Gas chromatography and

high-performance
chromatography
Introduction
 Previously we have studied how and where chromatography is used.
We have seen that with the use of a chromatography paper, we can
separate mixtures in to its components.
 Previously it only involved the separation of liquid mixtures but now
we are going to look at the chromatography of mixture of gases to
separate the different gases making a mixture of gases.
Gas Chromatography
 Gas chromatography is a common type of chromatography used in
analytical chemistry for separating and analyzing compounds that can
be vaporised without decomposition. In another words, gas
chromatography is analytical technique that separates components in a
mixture between a mobile phase and a stationary phase
 In gas chromatography, the mixture to be separated is dissolved in a
fluid ( remember fluids can be both liquids and gases) called the
Mobile phase. The mobile phase is an unreactive carrier gas such
as nitrogen, helium, argon and Carbon dioxide.

 Stationary phase The solid or liquid phase of a chromatography

system on which the materials are to be separated or selectively
adsorbed.
 This type of chromatography is better known as gas-liquid
chromatography.
Procedure
 Gas-liquid chromatography involves the injection of a tiny amount of
the sample through the injection port with a microsyringe. The sample
is then vaporised and the gaseous sample is carried through the
column by the flow of the inert gas, which acts as the mobile phase.
 The column itself contains a liquid, which is the stationary phase – this
liquid is adsorbed on to the surface of an inert solid. There are different
types of columns but a common arrangement is the ‘ packed column’
where the column itself contains a finely divided, inert, solid support
material coated with liquid stationary phase.
 Most packed columns are 1-10 metres long and have a 2-4 mm internal
diameter.
 As the sample passes through the column, the different components of
the sample separate because they move at different rates.
 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.
Types of HPLC
There are two types of HPLC:
Normal phase HPLC
 This is essentially just the same as you will already have read about in thin layer
chromatography or column chromatography.
 The column is filled with tiny silica particles, and the solvent is non-polar – hexane.
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.
Retention Time
 Retention time is a measure of the time taken for a solute to pass
through a chromatography column. It is calculated as the time from
injection to detection.
 Different compounds have different retention times. For a particular
compound, the retention time will vary depending on:

i. the pressure used (because that affects the flow rate of the solvent)
ii. the nature of the stationary phase (not only what material it is made
of, but also particle size)
iii. the exact composition of the solvent
iv. the temperature of the column
Detector
 There are many detectors that can be used but they all identify
separate substances leaving the column. Different detectors have
different ranges of selectivity:
i) A non-selective detector responds to all compounds except the
carrier gas.
ii) A selective detector responds to a range of compounds with a
common physical or chemical property.
iii) A specific detector responds to a single chemical compound.
 After the detector, there is a recorder which plots a chromatogram with
retention time on the x-axis and abundance on the y-axis. The area
under the graph represents of how much (the amount) the substance is
there.
 In this type of detector, the gases leaving the column are mixed with hydrogen and air, and
burnt. Organic compounds burn in the flame to produce ions and electrons which can
conduct electricity through the flame. A large electrical potential is applied at the burner
tip, and a collector electrode is located above the flame. The current resulting from any
organic compound is measured.
 This type of detector is good for detecting organic compounds as its robust, easy to use and
can measure the mass of each substance but it destroys the sample which can’t be beneficial.
 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.
 Coupling HPLC to a mass spectrometer
 When the detector is showing a peak, some of what is passing through the
detector at that time can be diverted to a mass spectrometer. There it will
give a fragmentation pattern which can be compared against a computer
database of known patterns. That means that the identity of a huge range
of compounds can be found without having to know their retention times.

Limitations of Gas chromatography

 Not suitable for detecting semi-volatile compounds
 Only indicates if volatile organic compounds are presents.