The coating of smart pH-responsive polyelectrolyte brushes in capillary and its application in CE

1352 Electrophoresis 2013, 34, 1352–1358

Jing-Xin Liu1

Ming-Zhe Zhao1

Yan Deng1

Cai Tie1

Hong-Xu Chen2

Ying-Lin Zhou1

Xin-Xiang Zhang1

1Beijing National Laboratory forMolecular Sciences (BNLMS),Key Laboratory of BioorganicChemistry and MolecularEngineering of Ministry ofEducation, Institute of AnalyticalChemistry, College ofChemistry, Peking University,Beijing, P. R. China

2Beckman Coulter CommercialEnterprise (China) Co., Ltd,Beijing, China

Received September 24, 2012Revised January 2, 2013Accepted January 10, 2013

Research Article

The coating of smart pH-responsivepolyelectrolyte brushes in capillary andits application in CE

A novel pH-responsive coating technique was developed and applied to CE successfullyin this paper. The coating was formed by bonding mixed opposite charge poly(acrylicacid) and poly(2-vinylpyridine) randomly onto the inner wall of a silica capillary. Thecoating processes were first characterized by ellipsometry and atomic force microscopyat macroscale and microscale, respectively. Measurements of EOF were implemented toconfirm the coating. Direction and velocity of EOF became controllable from negativeto positive, showing a perfect sigmoidal curve as the coating net charges alternated bythe pH of BGE. The control of the EOF makes it possible to analyze different kinds ofsmall molecules, peptides, and proteins successfully in the same capillary. Results showedthat the stability and reproducibility for separations of fluoroquinolone standards weresatisfactory for more than a hundred separations. A series of basic and acidic proteinstandards were separated with admirable efficiency and minimal adsorption using bothpolarities. The separation of tryptic BSA digest showed that the prepared capillary hasimmense potential in analyzing a single sample with both acidic and basic separations,which achieved the expectation in proteomics study by CE-MS.

Keywords:

Capillary electrophoresis / Permanent coating / pH-responsive / Polyelectrolytebrush DOI 10.1002/elps.201200518

� Additional supporting information may be found in the online version of thisarticle at the publisher’s web-site

1 Introduction

EOF is a key parameter influencing efficiency and repeata-bility of the separation in CZE. EOF can be manipulated bychanging the concentration and/or pH of BGE. Since thedistribution and construction of a double layer between thecapillary wall and the buffer solutions are the main parame-ters in the operation, the control of both solid phase (surface)and solution becomes very important. The absolute value ofEOF is also affected by changes in the separation voltage,capillary length, or operation temperature [1]. The fluctua-tion of electroosmotic mobility is related to the adsorption of

Correspondence: Professor Xin-Xiang Zhang, College of Chem-istry, Peking University, Beijing, P. R. ChinaE-mail: [email protected]: +86-10-62754680

Abbreviations: AFM, atomic force microscopy; CIP,ciprofloxacin; ENO, enoxacin; ENR, enrofloxacin; GAT,gatixacin; GPS, 3-glycidoxypropyltrimethoxysilane; LOM,lomefloxacin; OFL, ofloxacin; PAA, poly(acrylic acid); PEF,pefloxacin; P2VP, poly(2-vinylpyridine)

analytes with high positive charge densities to the negativelycharged bare fused silica capillaries, which cause changes tosurface structure and subsequently migration times, and alsolead to significant peak broadening and a decrease in sepa-ration efficiency. Quantification and quantitation would be-come difficult in the presence of irreversible adsorption andthe reliable control of surface structure of the capillary coatingis critical for the separation. Different kinds of coating tech-niques have been proposed and applied to the modificationof capillary and improvement of CE performance. The coat-ing techniques can be divided into three categories: dynamic,adsorbed-permanent, and permanent covalent wall coatings[2–4]. Dynamic coating is typically carried out by rinsing thecapillary with a solution containing coating agent, which iseither a polymer or a small molecule. It could be simple andprecise but addition of the modifiers in the separation bufferis needed because it is based on reversible equilibrium. Thedisadvantage of this mode is its incompatiblity with MS detec-tion [2,5]. Permanent coating would be preferable, especiallyfor compatibility with MS detection. The physical adsorbed

Colour Online: See the article online to view Figs. 1–3 in colour.

C© 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.electrophoresis-journal.com

Electrophoresis 2013, 34, 1352–1358 CE and CEC 1353

Figure 1. pH-Responsive mix-ed brushes prepared from PAA(in blue) and P2VP (in red).

coating is a common strategy to accomplish a simple semiper-manent coated capillary, but regeneration is sometimesneeded because of its limited lifetime [6]. This coating tech-nology is flexible because various coating materials are able toproduce anionic, cationic, and neutral surfaces. But the leak-age of the coating materials will limit the hybridizations be-tween CE and other techniques such as CE-MS [7–10]. Com-pared to the above-mentioned methods, permanent-covalentcoating is a preferred method because of its superior perfor-mance in repeatability and stability [11]. Although it is farfrom the ideal methodology because of the laborious proce-dure involved, the majority of published methods use neutralor cationic coatings that are compatible with CE-MS [12]. Dif-ferent kinds of coated capillaries are currently commerciallyavailable for different separation modes in CE.

In this study we present a novel, “smart,” permanentcovalent coating using mixed polyelectrolyte brushes withinthe capillary. The “grafting to” technique, which involves poly-mers with functional end chains covalently bond to substratesand widely applied in the polymer modification of solid sur-faces [13], was introduced in the inner wall modification ofcapillary in our work. When grafting density gets thicker, de-fined by when the distances between grafted polymer chainsare smaller than average end-to-end polymer chain distance.The soft chains “stand up” like bristles on a brush. Variousstudies have been reported on the development of polymerbrushes consisting of polyelectrolytes in recent years [14,15].In 2003 [15, 16], Houbenov et al. brought about the ideaof fabrication of mixed brushes with polyelectrolyte brushescomposed of two incompatible polymers randomly grafted tothe same substrate. Two polyelectrolytes in this novel brush,poly(acrylic acid) (PAA) and poly(2-vinylpyridine) (P2VP),were grafted onto the same silicon (Si) wafer. The surfaceof the coated Si wafer carried different charges when placedinto solutions at different pHs.

Some reports have been published on the applicationsof polyelectrolyte coatings in CE. But most of the reports arelimited to adsorbed coatings [9,17–19]. In this report, we firstapplied the binary mixed brushes into the field of capillarychemical coating. Utilizing the “grafting to” principle, a smartcapillary surface was constructed with a mix of PAA andP2VP polyelectrolytes in a liquid reaction state (Fig. 1). Thesmart polyelectrolyte brushes were covalently bonded to thecapillary surface and could show cationic, neutral, or anionicnet charges over a wide pH range. The EOF of the obtainedcapillary became controllable in both direction and velocity,which was imperative for the optimization of separation in

CZE. It also has a great advantage of long-term stability, whichis one of the most desirable properties of CE-sheathless ESI-MS analysis, especially for the nondisposable sprayer system.Different CE separations under different pHs demonstratedthat the smart coating was satisfactory for use in the analysesof several different classes of analytes in the same capillary.

2 Materials and methods

2.1 Chemicals and reagents

Carboxyl-terminated poly(tert-butyl acrylate) (PtBA-COOH,Mn = 42 000 g/mol, Mw = 47 000 g/mol) andpoly(2-vinylpyridine) (P2VP-COOH, Mn = 39 200 g/mol,Mw = 41 500 g/mol) were purchased from PolymerSource, Inc. Canada (synthesized by anionic polymerization).3-Glycidoxypropyltrimethoxysilane (GPS), trypsin (proteomicgrade), �-chymotrypsinogen A (bovine pancreas), lysozyme(chicken egg white), ribonuclease A (bovine pancreas),cytochrome C (bovine heart), horseradish peroxidase,�-lactoglobutin A (bovine milk), and BSA were obtained fromSigma-Aldrich (St. Louis, MO, USA). Formic acid and am-monium formate were purchased from TCI (Tokyo, Japan)for MS analysis. Seven fluoroquinolone standards, gatixacin(GAT), lomefloxacin (LOM), enoxacin (ENO), ciprofloxacin(CIP), ofloxacin (OFL), enrofloxacin (ENR), and pefloxacin(PEF) were purchased from National Institutes for Food andDrug Control (Beijing, China). All other reagents were an-alytical grade and obtained from Beijing Chemicals (Bei-jing, China). All solutions were prepared with ultrapurewater (18.2 M�·cm) by a Milli-Q system (Millipore, Bed-ford, MA, USA). Toluene and THF were distilled afterdrying over sodium. Highly polished silicon wafers wereobtained from Semiconductor Processing (Union Miniere,USA). The bare fused silica capillaries (365 �m od × 50 �mid) were purchased from Sino Sumtech (Hebei, China).Bare fused silica capillaries (150 �m od × 30 �m id)etched with a porous tip used in the CE-MS experiment weremade available by Beckman Coulter (Brea, CA, USA).

2.2 Coating procedure

Before coating, the capillary was treated with 1 M NaOHfor 30 min and rinsed with pure water for 30 min. Themixed brush coating was synthesized with a four-step


1354 J. -X. Liu et al. Electrophoresis 2013, 34, 1352–1358

Figure 2. Grafting of themixed brush onto the capil-lary inner wall: grafting GPS,grafting PtBA, grafting P2VP,and hydrolysis of PtBA toform PAA brush.

procedure as shown in Fig. 2. Dried fused silica capillary wasrinsed with 1% (v/v) GPS solution in toluene and reacted for16 h at room temperature. After the silanization reaction, thecapillary was rinsed several times with dried toluene to re-move nonspecifically adsorbed reagent. Then, PtBA-COOHwas selected to be the first grafted polymer. With nitrogen-driven rinse, the polymer was grafted on the GPS-modifiedcapillary inner wall and followed by annealing under ni-trogen atmosphere at 150�C for 20 min. After the graftingprocedure, unreacted polymers were removed by toluene.The second polymer P2VP-COOH was then grafted usingthe same procedure, and the grafting time was 15 h at100�C. Finally, the PtBA component of the mixed brushwas hydrolyzed by treatment in benzene saturated withp-toluenesulfonic acid monohydrate at 55�C for 1 h to yieldPAA.

2.3 Ellipsometry

Ellipsometry was applied to determine the thickness of thechemical coated GPS and grafted polymers. All measure-ments were carried out at a fixed incident angle of 70� forthe Si wafers with a multi-wavelength (370-1680 nm) rotatingcompensator ellipsometer M-2000VI (J. A. Woollam, USA).Data analysis was carried out using the manufactures soft-ware package Complete EASETM.

2.4 Atomic force microscopy (AFM)

Tapping mode AFM images were obtained using aNanoScope VIII MultiMode AFM (Digital Instruments, USA)at ambient conditions. Si cantilever tips (TESP, USA) with aresonance frequency of approximately 300 kHz and a springconstant of about 40 N/m were used. The scan rate variedfrom 0.7 to 1.2 Hz. The scanning density was 512 lines/frame.

The root-mean-square (rms) roughness was calculated over1 × 1 �m2 scanned area using commercial software.

2.5 CE measurements

CE system from Beckman Coulter equipped with a temper-ature controlled auto-sampler and a power supply able todeliver up to 30 kV. Data were collected and processed by 32Karat software (Beckman Coulter).

EOF measurements were performed through a 7 nL in-jection of 1% acetone as a neutral marker from pH 2 topH 9 and calculated from the migration times of acetone.Phosphate buffers at pH 2–3 and 6–7, acetate buffers atpH 4–5, and borate buffers at pH 8–9 were used.

Seven fluoroquinolone standards (GAT, LOM, ENO, CIP,OFL, ENR, and PEF) were studied at a concentration of1 mg/mL and a 50 mM borate buffer was used as BGE.Four basic proteins (�-chymotrypsinogen A, ribonuclease A,lysozyme, cytochrome C) were separated in an acid buffersystem (pH 3) using reversed polarity of CE, and three acidicproteins (HRP, �-lactoglobutin A, BSA) were studied in abasic buffer system (pH 9) using normal polarity of CE, todemonstrate the pH-responsive property of the binary brushcoating. A tryptic BSA digest was performed to test the bi-nary characterization of the coated capillary under pH 3and 9, respectively.

2.6 CE-sheathless ESI-MS

CE-ESI-MS experiments were performed on the prototypesheathless CE-MS interface (Beckman Coulter), which cou-pled CE to a 6320 Ion Trap mass spectrometer (AgilentTechnologies, Palo Alto, CA, USA) via a sheathless sprayer[20, 21].



Spectra were collected over the mass range of 50–2200 m/z at an Ultra Scan (23 000 m/z/s) mode. The fol-lowing spray conditions were used: an electrospray voltagefrom −1.0 to −1.5 kV; dry gas, 2 L/min; source temperature,160�C.

3 Results and discussion

3.1 Coating procedure

The synthesis procedures of mixed polyelectrolyte brusheshave been reported, and the coating procedure was appliedwith a slight change in this report [16,22,23]. Unlike the “graft-ing to” reactions done on the planar substrate through spincoating in a vacuum oven, the delivery of different reagentsfor the capillary coating was controlled by a custom-madepressure control reaction vial, which can pump reaction solu-tion into the capillary under different nitrogen pressures. Adried and limited oxygen environment was obtained to makeit accessible to the grafting reaction. The coatings were an-chored to the capillary surface by reacting GPS with silanolgroups at first, retaining high coverage of epoxy groups. Then,two incompatible polyelectrolytes (PtBA-COOH and P2VP-COOH) were grafted sequentially using an esterification re-action. PtBA-COOH shows a strong hydrophobic property inthe presence of main chains and the tertbutyl groups. Theopposite order of grafting did not yield reproducible resultsbecause of the high affinity of P2VP-COOH chains to the sub-strate (epoxy-rich), which essentially suppressed the penetra-tion of the hydrophobic polymer through P2VP brush. Theprocedure included four key steps: (i) silanization reaction ofGPS on the inner wall surface of bare silica capillary, (ii) graft-ing of PtBA-COOH, (iii) grafting of P2VP-COOH, and (iv)hydrolysis of PtBA yielding PAA. In step 4, tert-butyl groupswere removed from PtBA and the main chains of polymerfunctionalized fully with carboxyl groups. Thus, oppositelycharged mixed brushes (PAA and P2VP) were completelysynthesized. The coating reactions in each step were quitesimple and efficient, so no more optimization was needed.

3.2 Characterization of mixed brushes

Since coating inside the capillary is difficult to be charac-terized by the techniques such as AFM and ellipsometry, ahighly polished single-crystal silicon (Si) wafer was used asa substrate instead of the capillary for the characterization.A self-assembly strategy immersing Si wafers in polymer so-lutions and grafting the mixed brushes in a liquid state waschosen, which was believed to mimic the conditions in a capil-lary. All the samples characterized by ellipsometry and AFMwere synthesized with this strategy, but the measurementswere carried out in a dry state. An ellipsometric study showedmacroscopic homogeneity of the mixed brushes with an av-erage of five measurements for each Si wafer. For the mixedbrush, a three-layer model was used, with SiO2 and GPS con-

sidered as an effective optical layer. The Cauchy model inEq. (1) below has been used to fit the real part of the refractiveindex n(�) of the grafted layer [24]:

n(�) = A + B

�2+ C

�4(1)

where � is the wavelength of radiation in micrometers and A,B and C are the Cauchy coefficients.

The thickness of the GPS layer was about 1.6 nm mea-sured with a three-layer model: Si/SiO2/GPS for a GPS refrac-tive index equal to 1.45 [16]. The thickness of PtBA as the firstgrafted layer was evaluated to be 5.9 nm, with n = 1.46 forPtBA. The whole polymer film after the grafting of the secondpolymer (P2VP) was at a thickness of 9.8 nm (n = 1.59), whichproved that binary polymer chains were at a high graftingdensity and might stretch away from the substrate because ofthe electrostatic interactions and the excluded-volume effect[13,16]. After hydrolysis, a nonsignificant decrease (less than5%) of the brush thickness was observed. The results showedthat thicker brushes (9.2 nm) were synthesized compared to6.4 nm, which was mainly because the grafting procedure weutilized was at a liquid reaction state [16].

The microscopic morphology of the binary brushes wasobserved with AFM in tapping mode. The result revealed lat-eral miscible property as a homogeneous and smooth filmwith root-mean-square roughness of 0.98 nm in the mixedbrush in a dry state. Combining results of ellipsometry andAFM, it can be concluded that a successful grafting proce-dure was applied to synthesize the nanoscale patterned binarybrushes on Si wafer, and predictably inside the capillary.

Grafting patterns of mixed PAA and P2VP are innanoscale, so the upper layer in certain pHs seems to behomogeneous and achieves full coverage of the silica surface,which could obtain reproducible results as other homopoly-mer coatings in CE analysis [7].

3.3 Measurement of pH-responsive EOF

Electroosmotic mobility in a bare fused silica capillary isknown to have a sigmoidal relationship with pH [1]. At a lowpH, ionization of the surface silanol groups is suppressed,and EOF approaches zero. Under alkaline conditions, silanolgroups are fully charged, and EOF reaches a positive plateauvalue. Therefore, EOF is an important parameter in the eval-uation of the coating process with respect to CE analysis,corresponding to Zeta potential in polymer science. In ourcoated capillary grafted with mixed polyelectrolyte brushes,the EOF followed the sigmoidal relationship and involved awider range of mobility as expected, from negative EOF topositive EOF accompanied by a zero at a certain pH, andshowed quite a difference from homopolymer[7] or homo-charged coatings [6], which exhibited pH-independent EOF.Moreover, the zero EOF was related to the isoelectric pointof the mixed polyelectrolyte brushes as well as the ionic con-centration of the solution. Each buffer was prepared at a con-centration of 10 mM, and ionic strengths of all the buffers



Figure 3. Effect of pH on electroosmotic mobility using CE-UV inPAA-P2VP coated capillary; the star shows zero EOF at pH 4.6.

were equalized by the addition of KCl. Thus, electroosmoticmobility was only determined by pI of the grafting reagents.A sigmoidal relationship was found covering a wide pH rangeof 2–9 (Fig. 3), and an isoelectric point at about pH 4.6 wasshown, which was comparable to the measurement of Zeta-potential result (pI 4.9) of the mixed PAA-P2VP brushes [16].That data demonstrated amphiphilic properties of the mixedbrushes when the isoelectric point was located between thevalues of each homopolymer brushes (P2VP, pI 6.7; PAA,pI 3.2). It can be seen from Fig. 3 that at low pH (pH 2.0–4.6),negative EOF was observed; positive EOF was shown at highpH (pH 4.6–9.0). It was demonstrated that the binary graftingpolymers were successfully coated on the surface of the capil-lary with a sharp linear change of charges in the wide range ofpH, and opposite velocities of EOF were displayed in such acapillary, which was impossible with homopolymer coatings.Capillary-to-capillary coating repeatability in preparation wasnecessary for the practical application. Three coated capillar-ies were evaluated based on RSD of EOF. Compared witha smaller RSD of 0.58% of EOF reported by multiple ioniclayers absorbed coatings [6], yet still acceptable, an RSD of1.37% (n = 3) was obtained.

3.4 CE with UV detector (CE-UV) analyses by coated

capillaries

To avoid physical adsorption, the inner interface of the cap-illary is preferred to be coated with cationic coating whenconfronting cationic analytes and anionic coating for analysisof anionic species. In most coated capillaries, it is impossibleto achieve contrary charges at different pH conditions in thesame capillary, which limits its application in the separationof different kinds of analytes. [6,25] With this pH-responsivebinary brush coating, on the other hand, any polar applica-tions could be accomplished by simply changing the pH ofBGE in a single capillary.

Various kinds of analytes were tested to show the ease ofEOF change, stability, and reproducibility of the novel coating

Figure 4. Protein analysis using CE-UV in PAA-P2VP coated capil-lary (60 cm total length, 50 �m i.d.). Electropherograms obtainedin 20 successive injections (A) 1, �-chymotrypsinogen A; 2, ri-bonuclease A; 3, lysozyme; 4, cytochrome C at pH 3 (50 mMphosphate buffer); and (B) 1, HRP; 2, �-lactoglobutin A; 3, BSAat pH 9 (50 mM borate buffer).

capillary. Long-term stability of the coating was examined byperforming over a hundred replicate analyses of an anionictest mixture of seven fluoroquinolone standards (GAT, LOM,ENO, CIP, OFL, ENR, PEF), under condition of anionic coat-ings (PAA brushes stretched out in capillary inside) in boratebuffer, pH 9 (Supporting Information Fig. S1). As an approx-imate estimation, the endurance of this coating was morethan 500 runs. Repeatable analyses were obtained with RSDtm

(RSD of time migration) <1.19% of seven fluoroquinolones(n = 60) (Supporting Information Table S1). By comparison,the same mixture was operated within a bare-silica capillaryunder the same separation condition with a low-resolutionresult as shown in Supporting Information Fig. S2.

Proteins were separated successfully, which demon-strated the efficient prevention of macromolecule adsorp-tion onto capillary walls. Four basic protein samples(�-chymotrypsinogen A, ribonuclease A, lysozyme, cyto-chrome C) were separated under acid buffer pH 3 (Fig. 4A).Additionally, three acidic proteins, HRP, �-lactoglobutin A,and BSA, were analyzed with the same capillary under basiccondition, pH 9 (Fig. 4B). As shown in Supporting Informa-tion Table S2, good reproducibility was obtained with RSDtm



<1.07% for basic proteins and RSDtm <1.38% for acidic pro-teins. The eluted order of basic proteins and migration timeRSDs, whereas peak area RSDs (less than 10%) were compa-rable to SMIL-PB (3), another more commonly used cationiccoating for basic proteins separation [25, 26]. In conclusion,our smart coating provided an extremely effective surface foradsorption prevention in protein separation. It was empha-sized that utilizing this novel coating technique, differenttypes of samples could be separated in one column by simplychanging pH. Thus, it has great potential for the analysis ofcationic and anionic samples, providing much more infor-mation than other methods.

To test the binary characterization of this coating method,two polarities of BSA digest analyses on the same columnwith acidic and basic functionality were implemented. Asshown in Supporting Information Fig. S-3, three separationswere loaded for CE-UV detection. Nondigest BSA showed aGaussian peak when detecting in reverse mode. Meanwhile,the tryptic digest in both acidic and basic buffers showed adifference of well separation in CE-UV spectra as expected,demonstrating the binary ability of our mixed brush coatingcapillary.

3.5 CE–sheathless ESI-MS analyses of a BSA tryptic

digest

A digest experiment was carried out here to demonstratethe superior stability and great potential of this coating inCE-MS. The sheathless interface we used is a prototype plat-form developed by Beckman Coulter, which has a poroussection of separation capillary outlet etched with hydrofluo-ric acid (HF) [27]. Nanospray source was used with no postcolumn dilution to analytes. It offers improved sensitivitydue to more efficient ion formation, transport, and sampling,but this interface is limited by the variations of BGE com-ponents and capillary coating techniques, which need to bemore compatible with MS detection. A tryptic digest of BSAwas studied in the mixed polyelectrolyte brush capillary viaour CE-sheathless nanospray MS system. Separations wereperformed in a 90 cm-long PAA-P2VP–coated capillary with aterminal 3 cm-long porous segment inserted into the sprayerinterface enabling electric contact via a secondary bare silicacapillary filled with conductive liquid. When using this coatedcapillary, maximum resolution was achieved at a pH lowerthan the pI of the peptides because the direction of analytemigration is then opposed to the anodal EOF. This resolvingpower, in combination with high efficiencies achieved, madethe binary brush coating feasible in the analysis of complexsamples. A BGE used in the porous separation capillary andthe buffer in the conductive liquid capillary were both com-posed of 1% v/v formic acid (pH 3). A voltage of –20 kV wasapplied during separation yielding a stable current of approx-imately 4 �A, which indicated that the coating materials didnot pose a negative effect on the conduction of CE.

A typical base peak electropherogram (BPE) correspond-ing to the sheathless CE-ESI-MS analysis of a BSA trypticdigest is shown in Fig. 5A. It was referred to as a high effi-

Figure 5. CE-sheathless ESI-MS analysis of a tryptic digest ofBSA using PAA-P2VP coated capillary with a porous sprayer, to-tal length 90 cm × 30 �m id × 150 �m od; BGE and conductiveliquid, 1% formic acid. (A) Base peak electropherogram. (B) Ex-tracted ion electropherogram of m/z 536.7. (C) Extracted ion elec-tropherogram of m/z 583.9. (D) Extracted ion electropherogramof m/z 722.3.

cient separation with an enhanced peak capacity. Three ex-tracted ion electropherograms (EIE) were shown in Fig. 5B–Dcorresponding to three arbitrarily selected tryptic peptides. APoly E-323 capillary coating (a cationic adsorbed coating) us-ing CE-sheathless ESI-MS for analysis of a tryptic digest ofBSA was introduced in 2000. [28] Compared with 46% pep-tide determined by Mascot search, it is appreciated that anenhanced sequence coverage (63%) was obtained from sucha high efficiency electropherogram in our experiments.

4 Concluding remarks

In this study, a smart binary polymer brush coating inthe capillary was synthesized for CE analysis. A systematicstudy of the AFM and ellipsometry demonstrated a nanoscalemixed brush of oppositely charged polyelectrolytes, and var-ious applications were attempted to prove its pH-responsiveproperty, stability, repeatability, and potential use in CEanalysis.

An EOF-pH sigmoidal curve showed the amphiphilicproperties of the mixed brush coating. Favorable migrationtimes and peak area reproducibility were obtained for sevenfluoroquinolone standards, four basic proteins, and threeacidic proteins using CE-UV analysis. Also, a coated capil-lary with a porous tip sprayer in CE-sheathless ESI-MS hadbeen evaluated for proteomic profiling of a tryptic digest ofBSA. A good BPC was achieved because of the coating andthe sheathless interface. The results proved that the smartbrushes provide a wide range of potential uses in CE andCE-MS analyses. Under binary polarity conditions of elec-trophoresis, much information might be obtained for fur-ther analysis in the field of peptide profiling. Furthermore,it is possible to apply this coating method in CE analysis



of metabolomics as well as in pharmaceutical and foodanalysis.

This work was supported by National Natural Science Foun-dation of China (No. 30890142 and 20975007) and the NationalScientific Support Project 2009CB320305 (MOST, China). Theauthors thank Jordan Aerts and Supida Monaikul from Univer-sity of Illinois, Urbana-Champaign for polishing language.

The authors have declared no conflict of interest.

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The coating of smart pH-responsive polyelectrolyte brushes in capillary and its application in CE