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Covering the container ensures that the atmosphere in the beaker is saturated with solvent vapour.
Saturating the atmosphere in the beaker with vapour stops the solvent from evaporating as it rises up the
paper. As the solvent rises up the paper, the components of the mixture that are soluble rises with the
solvent (mobile phase) as well and travels at different rates so the mixtures are separated into different
coloured spots. When the solvent stops moving, the solvent front forms and a chromatograph develops.
Some compounds in a mixture travel almost as far as the solvent does; some stay much closer to the base
line. The distance travelled relative to the solvent is a constant for a particular compound as long as allother factors are held constant, that is the type of paper and the exact composition of the solvent. The
distance travelled relative to the solvent is called the R f value. For each compound it can be worked out
using the formula:
Rf =Distance travelled by compound
Distance travelled by solvent
Question: If one component of a mixture travelled 9.6 cm from the base line while the solvent hadtravelled 12.0 cm, what is the R f value for that component?
Rf =Distance travelled by compound
Distance travelled by solvent=
9.6 cm
12 cm= 0.8
In some cases, the separated components are colourless and thus a visualizing agent is needed. Thevisualizing agent functions to convert the colourless components into coloured spots. Amino acids may
be identified in this way. A small drop of a solution of the amino acid mixture is placed on the base line
of the paper, and similar spots of the known amino acids are placed alongside it. The paper is then stoodin a suitable solvent and left to develop. The position of the solvent front is marked in pencil and the
chromatogram is allowed to dry and is then sprayed with a solution of ninhydrin. Ninhydrin reacts with
amino acids to give coloured compounds, mainly brown or purple. Paper chromatography is from of
partition chromatography.
Thin-layer Chromatography
In thin-layer chromatography the stationary phase solid and is usually a thin, uniform layer of silica gel(silicon dioxide, SiO2) or alumina (aluminium oxide, Al2O3) coated onto a piece of glass, metal or rigid
plastic. The mobile phase is a suitable liquid solvent or mixture of solvents. The procedure is similar to
paper chromatography.
In the case of components of the mixture being colourless, the stationary phase is made of a substance
that fluoresces (glows) in UV light. When UV light is shun on the chromatogram, dark spots will beshown to indicate the position of the component on the chromatograph. In the case of amino acids, the
chromatogram is sprayed with a solution of ninhydrin, as in paper chromatography.
More solvent is then added to the column and allowed to drain through the stationary phase under gravity
by opening the tap at the base of the column.
The components of the mixture adsorbs to the surface if the stationary phase to different extents. This
process of washing a compound through a column using a solvent is called elution and the solvent iscalled an eluting solvent or eluent. The time taken a component to emerge at the bottom of the column is
called the retention time. This type of chromatography is most commonly used to:
Extract useful compounds from plant material
Isolate intermediates in organic reactions
Purification of biomolecules Identify and isolate amino acids, peptides and nucleotides
High-performance Liquid Chromatography (HPLC)
This technique is similar to column chromatography but used a high pressure (400 atmosphere) toincrease the rate at which the solvent (mobile phase) passes through the stationary phase unlike columnchromatography that flows due to gravity. HPLC allows for the use of a much smaller particle size for
the column packing material which gives a much greater surface area for interactions between the
stationary phase and the molecules flowing past it. This allows a much better separation of thecomponents of the mixture. The components, once separated is analysed using UV spectroscopy.
The retention time for HPLC will vary depending on:
the pressure used (because that affects the flow rate of the solvent)
the nature of the stationary phase (not only what material it is made of, but also particle size)
the exact composition of the solvent
the temperature of the column
HPLC may be used commercially for the identification of the stimulants theobromine and caffeine.Calibration allows for the concentrations of theobromine and caffeine to be found.
Gas-liquid Chromatography
In gas-liquid chromatography, the mobile phase is a gas such as helium or nitrogen and the stationary
phase is a high boiling point liquid adsorbed onto a solid. The sample is injected into a a heated entrance port using a small syringe, where vaporization occurs. The vapour is carried into the column by the
mobile phase (carrier gas). The syringe needle passes through a thick rubber disc (known as a septum)which reseals itself again when the syringe is pulled out.The injector is contained in an oven whose
temperature can be controlled. It is hot enough so that all the sample boils and is carried into the column
as a gas by the helium (or other carrier gas).
In GLC Different compounds have different retention times. For a particular compound, the retentiontime will vary depending on:
the boiling point of the compound. A compound which boils at a temperature higher than the
column temperature is going to spend nearly all of its time condensed as a liquid at the beginningof the column. So high boiling point means a long retention time.
the solubility in the liquid phase. The more soluble a compound is in the liquid phase, the lesstime it will spend being carried along by the gas. High solubility in the liquid phase means a high
the temperature of the column. A higher temperature will tend to excite molecules into the gas
phase - either because they evaporate more readily, or because they are so energetic that theattractions of the liquid no longer hold them. A high column temperature shortens retention times