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Paper Chromatography
Paper chromatography is one of the types of chromatography procedures which
runs on a piece of specialized paper. It is a planar chromatography
systems wherein a cellulose filter paper acts as a stationary phase on which
separation of compounds occurs.
Descending technique :
Just like in ascending technique, the solvent is placed in the base of a sealed
tank or glass jar to allow the chamber to become saturated with the solvent
vapour.
The end of the paper near which the samples are located is held in a trough
at the top of the tank and the rest of the paper allowed to held vertically but
not in a contact with the solvent in the base of the tank.
Development is started by adding the solvent to the trough. Separation of the
sample is achieved as the solvent moves downward under gravity.
Under the influence of these two forces, the sample components are
resolved better than in the descending technique.
Disadvantage:
Based on the above advantages, one can choose the technique which suits
ones purpose most.
Radial Chromatography
In this method, the sample is spotted at the center of a circularly cut disc
of paper which is placed horizontally.
The center of the paper is connected with a wick to the solvent, which is
placed at the base of a jar.
The solvent rise up the wick and thence onto the paper through capillary
action.
The sample components now move outward radially forming concentric
circles of increasing diameters.
If the components to be separated are colored, the chromatogram
developed by this method looks pleasing to the eye.
The resolution of components by this technique is sharper.
The apparatus also is simple.
One way to press the paper between two glass plates with a hole in the
center through which the wick can be connected for solvent supply.
Alternatively, a large circular glass jar covered with a glass plate serves
as a very good chamber.
Developing Solvent
1. Usually in paper chromatography, the stationary phase is water since it is
very well adsorbed by cellulose.
2. The mobile phase, which is less polar than water flows over the stationary
phase.
3. The mobile phase is usually a mixture of various solvents such as alcohols,
acids, esters, ketones, phenols, amines and hydrocarbons, etc.
4. The solvents are selected in such a way that the resolution of sample
components is satisfactory.
5. Apart from optimum separation, other factors that must be considered before
the choice of a solvent system is made is that the number of components in
the solvent system used should be kept as minimum as possible. This is
because the more components a solvent system contains, the more difficult it
will be to maintain a saturated atmosphere in the chamber.
6. In addition, the components of the solvent should be chosen in such a way
that the extent of evaporation of each individual component is more or less
similar. Differing extent of evaporation of the solvent components can
change the composition of the solvent and can lead to serious anomalies of
separation.
7. The temperature chosen for development should also be decided after
careful consideration since individual components of the solvent will differ
in their extent of evaporation at different temperatures.
8. The temperature, therefore, must be maintained within strict limits during
the entire experiment.
9. The solvent system should be so chosen that the two phases are immiscible.
Moreover, the sample components should have differing solubilities in the
two phases. Such a choice would lead to maximum separation. The time
required will also be short and the spreading of the separated zones will also
be minimal.
Detection method
If the substances are colored they are visually detected easily.
But for colorless substances various physical and chemical methods are
employed to detect the spot.
Chemical method: When the components are colorless they can be sprayed
with color producing reagents. For example: in case of amino acids
detection, ninhydrin reagent sprayed on paper reacts with amines and amino
acids to form a blue or purple color. Another
chemical method of detection involves the spraying of iodine. In this
method, presence of lipophilic substance cause the iodine to concentrate in
the substance zone thus giving rise to distinct yellow- brown
chromatographic zones on a lighter yellow background. Apart from these
two reagents there are various chemical reagents such as Dragendroffs
reagent, 3,5 dinitro benzoic acid, etc.,which could be used for the detection
of various alkaloids and cardiac glycosides respectively.