TEMPERATURE AS A VARIABLE IN REVERSED-PHASE HIGH-PERFORMANCE LIQUID-CHROMATOGRAPHIC SEPARATIONS OF PEPTIDE AND PROTEIN SAMPLES .2. SELECTIVITY EFFECTS OBSERVED IN THE SEPARATION OF SEVERAL PEPTIDE AND PROTEIN MIXTURES

Changes in band spacing as a function of temperature and/or gradient s teepness were investigated for four peptide or protein samples. Revers ed-phase HPLC in a gradient mode was used to separate tryptic digests of tissue plasminogen activator and calmodulin. Additionally, a synthe tic peptide mixture and a storage protein sample from wheat were studi ed. Simultaneous changes in gradient steepness and temperature were fo und to provide considerable control over band spacing and sample resol ution. The effects of temperature and gradient steepness on selectivit y in these systems appear to be complementary. Simultaneous optimizati on of both temperature and gradient steepness thus represents a powerf ul and convenient means of controlling band spacing and separation. Be cause of the complexity of these sample chromatograms, computer simula tion proved to be a useful tool in both interpreting these experiments and in optimizing final separations.