High Performance Liquid Chromatography is a Kind of Column chromatography that pumps a sample mix or analyte in a solvent called the mobile phase at high pressure through a column with chromatographic packing material stationary phase. The sample is carried by a moving carrier gas flow of helium or nitrogen. HPLC has the capacity to separate, and identify substances that are found in any sample which could be dissolved in a liquid in trace concentrations as low as parts per trillion. As a result of this versatility, HPLC is used in many different industrial and scientific applications, such as pharmaceutical, environmental, forensics, and compounds. Sample retention period will vary depending upon the interaction Between the stationary phase, the molecules being examined, and the solvent, or solvents used.
As the sample passes through the column it interacts between the two stages at different speed, primarily as a result of different polarities from the analytes. Analytes having the least amount of interaction with the stationary phase or the maximum amount of interaction with the mobile phase will exit the column faster. Main elements in an HPLC system comprise the solvent reservoir, or several reservoirs, a high-pressure pump, a column, injector system and the sensor. The reservoir holds the solvent, which can be referred to as the Mobile phase since it moves. There are normally a minimum of two reservoirs in a system, with each holding up to 1000 cc of solvent and generally fitted with a gas diffuser through which helium could be bubbled. A pump is used to create a predetermined flow of the mobile phase. Although manual injection of samples remains possible, most HPLCs are now fully automated and controlled by computer.
The injector, or auto sampler, introduces the solvent into a stage stream that carries the sample to the high pressure up to 400 bar column, which contains specific packing material required to effect separation. The packing material is known as the stationary phase since it is held in place from the column hardware. A sensor is required to observe that the split compound bands as they elute from the hplc testing. The data is sent from the sensor to a computer that generates the chromatogram. The mobile phase exits the sensor and can be sent to a waste, or accumulated, as desired. Helium sparging is an effective method of degassing the cellular Phase to prevent unstable baselines due to dissolved air. Nitrogen is used as a nebulisation gas in Evaporative Light Scattering Detector ELSD in which the solvent is evaporated from the sample leaving a mist as is quantified. Where standard HPLC typically uses column particles with sizes from 3 to 5µm and pressures of around 400 bar, hulk use specifically designed columns with Particles down to 1.7µm in proportion, at pressures in excess of 1000