Gas

Chromatography
and HPLC

By: Avipsita, Sid and Momina

 Introduction

Gas Chromatography

High Performance Liquid

Chromatography (HPLC)
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Gas ChromatographyResults
Injector
Port

Detector

Column
Carrier Simply Back to
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 Injector Port
The process of gas chromatography involves the
injection of tiny amount of the sample into the
head of a chromatography column, usually done
by use of a micro syringe.

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 Carrier Gas
The sample that is put into the machine via the
injector port is vaporised. The vaporised gaseous
sample is carried through the column by the flow
of an inert (carrier) gas, which acts as the mobile
phase.

Suitable
gases
include:
1. Nitrogen
2. Helium
3. Argon
4. Carbon
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 Column
The column contains liquid, which is in
stationary phase- this liquid is
absorbed onto the surface of a inert solid.
The different components of the sample
separate as it passes through the column
because they move at different rates.

Simple Analogy: Think of a swarm of bees and wasps moving

over a field of flowers. The bees would tend to stop to pick up
nectar from the flowers, but the wasps would not. Therefore,
at the other side of the field the wasps would be detected
first, and then the bees.
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 Detector
There are many types of detector that can be used but they all identify
the separate substances leaving the column.
Different detectors have different ranges of selectivity:
1. A non selective detector- responds to all compounds except the carrier
gas
2. A selective detector- responds to a range of compounds with a
common physical or chemical property
3. A specific detector responds to a single chemical compound.
4. Flame ionisation detector

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 Flame Ionisation detector
The gases leaving the column are
mixed with hydrogen and air, and
burnt.
Organic compounds burn in the flame
producing ions and electrons that
conduct electricity.
A large electrical potential is applied
at the burner tip and a collector
electrode is located above the flame.
The resulting current of the burning
organic compound is measured.

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 Why use that type of
method?
It is robust
It is relatively easy to use
It can measure the mass of each substance identified.
So what is the drawback?
It destroys the sample.

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 Results
The results are displayed on a
monitor or printout.

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 High Performance Liquid
Chromatography (HPLC)
First you need to know what column
chromatography is
i. It involves having a column packed with a solid phase
(such as alumina or calcium carbonate). A solution of the
plant pigment is added to the top of the column.
ii. Solvent is added to the column to make the mobile phase.
For instance, addition of a plant pigment solution
iii. Tap at the bottom is always open allowing liquid to
continually flow out.

Check this out:

http://www.chem-ilp.net/labTechniques/ChromatographyAnimati
on.htm
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So what is HPLC? Introducti
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 What is HPLC?
HLPC is basically highly improved form of column
chromatography.

Instead of the solvent being allowed to drip through a column

under gravity, it is forced through high pressure; making the
process much faster. A very small particle size for the column
packing material is used. This gives greater surface area for the
interactions between stationary phase and the molecules
flowing past; allowing better separation of the components.

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The two types of HPLC:

Normal Phase HPLC

Reversed Phase HPLC

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 Normal Phase HPLC
Column is filled with silica particles and the
solvent is non-polar. So polar molecules passing
through the column will stick for longer to the
polar silica than the non polar molecules. This
means that the non-polar ones will pass through
the column more quickly.

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 Reversed Phase HPLC

- In this process the silica is modified to make it non polar by

attaching a long hydrocarbon chain to its surface.
- A polar solvent, for example a mixture of water and ethanol, is
used.
- This means that there will be a strong attraction between the
polar molecules in the test sample. The attraction between the
hydrocarbon chains attached to the silica (stationary phase)
and the polar molecules in the solution is very weak. Polar
molecules spend most of their time moving with the solvent;
meaning that theWhat is Retention
polar molecules will travel through the
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column more quickly.
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 Retention Time
It is the time taken for a compound to travel
through the column and reach the detector. Time
is measured from the point 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 times as retention times
depend on:
The pressure used-as it affects the rate of flow
The nature of the stationary phase- what the material is and
how big the particles are
The exact composition of the solvent
The temperature of the column

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 Identifying Substances
There are many ways to identify the substance
which is passed through the column

For example:
UV absorption is one way as many organic compounds absorb
UV light of various wavelengths. The amount of light absorbed
will depend on the amount of a particular compound that is
passing through the light beam at a time.

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