Packing Capillary Columns for RP-HPLC
This protocol was adapted from “Reversed-Phase High-Performance Liquid Chromatography,” Chapter 5 in Proteins and Proteomics (ed. Simpson). Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USA, 2003.INTRODUCTION
Capillary or microcolumn (<0.5-mm internal diameter [I.D.]) high-performance liquid chromatography (HPLC) is an extremely powerful technique for separating small quantities of proteins and peptides. This protocol describes a procedure for adapting conventional HPLC systems to provide accurate low-flow rates (0.4-4 μl/min) and gradients required to operate slurry-packed capillary columns. A key component of this system is a commercial axial-beam longitudinal flow cell that can be fitted to a number of commercial UV detectors. Procedures are described for the fabrication of 0.32-mm I.D. polyimide-coated fused-silica columns, slurry-packed with reversed-phase chromatographic supports.
