High Performance Liquid Chromatography (HPLC) Theory

High-performance liquid chromatography (sometimes referred to as high-pressure liquid chromatography), HPLC, is a chromatographic technique used to separate a mixture of compounds in analytical chemistry and biochemistry with the purpose of identifying, quantifying or purifying the individual components of the mixture.


HPLC is accomplished by injection of a small amount of liquid sample into a moving stream of liquid (called the mobile phase) that passes through a column packed with particles of stationary phase. Separation of a mixture into its components depends on different degrees of retention of each component in the column. The extent to which a component is retained in the column is determined by its partitioning between the liquid mobile phase and the stationary phase. In HPLC this partitioning is affected by the relative solute/stationary phase and solute/mobile phase interactions.

Thus, unlike GC, changes in mobile phase composition can have an enormous impact on your separation. Since the compounds have different mobilities, they exit the column at different times; i.e., they have different retention times, tR. The retention time is the time between injection and detection. There are numerous detectors which can be used in liquid chromatography. It is a device that senses the presence of components different from the liquid mobile phase and converts that information to an electrical signal.

For qualitative identification one must rely on matching retention times of known compounds with the retention times of components in the unknown mixture.Quantitative analysis is often accomplished with HPLC. An automatic injector providing reproducible injection volumes is extremely beneficial, and are standard on modern commercial systems.

HPLC is just one type of liquid chromatography, meaning the mobile phase is a liquid. In this lab you will use what is called reversed phase HPLC. Reversed phase HPLC is the most common type of HPLC. What reversed phase means is that the mobile phase is relatively polar, and the stationary phase is relatively non-polar. Thus non-polar compounds will be more retained (i.e. Have longer retention times) than a polar compound. In normal phase HPLC, the mobile phase is relatively non-polar and the stationary phase is relatively polar. Other more general types of HPLC include partition, adsorption, ion-exchange, size-exclusion, and thin-layer chromatography.

Application :

  • Quantitative/qualitative analyses of amino acids, nucleic acids, proteins in physiological samples.
  • Measuring levels of active drugs, synthetic by-products, degradation products in pharmaceuticals.
  • Measuring levels of hazardous compounds such as pesticides and insecticides.
  • Monitoring environmental samples.
  • Purifying compounds from mixtures.

HPLC is unique highly sophisticated chromatographic equipment used to separate compounds that are dissolved in a solution. This is the reason why it is called High performance LIQUID chromatography. Also it is termed as High PRESSURE liquid chromatography is due to its mobile phase. Its mobile phase moves due to gravity. Unlike other methods HPLC includes all three sections in chromatographic equipment which are separation, identification and quantification. HPLC is an instrument or a machine which has all these three sections itself. At present HPLC methods are used that are controlled by software.

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