Open tubular capillary column for the separation of cytochrome C tryptic digest in capillary electrochromatography

J. Sep. Sci. 2015, 00, 1–10 1

Faiz AliWon Jo Cheong

Department of Chemistry, InhaUniversity, Namku, Incheon,South Korea

Received July 11, 2015Revised July 23, 2015Accepted August 8, 2015

Research Article

Open tubular capillary columnfor the separation of cytochrome C trypticdigest in capillary electrochromatography

A silica capillary of 50 �m internal diameter and 500 mm length (416 mm effective length)was chemically modified with 4-(trifluoromethoxy) phenyl isocyanate in the presence ofdibutyl tin dichloride as catalyst. Sodium diethyl dithiocarbamate was reacted with theterminal halogen of the bound ligand to incorporate the initiator moiety, and in situ poly-merization was performed using a monomer mixture of styrene, N-phenylacrylamide, andmethacrylic acid. The resultant open tubular capillary column immobilized with the copoly-mer layer was used for the separation of tryptic digest of cytochrome C in capillary elec-trochromatography. The sample was well eluted and separated into many components. Theelution patterns of tryptic digest of cytochrome C were studied with respect to pH andwater content in the mobile phase. This preliminary study demonstrates that open tubularcapillary electrochromatography columns with a modified copolymer layer composed ofproper nonpolar and polar units fabricated by reversible addition-fragmentation transferpolymerization can be useful as separation media for proteomic analysis.

Keywords: Capillary electrochromatography / Copolymer layer / Open tubularcolumn / Peptides / Tryptic digestDOI 10.1002/jssc.201500765

1 Introduction

High-throughput and selective analytical methods are highlyappreciated for tracking disease biomarkers since on-target(personalized) treatment for disease like cancer is gettingpopular [1] and protein identification is critical to know therelation between genes, gene variants and their contribu-tion to the disease [2]. Digestion of proteins into MS-friendlypeptides is generally carried out and subsequent analysis isdone by LC–MS, which is one of the best techniques forproteomics, and it requires the development of high reso-lution LC columns to separate as many peptides as possi-ble [3]. Monolithic columns [4], columns based on fused coreparticles [5], and UHPLC columns [6–8] are some examplesof novel chromatographic approaches of attracted interest.Especially, UHPLC may be the most effective technique inprotein, peptide and biomarker analysis. Columns packed

Correspondence: Professor Won Jo Cheong, Department ofChemistry, Inha University, 100 Inharo, Namku, Incheon 402–751,South KoreaE-mail: [email protected]: +82 328675604

Abbreviations: ACN, acetonitrile; DBTDC, dibutyl tin dichlo-ride; FE-SEM, field emission scanning electron microscopy;MAA, methacrylic acid; MIP, molecularly imprinted poly-mer; OT-CEC, open tubular capillary electrochromatogra-phy; RAFT, reversible addition-fragmentation chain transfer;SDEDTC, sodium diethyl dithiocarbamate; THF, tetrahydrofu-ran; 4-TPI, 4-(trifluoromethoxy)phenyl isocyanate

with very small (less than 3 �m) particles are employed undera very high pressure (up to 1000 bar) in UHPLC. In a recentreview of current and future trends of UHPLC [9], not onlythe merits of UHPLC such as dramatic increase of through-put, fast separation, and good resolution but also other trendssuch as high resolution analysis of complex samples, appli-cation in different chromatographic modes, methods for fur-ther improvement of kinetic performance, etc., have been wellintroduced.

Various CEC-based approaches for the separation ofpeptides and proteins have also been reported in theliterature such as granular packed CEC columns [10],monolith-based CEC columns [11, 12], pressurized CECcolumns [13] and open tubular capillary electrochromatog-raphy (OT-CEC) columns [10, 14–21]. It may be notedthat an OT-CEC column with poly(butyl methacrylate-co-ethylenedimethacrylate) showed excellent separation (up to400 000 plates/m) for alkylbenzenes [22].

Exploration of complex biological systems by analysis ofprotein function, expression, interaction, and modificationdemands sophisticated proteomic studies [23]. In the bottomup proteomics approach, peptides are generated by digestionof a protein mixture and those peptides are separated by LCfollowed by MS/MS while intact proteins or large proteinfragments are subjected to MS analysis in the top-down pro-teomics [23–25]. Before MS analysis, separation of peptidesor intact proteins by LC is very important [23,26]. Both 1-D LCand 2-D LC pre-separation can be incorporated either in online or offline mode of separation. To achieve improved sep-aration efficiency and peak capacity for proteomic analysis,

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2 F. Ali and W. J. Cheong J. Sep. Sci. 2015, 00, 1–10

long (3–5 m) and narrow open tubular LC columns with thinpolymer layers have been developed to show good results forproteomic analysis [27–31]. However, this approach requireslong analysis time and an extreme care is required to prepareand maintain the column to prevent column clogging. Thetypical internal diameter (id) of the capillary column is 10 �m.On the other hand, open tubular LC columns of wider id re-sult in band broadening and unacceptable chromatographicperformance.

For the purpose of overcoming the above problem,OT-CEC is a very good alternative where columns of widerid can be used to result in nice peak shape due to uniformvelocity distribution across the capillary diameter [32]. TheCEC–MS system accounts for excellent performance. OT-CEC columns for proteomic analysis are getting more at-tractive, and have been reported to show good separationefficiency for proteins and peptides [10] although this ap-proach has not been in wide practice for proteomic analy-sis yet. CEC columns can also be employed in multidimen-sional separation [33]. Our research group has reported theapplication of molecularly imprinted polymer (MIP) incorpo-rated OT-CEC column for proteomic analysis of cytochromeC tryptic digest [34] with excellent separation efficiency, butthe peptide peaks were assembled in a narrow range of re-tention time and the peak capacity was rather limited inthat study.

The goal of this study is to introduce the methodologyof preparation of new OT-CEC columns to contribute to pro-teomic research. As an attempt of such intention, currentstudy introduces the development of a new OT-CEC capil-lary column immobilized with a copolymer layer based onthree monomers for separation of various peptides presentin the tryptic digest of cytochrome C. We adopted reversibleaddition-fragmentation chain transfer (RAFT) polymeriza-tion for the preparation of OT-CEC column to ensure an-choring long polymer chains upon the surface as in ourprevious studies [35, 36]. In RAFT polymerization, the sur-face is modified with a ligand having a halogen terminal,then an initiator moiety is introduced upon it, thus the poly-mer chains are grown from the surface. Details of RAFTpolymerization may be found in refs. [35, 36] and the lit-erature cited there. The copolymer is composed of styrene,methacrylic acid (MAA), and N-phenylacrylamide. The pep-tides have been well eluted and separated. The effects of mo-bile phase composition and pH on separation performancehave been studied. Unfortunately, CEC–MS has not been in-corporated in this study owing to limited availability of such asystem to us.

2 Materials and methods

2.1 Chemicals and materials

Sodium hydroxide, glacial acetic acid, 4-(trifluoromethoxy)phenyl isocyanate (4-TPI), dibutyl tindichloride (DBTDC), sodium diethyl dithiocarbamate

(SDEDTC), anhydrous tetrahydrofuran (THF), styrene,MAA, anhydrous toluene, N-phenylacrylamide, trypsin,ammonium bicarbonate, urea, bovine cytochrome C, andstandard peptides (THR-TYR-SER, PRO-PHE-GLY-LYS,TYR-ILE-GLY-SER-ARG, PHE-LEU-GLU-GLU-ILE, VAL-GLU-PRO-ILE-PRO-TYR) were purchased from Sigma–Aldrich (St. Louis, MO, USA). HPLC-grade methanol,acetonitrile (ACN), acetone, and water were obtainedfrom Mallinckrodt Baker (Phillipsburg, NJ, USA). All thereagents were used as received. Silica capillaries with 50 �minternal diameter (id) and 365 �m outer diameter (od) werepurchased from Grace (Deerfield, IL, USA).

2.2 OT-CEC column immobilized with

styrene-methacrylic acid-N-phenylacrylamide

copolymer

2.2.1 Pretreatment

Pretreatment of the fused-silica capillary (50 cm length,360 �m od, and 50 �m id) was carried out according to thereported procedure [33, 34]. Briefly, 0.1 M NaOH solutionwas filled in the capillary, end-capped with septa and keptfor 24 h at room temperature, then at 55�C overnight. Thecapillary was washed with water, 0.1 M HCl, and again waterin sequence till neutrality, flushed with acetone for 1 h, anddried with N2 gas at 120�C for 2–3 h to expose the silanolfunctionalities.

2.2.2 Binding of initiator moiety onto inner capillary

surface

A solution composed of 4-TPI (40 �L), anhydrous toluene(2.5 mL), and DBTDC (30 mg) was kept flowing throughthe pretreated capillary at 90�C for 5–6 h at a flow rate of0.35 mL/h with a Harvard (Holliston, MA, USA) 11 Elitesyringe pump. The capillary was flushed with toluene for 8 hthen with acetone for 2 h, and dried with N2 gas for 10 min.The reaction scheme is given in Fig. 1A. The capillary wasthen subjected to the flow of a solution containing SDEDTC(100 mg) in anhydrous THF (3 mL) at 55�C for 5 h at a flowrate of 0.4 mL/ h. The capillary was washed with methanolovernight followed by acetone for 2 h, and dried with N2 for30 min. The reaction scheme is shown in Fig. 1B.

2.2.3 In situ co-polymerization

RAFT in situ copolymerization was performed onto thebound initiator on the capillary inner surface using apolymerization mixture containing styrene (0.65 mL), MAA(0.3 mL), N-phenylacrylamide (70 mg) and anhydrous toluene(2 mL) at 100�C for 8 h. The solution of reaction mixture wasdegassed by sonication and N2-purging for 10 min each andwas allowed to flow through the capillary via a 0.2 �m What-man (Maidstone, UK) syringe filter at a flow rate of 0.25 mL/h.


J. Sep. Sci. 2015, 00, 1–10 Electrodriven Separations 3

Figure 1. Scheme of reactions: (A) attachmentof ligand to OH-functionalities on the inner sur-face of pretreated fused-silica capillary; (B) ini-tiator binding to the ligand; (C) RAFT in situco-polymerization. R collectively denotes the differ-ent substitutes of vinyl monomers.

After polymerization the column was immediately flushedwith various washing solvents in sequence to remove the freeoligomers and residual reaction mixture. The column waswashed with toluene at room temperature for 6 h, then at50�C overnight, 2-propanol for 6 h, 60:40 v/v methanol/waterat 50�C for 10 h, and acetone for 2 h. The scheme of reactionfor polymerization is given in Fig. 1C.

The column was flushed with the mobile phase to getthe stationary phase in equilibrium with the mobile phasefor 2–3 h before CEC operation. A detection window was cre-ated at a distance of 84 mm from the outlet end by burningthe polyimide coating. The capillary column was finally in-stalled in the CEC instrument for analysis. The SEM imagesof the cross-section of the capillary CEC column were takenby a HITACHI (Tokyo, Japan) S-4200 field emission scanningelectron microscope (FE-SEM).

2.3 Tryptic digestion of cytochrome C

The intact protein (5 mg bovine cytochrome C) was digestedin the solution made by mixing trypsin (4 mg), 4.0 M urea(2 mL), and 0.2 M ammonium bicarbonate (2 mL). The solu-tion of reaction mixture was stirred by vortex for 10 min andkept in water bath at 37�C for 48 h. Then it was quenchedwith 1 mL 0.1% TFA, and the solution was filtered through a0.2 �m syringe filter, and the stock sample solution was storedbelow 4�C.

2.4 Instrumentation

CEC experiments were performed on an Agilent (Waldbronn,Germany) HP3D CE system with a diode array detector andthe Chemstation data processing software. Two stock buffersolutions (25 mM sodium phosphate and 25 mM sodiumacetate) were prepared in distilled water and kept in arefrigerator. The buffer pH was adjusted to the desired valueby addition of NaOH solution to the stock phosphate buffer

solution or acetic acid to the stock acetate buffer solution fol-lowed by addition of acetonitrile to get the final mobile phase.A fresh sample of cytochrome C tryptic digest was preparedeach time when a new mobile phase was used while all thesamples and eluents were filtered through a 0.2 �m What-man (Maidstone, UK) syringe filter. Samples were injectedin the positive potential mode (10–15 kV) with injection timeof 6–10 s. The detection wavelength, capillary temperature,and applied potential were set to 214 nm, 25�C, and 30 kV,respectively. The total and effective length of the open tubularcapillary column was 500 and 416 mm, respectively.

3 Results and discussion

3.1 Morphology of the stationary phase

The SEM images of styrene-methacrylic acid-N-phenylacrylamide copolymer layer immobilized on theinner surface of capillary column of current study are givenin Fig. 2, which shows that the polymer layer is thin andrugged. The thin (0.8–1.2 �m) and compact distributionof the polymer layer is attributed to the high density ofsurface initiator owing to the hydroxyl-isocyanate reactionthat occurs in the presence of catalyst [35, 36].

3.2 Separation performance for the tryptic digest

by CEC

3.2.1 Effect of pH on separation of cytochrome

C tryptic digest

Peptides may be positively charged, negatively charged orneutral by nature at pH 7. The neutral peptides occur in thezwitterion form at pH 7. However, the neutral peptides arealso positively charged at lower pH, and negatively chargedat higher pH. Thus the retention trends of peptides are verysensitive to pH. The pH effect is of vital importance when



Figure 2. Wide view (left) and close view (right) of cross-sectional FE-SEM images of the capillary column where the thin and compactbound polymer layer can be seen. An applied potential of 15 kV was used for the electron beam. The magnification was 10 000 and 30 000and the scale bar size was 5 and 1 �m for the left and right photos, respectively.

Figure 3. The pH effect on separation of tryptic digest of cytochrome C using 60:40 v/v ACN/ 25 mM sodium acetate buffer. (A) pH 4.5;(B) pH 6.8; (C) pH 9.0. Sample injection: 10 kV for 7 s; Applied potential: 30 kV; Current: 10–15 �A. The EOF marker (acetone) is denoted inthe electrochromatogram below the electrochromatogram of the tryptic digest in each case.

peptides and proteins are separated on OT-CEC stationaryphases with some polar functional groups. At lower pHs,MAA moieties in the stationary phase are less ionized result-ing in lower EOF leading to longer retention times of peptideswith increased band broadening (Fig. 3A). Similarly retentiontimes of peptides are decreased with narrow peak widths athigher pH due to increased EOF as shown in Fig. 3B. Further

increase in pH leads to peak congestion as shown in Fig. 3C.The EOF can be monitored in the electrochromatogramsof the EOF marker (acetone) as seen in the lower part ofFig. 3A, B, and C. Peaks (peptides) eluted before acetone arealso monitored, and they are probably positively charged pep-tides. The optimized pH value was concluded to be 6.8 inview of the number of peaks and separation efficiency.



Figure 4. Effect of water content on separation of tryptic digest ofcytochrome C using ACN/25 mM sodium phosphate buffer pH at6.8. The volume based water composition is 30% for A, 40% for B,and 50% for C. Sample injection: 10 kV for 7 s; Applied potential:30 kV; Current:10–15 �A. D is the EOF marker (acetone) obtainedunder optimized elution conditions used for B, i.e., 60:40 v/v ACN/25 mM sodium phosphate pH 6.8.

3.2.2 Effect of water content on separation of tryptic

digest of cytochrome C

Hydrogen bond interaction between the peptides and themobile phase increases with increase of water content in theeluent, and the disturbing effect of water on hydrogen bond-ing between the peptides and the stationary phase bearingMAA and N-phenylacrylamide is also enhanced. Thus, theretention of a peptide is decreased with increase of watercontent in the mobile phase (Fig. 4). Based on the results ofFig. 4, it is not straightforward to decide which compositionof water content is the optimum. As a compromise betweenseparation performance and analysis time, a water content of40% was selected to be optimized in this study.

3.2.3 Column to column repeatability

The electrochromatograms of tryptic digest of cytochromeC taken with three different capillary columns prepared bythe same protocol are given in Fig. 5. The number of pep-tide peaks in each of the electrochromatograms A, B, and Cranges from 30 to 32 as counted in the expanded view of theseelectropherograms. As shown in Fig. 5, the elution trends ofchromatograms A, B, C show some discrepancies in detailsalthough they are quite similar in general.

In this situation, two solutes were selected to estimate thecolumn to column repeatability: acetone (EOF marker) andthe last second peptide (nicely separated without any inter-ference). The retention and separation efficiency (number oftheoretical plates/m) data are assembled in Table 1. As shownin Table 1, repeatability in retention time and separationefficiency of both acetone and the selected peptide was good,repeatability for acetone being much better. The separationefficiency of acetone was very good while that of the selected

peptide was only marginal. As shown in Fig 4, the selectedpeptide peak was rather broad, and much better separationefficiency was obtained for most of the early eluting peptides(more than 100 000 plates/m).

Despite the good repeatability for acetone and the selectedpeptide, some discrepancies were observed in elution trendsamong different columns. We suspect that some peptides inthe tryptic digest are very sensitive to subtle change in thelocal environments of the stationary phase to show differentelution behaviors while most peptides and common neutralmolecules such as acetone are insensitive to such local varia-tions to show stable elution trends.

3.2.4 Comparison of separation performance

with those of previous researches in the literature

There have been some studies where superior separa-tion efficiency was demonstrated for a single or a fewproteomic (protein or peptide) components in OT-CEC[10, 17, 32]. For example, an extensive review on silicahydride stationary phases (etched and chemically modified)was published in 2009 where OT-CEC was also well in-troduced, and etched chemically modified capillaries wereclaimed useful for proteomics [17]. For example, the OT-CEC results for carbonic anhydrase under acidic conditionsobtained on an etched C5-modified capillary exhibitedexcellent peak shape and high separation efficiency (N >

1 000 000 plates/m) [18]. The stationary phases of re-cent OT-CEC studies include branched polyethyleneimine-bonded tentacle-type polymer (97 000–189 000 plates/m)[14], phenylalanine-functionalized tentacle-type polymer(13 000–182 000 plates/m) [15], triamine-bonded phase(87 000–110 000 plates/m) [16], gold-nanoparticle-basedphases [19, 20], and carboxymethychitosan (97 000–182 000 plates/m) [21].

However, some different components might show verypoor separation efficiency under the same experimental con-ditions, or the same component of very high separation effi-ciency might yield a very broad peak in different conditions(different pH values of mobile phase or different stationaryphases) [15, 18–20]. It is unlikely that a combination of afixed mobile phase and a fixed stationary phase serves as theoptimized separation environment for all the components ina complicated sample such as a tryptic digest that is com-posed of many components of different properties (polarity,pI, amount of charges, molecular weight, etc.). Thus, thereshould be some level of peak congestion for analysis of such acomplicated tryptic digest as demonstrated by the electrochro-matograms for BSA or HSA tryptic digests [10, 19, 20].

To unambiguously compare chromatographic separationperformances, recent studies on separation of cytochrome Ctryptic digest have been surveyed. There have been gradientLC studies by using monolithic capillary columns [37–40] andpacked columns with C18-modified silica particles [41–43],and CEC studies by monolithic capillary columns [44–46]and packed columns with C18 particles [44]. The OT-CECcolumn of this study shows comparable performance. An



Figure 5. Electrochromatograms (A, B, C) of tryp-tic digest of cytochrome C taken with three dif-ferent capillary columns prepared by the sameprotocol. Eluent: 60:40 v/v ACN/ 25 mM sodiumphosphate at pH 6.8; Sample injection: 10 kV for7 s; Applied potential: 30 kV; Current: 10–15 �A. Dis the EOF marker (acetone).

Table 1. Repeatability in separation efficiency (plates/ m) andretention time for acetone and the second last peptidechecked with three batches of columns using 60:40 v/vacetonitrile/ 25 mM sodium phosphate pH 6.8 as themobile phase

N-value Retention time (min)

Average %RSD Average % RSD

Acetone 473 000 2.2% 7.09 1.2%The second peak from the end 76 500 4.2% 25.24 2.7%

OT-CEC column has been recently prepared in our labo-ratory to show very high separation efficiency (more than200 000 plates/m) in separation of cytochrome C tryptic di-gest where the MIP technique was incorporated in formationof an organic monolith layer on the inner wall of the silicacapillary [34]. The separation performances of some of theabove studies and the current study are collectively comparedin Fig. 6.

Gradient elution can be freely used in LC while it is notin CEC. Packed or monolith LC columns coupled to gradientelution have been known to be very useful to enhance the peakcapacity. However, the peak capacity may be reduced depend-ing upon the choice of stationary and mobile phases or thesample on account of reduction of selectivity or band broad-ening followed by congestion of peaks. A leading strategy toincrease the peak capacity up to date is to use long and narrowopen tubular LC columns [27–31], but this approach may suf-fer from maintenance difficulties such as column cloggingand too long analysis time. The CEC systems with packed ormonolith columns show comparable but more or less inferiorseparation performance and peak capacity to the gradient LCsystems as demonstrated in ref. [44] since the CEC systemsuse isocratic elution despite the merit of uniform eluent flowvelocity distribution across the column diameter.

As shown in Fig. 6, more than 30 peaks were observedin current study (Fig. 6A) while 20 peaks at most wereobserved in other studies. In our previous MIP incorpo-rated OT-CEC study, the peptide peaks were crowded in anarrow retention time range owing to the low �tr/to value(0.53) despite the very good separation efficiency (more than200 000 plates/m) and the observed peak capacity was ratherlimited (Fig. 6B). In the above description, to is the retentiontime of EOF marker and �tr is the retention time span fromthe first peak to the last peak. The �tr/to value in this studyis 4.1. Gradient elution with C18 LC columns may result inagreeable separation performance at the price of somewhatlonger analysis time as shown in Fig. 6C and E. Analysis timemay be reduced when a UHPLC system is employed as shownin Fig. 6F, but the peptide peaks were rather assembled in anarrow retention time window in this case, and such trendwas quite similar to that of Fig. 6B except for the fact that thenumber of peaks of Fig 6F was less than that of Fig. 6B. Notethat Fig. 6B was obtained at 25�C by isocratic elution whileFig. 6F was obtained at 60�C by gradient elution. Organic-monolith-based LC columns have not been so successful inseparation of tryptic digests despite incorporation of gradientelution.

Bovine cytochrome C is composed of 104 amino acidunits including 18 lysine units and 2 arginine units [47]. Upondigestion with trypsin, 21 peptide fragments were observedand characterized for the sequence determination [47]. Thus,21 peaks may be observed for the tryptic digest of bovine cy-tochrome C if digestion is complete, self-digestion of trypsinis prevented, and all the peptides are perfectly resolved inthe chromatography system. The number of peaks may beincreased if digestion of cytochrome C is imperfect (additionalformation of larger fragments) or self-digestion of trypsinoccurs. Thus, observation of the number of peaks less than21 may be due to overlap of peaks in chromatographic separa-tion. The reason for observation of the number of peaks over30 in current study might be owing to self-digestion of trypsin



Figure 6. Comparison of separation performance of cytochrome C tryptic digest among different types of columns: (A) OT-CEC col-umn (0.05 mm × 416 mm) of this study; (B) OT-CEC column (0.05 mm × 416 mm) of ref [34]; (C) Gradient elution of commer-cial LC column (0.3 mm × 150 mm, 3.5 um C18 phase) from ref [41]; (D) Monolith CEC column (0.1 mm × 260 mm) from ref [45];(E) Gradient elution of commercial C18 silica monolith LC column (0.1 mm × 500 mm) from ref [37]; (F) Gradient elution of commercialLC column (0.75 mm × 150 mm, 2 um C18 phase) of ref [42] at 60�C. Reproduced with permission from refs. 34, 37, 41, 42, 45. Elutionconditions: (A) 60:40 v/v ACN/ 25 mM sodium phosphate at pH 6.8; (B) 60:40 v/v ACN/50 mM phosphate buffer at pH 7; (C) Linear gradient2–62% B over 60 min where eluent A is 0.1% formic acid in water and eluent B is 0.1% formic acid in ACN; (D) 60:40 v/v acetonitrile/80 mMTris-phosphate buffer pH 2.5; (E) Gradient 1% increase of B per min where eluent A is 0.1% formic acid in water and eluent B is 0.1% formicacid in acetonitrile; (F) Gradient 1% increase of B (1–55%) per min where eluent A is 0.05% formic acid in water and eluent B is 0.04% formicacid in 80:20 v/v ACN/H2O.

since we found that the number of peaks was decreased withreduction of digestion time. The number of observed peaksafter 6 h digestion was 22–24.

A very interesting observation was reported in ref. [41]where 33 peptides were identified by LC–MS/MS for the chro-matogram shown in Fig. 6C. The apparent number of peaksin Fig. 6C is less than 20. Thus their observation certainlydemonstrated the occurrence of peak overlaps in chromato-graphic analysis of tryptic digest of cytochrome C.

To clearly demonstrate the performance of the OT-CECcolumn of this study, a synthetic peptide mixture was pre-pared and separated, and the resulting electrochromatogramis shown in Fig. 7. The separation efficiency was better than300 000 plates/m (Fig. 7). The peptides 3 and 5 were old ones,and the side peaks might be formed for such a reason. Based

on the above discussion and results in Figs. 6 and 7, thisstudy offers some promising possibility of new types of OT-CEC columns although the performance is inferior to that ofthe state-of-the-art gradient HPLC columns at present.

3.2.5 Factors of the stationary phase of current study

The factors contributing to the promising performance of theOT-CEC columns of this study may be as follows. The firstfactor is adoption of long linear polymer chains bound to theinner surface of the silica capillary instead of 3-D cross-linkedmonolith layer. There are two factors of chromatographicseparation performance concerning stationary phases: reso-lution and separation efficiency [32]. Resolution is generated



Figure 7. Electrochromatograms of a mixture of five peptides(A) and acetone as EOF marker (B) where A1 is the expandedview of A. Some peptides have impurity peaks. Major peptidepeaks are denoted by asterisks and numbered 1–5. 1: THR-TYR-SER, 2: PRO-PHE-GLY-LYS, 3: TYR-ILE-GLY-SER-ARG, 4: PHE-LEU-GLU-GLU-ILE, 5: VAL-GLU-PRO-ILE-PRO-TYR. The peaks denotedby “a” and “b” are the side peaks for peptide 3 and 5, respec-tively. Eluent: 60:40 v/v ACN/ 25 mM sodium phosphate at pH 6.8;Sample injection: 10 kV for 7 s; Applied potential: 30 kV; Current:10–15 �A. The numbers of theoretical plates/m for peaks 1–5 are:321 000; 300 000; 352 000; 431 000; 407 000.

and increased by gradual increase of stationary phase volumein the chromatography system while separation efficiency isdecreased with increased stationary phase volume [32]. Goodseparation efficiency is obtained by fast mass transfer kinet-ics that is secured by maintaining thin and/or flexible (freemotion) stationary phase or by forming sufficient numbersof well-connected mesopores in the rather thick stationaryphase [32]. It seems that the former strategy is better than thelatter for proteomic samples. Thus formation of monolithlayer by cross-linking was avoided in this study. Flexible butuniform thin layer of linear polymer chains can be well pre-pared by RAFT polymerization that was adopted in this study.

The second factor is proper combination of polar and non-polar monomers, that is, assembling of a nonpolar monomerstill capable of some interaction with peptides (styrene), apolar monomer for EOF generation (MAA), and a monomer(N-phenylacrylamide) capable of good interaction with pep-tides yet having good compatibility with other monomers tomake useful three-component copolymer on the inner sur-face of silica capillary. The aromatic group has been knownto be more useful to differentiate structural isomers thanthe conventional C18 ligand [35, 48]. Tryptic digests are com-posed of many peptides, thus adoption of aromatic rings inthe stationary phase may be useful to incorporate improvedselectivity. Incorporation of a polar group is also required toincrease retention of polar peptides since increased retentioninduces improved resolution of peaks. Peptides have amidegroups, thus the amide group is selected as the polar group.The phenyl group is also incorporated in the polar monomerto improve the compatibility of the polymerization reactionmixture.

In addition, catalyst assisted hydroxyl-isocyanate re-action was adopted in the procedure of introduction ofinitiator moieties on the surface to result in anchoringof a maximum number of initiator moiety and subse-quent formation of copolymer chains of similar lengthsthat may swell and spread open easily in the eluent ofhigh acetonitrile content, which enables high mass transferkinetics.

Let us discuss about the factors that govern the perfor-mance of stationary phase in view of parameters of prepara-tion. The possible polymerization temperature was in a verynarrow range (90–100�C). It was impossible to make usefulstationary phase when the temperature was off the range.The necessary initial radicals for RAFT polymerization aregenerated by thermal self initiation of styrene [49]. This pro-cess requires a quite high temperature that is at least over90�C. The polymerization mixture should be flowed throughthe silica capillary to induce effective growth of the polymerchains. The boiling point of the solvent (toluene) is ca. 110�C.The temperature should be maintained to the level some-what below the boiling point to prevent accidental drying outin the capillary. Thus 100�C was chosen as the polymerizationtemperature.

The formulation of the polymerization mixture is alsovery critical. First, the major component of the monomersshould be styrene since only styrene can generate the neces-sary initial radicals at the reaction temperature. Fortunately,this requirement matched well with the desirable propertiesof stationary phase. The stationary phase should be com-posed of mostly nonpolar groups with some appropriatepolar functional groups to show proper retention and res-olution for peptides. Next, MAA was chosen for the EOFmonomer since it is common and easily available. The ad-dition of N-phenylacrylamide was essential although therequired amount in the formulation was small. WithoutN-phenylacrylamide, the resultant stationary phase did notshow enough retention and resolution. On the other hand,the solute retention became too long and the bandwidthwas too broad when the amount of N-phenylacrylamide wasdoubled.

Another factor is compatibility of the monomers in thereaction mixture. The use of N-phenylacrylamide instead ofacrylamide was helpful. When acrylamide was incorporatedin the reaction mixture instead of N-phenylacrylamide, theresultant stationary phase showed not only bad separationperformance but also poor reproducibility in preparation. Wethink that the formulation of this study is close to the opti-mum one although further improvement is required.

The separation efficiency (plate numbers/m) of the OT-CEC column of this study is certainly inferior to that ofthe OT-CEC column of the previous study [34] and thatof the state-of-the-art gradient LC system. Thus, our futurestudy will focus on development of OT-CEC columns havingimproved repeatability and bearing both merits: increasedrange of retention times of this study and high separationefficiency of the previous study. Incorporation of CEC–MSwork will be also pursued.



4 Concluding remarks

A special type of OT-CEC column has been developed forthe separation of various peptides in the tryptic digest ofcytochrome C. 4-TPI was bound to the inner surface of silicacapillary in the presence of DBTDC catalyst, then an ini-tiator moiety was incorporated to the ligand. RAFT in situco-polymerization was taken place onto the bound initiatorthat resulted in the formation of a thin three-componentcopolymer film on the inner surface of the capillary wall.Many peptides of the cytochrome C tryptic digest were sep-arated by this column using 60:40 v/v acetonitrile/25 mMsodium phosphate at pH 6.8 as the mobile phase. This studyoffers open tubular CEC columns with a proper copolymerlayer fabricated by reversible addition-fragmentation trans-fer polymerization as useful separation media for proteomicanalysis.

This research was supported by Basic Science Research Pro-gram through the National Research Foundation of Korea (NRF)funded by the Ministry of Science, ICT & Future Planning(2013R1A2A2A01067201).

The authors have declared no conflict of interest.

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Open tubular capillary column for the separation of cytochrome C tryptic digest in capillary electrochromatography