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Biology LibreTexts

9.6: High Performance Liquid Chromatography (HPLC)

HPLC (also sometimes called High Pressure Liquid Chromatography) is a powerful tool for separating smaller molecules based on their differential polarities. It employs columns with supports made of very tiny beads that are so tightly packed that .ow of solvents/buffers through the columns requires the application of high pressures (hence the name). The supports used can be polar (normal phase separation) or non-polar (reverse phase separation). In normal phase separations, non-polar molecules elute first followed by the more polar compounds. This order is switched in reverse phase chromatography. Of the two, reverse phase is much more commonly employed to due more reproducible chromatographic profiles (separations) that it typically produces.