High Efficiency Robust Open Tubular Capillary Electrochromatography Column for the Separation of Peptides Faiz Ali † and Won Jo Cheong * Department of Chemistry, Inha University, Incheon 402-751, South Korea. *E-mail: wjcheong@inha.ac.kr Received May 3, 2016, Accepted June 2, 2016, Published online July 22, 2016 Keywords: Open tubular capillary column, High separation efficiency, Peptides, Tryptic digest In-depth proteomic profiling is important for exploration of disease mechanisms and biomarker discovery for clinical diagnostics. 1–5 Acquisition of the full knowledge on pro- teins, their expression, interactions, functions, and modifica- tions requires a sophisticated and vast proteomic analysis. 1–5 Separation of intact proteins or peptides by more advanced LC enables better feasibility of LC/MS analysis resulting in better detailed information of proteomic sam- ples. 5,6 The analytical power of LC/MS could be improved by development of the LC stationary phases of better selec- tivity, resolution, and separation efficiency. 7 Useful prevail- ing chromatographic columns for separation of peptides are monolith columns, ultra-high performance liquid chroma- tography (UPLC) columns with sub-3 μm porous particles or fused core particles along with various CEC columns including open tubular capillary electrochromatography (OT-CEC) columns. 8 CEC columns as well as LC columns may be used in multidimensional separation. 9 Long (3–5 m) open tubular LC columns of narrow id with thin polymer layers on the inner surface were reported to achieve enhanced chromatographic performance in view of separation efficiency and peak capacity for separation of pep- tides and proteins. 10–14 The typical inner diameter of those columns was 10 μm and a heavy care should be required to prevent column clogging in those studies. Long analysis time was also required. On the other side, inferior chromatographic performance was obtained when LC columns (open tubular) with wider id were used owing to band broadening. OT-CEC is a good strategy to achieve narrow peak band- widths with columns of wider id owing to the flat velocity profile along the capillary cross-section. 15 An OT-CEC col- umn immobilized with a carefully designed 3-component copolymer layer has been prepared in current study for nice separation of different peptides in digested cytochrome C with enhanced separation efficiency (N = 500 000 plates/col- umn). Copolymer chains were chemically attached onto the capillary inner surface via reversible addition-fragmentation chain transfer (RAFT) polymerization. Readers are recom- mended to view the literature and the references quoted there 16,17 for detailed information of RAFT polymerization. The monomer mixture of this study was modified to a dif- ferent formulation from those of Refs.8 and 18 to fit the purpose of this study. Some modifications have been also made in preparation steps of OT-CEC columns. In this study, the carefully designed tri-component copol- ymer layer was fabricated onto the inner surface of a pre- treated silica capillary (52 cm effective length, 50 μm id). The initiator moieties were incorporated onto the capillary inner surface by reaction with 4-chloromehtylphenyl iso- cyanate followed by sodium diethyl dithiocarbamate. Next, RAFT copolymerization was held upon the initiator moi- eties and a thin polymer film was made. The CEC analysis of a tryptic digest of cytochrome C by the copolymer immobilized OT-CEC column resulted in nice separation of about 20 peptides with high separation efficiency close to 500 000 plates/column (950 000/m) and acceptable peak capacity (over 220). Even better separation efficiency (over 1 300 000/m) was observed for a synthetic mixture of five standard peptides. The repeatability of column preparation was good since a good reproducibility (better than 3%) was observed in both retention time and number of plates for three batches of columns. The SEM photographs of the cross-section of our CEC column are given in Figure 1. As shown in Figure 1, the stationary phase seems to be thin and compact with irregu- lar rises and sinks. About 20 peptide peaks were separated from the tryptic digest sample of cytochrome C, as shown in Figure 2(a). The average separation efficiency of the pep- tide peaks was found to be close to 500 000 plates/column (950 000 plates/m). A synthetic mixture of five standard peptides was also well separated with even higher separa- tion efficiency as shown in Figure 2(b). The average sepa- ration efficiency was 1 343 000 plates/m (Table 1). A good column-to-column repeatability was also obtained, as sum- marized in Table 1. Three different columns were made and examined for repeatability. The above excellent separation performance (ca. 1 343 000 plates/m) for a peptide mixture has never been obtained in the isocratic elution mode either in HPLC or in CEC. Such excellent performance of current CEC columns should be partially attributed to the following factors as †Present Address: Assistant Professor, University of Malakand, KPK, Pakistan. Note DOI: 10.1002/bkcs.10859 F. Ali and W. J. Cheong BULLETIN OF THE KOREAN CHEMICAL SOCIETY Bull. Korean Chem. Soc. 2016, Vol. 37, 1374–1377 © 2016 Korean Chemical Society, Seoul & Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim Wiley Online Library 1374