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Mechanism of Separation of Components by HPLC

HPLC is used in various industries to analyze different products by their separation into its components. Separation technique is described in this article.
HPLC stands for High Performance Liquid Chromatography system. This is an analytical chemistry technique that is used to separate, quantify and identify every component that is in a mixture. The technique depends on pumps to push some constrain liquid solvent which contains a mixture of the sample through a column containing a solid absorbent material.

Each of the components in the sample will interact with the absorbent material in a slightly different way. This will cause different flow rates in the various components hence causing the components to separate as they flow out of the column. High Performance Liquid Chromatography system is used mainly for manufacturing, medicine and research purposes.

Separation by HPLCIn manufacturing, HPLC is mostly used in the process of production of biological and also pharmaceutical products. In medicine, it is used in detecting the level of vitamin D in blood serum. When it comes to research, HPLC is used for a couple of purposes. A good example is; separating components in biological samples, most of which are complex.

High Performance Liquid Chromatography is used in separating and purifying compounds according to their polarity. To understand polarity, we can use a simple example of oil, water and ethanol. Ethanol is polar. Thus it will mix properly with water. On the contrary, oil is not polar, so it does not mix well with water.

The HPLC column is as well called the stationary phase. It is made from different substances, mostly silica, and it is highly compact in nature. Long carbon chains functionalize the silica particles. Carbon chains are non-polar. Thus, the longer the chain is, the more non-polar the column will become. C18 columns are the ones that are mostly used and contain 18 carbon chains.

High Performance Liquid Chromatography system can be used in separating compounds in environmental samples, medical chemistry, microbiology and also environmental samples. The compounds are injected into the HPLC column. The mobile phase, which is practically a mixture of water and an organic solvent is then introduced. The organic solvent used is mostly methanol or acetonitrile. This is called the mobile phase since the mixture moves all through the HPLC column and still flushes out the compounds in the column at the same time. The process of flushing out is also known as eluting.
Related: Difference between C8 and C18 Columns Used in HPLC System

Compounds are frequently eluted alongside a concentration gradient. In simpler terms, the amount of water which is in the mobile phase decreases after some time, as the non-polar solvent percentage increases synchronously. The components in the mobile phase will then become more non-polar. The compounds that are contained in the sample used will stick to the carbon chain in the column. The compounds that are most non-polar will stick strongest while the most polar compounds will weakly.
Related: 10 Tips for HPLC Analysis In Pharmaceuticals

Basically, High Performance Liquid Chromatography separates components from their compounds according to their polarity. HPLC is also used as an analytical tool to inspect environmental and biological samples for the presence or even the absence of known compounds. Such compounds include drugs, toxins, and pesticides. It can as well assist in the identification of unknown compounds.

Ankur Choudhary is India's first professional pharmaceutical blogger, author and founder of, a widely-read pharmaceutical blog since 2008. Sign-up for the free email updates for your daily dose of pharmaceutical tips.
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