High Performance Liquid Chromatography
HPLC, or High Performance Liquid Chromatography is used to separate out the components of a mixture in a way that they can be analysed and quantified. It is very useful when the sample consists of a mixture of different proteins or different sizes of molecule such as DNA. It is used extensively in both biochemistry, for analytical purposes, and chemistry, for the extraction of dyes or different chemical compounds.
HPLC has developed rapidly since its original inception in the 1920s. The first commercially available equipment was introduced in 1969. The technological advancements of the past fifty years mean that HPLC is the preferred method for separating a huge range of samples quickly and efficiently.
There are many reasons that a mixture would need to be separated by HPLC methods. It might be too complex for analysis via other methods. If there are a large number of compounds present, then separation of each compound by other methods might prove too costly or time-consuming. In other circumstances, the components could be too similar so that other methods would simply not have the sensitivity to separate out the different parts. Or, the analyte might be present in very small quantities and so HPLC would be the method of choice.
In the pharmaceutical industry, all aspects of drug discovery and development use HPLC as a major analytical tool. The effective use of robust HPLC methods is of paramount importance during all stages of compound identification, separation, analysis and production. It is vital in the separation of a host of unknown compounds from a myriad of new discoveries.
Separations are performed in steel or glass tubes which are called columns. These are tightly packed with very small particles of between 1-5microns. Highly specialised separations can also employ titanium columns packed with even smaller particles in the region of 0.2-0.5micron diameter. All the separation of compounds occurs here.
A pump is used to provide a constant flow of the mobile phase containing the mixture under examination. Pumps can pull the mobile phase at constant or variable speeds. They can also be used to mix different solvents or to control solvent delivery at different speeds and pressures. The mixture is injected into the stream of the mobile phase before it enters the column. Different solutes within the mixture travel at different speeds within the column and emerge at different rates where they are picked up by sensitive detectors.
HPLC is an elegant and sophisticated way to separate unknown compounds out into their elementary components and is used with great efficacy in the modern pharmaceutical world.
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