What is High Performance Liquid Chromatography?
High performance liquid chromatography (HPLC
) is a highly efficient and rapid analytical separation technique that uses a column to separate a mixture, followed by spectroscopy for identification and quantification. HPLC uses high-pressure metal columns developed from column chromatography LC uses low-pressure glass columns. Therefore, HPLC is essentially a highly modified column liquid chromatography method. Instead of passing the solvent through the column by gravity, it is fed into the column using an infusion pump with up to 400 atmospheres of pressure. HPLC can analyze a wider range of compounds, including thermally labile or nonvolatile compounds. This characteristic makes HPLC an invaluable tool for the analysis of complex samples that cannot be adequately assessed using other analytical methods.
How Does High Performance Liquid Chromatography Work?
The working principle of HPLC is that the components of the measured mixture are separated and purified in the chromatographic column through the interaction with the stationary phase and the mobile phase.
The stationary phase is a very small porous particulate material, such as silica or polymer, present in the column, called the filling material or chromatographic carrier. The mobile phase is a solvent or mixture of solvents, commonly consisting of water, acetonitrile, methanol, or a mixture thereof, that is forced through the column under high pressure. The sample is injected with a syringe during the assay and mixed with the mobile phase through a valve attached to the sample ring. Subsequently, each component of the sample passes through the column at different rates, due to the different adsorption capacity of each component to the stationary phase, the eluting time of each component from the column is also different. The component concentrations can be converted into electrical signals which are transmitted to the computer's HPLC software via suitable detectors. At the end of the run, the chromatogram is available in the HPLC software.
Types of HPLC
There are two primary types of HPLC commonly employed in practice: normal-phase and reversed-phase HPLC.
Reversed-Phase HPLC
Reverse-phase chromatography is the most widely used type of HPLC. It uses a non-polar stationary phase and a polar mobile phase (such as a mixture of water and an alcohol like methanol). Nonpolar stationary phases (such as silica gel particles), interact with nonpolar or slightly polar analytes in the sample mixture. As a result, more polar compounds elute faster due to their greater attraction to polar solvents, while less polar compounds elute due to interactions with hydrocarbon groups, mainly through van der Waals and hydrophobic interactions. Moves through the column more slowly. Reverse-phase HPLC is commonly used for the analysis of organic compounds, pharmaceuticals, and biomolecules.
Normal-Phase HPLC
In contrast to reversed-phase HPLC, normal-phase HPLC has a polar stationary phase (such as tiny silica particles) that packs the column and uses a nonpolar solvent (such as hexane). In this technique, non-polar compounds in the mixture pass through the column at a faster rate because they have a lower affinity for polar silica compared to polar compounds. Normal phase HPLC is commonly used to separate polar compounds such as amino acids, vitamins, and pesticides.
In conclusion, high performance liquid chromatography (HPLC) has emerged as a powerful technique for separating, identifying, and quantifying components in complex mixtures. HPLC has excellent sensitivity and is widely used in many fields, including pharmaceuticals, environmental analysis, food and beverages, forensics, and more.
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