Introduction 1 4 5 6 7 8 9 10 11 12 13 10 14 16 13 10 17 10 17 18 http://www.matrixscience.com 19 In this paper the evaluation of capillary monolithic silica columns of different lengths for the LC-UV-MS analysis of a bovine serum albumin (BSA) tryptic digest is described. Columns of 150- and 750-mm length were investigated using gradient times varying from 3 to 75 min. Chromatographic peak capacities, based on UV detection, and protein identification, based on Mascot scoring data of the MS detection, were determined as a measure for the efficiency of peptide separation. Theoretical aspects R s 20 1 1 \documentclass[12pt]{minimal}
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\begin{document}$$ \frac{{\Delta \phi t_{0} }} {{t_{g} }}, $$\end{document} ϕ ϕ t 0 t 0 t g Experimental Materials and reagents 4 3 4 3 4 3 Apparatus and LC columns All analyses were performed with an Agilent 1100 nanoLC system (Agilent Technologies, Waldbronn, Germany), consisting of a vacuum degasser, a binary Nano-Pump, a μ-well plate sampler and a column switching module with a trapping column in the 1–4 position of the six-port column-switching valve. The trapping pump was a Gynkotek model 480 (Gynkotek, Germering, Germany). Detection was performed by UV and MS detection, with the detectors connected in series. The UV detector was an MU 701 UV–VIS detector (ATAS GL International, Veldhoven, the Netherlands), equipped with an external optical-fibre flow cell (6 nl, 3-mm light path); peptides were detected at 215 nm. The mass spectrometer was an Agilent LC/MSD Trap XCT (Agilent Technologies, Waldbronn, Germany) ion-trap mass spectrometer, equipped with an orthogonal electrospray ionisation (ESI) interface. The external flow cell of the UV detector allows minimal time delay and band-broadening between UV and MS detection. The monolithic columns were provided by GL Sciences (Tokyo, Japan). The columns were a 150 mm × 0.1 mm MonoCap for nano-flow C18-silica monolithic column and a 750 mm × 0.2 mm MonoCap high resolution C18-silica monolithic column. For trapping of the digest a 5 mm × 0.3 mm column packed with 5 μm Zorbax 300 SB-C18 (Agilent Technologies, Waldbronn, Germany) was used. Method and data analysis m z m z 19 Results and discussion Liquid chromatography–UV analysis Because of the difference in diameter, the 150 mm × 0.1 mm and the 750 mm × 0.2 mm columns were used with different flow rates. For the 150- and the 750-mm columns, the flow rates were 0.5 and 2.0 μl/min, respectively, resulting in a linear flow rate of 1.06 mm/s. Injection volumes were also proportional to the square of the column diameter, 0.25 μl of the digest for the 0.1-mm column and 1.0 μl onto the 0.2-mm column. During the gradient, the maximum back-pressure of the 750-mm column was below 20 Mpa, which is well below the manufacturers limit of 30 Mpa. 1 1 1 1 21 22 10 t g 10 Fig. 1 a c b d a b c d  Table 1 Chromatographic parameters from liquid chromatography–UV analysis   150 mm × 0.1 mm 750 mm × 0.2 mm t g w av t PC** w av t PC 3 0.24 3.05 12.6 0.28 3.16 11.2 15 0.44 11.0 25.0 0.39 11.6 29.7 75 1.27 37.6 29.6 0.77 31.7 41.0 t g w av t PC t w av 11 12 Liquid chromatography–mass spectrometry analysis 2 Table 2 Mascot® database search results   150 mm × 0.1 mm 750 mm × 0.2 mm t g Mascot score a b Mascot score a b c 317 9 17 376 10 18 3 min 641 14 28 787 13 25 15 min 750 16 29 1,376 24 38 75 min 693 16 29 993 19 29 a b c 2 2 Fig. 2 a b a b  3 Fig. 3 m z 2+ a b  Conclusions The use of long silica-based capillary monolithic columns provides a clear advantage over use of shorter columns, i.e. an increase of chromatographic efficiency and reliability of protein identification. As expected from chromatography theory, a factor 5 longer column gives a 1.6–2.4 times increase in peak capacity for separations with similar gradient slope. The use of longer gradients also leads to an initial improvement of the protein identification score, but the score seems to have a maximum at longer gradient times. While the use of longer columns for the separation of peptides has a clear advantage because of the gain in chromatographic efficiency, this also gives a longer analysis time. As maximum protein identification scores for rather simple digests are reached at relatively short gradient times, it is important to find a compromise between chromatographic efficiency and analysis time. However, if the sample is more complex, the use of longer columns is more attractive as longer gradients are necessary to achieve sufficient separation. In the near future, short and long columns of the same diameter (0.1-mm inner diameter) will be compared. Further improvement of the separation might be obtained by optimisation of the combination of the trap column and the analysis column. Moreover, the potential of longer monolithic capillary columns will be demonstrated by the analysis of more complex and real samples.