High-performance liquid chromatography(HPLC), formerly referred to as High-pressure liquid chromatography, is a powerful technique used to separate, identify, and quantify components in complex mixtures. The term "high pressure" refers to using high pressure to push the liquid mobile phase through the tightly packed columns.
In HPLC, two phases play a critical role in the separation process:
Separation occurs as the analytes interact differently with both the mobile and stationary phases, depending on their chemical properties, which leads to differential migration through the column.
Sometimes, solute molecules bind irreversibly to the stationary phase, clogging the analytical column and decreasing its performance. To prevent this, a guard column is placed before the analytical column. A precolumn called a scavenger column may also be placed between the mobile phase reservoir and the injector. This ensures that the solvent enters the analytical column, which is already saturated with silica from the scavenger column, preventing unwanted reactions and extending the lifetime of the analytical column.
After the separation, analytes are detected by various detectors, depending on the characteristics of the compounds. The results are visualized as chromatograms, with peaks corresponding to the different components of the mixture. This provides both qualitative and quantitative data about the sample.
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