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High-performance liquid chromatography (or High pressure liquid chromatography, HPLC) is a form of column chromatography used frequently in biochemistry and analytical chemistry to separate, identify, and quantify compounds. HPLC utilizes a column that holds chromatographic packing material (stationary phase), a pump that moves the mobile phase(s) through the column, and a detector that shows the retention times of the molecules. Retention time varies depending on the interactions between the stationary phase, molecules being analyzed, and the solvent(s) used.

The sample to be analyzed is introduced in small volume to the stream of mobile phase and is retarded by specific chemical or physical interactions with the stationary phase as it traverses the length of the column. The amount of retardation depends on the nature of the analyte, stationary phase and mobile phase composition. The time at which a specific analyte elutes (comes out of the end of the column) is called the retention time and is considered a reasonably unique identifying characteristic of a given analyte. The use of pressure increases the linear velocity (speed) giving the components less time to diffuse within the column, leading to improved resolution in the resulting chromatogram.

A further refinement to HPLC has been to vary the mobile phase composition during the analysis, this is known as gradient elution. A normal gradient for reversed phase chromatography might start at 5 % methanol and progress linearly to 50 % methanol over 25 minutes, depending on how hydrophobic the analyte is. The gradient separates the analyte mixtures as a function of the affinity of the analyte for the current mobile phase composition relative to the stationary phase. The choice of solvents, additives and gradient depend on the nature of the stationary phase and the analyte. Often a series of tests are performed on the analyte and a number of generic runs may be processed in order to find the optimum HPLC method for the analyte - the method which gives the best separation of peaks.