High performance liquid chromatography (HPLC) is currently one of the most powerful analytical tools that has revolutionized the field of proteomics. Formerly known as high pressure liquid chromatography, this technique was introduced in the early 1960s to improve the efficiency of liquid chromatography separations using small stationary phase particles packed in columns. Since its introduction, continued advancements in column technology, development of different stationary phase materials and improved instrumentation has allowed the full potential of this technique to be realized. The various modes of HPLC in combination with mass spectrometry has evolved into the principal analytical technique in proteomics. It is now common practice to combine different types of HPLC in a multidimensional workflow to identify and quantify peptides and proteins with high sensitivity and resolution from limited amounts of samples. More recently, the introduction of Ultra High Performance Liquid Chromatography (UHPLC) has further raised the level of performance of this technique with significant increases in resolution, speed and sensitivity. The number of applications of HPLC and UHPLC in proteomics has been rapidly expanding and will continue to be a pivotal analytical technique. The aim of the following sections is to familiarize the beginner with the various HPLC methods routinely used in proteomics and provide sufficient practical knowledge regarding each of them to develop a separation and analytical protocol.
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