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High Performance Liquid Chromatography

High performance liquid chromatography (HPLC) is a powerful analytical technique that uses high pressure to force a solvent through a column containing tightly packed adsorbent particles. This allows for faster separation and analysis of sample components compared to traditional column chromatography. There are two main types of HPLC - normal phase uses a non-polar stationary phase and solvent, while reversed phase (more common) uses a polar stationary phase and non-polar solvent. Different compounds have varying retention times depending on their interactions with the stationary and mobile phases, allowing identification.

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83 views4 pages

High Performance Liquid Chromatography

High performance liquid chromatography (HPLC) is a powerful analytical technique that uses high pressure to force a solvent through a column containing tightly packed adsorbent particles. This allows for faster separation and analysis of sample components compared to traditional column chromatography. There are two main types of HPLC - normal phase uses a non-polar stationary phase and solvent, while reversed phase (more common) uses a polar stationary phase and non-polar solvent. Different compounds have varying retention times depending on their interactions with the stationary and mobile phases, allowing identification.

Uploaded by

Ravin Kumar
Copyright
© © All Rights Reserved
We take content rights seriously. If you suspect this is your content, claim it here.
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HIGH PERFORMANCE LIQUID CHROMATOGRAPHY –

HPLC
High performance liquid chromatography is a powerful tool in analysis. 

Introduction

High performance liquid chromatography is basically a highly improved


form of column chromatography. Instead of a solvent being allowed to
drip through a column under gravity, it is forced through under high
pressures of up to 400 atmospheres. That makes it much faster.

It also allows you to use a very much smaller particle size for the
column packing material which gives a much greater surface area for
interactions between the stationary phase and the molecules flowing past
it. This allows a much better separation of the components of the
mixture.

The other major improvement over column chromatography concerns


the detection methods which can be used. These methods are highly
automated and extremely sensitive.

The column and the solvent

Confusingly, there are two variants in use in HPLC depending on the


relative polarity of the solvent and the stationary phase.

Normal phase HPLC

This is essentially just the same as you will already have read about in
thin layer chromatography or column chromatography. Although it is
described as "normal", it isn't the most commonly used form of HPLC.

The column is filled with tiny silica particles, and the solvent is non-
polar - hexane, for example. A typical column has an internal diameter
of 4.6 mm (and may be less than that), and a length of 150 to 250 mm.
Polar compounds in the mixture being passed through the column will
stick longer to the polar silica than non-polar compounds will. The non-
polar ones will therefore pass more quickly through the column.

Reversed phase HPLC

In this case, the column size is the same, but the silica is modified to
make it non-polar by attaching long hydrocarbon chains to its surface -
typically with either 8 or 18 carbon atoms in them. A polar solvent is
used - for example, a mixture of water and an alcohol such as methanol.

In this case, there will be a strong attraction between the polar solvent
and polar molecules in the mixture being passed through the column.
There won't be as much attraction between the hydrocarbon chains
attached to the silica (the stationary phase) and the polar molecules in
the solution. Polar molecules in the mixture will therefore spend most of
their time moving with the solvent.

Non-polar compounds in the mixture will tend to form attractions with


the hydrocarbon groups because of van der Waals dispersion forces.
They will also be less soluble in the solvent because of the need to break
hydrogen bonds as they squeeze in between the water or methanol
molecules, for example. They therefore spend less time in solution in the
solvent and this will slow them down on their way through the column.

That means that now it is the polar molecules that will travel through the
column more quickly.

Reversed phase HPLC is the most commonly used form of HPLC.


Injection of the sample

Injection of the sample is entirely automated, and you wouldn't be


expected to know how this is done at this introductory level. Because of
the pressures involved, it is not the same as in gas chromatography (if
you have already studied that).

Retention time

The time taken for a particular compound to travel through the column
to the detector is known as its retention time. This time is measured
from the time at which the sample is injected to the point at which the
display shows a maximum peak height for that compound.
Different compounds have different retention times. For a particular
compound, the retention time will vary depending on:

 the pressure used (because that affects the flow rate of the solvent)
 the nature of the stationary phase (not only what material it is made
of, but also particle size)
 the exact composition of the solvent
 the temperature of the column

That means that conditions have to be carefully controlled if you are


using retention times as a way of identifying compounds.

The detector

There are several ways of detecting when a substance has passed


through the column. A common method which is easy to explain uses
ultra-violet absorption.

Many organic compounds absorb UV light of various wavelengths. If


you have a beam of UV light shining through the stream of liquid
coming out of the column, and a UV detector on the opposite side of the
stream, you can get a direct reading of how much of the light is
absorbed.

The amount of light absorbed will depend on the amount of a particular


compound that is passing through the beam at the time.

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