Year 12 Chemistry: Chapter 6 Chromatography
Paper Chromatography
Thin-layer Chromatography
Define viscous: ___________________________________________________
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6.3 Column Chromatography
The stationary phase is a solid, or a solid that has been thinly coated in a viscous liquid
and packed into a column. The sample is applied, which is the mobile phase and is dripped
slowly onto the column from a reservoir above. A tap at the bottom of the column allows
the solvent, which is called the eluent, to leave the column at the same rate as it enters
it at the other end.
Define eluent: ____________________________________________________
________________________________________________________________
There are two instrumental chromatographic techniques based on column
chromatography:
 High performance liquid chromatography (HPLC)
 Gas chromatography (GC)
High Performance Liquid Chromatography
High performance, or high pressure, liquid chromatography (HPLC) is an extremely
sensitive analysis. Routinely used for pharmaceutical and industrial analyses eg, can be
used to detect barbiturates (sleeping tablets) in the blood.
HPLC differs from traditional chromatography:
 The size of the particles in the solid (stationary phase) is often _________ than
in column chromatography.
 More frequent adsorption and desorption, giving better separation.
 Solvent in pumped under high pressure (can be up to 14 000 kPa)
 A range of solids are available for use in HPLC columns, some with chemicals
specially bonded to their surfaces to improve separation of particular classes of
compounds.
In HPLC, the components are usually detected by passing the eluent stream through a
beam of UV light, the signal is picked up by the detector and the result is a
chromatogram.
The time taken for a component to pass through the column is called the retention time
(Rf). The retention times are used to identify the components associated with the peaks
on a chromatogram. The relative amounts of each component in a mixture may be
determined by comparing the areas under each peak with areas under peaks for
standard samples.
Gas Chromatography
The most sensitive of the chromatographic techniques is gas chromatography (GC). It is
limited to compounds that can be readily vapourised without decomposing, usually with
relative molecular masses less than 300. HPLC can separate compounds with relative
molecular masses of 1000 or more.
The extreme sensitivity of gas chromatography makes it ideal for the analysis of trace
contaminants in samples or for the detection of tiny amounts of very potent compounds.
For example, urine samples for identification of illegal or performance enhancing drugs.
There are two types of gas chromatography:
 Gas-liquid chromatography
 Gas-solid chromatography
Gas chromatography has the following features:
 The mobile phase is generally a gas (nitrogen) called the carrier gas.
 Injection port, where the sample is added.
 The injection port is heated to vapourise the sample
 The column is a loop, or series of loops, of glass that has an internal diameter of
amount 4mm and 2-3 meters long.
 The column is mounted and heated in an oven
 The least soluble are swept out first.
One of the most useful detectors is the flame ionisation detector, invented by an
Australian, Ian McWilliam. Ions produced in the flame are attracted to electrodes and
cause a current to flow.
Interpreting the Chromatogram
The chromatogram shoes the response of the detector against time. As components pass
through the detector the response changes and a peak is formed. Different chemicals
show differing strengths of attraction for the stationary phase and the mobile phase,
and so pass through the column at different rates. The time a component takes to pass
through the column is called the retention time, Rf. The same compound will five the
same retention time if the conditions are the same.
Each component forms one peak in the chromatogram. The peaks can be identified (
____________ analysis) by running a sample containing known pure compounds under
the exact same conditions as the sample.
A compound can also be identified by ‘spiking’ the sample with a known compound.
The concentration of a component in the sample ( _____________ analysis) can be
determined by running a series of standards of known concentration under the same
conditions as the sample. The area under the peak is proportional to the amount of the
substance present.
Example: The concentration of benzene in a sample of petrol was determined by gas
chromatography. A series of standards with an accurately known concentration of
benzene were run under the same conditions as the sample.
The areas under the peaks were measured and a calibration curve was plotted. A line can
be drawn on the calibration graph corresponding to the peak area of the sample and the
concentration read off from the graph. What is the concentration? ____________
Questions: 1, 3, 8, 10, 14,
16, 17, 19 & 20.