Two-dimensional capillary liquid chromatography: pH Gradient ion exchange and reversed phase chromatography for rapid separation of proteins

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• 作者：Milaim Pepaj ; Steven R. Wilson ; Katerina Novotna ; Elsa Lundanes and Tyge Greibrokk
• 关键词：pH Gradient ion exchange chromatography ; pI ; Proteins ; Two-dimensional capillary liquid chromatography
• 刊名：Journal of Chromatography A
• 出版年：2006
• 出版时间：7 July 2006
• 年：2006
• 卷：1120
• 期：1-2
• 页码：132-141
• 全文大小：267 K

文摘

In the present work, an orthogonal two-dimensional (2D) capillary liquid chromatography (LC) method for fractionation and separation of proteins using wide range pH gradient ion exchange chromatography (IEC) in the first dimension and reversed phase (RP) in the second dimension, is demonstrated. In the first dimension a strong anion exchange (SAX) column subjected to a wide range (10.5-3.5) descending pH gradient was employed, while in the second dimension, a large pore (4000 Å) polystyrene-divinylbenzene (PS-DVB) RP analytical column was used for separation of the protein pH-fractions from the first dimension. The separation power of the off-line 2D method was demonstrated by fractionation and separation of human plasma proteins. Seventeen pH-fractions were manually collected and immediately separated in the second dimension using a column switching capillary RP-LC system. Totally, more than 200 protein peaks were observed in the RP chromatograms of the pH-fractions. On-line 2D analysis was performed for fractionation and separation of ten standard proteins. Two pH-fractions (basic and acidic) from the first dimension were trapped on PS-DVB RP trap columns prior to back-flushed elution onto the analytical RP column for fast separation of the proteins with UV/MS detection.