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Chromatography
Vocabulary
adsorption The attraction of one substance to the surface of another. It can be
also said that it is the molecules in the mixture that adhere to the stationary
phase due to the intermolecular forces.
desorption The breaking of the bonds between a substance and the surface to
which the substance is adsorbed. It is can also be said that molecules in the
mixture detaches from the stationary phase surface to the mobile phase solvent
due to the attraction with the molecules of the mobile phase continuously
moving upwards.
mobile phase The phase that moves over the stationary phase in
chromatography (mixture/solvent)
stationary phase A solid, or a solid that is coated in a viscous liquid, used in
chromatography. The components of a mixture undergo adsorption to this phase
as they are carried along by the mobile phase.
eluent A liquid used as the mobile phase in chromatography.
retention time The time taken for a component to pass through a
chromatography column.
carrier gas The gas used as the mobile phase in gas chromatography.

This process of deferential adsorption and desorption leads to the

components travelling at different speed and thus they separate out.
The rate of movement of each compound depends mainly on how strongly
it adsorbs (affinity) onto the stationary phase and how readily it dissolves
(solubility) on the mobile phase.

Thin Layer Chromatography (TLC)

Stationary phase is a thin layer of a fine powder (alumina or silica)

spread on a glass or plastic plate
Mobile phase is the suitable solvent

Qualitative analysis

The ideal chromatogram has good resolution (the right stationary and
mobile phases must be chosen) and the different components of the subject
mixture should be clearly separated and sharply defined. The long streaking
(tailing) should not be present. Streaking may be caused due to the initial spot
placed on the origin as large in diameter.

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Methods of Analysis/ Interpreting

results
-

Run standards of known chemicals on

the same chromatogram and compare
for similar substances.

Rf=

Distance moved
Distance moved
origin by component origin by solvent

Rf value depends on the compound, the eluent, the stationary phase

and temperature.

If the sample is solid, dissolve the sample in a suitable solvent (mobile

phase)
A line is drawn above the edge of the paper (and above the level of water)
which is called the origin
o A pencil is used due to graphite being insoluble
The sample of solution to be analysed is places on the origin line, and as
small spot possible.
o A small spot achieves better separation without streaking, then the
Rf value will not be an estimation
Dry the spots with a hand dryer
Hang the paper in the solvent jar, as the edge of the paper just dip in the
solvent
o If the origin with the component is drowned, then components will
dissolve in solvent and disappear (No adsorption/desorption
process)

Comparison of paper and thin-layer

Paper Chromatography
Cheap
Little Preparation
More efficient for polar and water
soluble compounds
Easy to handle and store
A wide range of stationary phases is

chromatography
Thin Layer Chromatography
Faster
Detects smaller amounts
Better separation of less polar
compounds
Corrosive materials can be used

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available

High Performance Liquid Chromatography (HPLC)

Stationary phase is a solid hydrocarbon (non-polar granules), and

packed into a column.
Mobile phase is the suitable solvent
Column Chromatography (Traditional
Chromatography)

Component 1: highest affinity, highest Mr,

most non-polar, least solubility (swept out
by solvent first)
Component 3: least affinity, lowest Mr, least
non-polar, highest solubility (swept out by
solvent first)
Solid stationary phase: hydrocarbon
granules
First drop of eluent contain pure solvent
(mobile phase)

HPLC technique differs from column

chromatography as follows

Size of granules are smaller (High SA)

Advantage: smaller particles allow for more
frequent adsorption and desorption, giving a
better separation
Disadvantage: smaller particles create a
considerable resistance to the flow of the
mobile phase and the solvent is pumped under high pressure (risky and
expensive)
High performance Liquid
Chromatography (Modern
Chromatography)

Uses a column to separate the

components being analysed. In this
case, the sample remains as a liquid and
flush through the column by another
liquid (mobile phase)
HPLC can separate compounds with
relatively high molecular masses of
1000 or more, as the sample remains
as liquid (advantage over GLC)
Least soluble component swept out last
has the highest retention (Rt) time
Components are detected by passing
the mixture of components though a UV

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light beam. Many compounds absorb UV light (organic appear

red/inorganic appear blue), thus when an organic compound passes in
front of the beam, its signal is picked up by a detector.

Gas Chromatography (GC)

Gas Liquid Chromatography (GLC)/Gas Solid Chromatography (GSC)

Stationary phase is a high-boiling point liquid hydrocarbon or ester (nonpolar) coated onto fine granules of an inert solid (granules packed into
column)
Mobile phase is a gas (usually nitrogen), the injected sample (liquid)
must be vaporised in order to be carried out by the gaseous mobile phase

After sample is injected

through injection port, it is
then heated to a
temperature that will
instantly vaporise the
sample (making it a gas)

GLC

packed with

porous solid

GSC

packed with

adsorbent solid (able to

adhere to)

silica/alumina
Column is mounted in an
oven to be heated, and components of sample are repeatedly passed into
and out of the solution with the stationary phase.
GC is limited to compounds that can be readily vaporised without
decomposing and has a relative mass less than 300 (Mr)

Qualitative and Quantitative analysis for HPLC & GLC

The time taken from the injection of a sample until the detection of a
component at the end of a column is referred to as that components
retention time (Rt)
The position of the peak = components retention time
Rt is the instrumental equivalent to Rf values

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Position of the peak (Rt) identifies each component

analysis
Height of the peak or the area of the peak proportional to concentration

qualitative

quantitative analysis

Analysis of Petrol sample (Qualitative analysis)

Inject pure standard solution and obtain chromatogram (reference)

Inject the petrol sample (NOTE: rinse before the use of column, and
maintain same conditions)
Compare the standards and sample chromatograms with the R t

Quantitative analysis

Prepare standard solutions (v/v%)

Inject standard solutions from low to high concentration and obtain peak
area
Plot calibration curve (y-axis: peak area/x-axis: concentration)
Use peak area of sample to find concentration

Conversions
1% (w/v) = 0.01g/mL = 1 g/(100 mL)
1 ppm = 1

g/mL = 1 mg/L

1 ppb = 1 ng/mL = 1

g/L