ABSTRACT
The flexibility of reversed-phase chromatography (RPC) makes it the obvious choice for an initial high-performance liquid chromatography (HPLC) run to gauge the complexity of a peptide mixture. This chapter examines the effect of flow- and gradient-rate on the reversed-phase elution profile of a mixture of closely-related peptide analogs. The effect on peptide retention times, peak height, peak width, and overall peptide resolution is clearly demonstrated. In addition, the effect of varying the detection wavelength on the sensitivity of peak detection is illustrated. Detection of peptides during HPLC is based on peptide bond absorbance at low UV wavelengths, usually over the 205 to 215 nm wavelength range. The RPC elution profiles demonstrate the effect of varying detection wavelength on the sensitivity of detection of the decapeptide standards. The sensitivity of peak detection decreases dramatically as the detection wavelength increases from 200 nm to 230 nm. On each 10 nm increase in detection wavelength, there is an approximate fourfold decrease in sensitivity.
