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Gas ChromatographyThere an be many parts to a gas chromatography system but the

basic components include:

An injection system.A column (controllable oven)A detector.

Gas chromatography can be used for the analysis of most environmental samples.

GC is a very efficient technique, (has very low plate heights ‘H’) and with environmental samples being complex this has obvious advantages.

Many pollutants are volatile such as pesticides and PCB’s and can be turned into gaseous form easily, others such as PAH’s are analysed with some additional sample preparation.

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The efficiency of GC can be clearly seen by the narrow and tall peaks observed.

This allows many more peaks to fit into a time period and still be resolved from each other.

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Before introducing such samples into a GC column it is necessary to remove the analytes from the mass of air or water or ‘concentrate’ the

sample.

For water samples this is often done using ‘purge and trap’

apparatus.

In environmental analyses the most common crude sample will consist of ‘air’ or ‘water’.

HRGC is a very sensitive technique which can identify very low concentration.

When coupled to suitably sensitive AND fast detectors

e.g. FID, ECD, MS…………

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After pre-concentration the analytes are introduced

to the column using an injector valve.

Analytes stick to a cold trap while the sample air passes

through, heating the trap removes the concentrated analytes.

For air samples the analytes are trapped

using thermal adsorption/desorption.

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The injector valve in GC

has two positions.

‘Position one’ to collect

the analytes on a ‘cold’

trap before injection.

The valve turns changing the

direction of the mobile

phase through the trap.

In ‘Position two’ the trap

heats up and the analytes

injected onto the column.

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Are of different dimensions depending on how much sample you want to analyse on the column.

These contain different stationary phases which are selective toward different analytes.

There are many types of GC column which can be chosen for the separation of a particular mixture.

And some of them have packed columns similar to those used in liquid chromatography.

It is normal to increase the oven temperature with time in order to speed up the movement of the slower (less volatile) analytes through the column.

The other main part of the apparatus used in GC is the oven.

These are often computer controlled so that the temperature may be changed precisely over the analysis time.

These are mostly widely separated by differences in retention and so speeding up the chromatographic process does not reduce the resolution of such

analytes.

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At low temperatures the last peaks are seen very late and

are fat and so not too identifiable.

Peaks are sharper

but some become

unresolved .

Higher temperatures bring out the analytes much quicker.

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Programming a computer to increase the temperature while

the separation takes place makes for a short analysis time

with sharp peaks and the required resolution.

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High Pressure Liquid Chromatography (HPLC)

There are two main modes of LC which differ in the way that

analytes interact with the stationary and mobile phases, these

are known as ‘Normal’ and ‘Reverse’ phase modes (see later).

HPLC is widely used in environmental analysis, although is also commonly

used in pharmaceuticals and biomedical applications.

Samples that contain analytes which are not stable when

heated up to the gaseous phase, and high molecular

weight compounds are commonly analysed by HPLC.

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Similar to GC, HPLC instrumentation consists of a sample introduction,

mobile phase source, separating column and a detector.

The components themselves are somewhat different to

those in GC due to the different nature of analytes and

mobile phases in a liquid form.

First let us start with the sample introduction.

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As in GC the analyte is introduced using a two position valve.With mobile phase passing

through the column, sample is loaded into loop of known volume.

Now the mobile phase passes through the sample loop flushing out the sample in to the column.

The internal diameter of the column is far wider than the sample loop and so the analyte band

narrows on introduction – VERY FAVOURABLE!

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There are two kinds of interactions in LC namely ‘polar’ and ‘non-polar’ interactions.

All compounds having electrons have a certain polarity, which is created by uneven distributions of that compounds electrons.

Alcohols and chlorinated compounds have a high

polarity:

Other compounds have evenly distributed electrons, so are non-polar:

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Non-polar compounds interact with other non-polar compounds in the same manner.

In HPLC, it is these interactions which cause retention of the analyte molecules by the stationary phase, and it is these two types of interaction which bring about the two modes

‘normal phase’ and ‘reverse phase’ LC.

Polar compounds interact with other polar compounds, the stronger the polarity the stronger the interaction.

‘Reverse phase’ HPLC has a ‘non-polar’ stationary phase comprising of organic molecules bonded to the silica.

The two modes of HPLC, differ in the ‘polar’ nature of the stationary phase within the column and the nature of the

mobile phase used.

In ‘Normal phase’ HPLC the stationary phase is of a ‘polar’ nature, and so interacts with polar analytes molecules.

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This is why (unlike in GC), interaction of analytes with the mobile phase is just as important as interactions with the stationary

phase.

The mobile phase in HPLC can also be polar and non-polar, comprising of a mixture of ‘solvents’ with different polarities.

As a result interaction between analyte and the stationary phase will be reduced so reducing retention of the

analyte.

Making the mobile phase stronger in polarity will increase its interaction with the analyte.

In Normal phase HPLC a polar analyte will interact with a polar mobile phase as well as the stationary phase.

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Chloroform, methanol or water all have different strengths of polarity:

Mixtures of two or more of such solvents can create mobile phases of intermediate polarity.

Analyte retention is significantly effected by the polarity. Polarity of a mobile phase can be changed by altering its composition.

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By altering the percentage composition of a mobile phase made from two solvents of different polarities while a separation takes place, the interaction of

highly retained compounds with the stationary phase can be changed.

In HPLC a ‘gradient’ mobile phase is used in the same manner.

We have seen that in GC gradient temperature programs are used to improve analysis times and efficiency of highly retained

compounds.

Mixing takes place in a ‘T’ before mixing with the sample.

The system which changes the composition of the mobile phase comprises of two

pumps controlled by a computer.

Speeding one pump and slowing the other can quantitatively change the

percentage of the two solvents.

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Here five of seven compounds are separated using an ‘isocratic’ mobile phase of 70% water and 30% methanol on a reverse phase (non-polar) column.

•First 2 peaks need MORE separation

•Peaks 3, 4 & 5 OK

•Peaks 6 & 7 too retained (broad)

% M

ethan

ol

Increasing the methanol from 10-100% over the analysis resolves all 7 peaks,

reduces analysis time, and better peaks.

•First 2 peaks – more time interacting with stationary phase

•Peak 3 – about the same

•Peaks 4 & 5 more interaction with mobile phase (faster)

•Peaks 6 & 7 much more interaction with mobile phase (much improved peak shape)

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Decreasing the polarity of mobile phase shifts the equilibrium toward interaction with the solvent so speeding up the later

eluting compounds.

With 10% methanol interaction with the non-polar stationary phase is high so

separating the first two peaks.

Water is more polar than methanol and so the polarity of the mobile phase is reduced through the analysis.

The normal and reverse phase modes of HPLC become a little more complex when we consider that most analyte molecules have both polar and non-

polar sections.

As a result these compounds interact with polar stationary phases and mobile phases as well as non-polar ones.

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Many choices of both column packing and mobile phase to decide on when planning an analysis using HPLC.

Packing material may be silica or alumina, but on to which many

different coatings may be bonded.

As interaction between analytes and the stationary phase is dictated by

these bonded compounds, chemists may ‘design’ packing materials specifically to separate certain

mixtures of analytes.

The mobile phases possible for HPLC may be a mixture of any number of solvents with any percentage composition, optimum

compositions may be found for any particular separation.

MonolithicTraditional

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The power of POLARITY!

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Other Chromatographic Methods.

GC and HPLC are to most common methods employed in the lab BUT there are a number of other chromatographic techniques available to the analyst:

• Liquid Chromatography (LC)

• Thin Layer Chromatography (TLC)

• Ion Chromatography (IC)

• Super Critical Fluid Chromatography (SFC)

• Gel/Size Exclusion Chromatography (SEC)

• Capillary Electro Chromatography (CEC)

• Capillary Electrophoresis (CE)