High Performance Liquid Chromatography HPLC

High Performance Liquid Chromatography HPLC

August 27, 2019 100 By Stanley Isaacs


HPLC stands for high-performance liquid
chromatography, but could equally well stand for high pressure liquid
chromatography. It is used for separating mixtures either to analyze the mixture or to separate a
required product from others in a reaction mixture. It can also
be used to find the relative amounts of different components in a mixture. HPLC works on the same principle as paper
chromatography, here shown speeded up. A liquid, called the mobile phase, moves
past a solid, the stationary phase. In paper
chromatography the stationary phase consists of water
molecules bound to the cellulose in the paper. The mobile phase carries different components of a mixture, called the sample, along with it at different rates. How fast each one moves depends on its relative affinity for the
mobile in the stationary phases. For example, if the mobile phase is more polar than
the stationary phase the more polar components of a mixture
will tend to move more quickly than the less polar ones. In HPLC the stationary phase is a solid packed
into a column like one of these. This particular column contains silica
particles to which C8 hydrocarbons are attached making the
stationary phase nonpolar. In paper chromatography the solvent moves along the paper by capilliary action. In HPLC the liquid is forced through the column
by high-pressure pumps. The whole apparatus looks like this. These bottles contain solvents. Two solvents can be mixed in any
proportions to give a mixture, the liquid phase, of suitable polarity for
the separation that is being done. In this case one solvent is water, very polar, and the other, ethane nitrile, less polar. The operator can decide on a mixture with the correct polarity for the separation she is doing. These are the pumps. They produce a pressure of fifteen
thousand kilopascals, 150 times that of the atmosphere, hence
the name high pressure liquid chromatography. If a single sample is to be run, it is injected into the solvent stream here in the injection port via a hypodermic
syringe. Alternatively, several samples can be run in succession
by loading them into this auto sampler which will run them in
order without any human intervention. The pumps force the mixed solvents through the column. The solvent emerging from the column and carrying the separated components of
the mixture passes into the detector. Here a beam of ultraviolet light shines
through it. This light is set at a wavelength that is absorbed by all the components
to be separated. When the detector reading drops, the component that is absorbing UV light
is coming out of the column and passing through the detector. Many
alternative types of detector are possible. This one measures refractive index. The
time that each component takes to come off the column is called its retention time and can be
used to help identify it. Here the HPLC instrument is being used to
separate a mixture of two steroids used in a pharmaceutical
preparation. The column chosen is packed with a
nonpolar solid. The tails of the molecules represent
hydrocarbon chains C8H17. Having chosen the solvents, detector wavelength and flow rate, a single
sample is run by injecting about 20 microlitres
into the injection port. The more polar component comes off the
column first, followed by the less polar. The peak at retention time 1.5 minutes represents other ingredients used in
formulating the product. This is the pharmaceutical product and behind it, its chromatogram.