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.