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Journal of Chromatography A, 1446 (2016) 125–133

Contents lists available at ScienceDirect

Journal of Chromatography A

j o ur na l ho me page: www.elsev ier .com/ locate /chroma

ilica-based polypeptide-monolithic stationary phase for hydrophilichromatography and chiral separation

icong Zhaoa, Limin Yanga,∗∗, Qiuquan Wanga,b,∗

Department of Chemistry & the Key Laboratory of Spectrochemical Analysis and Instrumentation, College of Chemistry and Chemical Engineering, Xiamenniversity, Xiamen 361005, PR ChinaState Key Laboratory of Marine Environmental Science, Xiamen University, Xiamen 361102, PR China

r t i c l e i n f o

rticle history:eceived 20 December 2015eceived in revised form 16 March 2016ccepted 4 April 2016vailable online 7 April 2016

eywords:apillary electrochromatographyilica-based polypeptide-monolithictationary phaseydrophilic interactionhiral separationold nanoparticles

a b s t r a c t

Glutathione (GSH)-, somatostatin acetate (ST)- and ovomucoid (OV)-functionalized silica-monolithicstationary phases were designed and synthesized for HILIC and chiral separation using capillaryelectrochromatography (CEC). GSH, ST and OV were covalently incorporated into the silica skele-ton via the epoxy ring-opening reaction between their amino groups and the glycidyl moiety in�-glycidoxypropyltrimethoxysilane (GPTMS) together with polycondensation and copolymerization oftetramethyloxysilane and GPTMS. Not only could the direction and electroosmotic flow magnitude onthe prepared GSH-, ST- and OV-silica hybrid monolithic stationary phases be controlled by the pH of themobile phase, but also a typical HILIC behavior was observed so that the nucleotides and HPLC peptidestandard mixture could be baseline separated using an aqueous mobile phase without any acetonitrileduring CEC. Moreover, the prepared monolithic columns had a chiral separation ability to separate dl-amino acids. The OV-silica hybrid monolithic column was most effective in chiral separation and couldseparate dl-glutamic acid (Glu) (the resolution R = 1.07), dl-tyrosine (Tyr) (1.57) and dl-histidine (His)(1.06). Importantly, the chiral separation ability of the GSH-silica hybrid monolithic column could beremarkably enhanced when using gold nanoparticles (AuNPs) to fabricate an AuNP-mediated GSH-AuNP-GSH-silica hybrid monolithic column. The R of dl-Glu, dl-Tyr and dl-His reached 1.19, 1.60 and 2.03. This

monolithic column was thus applied to separate drug enantiomers, and quantitative separation of allfour R/S drug enantiomers were achieved with R ranging from 4.36 to 5.64. These peptide- and protein-silica monolithic stationary phases with typical HILIC separation behavior and chiral separation abilityimplied their promise for the analysis of not only the future metabolic studies, but also drug enantiomersrecognition.

© 2016 Elsevier B.V. All rights reserved.

. Introduction

With the advantages of excellent permeability, versatile surfaceodification and ease of preparation, the monolithic station-

ry phase has attracted increasing attention since the 1990s1,2]. As an alternative to the microparticle-based technique, it is

sed in capillary electrochromatography (CEC) and capillary liq-id chromatography for the separation of small molecules and

arge biomolecules [3,4]. Rapid progress has been made in column

∗ Corresponding author at: Department of Chemistry & the Key Laboratory ofpectrochemical Analysis and Instrumentation, College of Chemistry and Chemicalngineering, Xiamen University, Xiamen 361005, PR China.∗∗ Corresponding author.

E-mail addresses: lmyang@xmu.edu.cn (L. Yang), qqwang@xmu.edu.cnQ. Wang).

ttp://dx.doi.org/10.1016/j.chroma.2016.04.014021-9673/© 2016 Elsevier B.V. All rights reserved.

efficiency, modulation of selectivity and applications of the state-of-the-art monoliths [5]. The surface chemistry of the monolithicstationary phase can be adjusted by the selection of monomers[6–12] and cross-linkers [13–16], as well as further modifica-tion of the nanoparticles [17–26] which differ in hydrophobicity,hydrophilicity and ionizability under a certain mobile phase, but, ingeneral, one type of the stationary phase currently used emphasizesa given chromatographic mode. Mixed-mode chromatographyemploying multifunctional stationary phases often provides res-olution that far exceeds that observed with a single-mode process[27–33], for example, the separation of basic chitonoligosaccha-rides, strongly acidic carrageenan oligosaccharides and standardpeptides on cation-exchange chromatography (CEX) were much

improved on HILIC/CEX [30]. In addition to target analytes, theseparation efficiencies of a monolithic column are largely depen-dent on the physicochemical properties of the precursors that

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26 L. Zhao et al. / J. Chroma

orm the column structure and control the Kinetics and ther-odynamic properties. Peptides and/or proteins are naturally

witterionic and chiral compounds with hydrophilic or hydropho-ic properties and various spatial structures dedicated by theirorresponding amino acid compositions and sequence. They coulde modified on the monolithic columns by post-modification16,34–37] or incorporated in monolithic skeleton by one-pottrategy [31,38–41]. However, systematic evaluation of both HILICehavior and chiral separation ability of such monolithic columns

s very scarce. We aimed to design and synthesize a mono-ithic stationary phase using tetramethyloxysilane (TMOS) and-glycidoxypropyltrimethoxysilane (GPTMS) as co-precursors touild the silica monolithic skeleton, while peptide such as glu-athione (GSH) and/or somatostatin (ST) or a protein such asvomucoid (OV) as exemplified was decorated simultaneously inhe silica monolithic skeleton via the epoxy ring-opening reac-ion between the glycidyl in GPTMS and the amine of the peptidend/or protein, alongside polycondensation and copolymerizationf TMOS and GPTMS in the so-called one-pot manner. These mono-ithic stationary phases designed and synthesized were expectedo demonstrate the pH-switchable characteristics of electroos-

otic flow (EOF) direction and magnitude, HILIC behavior andhiral separation ability during CEC. Moreover, gold nanoparticlesAuNPs) were used to fabricate an AuNP-mediated GSH-AuNP-GSH

onolithic stationary phase via the strong interaction betweenulfhydryl and Au to improve its chiral separation ability.

. Materials and methods

.1. Chemicals and reagents

TMOS, GPTMS, polyethylene glycol (PEG, Mw = 10,000 Da),PLC peptide standard mixture (Gly-Tyr, Val-Tyr-Val, Metnkephalin, Leu enkephalin and angiotensin II), OV (Type III-O,ree of ovoinhibitor, 186 amino acids, pI 4.71, polarity −0.29http://web.expasy.org/protparam/), Mw = 20476.7 Da), tris(2-arboxyethyl)phosphine (TCEP) and HPLC-grade acetonitrile (ACN)ere purchased from Aldrich (Milwaukee, WI, USA). GSH (pI 5.93,olarity −0.49 (http://www.hilic.com/), Mw = 307.3 Da), adeno-ine 5′-monophosphate (AMP), cytidine 5′-monophosphate (CMP),uanosine 5′-monophosphate (GMP), uridine 5′-monophosphateUMP), tyrosine (Tyr), glutamic acid (Glu) and histidine (His)ere purchased from Aladdin (Shanghai, China). Somatostatin

cetate (ST, a cyclopeptide containing 14 amino acids A-G-C--N-F-F-W-K-T-F-T-S-C, pI 8.91, Mw = 1637.9 Da, polarity 0.029)as purchased from Sangon (Shanghai, China). Chloroauric acid

HAuCl4), trisodium citrate, sodium borohydride and disodiumydrogenphosphate were obtained from the Sinopharm Chem-

cal Reagent Corporation (Shanghai, China). Drug enantiomers:imodipine (Yabao Phamaceutical Ltd., Shanxi, China), amlodip-

ne and lansoprazole (Kailun New Chemical Materials Ltd., Hubei,hina), and omeprazole (Huaxin Phamaceutical Ltd., Guangxi,hina) were gifted by Prof. Hui Zhang. All other reagents used inhis study were of at least analytical grade. Water used in all exper-ments was purified using a Milli-Q system (Millipore, Milford, MA,SA). Fused-silica capillary with 75 �m i.d. and 375 �m o.d. wasurchased from Refine Chromatography Ltd. (Hebei, China).

.2. Instruments

CEC experiments were carried out on a P/ACE MDQ system

Beckman, USA) equipped with a UV–vis detector at 25 ◦C. AITACHI S-4800 SEM and energy dispersive X-ray spectrometry

EDS) instrument (Hitachi, Japan) was used to study column mor-hology and to determine the elemental content of Au and S. A

1446 (2016) 125–133

JEM-1400 TEM instrument (JEOL, Japan) was used to study goldnanoparticle morphology. The UV–vis spectra were recorded ona UV-2550 (Shimadzu, Japan). All IR measurements were carriedout on a Nicolet IR200 Spectrometer (Thermo Electron, USA). AVario EL III (Elementar, Germany) was used for elemental analy-sis. The macropore size distribution of the monoliths synthesizedwas measured on a Poremaster 60 mercury intrusion apparatus(Quantachrome, Boynton Beach, FL, USA), and the mesopore diam-eter and Brunauer-Emmett-Teller surface area were determined ona Micromeritics Tristar 3000 (Norcross, GA, USA) through nitrogenadsorption/desorption.

2.3. Electrochromatography procedures

Prior to CEC, the monolithic column placed in the CE cartridgewas preconditioned with an appropriate running buffer for 1 h witha syringe pump (Unimicro Technologies, Pleasanton, CA, USA), andthen equilibrated on the CE instrument by applying a low voltageof 5 kV until the current was stable before sample introduction.Both sampling and separation were performed at 25 ◦C. The ACN-aqueous mobile phase was prepared by mixing the desired amountof phosphate solution or ammonium formate buffer and ACN. Allthe mobile phases were filtered through 0.45 �m membranes anddegassed under sonication before use. All the hybrid monolithiccolumns used had a total length of 30 cm (effective length 20 cm).A detector window (2 mm) was created by removing the polyimidecoating of a fused-silica capillary with concentrated sulfuric acid.

2.4. Preparation of the peptide- and protein-silica hybridmonolithic columns

First, in order to clean and activate the inner surface of thecapillary for effective attachment of the silica skeleton, it was pre-treated by rinsing with 1 M HCl for 12 h, water for 30 min, 1 M NaOHfor 12 h, water for 30 min and acetone for 1 h in sequence using asyringe pump, and then dried using a nitrogen stream at room tem-perature overnight. Subsequently, a prehydrolyzed mixture wasprepared by mixing appropriate amounts of TMOS, GPTMS, HAcand PEG for 1 h under an ice bath to form a homogeneous solution.The molar ratio of TMOS and GPTMS monomer was optimized at 4:3under HAc/PEG = 1:2 and 25:36, and used for preparing each typeof hybrid monolithic column. Then, appropriate amounts of GSH,ST and OV as well as NaOH solution, which was to adjust the pH andincrease the peptide/protein solubility in the hydrolyzed mixture,were added into the hydrolyzed mixture (0.5 mL), followed by son-icating for 10 min. Afterwards, the resulting mixture was manuallyinjected into the pretreated capillary with a syringe, and both endsof the capillary sealed with rubber septa. The filled capillary wasplaced in a GC oven and kept at 40 ◦C for polycondensation (12 h)and then at 80 ◦C for copolymerization (12 h). The obtained mono-lithic columns were flushed with ACN/water (70:30, v/v) with anHPLC pump in order to remove the unreacted residuals. Becauseof the different physical and chemical properties of GSH, ST andOV, the amounts of the porogens HAc and PEG used were not thesame for each type of monolithic column synthesized. The finalpreparation conditions are listed in Table 1. The prepared hybridmonolithic columns were named GSH-, ST- and OV-silica hybridmonolithic columns, and were stored in a refrigerator at 4 ◦C topreserve them from possible bacterial growth before use. In par-allel, the corresponding bulk hybrid monoliths were prepared ina centrifuge tube under the same conditions for characterization

of the hybrid silica monoliths prepared. The bulk hybrid monolithwas cut into smaller pieces, extracted with ethanol overnight ina Soxhlet apparatus and then dried at 80 ◦C overnight for furthermeasurements.

L. Zhao et al. / J. Chromatogr. A 1446 (2016) 125–133 127

Table 1Optimum conditions for the preparation of GSH-, ST- and OV-silica hybrid monolithic columns.

Hybrid monolith Molar ratio NaOHa/H2O (�L/�L) pH Backpressureb (MPa) Mesopore/macropore (nm/�m) Surface area (m2/g)

TMOS/GPTMS HAc/PEG/polypeptide

GSH-silica 4/3 1/2/8 23/0 3.8 1.5 3.1/1.5 6.7ST-silica 4/3 25/36/10 0.8/3.2 5.1 4.0 3.2/1.0 7.5OV-silica 4/3 25/36/1 0.6/29.4 4.1 3.0 3.8/1.2 9.8

a 5 M.b At the flow rate of 19 mm/s ACN/H2O (70:30).

F ybridG

2m

patewtwfitTmtticwtwG(moim

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ig. 1. Schematic one-pot process for the preparation of peptide- and protein-silica hSH-AuNP-GSH-silica hybrid monolithic columns (b).

.5. Preparation of the auNP-mediated GSH-silica hybridonolithic column

The AuNPs were synthesized following the protocol reportedreviously [42] but with some modifications. Briefly, a 20 mLqueous solution containing 2.5 × 10−4 M HAuCl4 and 2.5 × 10−4 Mrisodium citrate was prepared in a round-bottomed flask at ambi-nt temperature, and then 0.6 mL of ice cold 0.1 M NaBH4 solutionas added dropwise to the solution under stirring, when the solu-

ion turned pink immediately and then wine-colored. The solutionas stirred for another half hour to obtain AuNPs. The AuNPs modi-ed GSH-silica hybrid monolithic column was then prepared usinghe post-modification method. Before modification with AuNPs,CEP solution (50 mM) was pumped through the GSH-silica hybridonolithic column at a flow rate of 1 �L/min for 3 h to guarantee

he cleavage of possible disulfide bonds and washed with watero remove any extra TCEP. The colloidal dispersion of AuNPs wasmmediately pumped through the GSH-silica hybrid monolithicolumn at a flow rate of 1 �L/min until the entire column turnedine-colored and AuNP solution was observed coming out from

he capillary outlet. The column was then rinsed thoroughly withater to remove the excess AuNPs to obtain the AuNP-modifiedSH-silica hybrid monolithic column. Subsequently, GSH solution

1 M) was pumped through the AuNP-modified GSH-silica hybridonolithic column with a syringe pump at a flow rate of 1 �L/min

vernight and it was rinsed with water to remove the excess GSHn order to obtain the AuNP-mediated GSH-AuNP-GSH-silica hybrid

onolithic column.

. Results and discussion

.1. Characterization of the peptide- and protein- as well as the

uNP-mediated GSH-AuNP-GSH-silica hybrid monolithic columns

Preparation of the peptide- and/or protein-silica hybrid mono-ithic stationary phase via the so-called one-pot process made use

monolithic columns (a); and AuNP-modified AuNP-GSH-silica and AuNP-mediated

of sol-gel chemistry, involving hydrolysis, polycondensation andcopolymerization of TMOS and GPTMS, as well as the epoxy open-ing reaction between the glycidyl in the GPTMS with the aminefrom the peptide and/or protein (Fig. 1a). The silica skeleton struc-ture is formed via the development of a transient structure ofphase separation induced by the hydrolysis and polycondensationof TMOS and GPTMS and subsequent freezing of the structure by thesol-gel transition. The size of thus formed interconnected macrop-ores is determined by the onset of phase separation relative to thegel formation [43]. As a monomer of the monoliths, GPTMS alsoplays the role of an organic additive that induces phase separation.When the molar ratio of TMOS/GPTMS was more than 4:4 whilethe amount of water for hydrolysis was kept constant, the onset ofearly phase separation relative to the sol-gel transition retarded,resulting small macropore size and thus high backpressure of theobtained monolith; when the molar ratio was 4:2, the synthesizedmonolith like open-tubular column, only formed a layer on the cap-illary wall. The molar ratio TMOS/GPTMS of 4:3 was thus chosento build the silica skeleton structure of the monolithic columnsunder 1 h ice-bath hydrolysis, 12 h polycondensation (40 ◦C) and12 h copolymerization (80 ◦C) [40]. Moreover, the porogenic sol-vents HAc (poor solvent) and PEG (good solvent), the peptide (GSHand/or ST) and OV are the other factors which determine the ulti-mate morphology and pore structures of the resultant monolithicmatrix [44]. For example, when the molar ratios of HAc/PEG/GSHwere 3:8:32 and 3.6:8:32, the onset of phase separation tendedto occur later and the silicon alkoxides might not hydrolyze com-pletely, forming water-rich phase and polymer-rich phase. Theprepared monolithic columns encountered shrinkage (Fig. 2a and b)after flushed out unreacted residual and solvents. As increasing HAccontent to the ratio 1:2:8 of HAc/PEG/GSH, the obtained monolithiccolumn had a reasonable distribution of mesopores and macrop-

ores as shown in Fig. 2c and bound with the capillary wall tightly(Fig. 2c1) besides the permeability and separation efficiency beingconsidered. Further increasing PEG content to the ratios 2:3:16 and2:5:16 of HAc/PEG/GSH leaded to retarded onset of phase separa-

128 L. Zhao et al. / J. Chromatogr. A 1446 (2016) 125–133

Fig. 2. SEM images of GSH-silica hybrid monolithic columns prepared at a 4:3 molar ratio of TMOS/GPTMS and 3:8:32 (a), 3.6:8:32 (b), 1:2:8 (c) and (c1), 2:3:16 (d), 2:5:16 (e),1 at a mc and (

twwwcimsrmsoraGtt1shhras

bvci91ilos

:2:7.3 (f), 1:2:9 (g) of HAc/PEG/GSH; ST-silica hybrid monolithic column preparedolumn at 25:36:1.0HAc/PEG/OV (i) and (i1). Magnifications: (a1, h1 and i1) ×4000

ion relative to the sol–gel transition. As a result, the phase domainsere frozen in the earlier stage of the coarsening, and finer domainsere observed and the macropore was small, thus the backpressureas high (Fig. 2d and e); moreover, decreasing or increasing GSH

ontent to the ratios 1:2:7.3 and 1:2:9 of HAc/PEG/GSH, resultingn shrinkage again (Fig. 2f) and small macropore (Fig. 2g) due to the

ore or less hydrolysis of all precursors. In the case of the GSH-ilica hybrid monolithic stationary phase obtained with the molaratio of HAc/PEG/GSH 1:2:8 at pH 3.8 (Fig. 2c, c1 and Table 1), theacropore size and mesopore size were 1.5 �m and 3.1 nm with

urface area 6.7 m2/g and backpressure 1.5 MPa at the flow ratef 5 �L/min (corresponding to 19 mm/s) ACN/H2O (70:30). Theseesults suggested that not only a suitable balance between HAcnd PEG was favorable to phase separation but also the amount ofSH, which was initially considered to be used for functionalizing

he silica backbone, affected the formation and physical proper-ies of the hybrid monolith. Similarly, ST-silica (macropore size.0 �m, mesopore size 3.2 nm, surface area 7.5 m2/g and backpres-ure 4.0 MPa at the flow rate of 19 mm/s 70:30 ACN/H2O) (Fig. 2h,1) and OV-silica (1.2 �m, 3.8 nm, 9.8 m2/g and 3.0 MPa) (Fig. 2i, i1)ybrid monolithic stationary phases were obtained using the molaratio of HAc/PEG/ST 25:36:10 at pH 5.1 and HAc/PEG/OV 25:36:1t pH 4.1 (Table 1), owing to the different amount of –NH2 and theolubility of ST and OV in the precondensation solution.

The peptide and/or protein were incorporated into the silicaackbone of the hybrid monolithic stationary phases synthesizedia the epoxy ring-opening reaction. Results from FT-IR studiesonfirmed the covalent binding of GSH, ST and OV into the sil-ca skeleton. The �–O– of the epoxy in GPTMS at 1254 cm−1 and10 cm−1 disappeared, and the �O C N at 1384 cm−1, �COOH at734 cm−1 and �–CON– at 1654 cm−1 in the polypeptides appeared

n the hybrid monoliths, indicating the formation of a C N cova-ent bond via the epoxy ring-opening reaction and the modificationf the polypeptides. Moreover, the sulfur content (S%)that repre-ents the contents of GSH, ST and OV in the stationary phase was

olar ratio of 25:36:10HAc/PEG/ST (h) and (h1); and an OV-silica hybrid monolithica–i) ×1000.

determined with elemental analysis. The results obtained of 4.03%GSH, 0.35% ST and 0.43% OV were well in accordance with the cor-responding theoretical values of 3.97, 0.37 and 0.41, which werecalculated assuming that copolymerization of the TMOS and GPTMSand the epoxy ring-opening reaction were completed.

In order to produce the AuNP-modified GSH-silica hybrid mono-lithic column, AuNPs were prepared. The size of the preparedAuNPs was 5.5 nm measured using TEM (Fig. S1a) and UV–visabsorption spectrometry (Fig. S1b). These AuNPs were first used tomodify the GSH-silica monolithic stationary phase via the stronginteraction between Au and the sulfhydryl in GSH to obtain theAuNP-GSH-silica monolithic stationary phase (Fig. 1b). The AuNPsmodified in the AuNP-GSH- silica monolithic stationary phasecould mediate further GSH assembly to enable the preparationof the GSH-AuNP-GSH-silica hybrid monolithic stationary phase(Fig. 1b). The elemental compositions in the AuNP-GSH-silica andGSH-AuNP-GSH-silica hybrid monolithic stationary phase weremeasured using EDS. The results confirmed that the AuNPs hadattached and more GSH assembled into the monolithic columnswith an Au content of 13.05% (w/w) and that of S% increasing from3.07 in the AuNP-GSH-silica monolithic column to 7.47 in the GSH-AuNP-GSH-silica monolithic column. The AuNP-GSH-silica andGSH-AuNP-GSH-silica monolithic stationary phase surface areaswere 8.1 and 9.0 m2/g, the macropore sizes both 0.7 �m, the meso-pore sizes 2.9 and 2.8 nm, and the backpressure 4.5 and 4.5 MPa atthe flow rate of 19 mm/s of ACN/H2O (70:30).

3.2. Mixed-mode electrochromatographic behavior

3.2.1. pH-switchable characteristics of EOF directionCEC represents a powerful hybrid technique of CE and LC, in

which the stationary phase plays a dual role, providing both thesites for the desired interaction with analytes and the sites for gen-erating EOF. Based on the fact that the covalently bonded peptide(or protein) existed in the monolithic stationary phase, which had

L. Zhao et al. / J. Chromatogr. A 1446 (2016) 125–133 129

Fig. 3. Effect of the running buffer pH on the EOF of the peptide- and protein-silicahybrid monolithic stationary phases. Conditions: running buffer, 20 mM PBS con-taining 60% v/v ACN and pH ranging from 2.6 to 8.0; applied voltage, −20 kV when pHsmaller than 3.4 and 20 kV pH when larger than 3.5 for the GSH-silica hybrid mono-lithic stationary phase; −20 kV when pH smaller than 6.5 and 20 kV pH larger than6.5 for the ST-silica hybrid monolithic stationary phase; −20 kV when pH smallerthan 4.5 and 20 kV pH larger than 4.5 for the OV-silica hybrid monolithic station-ary phase; sample injection, 8 psi for 5 s; void time marker of EOF, toluene (5 mM);d

boTwv�aLGotbnrEphscitooihtatpmpO1asl

Fig. 4. Effect of ACN content from 0 to 80% on the retention of the neutral compoundson the GSH-silica hybrid monolithic column using 20 mM ammonium formate buffer(pH 2.6) and applied voltage 20 kV (a), where k = (tr − t0) /t0, tr is the retention timeof an analyte, and t0 is the retention time of an unretained compound (t0: DMFwithout ACN in running buffer; t0: toluene with ACN from 20 to 80% in runningbuffer); HETP of toluene against the linear velocity of the mobile phase containing60% ACN and 20 mM ammonium formate buffer (pH 2.6) via increasing the applied

thiourea could not be baseline separated on the ST-silica monolithiccolumn using 60% ACN. Even increasing ACN to 90%, formamide and

etection wavelength, 214 nm.

oth amino and carboxylic groups, the magnitude and directionf EOF could be controlled via the pH of the running buffer used.he effects of the running buffer pH on EOF are shown in Fig. 3,here a negative value of EOF denotes anodic EOF and positive

alue cathodic EOF. EOF mobility was calculated with the equatione = IL/Vt0, where �e is the effective mobility, V is the applied volt-

ge, t0 is the migration time of the EOF marker (toluene), and I and are the effective and total length of the capillary. In the case of theSH-silica hybrid monolithic stationary phase, the absolute valuef anodic EOF decreased from 7.5 to 2.8 cm2/kV min together withhe increase in pH from 2.6 to 3.3. When the pH reached 3.4, the EOFecame zero. This meant that both the positively charged and theegatively charged groups on the surface of the stationary phaseemained equal, and the net charge was zero. Then the cathodicOF increased from 8.0 to 14.2 cm2/kV min as pH increased fromH 3.5 to 7.0. These results suggested that the prepared GSH-silicaybrid monolithic stationary phase behaved as a good zwitterionictationary phase, and both EOF direction and magnitude could beontrolled by the pH of the running buffer used (Fig. 3). However,t should be noted that the pH (3.4) at which the EOF was equalo zero was not the same as the pI of GSH (5.93), but nearly threerders of magnitude lower. This might be due to the epoxy ring-pening reaction with the only one NH2 group in GSH, resultingn the decrease in its actual pI in the stationary phase. On the otherand, such a phenomenon again confirmed the binding of GSH intohe silica skeleton. In the case of the ST-silica monolithic station-ry phase, similar pH-switchable EOF direction and a more thanwo orders of magnitude shift from ST’s pI (8.91) to the zero EOFH (6.5) were observed (Fig. 3). Compared with the peptide-silicaonolithic stationary phase, the OV-silica monolithic stationary

hase showed zero EOF at pH 4.5 that was almost the same as theV’s pI (4.71). This phenomenon might be because OV contains86 amino acids in its molecule, and the binding of NH2 did notffect very much the charge status at a given pH. All these results

uggested that the prepared GSH-, ST- and OV-silica hybrid mono-iths were good zwitterionic stationary phases, and that one could

voltage from 10 kV to 26 kV on the GSH-, ST-, OV-silica hybrid monolithic columns(b). The concentration of neutral compounds: 5 mM. Other conditions were the sameas in Fig. 3.

change the magnitude and direction of EOF and thus the selectivityof separation via the running buffer pH.

3.2.2. HILIC behaviorIn order to investigate the chromatographic proper-

ties of the hybrid monolithic stationary phases, four classictest compounds with different polarity (logP) in the orderthiourea (−1.1) > formamide (−1.0) > dimethylformamide (DMF)(−0.6) > toluene (2.52) were used. A typical HILIC mechanism wasobserved on the GSH-silica hybrid monolithic column (Fig. 4). Forexample, the retention factors (k) of the most polar thiourea amongthe four compounds and the least polar toluene improved from0.1 to 0.7 and non-retained to 0.4 as the percentage of ACN in themobile phase increased from 0 to 80 while the ammonium formateconcentration was kept constant at 20 mM (pH 2.6) as shown inFig. 4a. When toluene was used to determine its height equivalentto a theoretical plate (HETP) against the velocity of the mobilephase (60% ACN with 20 mM ammonium formate at pH 2.6) on theGSH-silica monolithic column (Fig. 4b), the lowest HETP reached5.1 �m at 0.76 mm/s with k = 0.13, and the four compoundscould be baseline separated (Fig. 5a) in the sequence toluene,DMF (k = 0.19; HETP, 7.3 �m), formamide (0.27; 8.8 �m) andthiourea (0.34; 8.0 �m) with the resolutions RDMF/toluene = 2.20,Rformamide/DMF = 2.57 and Rthiourea/formamide = 1.91.

The nearly flat profile of HETP against a mobile phase velocitymore than 0.76 mm/s (Fig. 4b) suggested a high mass transfer rate(C term, 1.86 ms) of the prepared GSH-silica monolithic column.

In comparison, ST, a 14 amino acids cyclopeptide containingthree phenylalanine and one tryptophan, is weaker in hydrophilic-ity (polarity 0.029) than GSH (−0.49), toluene, DMF, formamide and

thiourea still overlapped (Rformamide/thiourea = 1.08) except the base-line separation of toluene (k = 0.35; HETP = 11.4 �m)/DMF (0.41;

130 L. Zhao et al. / J. Chromatogr. A 1446 (2016) 125–133

Fig. 5. Electrochromatograms of neutral compounds (a–c); nucleotides (d–f) and the HPLC peptide standard mixture (g–i) on the GSH-, ST- and OV-silica hybrid monolithiccolumns. Experimental conditions: 20 mM ammonium formate buffer containing 60% ACN at pH 2.6 for GSH-silica hybrid monolithic column (a), 90% ACN at pH 2.6 forST- and OV-silica hybrid monolithic columns (b) and (c); applied voltage, 20 kV; detection wavelength: 190 nm (a–c); mobile phase, 20 mM PBS at pH 2.9; applied voltage,2 plied( ; (1) LI : 5 mM

1(R(toifMctsthaoftfap(4dlc

OccE

0 kV; detection wavelength: 260 nm (d–f); mobile phase, 20 mM PBS at pH 2.9; ap3) formamide and (4) thiourea (a–c); (1) UMP, (2) AMP, (3) CMP and (4) GMP (d–f)I (g–i). Sample injection, 8 psi for 5 s. The concentration of neutral compound each

0.4 �m)/formamide (0.51; 9.1 �m) and toluene/DMF/thiourea0.55; 9.5 �m) could be baseline separated with the resolutions

DMF/toluene = 1.58, Rformamide/DMF = 2.33 and Rthiourea/DMF = 3.23Fig. 5b). In the case of the OV-silica monolithic column, becausehe polarity of OV (−0.29) is higher than ST, baseline separationf the four neutral compounds could be achieved using 90% ACNn the order toluene (k = 0.17; HETP 10.8 �m), DMF (0.26; 6.0 �m),ormamide (0.45; 10.2 �m) and thiourea (0.52; 9.3 �m) (Fig. 5c).

oreover, the larger size and more amino acid residues and thusomplicated configuration of ST and OV leaded to higher massransfer resistance (C-term 4.19 and 12.01 ms) of the ST- and OV-ilica monolithic columns, resulting in longer retention time andhus, in turn, more serious longitudinal diffusion and higher plateeight (Fig. 4b). Run-to-run reproducibilities of GSH- and/or ST-nd/or OV-silica hybrid monolithic columns were evaluated in termf relative standard deviation (RSD%) of the retention time (n = 11)or the four compounds. They were 3.91%, 0.26% and 0.15% foroluene, 3.55%, 0.44% and 0.17% for DMF, 4.02%, 0.20% and 0.14%or formamide and 4.04%, 0.49% and 0.15% for thiourea, when using

mobile phase of 60% ACN with 20 mM ammonium formate atH 2.6. Both column-to-column and day-to-day reproducibilitiesn = 11) of the analysts were less than 5.52%, 4.61% and 6.20% and.51%, 2.12% and 2.60%, respectively. Besides, there was no obviousecline in the column efficiency after 30 days’ use (no test for a

onger time), suggesting that the prepared silica hybrid monolithicolumns have good stability and reproducibility.

In order to further demonstrate the advantage of GSH-, ST- and

V-silica monolithic columns for separating polar compounds, theolumns were applied to separate nucleotides, where it is diffi-ult to separate using RP-CEC because of the counter-directionalOF to electrophoresis [45]. The typical electrochromatograms of

voltage: 10 kV; detection wavelength: 190 nm (g–i). Solutes: (1) toluene, (2) DMF,eu enkephalin, (2) Met enkephalin, (3) Val-Try-Val, (4) Gly-Try and (5) angiotensin; nucleotide each: 0.3 mM and HPLC peptide standard mixture: 0.05 mg/mL.

UMP (pKa 1.0), AMP (pKa 2.35), CMP (pKa 2.65) and GMP (pKa

1.55) are shown in Fig. 5d–f. Surprisingly, the four nucleotidescould be baseline separated on all the three monolithic columnsusing 20 mM PBS aqueous running buffer (pH 2.9) without anyACN. Sharp and symmetric peaks for all four nucleotides wereobtained and eluted based on their hydrophilicity and chargesin the sequence of UMP (polarity, −3.27), AMP (−3.22), CMP(−3.31) and GMP (−3.84), demonstrating a hydrophilic reten-tion behavior. The resolutions between the adjacent nucleotideswere RAMP/UMP = 12.9, RCMP/AMP = 23.1 and RGMP/CMP = 6.29 on theGSH-silica hybrid monolithic column, and those on the ST-silicaand OV-silica monolithic columns were 5.27, 2.46, 1.65 and 9.88,19.75, 4.13. ST-silica hybrid monolith column efficiency and reten-tion time were a little poorer and shorter compared to the GSHand OV-silica monolithic columns, which was attributed to the factthat the polarity of ST is much weaker than GSH and OV althoughthe separation was still achieved using aqueous mobile phase. Thebaseline separation of the nucleotides with the column efficiencyup to 242 700 plates/m using CEC was much higher comparedto those (109 000 plates/m) on the HI-SAX monolith containingacrylic monomer ([2-(acryloyloxy)ethyl] trimethyl ammonium-methyl sulfate (AETA)) in pressurized CEC and on “thiol-ene” clickchemistry based glutathione-silica hybrid monolith in capillary liq-uid chromatography [33,41]. These monolithic columns were alsoused to separate a HPLC peptide standards mixture (Table S1) con-taining Gly-Tyr, Val-Tyr-Val, Met enkephalin, Leu enkephalin andangiotensin II. As can be seen from Fig. 5g–i, they were quantita-

tively separated using an aqueous running buffer of 20 mM PBS (pH2.9) under 10 kV. Leu enkephalin, Met enkephalin, Val-Tyr-Val andGly-Tyr (positively charged +1 at pH 2.9) and angiotensin II (+3)were eluted in sequence. The resolutions between the adjacent

L. Zhao et al. / J. Chromatogr. A 1446 (2016) 125–133 131

F prepaG g buffw acid e

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ig. 6. Enantioseparation of dl-tyrosine, dl-Histidine and dl-Glutamic acid on theSH-silica hybrid monolithic columns with CEC. Experimental conditions: runninavelength, 190 nm; sample injection, 8 psi for 5 s; the concentration of the amino

eptides were 5.2, 10.7, 15.3 and 6.0 on the GSH-silica mono-ithic column, and those on the ST-silica and OV-silica monolithicolumns 9.2, 2.2, 8.7, 4.5 and 9.8, 2.7, 6.3, 3.0. These column effi-iency and resolution obtained were much improved comparedo the l-Lysine–silica hybrid monolithic column that we reportedefore [40]. More importantly, quantitative separation of the fourypical nucleotides and the HPLC peptide standards mixture usingqueous mobile phase without any ACN is more close to greenhemistry. In parallel, separation of the four typical nucleotides andPLC peptide standards mixture were tried using a bare silica cap-

llary of the same size as the monolithic columns prepared underhe same conditions. The nucleotides were co-eluted (Fig. S2); Val-yr-Val and Gly-Tyr in the HPLC peptide standards mixture couldot be separated (Fig. S3). These obtained results suggested thathere must be chemical interactions between the analyte and the

onolithic stationary phase, in addition to electromigration. Theseynthesized peptide- and protein-silica hybrid monolithic station-ry phases with typical HILIC separation behavior and successfulpplication to separate the nucleotides and small peptides impliedheir promise for the analysis of the nucleotides pool and future

etabolic studies.

.2.3. Chiral separation ability

.2.3.1. Separation of dl-amino acids. Because GSH, ST and OV areatural chiral compounds, the GSH-, ST- and OV-silica hybridonolithic stationary phases were expected to have enantiosep-

ration ability. To evaluate the enantioseparation capability ofhese prepared monolithic columns, three pairs of the typical chiral

mino acids, dl-Tyr, dl-Glu and dl-His, were first selected. Typi-al electrochromatograms of dl-Tyr, for example, are presented inig. 6. Results obtained suggested that the OV-silica hybrid mono-ithic column was the best followed by the ST- and GSH-silica

red GSH-, ST- and OV-silica hybrid monolithic columns as well as the GSH-AuNP-er, 20 mM PBS containing 60% ACN at pH 6.8; applied voltage, −10 kV; detectionach was 1 mM.

monolithic columns with the R of dl-Tyr of 1.57, 0.76 and 0.74 using20 mM PBS (pH 6.8) containing 60% ACN (Fig. 6a–c). This observa-tion was well in accordance with our predication that the numberof chiral carbon atoms (171) in OV is much more than those in ST(13) and GSH (2), and thus multiple intermolecular interactions.Similar results were obtained in the case of dl-His and dl-GL withthe R of 1.06, 1.07 and 0.65, 0.93 on OV- and ST-silica hybrid mono-lithic columns (Fig. 6f, g, j and k), but their mutual separation wasnever achieved on a GSH-silica hybrid monolithic column (Fig. 6eand i). We thus switched to using the GSH-AuNP-GSH-silica hybridmonolithic column, when remarkable improvements in not onlydl-Tyr but also dl-His and dl-Glu separation (Fig. 6d, h, l) wereobserved with the R of 1.60 (dl-Tyr), 2.03 (dl-His) and 1.19 (dl-Glu) even better than those of the OV-silica monolithic column.The larger amount of GSH assembling on the AuNPs, resulting inmore chiral carbon atoms in the stationary phase, is responsiblefor the significant improvements in separation. Although OV hasmore chiral carbons and can provide more interaction sites, thenative protein structure might be highly folded and not all the chiralcarbons and structural domains could play a role in chiral separa-tion. Thus, when more full-exposed GSH molecules bonded ontothe AuNP-mediated GSH-silica monolithic column, the GSH-AuNP-GSH-silica monolithic column is more effective for chiral separationcapability than the OV-silica monolithic column. The four typicalnucleotides were used to evaluate the run-to-run reproducibility ofGSH-AuNP-GSH-silica hybrid monolithic column, and RSD% of theretention time (n = 11) were 0.64% for UMP, 0.68% AMP, 0.72% CMPand 0.64% GMP. Both column-to-column (n = 11) and day-to-dayreproducibilities (n = 11) were less than 6.14% and 4.20%. Next we

used the GSH-AuNP-GSH-silica hybrid monolithic column to sepa-rate drug enantiomers. Separation of the four drug enantiomers wasrealized (Fig. 7) using 20 mM PBS (pH 7.0) containing 60% ACN. The

132 L. Zhao et al. / J. Chromatogr. A

Fig. 7. Enantioseparation of drug enantiomers on the GSH-AuNP-GSH-silica mono-lithic column using CEC. Experimental conditions: running buffer, 20 mM PBScD0

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ontaining 60% ACN at pH 7.0; applied voltage: 10 kV; detection wavelength, 190 nm.rug enantiomers: nimodipine, amlodipine, lansoprazole and omeprazole each of.2 mM; sample injection, 8 psi for 5 s.

esolution R was 4.78 (R/S-omeprazole), 4.36 (R/S-lansoprazole),.19 (R/S-amlodipine) and 5.64 (R/S-nimodipine).

. Conclusions

GSH-, ST- and OV-silica hybrid monolithic capillary monolithicolumns were designed and prepared as well as evaluated in termsf physical structure and CEC performance. The incorporation ofhe peptide or protein into the silica monolithic skeleton makeshe monolithic stationary phase a typical HILIC and chiral station-ry phase. Its EOF can be easily controlled by adjusting the acidity ofhe running buffer. It could use aqueous solution as a mobile phaseo separate nucleotides and peptides. Successful applications to theeparation of dl-amino acids implied that this type of monolithicolumn is also promising for the separation of enantiomers. Furtherodification of AuNPs resulted in the AuNP-mediated GSH-AuNP-SH-silica monolithic capillary column, and its chiral separationbility was significantly improved. Natural peptides and proteinsre suitable molecules to be used as vital components in the prepa-ation of novel stationary phases to achieve various functions and toealize the different separation purposes desired, if their amino acidompositions and sequence as well as configuration are carefullyonsidered in advance.

cknowledgements

We thank the financial supports from the National Nat-ral Science Foundation of China (21535007, 21475108 and1275120), the National Basic Research 973 Program of China2014CB932004) and Naitonal Instrumentation Program of China

[

1446 (2016) 125–133

(2011YQ03012401) as well as the Foundation for InnovativeResearch Groups of the National Natural Science Foundation ofChina (21521004) and Program for Changjiang Scholars and Inno-vative Research Team in University (PCSIRT, IRT13036). We thankProf. Hui Zhang of Xiamen University for offering the drug enan-tiomers. We thank Professor John Hodgkiss of The University ofHong Kong for his help with the English.

Appendix A. Supplementary data

Supplementary data associated with this article can be found, inthe online version, at http://dx.doi.org/10.1016/j.chroma.2016.04.014.

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