کد مقاله | کد نشریه | سال انتشار | مقاله انگلیسی | نسخه تمام متن |
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1209433 | 965342 | 2006 | 11 صفحه PDF | دانلود رایگان |
Monolithic columns based on poly-(styrene-divinylbenzene) (PS-DVB) were utilized both for preconcentration (in 10 mm × 0.20 mm I.D. format) and analytical separation (in 60 mm × 0.20 and 0.10 mm I.D. format) of peptides and proteins in column switching micro-scale high-performance liquid chromatography. A special holder for short monolithic preconcentration columns was designed and pressure durability tests approved long-term stability up to 400 bar. An 11–20% decrease in the average peak widths of nine peptides was obtained upon combining a preconcentration column with an analytical column as compared with a setup using an analytical column only.Trapping efficiency, especially for small and hydrophilic peptides, was optimized by using 0.10% heptafluorobutyric acid instead of 0.050% trifluoroacetic acid as solvent additive during sample loading. Using a 10 mm × 0.20 mm I.D. preconcentration column, loadabilities between 0.5 and 1.6 μg were determined by frontal analysis of proteins and bioactive peptides, respectively. A 100-fold concentration followed by direct on-line intact mass determination is demonstrated for diluted (3 μmol L−1) protein solutions. The applicability of the monolithic preconcentration column for multidimensional chromatography was tested by off-line two-dimensional separation, combining strong cation-exchange chromatography and ion-pair reversed-phase chromatography. Peptide identification data from digested protein mixtures demonstrated reproducibilities of 46–75% in triplicate analyses, and confident peptide identifications of low abundant peptides even in the presence of a 650-fold molar excess of high abundant peptides.
Journal: Journal of Chromatography A - Volume 1136, Issue 2, 15 December 2006, Pages 210–220