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Journal of Chromatography A, 1325 (2014) 109– 114

Contents lists available at ScienceDirect

Journal of Chromatography A

jou rn al hom epage: www.elsev ier .com/ locate /chroma

“plug-and-use” approach towards facile fabrication of capillaryolumns for high performance nanoflow liquid chromatography

hiliang Xiao, Lin Wang, Ya Liu, Qiuquan Wang, Bo Zhang ∗

epartment of Chemistry and the MOE Key Laboratory of Spectrochemical Analysis and Instrumentation, College of Chemistry and Chemical Engineering,iamen University, Xiamen 361005, China

r t i c l e i n f o

rticle history:eceived 20 October 2013eceived in revised form8 November 2013ccepted 1 December 2013vailable online 11 December 2013

eywords:olumn technology

a b s t r a c t

Capillary columns used for nanoflow liquid chromatography play an important role in modern pro-teomics. High quality columns are needed to provide high peak capacity and highly reproducibleseparations. This is extremely important when multiple separations were compared in parallel in search-ing for potential biomarkers. Herein, we introduce a “plug-and-use” fritting technology for fabrication ofhigh quality and highly reproducible capillary columns. Due to the identical length, good permeability,and stability of the prefabricated frits adopted, the capillary columns presented excellent performanceconsistency in terms of retention time, peak width as well as peak capacity at a column-to-column level(relative standard deviations, RSDs, at 0.4–0.9%, 2.1–3.6%, and 2.7%, respectively, n = 6) for separations of

hromatographyroteomicsigh throughput screeningapillary column

complex mixtures of protein digest. For capillary columns packed with 5 �m particles, high separationefficiency was demonstrated by the minimum plate height of 11 �m, approaching the theoretical per-formance limit of such material. For separations of protein digests, the columns demonstrated excellentpeak capacities of 110 and 300 for 20 and 360 min gradients, respectively. The simple fabrication, goodperformance as well as consistent quality of such columns provide a reliable tool for high throughput

use o

separations requiring the

. Introduction

In 2010, Nature Biotechnology published guidelines for columnhromatography by Human Proteome Organization’s Proteomicstandards Initiative [1], which highlighted the demand for highuality microcolumns towards the standardization of proteomicnalysis. Capillary column used for nanoflow liquid chromatogra-hy (nanoLC) has become a key separation tool for biomolecules2,3]. Over the past ten years or so, many developments andmprovements for microcolumn technology have been introduced,oth in capillary [4–12], and microchip formats [13–18], for normalressure and recently also ultra high pressure nanoLC separations19–22].

As an indispensible consumptive material in modern pro-eomics, capillary columns need to be quality-controlled to a hightandard. In practice, however, quality control studies were sel-om seen over the microcolumns fabricated and used. A commonractice seen in many proteomics laboratories is that, one packs

single column and uses it for nanoLC–mass spectrometry (MS)xperiment straight away. The column is used until its perfor-ance degradation or column breakage, and then a new capillary is

∗ Corresponding author. Tel.: +86 592 2188691; fax: +86 592 2188691.E-mail address: bozhang@xmu.edu.cn (B. Zhang).

021-9673/$ – see front matter © 2013 Elsevier B.V. All rights reserved.ttp://dx.doi.org/10.1016/j.chroma.2013.12.002

f multiple high performance capillary columns in parallel.© 2013 Elsevier B.V. All rights reserved.

packed and used. Although MS as an information-rich detector cantolerate, to a great extent, the inconsistency of separation perfor-mance between columns, the column-to-column irreproducibilitymay become a crucial issue when large-scale screening proteomicsbased on parallel use of multiple columns [16–18,23–26] is per-formed, especially when optical detection is adopted.

At the turn of the century, the introduction of capillary arrayelectrophoresis using 96 or 384 capillaries greatly pushed for-ward the pace of genomic discovery [27]. From the viewpoint ofproteome-wide screening for biological discovery [28,29], thereneeds innovative developments in separation platforms and relatedconsumable devices. It is to this end that high quality capillarycolumns, with excellent performance consistency at a column-to-column level, are demanded by, but not limited to, high throughputproteomics.

To date, the most routinely used chromatographic medium formicroscale bioseparations is particulate packed capillary columns[2,3], although monolithic [30–35], and open-tubular capillarycolumns [36], have also been introduced. The aim of the presentwork is to develop a packed column technology with high stan-dard reproducibility to support high performance microcolumn

separations of complex mixtures. In resolving this issue, there aretwo aspects one needs to take into account: fritting and packing.Although column packing plays an important role in column perfor-mance [37,38], the main technical challenge is fabrication of high

dx.doi.org/10.1016/j.chroma.2013.12.002http://www.sciencedirect.com/science/journal/00219673http://www.elsevier.com/locate/chromahttp://crossmark.crossref.org/dialog/?doi=10.1016/j.chroma.2013.12.002&domain=pdfmailto:bozhang@xmu.edu.cndx.doi.org/10.1016/j.chroma.2013.12.002

110 Z. Xiao et al. / J. Chromatogr. A 1325 (2014) 109– 114

Fig. 1. A two-step “plug-and-use” fritting approach. Step 1: A large permeable porous (perfusive) silica bead is tapped onto one end of a capillary. Step 2: The captured silicab ca beaT y’s in

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ead was forced into the capillary. In the photos, the dark dots are the perfusive silihe perfusive silica bead has a nominal outer diameter of ∼110 �m and the capillar

uality frits inside capillary tube [39]. In this study, we introduce in “plug-and-use” approach, based on prefabricated frits with goodermeability and predetermined short length, to facilitate columnreparation. We will also interrogate the columns’ quality as wells performance consistency in separations of complex mixtures.

. Experimental

.1. Materials and apparatus

Polyimide-coated fused silica capillaries were purchased fromongnian Reafine Chromatography (Hebei China). The porous sil-

ca particles ∼110 �m in diameter with large throughpores about �m, to be used as prefabricated single particle frits, were providedy X-tec (Bromborough, UK). The packing material Ultimate XB-C185 �m, 300 Å) was obtained from Welch Materials Inc. (Shanghai,hina). Thiourea, NH4HCO3, methyl-, ethyl-, propyl-, and butyl-enzenes of analytical grade, dithiothreitol (DTT), iodoacetamideIAA) trifluoroacetic acid (TFA), trypsin of sequencing grade, stan-ard protein cytochrome C, lysozyme, ovalbumin, bovine serumlbumin, and transferrin were purchased from Sigma–Aldrich (St.ouis, MO). Acetonitrile and acetone of HPLC grade were providedy Merck (Darmstadt, Germany). An Elite P230 high pressure pumprom Dalian Elite Analytical Instruments (Dalian, China) was usedor column packing.

.2. Protein digestion

Complex peptide mixtures were prepared by tryptic diges-ion of standard proteins in solution. Generally, proteins wereolubilized in 8 M urea, 50 mM NH4HCO3. Then, the sample waseduced by DTT and alkylated by IAA. Finally, trypsin was added at

ds intentionally left in to show the relative size of the beads and the capillary tube.ner diameter is 100 �m.

a protein-to-enzyme ratio of 50:1, the digestion was incubated at37 ◦C over night.

2.3. Nanoflow liquid chromatography

NanoLC experiments were carried out on an Ultimate 3000nanoLC system (Thermo-Dionex, Amsterdam, The Netherlands),equipped with an autosampler and a variable wavelength UV–visdetector with a 3 nL flow cell. A 4 nL Valco nanovolume injector(VICI AG, Schenkon, Switzerland) was used for column performanceevaluation under isocratic condition. For large volume injectionsunder gradient elution, the autosampler with a 1 �L loop wasadopted.

2.4. “Plug-and-use” fritting and column packingA 25 cm long, fused silica capillary (100 �m I.D., 365 �m O.D.)

was chosen as the column tubing. As shown in Fig. 1, one end ofthe capillary was tapped into a micro centrifuge tube, in which asmall number of ∼110 �m perfusive silica beads were deposited.A single perfusive silica bead can be captured at the head of thecapillary (Step 1, Fig. 1), the single bead was then pushed into thecapillary by pressing the end of the capillary against a plane sur-face (Step 2, Fig. 1). This two-step process can be monitored andconfirmed by observation under a microscope. This single silicabead served as the outlet frit of the column. The capillary columnwas slurry-packed under high pressure. The packing material wassuspended in acetone at a concentration of 2 mg/mL and ultrason-icated for 15 min. The slurry was loaded into a reservoir (4.6 mmI.D., 15 cm long) attached to a high pressure pump. The one endfritted capillary tube was connected to the reservoir via the openend. Pressure was increased gradually (upto 6000 psi) until the col-

umn was packed. The column was cut to a desired length after thepacking system was fully depressurized. Finally, another single per-fusive silica bead was forced into the cut end serving as the inletfrit of the column. Before use, the column was mounted on to the

Z. Xiao et al. / J. Chromatogr. A 1325 (2014) 109– 114 111

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ig. 2. Single perfusive bead-fritted column based on keystone effect. A, formationnd their end frits using single perfusive beads, C, SEM of a single perfusive silica ben inner diameter of 100 �m.

anoLC system and fully equilibrated with the mobile phase underigh pressure.

For this study, twelve capillary columns, six for 15 cm long andhe other six 20 cm long, both 100 �m I.D., packed with UltimateB-C18, 5 �m, 300 Å, were fabricated and used for performance

nvestigations. The 20 cm long columns were mainly used for Vaneemter curve evaluation in isocratic mode, and the 15 cm longolumns were used for protein digest separations performed inradient elution mode.

. Results and discussion

.1. “Plug-and-use” approach for capillary column fabrication

The “plug-and-use” frit is formed using prefabricated singleerfusive silica beads, as we first introduced for electrochromatog-aphy (CEC) [40,41]. The beads have diameters of ∼110 �m, ashown in Fig. 2. A single bead was forced into one end of the cap-llary and lodged in the place due to keystone effect [6,40–44]. As

e demonstrated before [40,41], this keystone effect was formedetween the single silica bead and the fines and the capillary wallFig. 2A). The keystone effect is like a stone arch bridge in real life,hich can sustain heavy weight on its top but without pier under-eath [6]. In this case (Fig. 2A), the flow pressure applied on theingle bead was largely converted to pressures against capillaryall via keystone effect. At the same time, since the single bead

s perfusive (i.e., rich in throughpores) [40,41], liquid flow can go

hrough the bead itself. In this way, the perfusive silica bead canork effectively as a frit: it sustains high hydraulic pressure, allows

olvent go though and holds particulate material inside the column.reviously, Mann’s group reported formation of keystone effect

ystone effect between the bead, fines, and capillary wall, B, six columns fabricatedd D, SEM of a perfusive bead as an end frit inside a capillary. The capillary tube has

based on particulate packing material at tapered capillary end [6].In our case, the keystone effect was enabled straight after “plug-in”of a single perfusive bead. The capillary was used as native, no endshrinking (as for the frit-less columns) [6,44–46] or chemical reac-tion (as for Kasil [7,9,11,21,22] or polymer monolithic frits [47–50])was needed. In practice, such “plug-and-use” fritting operationtakes almost no labor or time, which greatly facilitates capillarycolumn fabrication. Six capillary columns fabricated through this“plug-and-use” approach are presented in Fig. 2B. The end fritsbased on single silica beads are clearly visualized. They have essen-tially the same topology and more importantly, consistently shortlength of ∼100 �m. This is crucial for separation reproducibility andperformance consistency between columns, as discussed below.

In this “plug-and-use” approach, a capillary column can be fab-ricated within one hour according to our experience, while themost time-consuming part is column packing. This feature signifi-cantly improved column production throughput. Most importantly,it makes column quality control in laboratory realistically feasible:one can fabricate a batch of capillary columns for certain set ofexperiments and all go through a quality control process beforeuse.

3.2. Operational aspects

Our previous work has proved such frit’s feasibility and stabil-ity in electrochromatography [40,41]. Obviously, CEC separation isdriven by electroosmosis rather than hydraulic pressure [51]. In the

present study, the top concern is that whether the frits can sustainhigh hydraulic pressure in pressure-driven liquid chromatogra-phy. Experimental result has shown that the “plug-and-use” frittedcolumns can be run up to 6000 psi (40 MPa) without problem,

112 Z. Xiao et al. / J. Chromatogr. A

Fig. 3. Van Deemter curve evaluation. Capillary column: Ultimate XB-C18, 5 �m,3dd

wtc1

FCfl

00 Å, 200 mm × 100 �m i.d.; mobile phase: 60% ACN; injection volume: 4 nL; UVetection: 214 nm. A retained neutral analyte, butylbenzene, was used as the stan-ard.

hich is the pressure uplimit of the Dionex nanoLC system. In fact,he columns have been exposed to 6000 psi during packing. Theolumns were also subjected to repeated routine use and storage for2 month under both isocratic and gradient elution conditions. All

ig. 4. Separations of tryptic digest of Cytochrome C on six capillary columns. A, columapillary columns: Ultimate XB-C18, 5 �m, 300 Å, 150 mm × 100 �m i.d.; mobile phase Aow rate: 350 nL/min; UV detection: 214 nm.

1325 (2014) 109– 114

the frits were stable during the whole time span, revealing excellentlong-term mechanical strength of the frits (and thus the columns)under pressure-driven chromatography.

The columns were also tested for sudden pressure drop. Sincethe columns were fritted at both ends, no leakage of packingmaterial was observed, the columns showed excellent stabilityagainst pressure pulsations. While many in-house fabricated capil-lary columns used for proteomics analysis [6–11] were only fritted(or tapered) at the outlet end, and the inlet end was left open.These one-end-fritted columns may be subject to the risk of pack-ing material loss when experiencing sudden pressure fluctuationduring running.

3.3. Column performance and consistency

To interrogate the quality of the columns fabricated, we useda mixture of neutral analytes (alkylbenzenes) to evaluate thecolumns’ separation efficiency. With a retained analyte (butyl-benzene) as the probe, Van Deemter curve was drawn in linearvelocity range 0.3–2.0 mm/s (corresponding to volumetric flowrate 100–650 nL/min), as shown in Fig. 3. The optimum plateheight (10.9 �m, equivalent to 92,000 plates/m, observed at lin-

ear velocity 1.1 mm/s) is approaching 10 �m, the theoretical limitof 5 �m packing material [52]. This is an important sign of thegood quality of the column. It is well documented that [39,53], thefrit length, as an additional section to the capillary column bed,

n-to-column reproducibility. B, 35 repeated injections performed on one column., H2O + 0.05% TFA, B, ACN + 0.05% TFA, 5–50% B in 20 min; injection volume: 0.2 �L;

Z. Xiao et al. / J. Chromatogr. A 1325 (2014) 109– 114 113

Fig. 5. Long gradient separation of tryptic digest of five proteins. Capillary column: Ultimate XB-C18, 5 �m, 300 Å, 150 mm × 100 �m i.d.; mobile phase A, H2O + 0.05% TFA, B,A , bovinU

cup1(ibewd

tohcmtmgaaactotmtcf

tMfiecMackp

CN + 0.05% TFA, 5–50% B in 360 min; proteins: cytochrome C, lysozyme, ovalbuminV detection: 214 nm.

ontributes significantly to the column bed’s overall non-niformity, and leads to decreased separation efficiency. Com-ared with other frits reported (commonly at lengths around000–5000 �m) [11,21,22,47–50], the consistently short frit length∼100 �m) in this “plug-and-use” strategy significantly dimin-shed frit effect and improved overall uniformity of the packeded. With the same neutral analytes in isocratic elution mode,xcellent column-to-column reproducibility of retention timesas observed (RSD < 1%, Fig. S1 and Table S1 in Supplementaryata).

The focus of our investigation is column performance consis-ency in gradient separation of complex mixtures. A tryptic digestf cytochrome C was used for this evaluation. As shown in Fig. 4A,ighly consistent chromatograms were recorded on all the sixolumns. To represent peaks of different eluting time and size, fivearker peaks (Fig. 4A) were picked up and traced for retention

ime and peak width at half height (Tables S2 and S3 in Supple-entary data). Between all the six columns, the five marker peaks

ave excellent reproducibility of RSD = 0.4–0.9% for retention times,nd RSD = 2.1–3.6% for peak widths at half height: both markedn extremely stable column-to-column consistency. In literature,n RSD

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14 Z. Xiao et al. / J. Chroma

ppendix A. Supplementary data

Supplementary material related to this article can beound, in the online version, at http://dx.doi.org/10.1016/.chroma.2013.12.002.

eferences

[1] A.R. Jones, K. Carroll, D. Knight, K. MacLellan, P.J. Domann, C. Legido-Quigley,L.H. Huang, L. Smallshaw, H. Mirzaei, J. Shofstahl, N.W. Paton, Nat. Biotechnol.28 (2010) 654.

[2] F. Xie, R.D. Smith, Y.F. Shen, J. Chromatogr. A 1261 (2012) 78.[3] S. Di Palma, M.L. Hennrich, A.J.R. Heck, S.J. Mohammed, Proteomics 75 (2012)

3791.[4] M.P. Washburn, D. Wolters J.R.III, Yates, Nat. Biotechnol. 19 (2001) 242.[5] A.W. Guzzetta, A.S. Chien, J. Proteome Res. 4 (2005) 2412.[6] Y. Ishihama, J. Rappsilber, J.S. Andersen, M. Mann, J. Chromatogr. A 979 (2002)

233.[7] A. Maiolica, D. Borsotti, J. Rappsilber, Proteomics 5 (2005) 3847.[8] L.C. Howells, A.O. Whelan, M.J. Woodward, N.G. Coldham, Proteomics 5 (2005)

3864.[9] L.C. Wang, C.Y. Okitsu, H. Kochounian, A. Rodriguez, C.L. Hsieh, E. Zandi, Pro-

teomics 8 (2008) 1758.10] F. Tan, S. Chen, Y.J. Zhang, Y. Cai, X.H. Qian, Proteomics 10 (2010) 1724.11] T. Masuda, N. Sugiyama, M. Tomita, Y. Ishihama, Anal. Chem. 83 (2011) 7698.12] S.D. Palma, S. Mohammed, A.J.R. Heck, Nat. Protoc. 7 (2012) 2041.13] H. Yin, K. Killeen, R. Brennen, D. Sobek, M. Werlich, T. van de Goor, Anal. Chem.

77 (2005) 527.14] A. Gaspar, M.E. Piyasena, F.A. Gomez, Anal. Chem. 79 (2007) 7906.15] W.D. Malsche, J.O.D. Beeck, S.D. Bruyne, H. Gardeniers, G. Desmet, Anal. Chem.

84 (2012) 1214.16] J.K. Liu, C.F. Chen, S. Yang, C.C. Chang, D.L. DeVoe, Lab. Chip 10 (2010) 2122.17] P. Mao, R.G. Sjoberg, D.J. Wang, Anal. Chem. 85 (2013) 816.18] J. Huft, C.A. Haynes, C.L. Hansen, Anal. Chem. 85 (2013) 1797.19] A. Motoyama, J.D. Venable, C.I. Ruse J.R.III, Yates, Anal. Chem. 78 (2006) 5109.20] J.W. Jorgenson, Annu. Rev. Anal. Chem. 3 (2010) 129.21] C.J. Chen, W.Y. Chen, M.C. Tseng, Y.R. Chen, Anal. Chem. 84 (2012) 297.22] F. Zhou, Y. Lu, S.B. Ficarro, J.T. Webber, J.A. Marto, Anal. Chem. 84 (2012) 5133.

23] A.V. Lemmo, S. Hobbs, P. Patel, Assay Drug Dev. Technol. 2 (2004) 389.24] X. Gu, C.H. Deng, G.Q. Yan, X.M. Zhang, J. Proteome Res. 5 (2006) 3186.25] E.A. Livesay, K.Q. Tang, B.K. Taylor, M.A. Buschbach, D.F. Hopkins, B.L. LaMarche,

R. Zhao, Y.F. Shen, D.J. Orton, R.J. Moore, R.T. Kelly, H.R. Udseth, R.D. Smith, Anal.Chem. 80 (2008) 294.

[[[[

1325 (2014) 109– 114

26] H. Wang, S.M. Hanash, J. Proteome Res. 7 (2008) 2743.27] N.J. Dovichi, J.Z. Zhang, Angew. Chem. Int. Ed. 39 (2000) 4463.28] R.F. Service, Science 294 (2001) 2074.29] S. Hanash, Nature 422 (2003) 226.30] A. Premstaller, H. Oberacher, W. Walcher, A.M. Timperio, L. Zolla, J. Chervet, N.

Cavusoglu, A. von Dorsselaer, C.G. Huber, Anal. Chem. 73 (2001) 2390.31] A.R. Ivanov, L. Zang, B.L. Karger, Anal. Chem. 75 (2003) 5306.32] Q. Luo, Y. Shen, K.K. Hixson, R. Zhao, F. Yang, R.J. Moore, H.M. Mottaz, R.D. Smith,

Anal. Chem. 77 (2005) 5028.33] K. Miyamoto, T. Hara, H. Kobayashi, H. Morisaka, D. Tokuda, K. Horie, K. Koduki,

S. Makino, O. Nunez, C. Yang, T. Kawabe, T. Ikegami, H. Takubo, Y. Ishihama, N.Tanaka, Anal. Chem. 80 (2008) 8741.

34] M. Iwasaki, S. Miwa, T. Ikegami, M. Tomita, N. Tanaka, Y. Ishihama, Anal. Chem.82 (2010) 2616.

35] H. Lin, J.J. Ou, Z.B. Zhang, J. Dong, M.H. Wu, H.F. Zou, Anal. Chem. 84 (2012)2721.

36] G. Yue, Q. Luo, J. Zhang, S.L. Wu, B.L. Karger, Anal. Chem. 79 (2007) 938.37] T.D. Maloney, L.A. Colon, J. Sep. Sci. 25 (2002) 1215.38] F.M. Lancas, J.C. Rodrigues, S.S. Freitas, J. Sep. Sci. 27 (2004) 1475.39] S.M. Piraino, J.G. Dorsey, Anal. Chem. 75 (2003) 4292.40] B. Zhang, E.T. Bergstrom, D.M. Goodall, P. Myers, Anal. Chem. 79 (2007) 9229.41] B. Zhang, Q. Liu, L.J. Yang, Q.Q. Wang, J. Chromatogr. A 1272 (2013) 136.42] G.A. Lord, D.B. Gordon, P. Myers, B.W. King, J. Chromatogr. A 768 (1997) 9.43] G. Choudhary, Cs. Horvath, J.F. Banks, J. Chromatogr. A 828 (1998) 469.44] M. Mayer, E. Rapp, C. Marck, G.J.M. Bruin, Electrophoresis 20 (1999) 43.45] E. Rapp, E. Bayer, J. Chromatogr. A 887 (2000) 367.46] J. Zheng, D. Norton, S.A. Shamsi, Anal. Chem. 78 (2006) 1323.47] J.R. Chen, M.T. Dulay, R.N. Zare, Anal. Chem. 72 (2000) 1224.48] M. Kato, M.T. Dulay, B.D. Bennett, J.P. Quirino, R.N. Zare, J. Chromatogr. A 924

(2001) 187.49] C. Legido-Quigley, N.W. Smith, J. Chromatogr. A 1042 (2004) 61.50] G. D’Orazio, S. Fanali, J. Chromatogr. A 1232 (2012) 176.51] K.D. Bartle, P. Myers, Capillary electrochromatography, RSC Cambridge (2001).52] L.R. Snyder, J.J. Kirkland, J.W. Dolan, Introduction to Modern Liquid Chromatog-

raphy, 3rd ed., Wiley, Hoboken, 2010, pp. p35.53] F. Lelievre, C. Yan, R.N. Zare, P. Gareil, J. Chromatogr. A 723 (1996) 145.54] R. Aebersold, M. Mann, Nature 422 (2003) 198.55] T.E. Angel, U.K. Aryal, S.M. Hengel, E.S. Baker, R.T. Kelly, E.W. Robinson, R.D.

Smith, Chem. Soc. Rev. 41 (2012) 3912.56] J.R. Yates, J. Am. Chem. Soc. 135 (2013) 1629.

57] Y.F. Shen, J.S. Page, R.D. Smith, Adv. Chromatogr. 47 (2009) 31.58] T. Köcher, R. Swart, K. Mechtler, Anal. Chem. 83 (2011) 2699.59] T. Köcher, P. Pichler, R. Swart, K. Mechtler, Nat. Protoc. 7 (2012) 882.60] J.C. Giddings, Unified Separation Science, Wiley, New York, 1991, pp. 26, Chap-

ter 5.

http://dx.doi.org/10.1016/j.chroma.2013.12.002http://dx.doi.org/10.1016/j.chroma.2013.12.002http://refhub.elsevier.com/S0021-9673(13)01866-9/sbref0005http://refhub.elsevier.com/S0021-9673(13)01866-9/sbref0005http://refhub.elsevier.com/S0021-9673(13)01866-9/sbref0005http://refhub.elsevier.com/S0021-9673(13)01866-9/sbref0005http://refhub.elsevier.com/S0021-9673(13)01866-9/sbref0005http://refhub.elsevier.com/S0021-9673(13)01866-9/sbref0005http://refhub.elsevier.com/S0021-9673(13)01866-9/sbref0005http://refhub.elsevier.com/S0021-9673(13)01866-9/sbref0005http://refhub.elsevier.com/S0021-9673(13)01866-9/sbref0005http://refhub.elsevier.com/S0021-9673(13)01866-9/sbref0005http://refhub.elsevier.com/S0021-9673(13)01866-9/sbref0005http://refhub.elsevier.com/S0021-9673(13)01866-9/sbref0005http://refhub.elsevier.com/S0021-9673(13)01866-9/sbref0005http://refhub.elsevier.com/S0021-9673(13)01866-9/sbref0005http://refhub.elsevier.com/S0021-9673(13)01866-9/sbref0005http://refhub.elsevier.com/S0021-9673(13)01866-9/sbref0005http://refhub.elsevier.com/S0021-9673(13)01866-9/sbref0005http://refhub.elsevier.com/S0021-9673(13)01866-9/sbref0005http://refhub.elsevier.com/S0021-9673(13)01866-9/sbref0005http://refhub.elsevier.com/S0021-9673(13)01866-9/sbref0005http://refhub.elsevier.com/S0021-9673(13)01866-9/sbref0005http://refhub.elsevier.com/S0021-9673(13)01866-9/sbref0005http://refhub.elsevier.com/S0021-9673(13)01866-9/sbref0005http://refhub.elsevier.com/S0021-9673(13)01866-9/sbref0005http://refhub.elsevier.com/S0021-9673(13)01866-9/sbref0005http://refhub.elsevier.com/S0021-9673(13)01866-9/sbref0005http://refhub.elsevier.com/S0021-9673(13)01866-9/sbref0005http://refhub.elsevier.com/S0021-9673(13)01866-9/sbref0010http://refhub.elsevier.com/S0021-9673(13)01866-9/sbref0010http://refhub.elsevier.com/S0021-9673(13)01866-9/sbref0010http://refhub.elsevier.com/S0021-9673(13)01866-9/sbref0010http://refhub.elsevier.com/S0021-9673(13)01866-9/sbref0010http://refhub.elsevier.com/S0021-9673(13)01866-9/sbref0010http://refhub.elsevier.com/S0021-9673(13)01866-9/sbref0010http://refhub.elsevier.com/S0021-9673(13)01866-9/sbref0010http://refhub.elsevier.com/S0021-9673(13)01866-9/sbref0010http://refhub.elsevier.com/S0021-9673(13)01866-9/sbref0010http://refhub.elsevier.com/S0021-9673(13)01866-9/sbref0010http://refhub.elsevier.com/S0021-9673(13)01866-9/sbref0010http://refhub.elsevier.com/S0021-9673(13)01866-9/sbref0015http://refhub.elsevier.com/S0021-9673(13)01866-9/sbref0015http://refhub.elsevier.com/S0021-9673(13)01866-9/sbref0015http://refhub.elsevier.com/S0021-9673(13)01866-9/sbref0015http://refhub.elsevier.com/S0021-9673(13)01866-9/sbref0015http://refhub.elsevier.com/S0021-9673(13)01866-9/sbref0015http://refhub.elsevier.com/S0021-9673(13)01866-9/sbref0015http://refhub.elsevier.com/S0021-9673(13)01866-9/sbref0015http://refhub.elsevier.com/S0021-9673(13)01866-9/sbref0015http://refhub.elsevier.com/S0021-9673(13)01866-9/sbref0015http://refhub.elsevier.com/S0021-9673(13)01866-9/sbref0015http://refhub.elsevier.com/S0021-9673(13)01866-9/sbref0015http://refhub.elsevier.com/S0021-9673(13)01866-9/sbref0015http://refhub.elsevier.com/S0021-9673(13)01866-9/sbref0020http://refhub.elsevier.com/S0021-9673(13)01866-9/sbref0020http://refhub.elsevier.com/S0021-9673(13)01866-9/sbref0020http://refhub.elsevier.com/S0021-9673(13)01866-9/sbref0020http://refhub.elsevier.com/S0021-9673(13)01866-9/sbref0020http://refhub.elsevier.com/S0021-9673(13)01866-9/sbref0020http://refhub.elsevier.com/S0021-9673(13)01866-9/sbref0020http://refhub.elsevier.com/S0021-9673(13)01866-9/sbref0020http://refhub.elsevier.com/S0021-9673(13)01866-9/sbref0020http://refhub.elsevier.com/S0021-9673(13)01866-9/sbref0020http://refhub.elsevier.com/S0021-9673(13)01866-9/sbref0020http://refhub.elsevier.com/S0021-9673(13)01866-9/sbref0025http://refhub.elsevier.com/S0021-9673(13)01866-9/sbref0025http://refhub.elsevier.com/S0021-9673(13)01866-9/sbref0025http://refhub.elsevier.com/S0021-9673(13)01866-9/sbref0025http://refhub.elsevier.com/S0021-9673(13)01866-9/sbref0025http://refhub.elsevier.com/S0021-9673(13)01866-9/sbref0025http://refhub.elsevier.com/S0021-9673(13)01866-9/sbref0025http://refhub.elsevier.com/S0021-9673(13)01866-9/sbref0025http://refhub.elsevier.com/S0021-9673(13)01866-9/sbref0025http://refhub.elsevier.com/S0021-9673(13)01866-9/sbref0025http://refhub.elsevier.com/S0021-9673(13)01866-9/sbref0030http://refhub.elsevier.com/S0021-9673(13)01866-9/sbref0030http://refhub.elsevier.com/S0021-9673(13)01866-9/sbref0030http://refhub.elsevier.com/S0021-9673(13)01866-9/sbref0030http://refhub.elsevier.com/S0021-9673(13)01866-9/sbref0030http://refhub.elsevier.com/S0021-9673(13)01866-9/sbref0030http://refhub.elsevier.com/S0021-9673(13)01866-9/sbref0030http://refhub.elsevier.com/S0021-9673(13)01866-9/sbref0030http://refhub.elsevier.com/S0021-9673(13)01866-9/sbref0030http://refhub.elsevier.com/S0021-9673(13)01866-9/sbref0030http://refhub.elsevier.com/S0021-9673(13)01866-9/sbref0030http://refhub.elsevier.com/S0021-967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A “plug-and-use” approach towards facile fabrication of capillary columns for high performance nanoflow liquid chromatography1 Introduction2 Experimental2.1 Materials and apparatus2.2 Protein digestion2.3 Nanoflow liquid chromatography

3 Results and discussion3.1 “Plug-and-use” approach for capillary column fabrication3.2 Operational aspects3.3 Column performance and consistency

4 ConclusionsAcknowledgementsAppendix A Supplementary dataAppendix A Supplementary data