds tR1 (min)

ip t

613

30

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621

Table 5. Relative fractions (in percentage) obtained by deconvoluting 29Si MAS spectra of 1.8 and 5 m

622

CSPs.

Fraction (%)

Fraction (%)

(1.8 m CPS)

(5 m CPS)

cr

ppm

us

Units

ip t

623

Q4

-110.1

72.0

Q3

-100.6

19.6

14.5

Q2

-91.9

2.1

4.6

an

M

d

te

Ac ce p

68.4

C

-83.6

3.3

---

T3

-64.1

2.0

6.6

T2

-54.8

1.0

5.9

624 625 31

Page 31 of 43

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cr

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Figure 1a

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Figure 1b

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Figure 1c

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Figure 2

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Figure 3

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Figure 4

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Figure 5

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Figure 6

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Figure 7

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Figure 8

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Figure 9

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Figure 10

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