ABSTRACT
This chapter examines the effect of varying gradient-rates and sample load, at a standard analytical flow-rate of 1 ml/min, on the yield of purified peptide from a peptide mixture. It describes procedures for rapid optimization of conditions for preparative reversed-phase chromatography (RPC) of peptides on analytical columns. The most common analytical method used in RPC of peptides involves linear gradient elution, where eluent A is 0.1% aq. trifluoroacetic acid (TFA) and eluent B is 0.1% TFA in acetonitrile. However, the required amount of purified peptide is generally in the range of 1 to 100 mg for most researchers, and the analytical conditions will not suffice for the great majority of such preparative applications. Preparative gradient elution chromatography is a compromise between run time, gradient-rate and sample load. If scale-up is required, the researcher can increase column length and/or increase column diameter.
