High-speed high-performance liquid chromatography of peptides and proteins

Over the last thirty years the name HPLC has been synonymous with high-speed liquid chromatography and during the last ten years we have experienced a dramatic increase in the speed of analysis particularly as far as the separation of biological macromolecules, such as proteins, is concerned. With a solid grounding in the chromatographic theories, column technology has been mainly responsible for the advances in this field. Recent development shows that columns packed with micropellicular or gigaporous stationary phases of the bidisperse or the bimodal type facilitate rapid mass transfer between the mobile and stationary phases and thus can deliver high resolution separations in a very short time. This suggest that HPLC has the potential to be the prime analytical technique for on-line monitoring of biotechnological processes in real time. Further enhancement of the speed of separation comes from the use of elevated temperatures. The role of temperature in HPLC has largely been ignored and most commercial instruments are not equipped with appropriate temperature control. Results presented here strongly suggest, however, that elevated column temperature may find increasing use in the HPLC of large molecules. In such analytical applications temperature programming may also play a major role provided columns with low heat capacity, such as packed fused-silica capillaries, gain wider employment in HPLC.