PEPTIDES | Liquid Chromatography

Abstract

High-pressure (or performance) liquid chromatography (HPLC) in its different modes of separation, reversed-phase (RP), ion exchange (IEX), size exclusion (SEC), hydrophobic interaction (HIC), and affinity (AC) chromatography, is the technique of choice for the separation of peptides. HPLC is an instrumental separation technique that is well suited for the separation of small as well as large peptides. The advantage of HPLC for peptide separation is that it affords the analyst the freedom to resolve simple peptide mixtures by one diemensional or complex mixtures, cell lysates, by multidimensional approaches. Also, the advantages of HPLC separations are automation, sensitivity, reproducibility, and systems including UV/Vis, conductivity, light scattering, mass spectrometry (MS), fluorescence and laser-induced fluorescence (LIF). This chapter presents a comprehensive discussion of different experimental parameters that affect peptide resolution, selectivity and detection, in addition to different parameters such as column selection, effect of column dimensions, packing materials properties, effect of column packing's chemistry, monolithic columns, cyclodextrin columns, effect of column temperature, mobile phase properties and elution, and multidimensional approaches that have been used for the separation of complex peptide mixtures.