Chromatography
Mr G. Pomerai
Bsc BCH (UZ)
5/22/2020 Riverside College 1
Chromatography
1. Paper Chromatography
2. Thin layer chromatography
3. High-perfomance liquid chromatography (HPLC)
4. Gas-liquid chromatography (GLC)
Paper chromatography
It is used to separate mixtures as a solvent moves up a piece of
absorbent paper.
The solvent is called the mobile phase, and water trapped between the
cellulose fibres of the paper is the stationary phase.
The substances in the mixture will have different affinities for
the solvent and for the water, and so they move at different
rates over the paper.
Paper chromatography
a Paper chromatography. b The chromatogram produced.
• Coloured substances can be seen directly on the paper but others are
sprayed with a chemical that forms coloured compounds on the
chromatogram
Calculating Rf values
• Components of the mixture can be identified by comparison
with pure reference compounds or by calculating Rf values
and comparing these values with those in tables of data.
Partitioning
• The solutes in the mixture being separated are partitioned to
different extents between the solvents in the mobile and
stationary phases.
• In paper chromatography the different partition coefficients
of the components in a mixture correspond to their relative
solubilities in the two solvents.
• The greater the relative solubility in the mobile phase, the
faster the rate of movement as the mobile phase passes over
the stationary phase.
Partition chromatography
Paper chromatography is a partition chromatography. The mobile
phase moves over the stationary liquid phase, carrying solute
particles with it.
The filter paper is the solid support in paper chromatography.
Thin-layer chromatography
• In thin-layer chromatography, referred to as TLC, the stationary phase is a
solid that adsorbs solute molecules onto its surface.
• TLC is an adsorption chromatography. The mobile phase moves over
the stationary solid phase.
TLC
• The solid stationary phase is usually alumina (Al2O3) or silica (SiO2)
coated onto a glass or plastic surface.
Thin-layer chromatography
• Polar molecules have a greater attraction for a polar solid
used as the stationary phase, and they are adsorbed more
strongly onto its surface.
• Polar molecules therefore they travel more slowly than non-
polar molecules up the thin layer of alumina or silica, and
separation occurs.
• Solutes are located on the chromatogram and identified by
comparing with standard known substances or by calculating
Rf values.
TLC
• Although TLC is normally described as adsorption
chromatography, some partitioning does occur if water is
present.
• Both dried alumina and silica can become rehydrated.
• When this happens, water acts as a partitioning stationary
phase together with the adsorbing stationary solid phase.
Advantages of TLC
• TLC is quicker than paper chromatography
• TLC can be used on smaller samples, making it useful in
forensic science to identify drugs.
High-performance liquid
chromatography (HPLC)
• HPLC uses partitioning to separate and identify the
components in a mixture.
• The stationary phase is a non-volatile liquid, such as a long-
chain hydrocarbon liquid, bonded onto a solid support, e.g.
small particles of silica. This is packed tightly into a column.
• The solvent chosen for the mobile phase is usually polar, e.g. a
methanol/water solvent. This has to be forced under pressure
through the densely packed column where separation occurs
HPLC
• The more polar components in the mixture have a greater
relative solubility in the polar solvent.
• Therefore they are carried through the column faster than
components whose molecules are more non-polar (which
dissolve better in the non-polar stationary phase in the
column).
• The detector records retention times, i.e. how long it takes
each component to pass through the column.
Apparatus for HPLC
HPLC
• The area under each peak recorded is proportional to the
amount of solute emerging from the column.
The chromatogram from a vitamin E HPLC analysis carried out by a food scientist
investigating chilli peppers.
Uses of HPLC
1. In medical research to separate peptides and proteins
2. To analyse urine samples from athletes for banned
substances such as steroids or stimulants
3. For monitoring pollutants in the atmosphere and in rivers, e.g.
measuring levels of pesticides
4. By food standards agencies to check the accuracy of the
data on food labels.
Gas–liquid chromatography (GLC)
• GLC is similar to HPLC but a gaseous sample enters the
column.
• The column contains the stationary phase and the sample is
moved through by an inert carrier gas.
• This method is used with gases, liquids and volatile solids (as
they must be in the form of a vapour).
Apparatus for GLC
• The oven maintains a constant temperature, higher than the boiling point of the
components in the mixture to be analysed.
GLC
• Conditions must be controlled in order to make comparisons with
published databases.
• The chromatogram must be obtained using the same carrier gas,
flow rate, stationary phase and temperature that were used
when the standard data was obtained.
• Analysis by gas–liquid chromatography does have some
limitations.
• For example, similar compounds will have similar retention times
and if a newly discovered compound is detected it will not have
a match in the computer’s database of retention times.
Determination of the percentage
composition of a mixture by GLC
• For quantitative analysis, the component peaks are first
identified and then the area of each is measured.
• The peaks are roughly triangular in shape so their area is
approximately:
• The GLC machine usually measures the area of the peak
automatically and can print the results with the
chromatogram.
• If the peaks are very narrow or have similar base widths, then
peak height may be used instead of peak area to estimate
the proportion of components in a mixture.
Determination of the percentage
composition of a mixture by GLC
For this method:
• the chromatogram must show peaks for all the components in
the mixture
• all the components of the mixture must be separated
• the detector must respond equally to the different
components so that peak area is directly proportional to the
component concentration.
Determination of the percentage
composition of a mixture by GLC
• The amount of each component in a mixture is found by
expressing it as a percentage of the sum of the areas under all
the peaks.
• For example, for a mixture of three esters A, B and C:
(approx.) % of ester A
Uses of GLC
• Used in testing for steroids in competing athletes
• For testing the fuels used in motor racing.
• It is also used for medical diagnosis to determine the
percentages of dissolved oxygen, nitrogen, carbon dioxide
and carbon monoxide in blood samples.
• GLC is often combined with mass spectrometry to separate
then rapidly identify the components of a mixture.
Exercise
1. Look at this paper chromatogram:
a. The solvent used was ethanol. Which sample of ink, A, B or C, has
the greatest relative solubility in ethanol?
b. Work out the Rf value of the ink whose partition coefficient in
ethanol and water lies between the values of the other two inks.
Exercise
2.
a. TLC can separate mixtures of components. What do we call
the mechanism of separation usually at work in TLC?
b. A mixture of propanone and hexane was separated on a TLC
chromatogram using alumina as the stationary phase and
methylbenzene as the solvent. Which substance would you
expect to rise further up the chromatogram? Explain why.
Exercise
3.
a. For GLC separations explain:
i how retention time is measured
ii how the areas under the component peaks
are used.
b. What can you use as an approximate measure of
the proportion of a component in a mixture from a
GLC chromatogram which produces sharp peaks?
