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.