Molecular Weight Estimation for Neuropeptides Using Size-Exclusion High Performance Liquid Chromatography

Abstract

Size-exclusion high performance liquid chromatography (HPLC) has enabled purification of peptides and proteins to be carried out 10–100 times faster than conventional gel filtration chromatography on soft gels. The prepacked columns (about 1 × 30 cm) contain small particles (13 µm or less), resulting in tens of thousands of theoretical plates per meter. They can be operated at flow rates of about 1 mL/min, giving run trmes of <20 min. The improved peak sharpness and speed have led to a resurgence of interest in the technique. As well as being a standard chromatographic mode for the purification of peptides and proteins, size-exclusion chromatography can be used for estimation of molecular weights. Polyacrylamide gel electrophoresis in sodium dodecyl sulfate-containing buffers is widely used for determining the molecular weight of protein subunits, but is operating near the limits of resolution of the technique in the molecular weight range below a few thousand Daltons (see Chapter 10).