Sei sulla pagina 1di 3

Chromatographic Methods of Separation:

 Chromatography is used to analyze small quantities of a mixture of substances which are


chemically similar.
 It involves the partition of components of the mixture between a stationary phase and a
mobile phase
 The mixture to be separated is introduced into the stationary phase, which stays still.
 The mobile phase is then allowed to move over the stationary phase for separation
 Partition depends on the different solubilities of the components in the mobile phase and
the different adsorption forces of the components in the stationary phase
o Adsorption – temporary attraction of molecules of a gas or liquid to a solid
surface
 Components with greater solubilities will dissolve in the mobile phase and move along
with it readily
 Components with stronger adsorption forces will be held onto the stationary phase and
not move along readily with the mobile phase
 The difference in solubilities and adsorption bring about separation
Paper Chromatography:
 A piece of filter paper or chromatography paper is used which consists of stationary
water molecules embedded in a cellulose matrix
 The paper acts as a stationary phase and the mobile phase consists of suitable solvent that
travels up the stationary phase
 The mixture to be separated is spotted up a short distance from one end of the paper (base
line)
 The end below the spot is placed in a suitable solvent, as the solvent moves along the
mixture, it carries the mixture with it.
 Solvent front – the distance thee solvent moves from the base line
 Components of the mixture will be separated readily according to how strongly they
adsorb on the stationary phase and how readily they dissolve in the mobile phase
 If the separated components are colorless, then a visualizing agent can be used to convert
them into colored spots
 The position of certain substances can also be determined by fluorescing under a UV
lamp
 Rf - The ratio of the distance moved by the component of the mixture to the distance
moved by the solvent.
ⅆ𝑖𝑠𝑡𝑎𝑛𝑐𝑒 𝑚𝑜𝑣𝑒ⅆ 𝑏𝑦 𝑎 𝑐𝑜𝑚𝑝𝑜𝑛𝑒𝑛𝑡
o 𝑅𝑓 = ⅆ𝑖𝑠𝑡𝑎𝑛𝑐𝑒 𝑚𝑜𝑣𝑒ⅆ 𝑏𝑦 𝑎 𝑠𝑜𝑙𝑣𝑒𝑛𝑡
 Each component has a characteristic 𝑅𝑓 for a given solvent under controlled conditions
i.e. the 𝑅𝑓 values of known substances can be used to identify components of a mixture
 Paper chromatography can be used to analyze mixtures such as dyes in inks, coloring in
food additives and amino acid from protein hydrolysis. A visualizing agent such as
ninhydrin is used to detect amino acids and amines
Thin Layer Chromatography:
 This method is similar to paper chromatography except the stationary phase is a thin
layer of powdered alumina or silica gel which is fixed onto a glass or plastic plate.
 The plates can be coated with a slurry of powdered adsorbent and then oven dried
 The mixture to be analyzed is spotted at the bottom of the plate and the end below the
plate is placed in a suitable solvent
 This solvent is the mobile phase and moves up the plate causing components of the
mixture to partition between the adsorbent on the plate and the moving solvent.
 The separated components may be removed for further analysis by scraping the spots off
the plate
 Thin layer chromatography has the advantage that a variety of adsorbents can be used for
separation. It is commonly used to separate amino acids in blood samples and for the
analysis of food dyes.
Column Chromatography:
 Method is like thin layer chromatography except the stationary phase is packed into a
vertical glass column (1-2cm in diameter)
 A slurry of silica gel or alumina is commonly used for column chromatography
 The mixture to be analyzed is placed at the top of the column
 The mobile phase is a suitable solvent which is added to the top of the loaded column
 The solvent flows down he column under gravity causing components of the mixture to
partition between the adsorbent and the solvent
 Each component emerges from the column at different times and can be collected
separately
 Retention Time – the between the addition of the sample at the top of the column and the
emergence of the component at the boom of the column
 Identical substances will have the same retention time under the same conditions thus
retention times can be used to identify substances
 Column chromatography has the advantage that larger quantities can be separated and
used to prepare compounds, in addition to analyzing them
 This method is used in biochemical research and in hospitals to identify amin acids,
peptides and nucleotides
Gas-Liquid Chromatography:
 This method uses a long column which is usually packed with a stationary phase (inert
powder coated with an involatile oil)
 The column is maintained at a constant, pre-set temperature in an oven
 The mobile phase is an unreactive gas, usually nitrogen or helium and is referred as the
carrier gas.
 The sample to be analyzed must be in the vapor state at the temperature at which the
column is operated
 The vaporized sample is carried through the column by the mobile phase
 The sample is partitioned between the oil and carrier gas and a detector records each
component as it leaves the column at different times
 Emerging components can be fed directly into a mass spectrometer for identification
 The area under each peak produced in the chromatogram of a substance is directly
proportional to the concentration of the component in the mixture
 GLC method of analysis is very sensitive and can be used in forensic testing, to monitor
air and water pollution, to detect and identify traces of pesticides or agricultural
chemicals in foodstuff and to check the dosage of drugs in blood or urine samples.

High performance Liquid Chromatography:


 Technique similar to column chromatography however instead of the gravity feed, high
pressure is used to force the solvent through the column
 Columns are smaller than those used in column chromatography, some being 10-30cm
long and 4mm in diameter
 Retention times are shorter thus rapid analysis is made
 HPLC is used in the industry and hospitals, it is also used to identify suspected
stimulants, doping and drugs that may be present in athletes or racehorses.

Potrebbero piacerti anche