In order to generate the flow which is required for liquid chromatography in packed columns, a high pressure is required or needed. This fact was referred to as the HPLC (High-Performance Liquid Chromatography). Earlier, High-Pressure Liquid Chromatography (HPLC) was termed to be as the pressure of 500psi (35 bars) which is generated with the help of instrument components that only have the capacity of producing such pressures (High Performance Liquid Chromatography (HPLC) Primer, 2008).
In 1970’s, due to the technological advancement, a new “HPLC” instrument was developed that was capable of generating 6,000psi (400 bar) of pressure along with improved detectors and columns. Now, in the field of analytical chemistry, HPLC is considered to be as a powerful tool that has the ability to separate, purify, quantify and identify the compounds present in any sample that could be dissolved in water or liquid.
HPLC is one of the sorts of column chromatography, which is derived by putting pressure in a solvent or substance. For the resolution of the complex mixtures, high sensitivity, shorter retention time and high selectivity are considered as the main advantages of HPLC. “Normal” and “Reverse” phases are the two distinct modes of HPLC methods. Normal Phase HPLC (NH-HPLC) is also known as the Normal Phase Chromatography. It is used to separate analytes that are based on polarity. A polar stationary phase and a non-polar mobile phase are used in this method. It is used when the analyte of interest is fairly polar in nature. When the solute is eluted by a polar mobile phase over a hydrophobic stationary phase at that time, reverse phase HPLC is used. A non-polar stationary phase and an aqueous, moderately polar mobile phase is used in RP-HPLC method. RP-HPLC operates on the principle of hydrophobic interactions.
In order to determine the identity of the mixture and possible irregularities for the analysis of small samples, HPLC is considered to be...