High-performance liquid chromatography (HPLC) is an advanced form of liquid chromatography used in separating the complex mixture of molecules encountered in chemical and biological systems, in order to understand better the role of individual molecules. In liquid chromatography, a mixture of molecules dissolved in a solution (mobile phase) is separated into its constituent parts by passing through a column of tightly packed solid particles (stationary phase). The separation occurs because each component in the mixture interacts differently with the stationary phase. Molecules that interact strongly with the stationary phase will move slowly through the column, while the molecules that interact less strongly will move rapidly through the column. This differential rate of migration facilitates the separation of the molecules.
The advantages of HPLC over other forms of liquid chromatography are several. It allows analysis to be done in a shorter time and achieves a higher degree of resolution, that is, the separation of constituents is more complete. In addition, it allows stationary columns to be reused a number of times without requiring that they be regenerated, and the results of analysis are more highly reproducible. A further advantage of HPLC is that it permits both instrumentation and quantitation to be automated.
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