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What is High-Performance Liquid Chromatography?

Chromatography is a chromatographic laboratory technique for the separation of a mixture, and there are several different types of chromatography, such as column chromatography, paper chromatography, gas chromatography and high-performance liquid chromatography, which will be focusing on here. The goal of chromatography is to separate, identify and quantify the components of a mixture. This is done by a solvent that moves through the mixture. The different components of the mixture will have different properties and as such separate at different points, allowing them to be identified. Chromatography gets its name because in basic chromatography you can actually see the different colors (chroma) separating from the mixture.

High-performance liquid chromatography (HPLC), formerly known as high-pressure liquid chromatography, relies on pumps to pass a pressurized liquid solvent containing the sample mixture through a column filled with a solid adsorbent material. The absorbent material causes the different components in the mixture to behave differently based on their properties. For example, components within the mixture will have different flow rates, causing them to be recorded separately as the flow out of the column.  The process is done at high pressure to allow for faster and more accurate separation.

Normal and Reverse phase HPLC are commonly used techniques. Normal phase is when the mobile phase is nonpolar and the stationary phase is polar. In this case, the least polar elements will come out first. Reverse phase is the opposite of this. The mobile phase is polar and the stationary phase is nonpolar. Here the opposite occurs, polar compounds will elute first, and less polar will elute at a slower rate.

Uses of HPLC

  • Pharmaceuticals: HPLC plays a critical role in the pharmaceutical industry since it is used to detect raw ingredients used to make products. The US Food and Drug Administration actually requires companies to use HPLC to detect the quality of their products.
  • Water Purification
  • Pre-concentration of trace components (the process of concentrating a sample before analysis, so that trace components won’t be overlooked.)
  • Other chromatography such as Ligand-exchange chromatography and Ion-exchange chromatography of proteins