Techniques in chromatography

There are two basic techniques of chromatography:

Plane chromatography: In plane chromatography the stationary phase is

coated onto a plane surface. There are two variants of plane
chromatography: paper chromatography and thin layer chromatography. In
paper chromatography, the stationary phase is supported by cellulose fibers
of the paper sheet. In thin layer chromatography the stationary phase is
coated onto a glass or plastic surface.
Column chromatography: In this case, the stationary phase is packed into a
glass or plastic column. Each of these techniques has their specific
advantages, application and modes of operation.

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.

Principle of paper chromatography

The principle involved is partition chromatography where in the substances are
distributed or partitioned between to liquid phases. One phase is the water which is
trapped in the filter paper used and other phase is that of mobile phase which
moves over the paper. The compounds in the mixture get separated due to
differences in their affinity towards water (in stationary phase) and mobile phase
solvents (i.e. organic solvent) during the movement of mobile phase under the
capillary action of pores in the paper.

The essential structure of paper

Paper is made of cellulose fibres, and cellulose is a polymer of the simple

sugar, glucose.
 Cellulose chains have -OH groups sticking out all around them. It presents the
same sort of surface as silica gel or alumina in thin layer chromatography.
The cellulose fibers in the paper hold moisture tightly through the formation of
hydrogen bonds. This moisture acts as stationary phase and the mobile phase
solvents moves over it.
The cellulose itself takes a negative charge in company of water. The paper
exhibits weak ion exchange and adsorptive properties.
Modified forms of paper have been produced in which the paper has been
impregnated with alumina, silica gel, ion exchange resin, etc.
While these modifications lead to different mechanisms of separation, the
technique remains the same.

Uses and applications of paper chromatography

1. Paper chromatography is specially used for separation of mixture having
polar and non polar compounds.
2. For separation of amino acids.
3. It is used to determine organic compounds, biochemicals in urine etc.
4. In pharma sector for determination of hormones, drugs.
5. Sometimes used for evaluation of inorganic compounds like salts and
complexes.

Choice of filter paper

Whatman filter papers of different grades like No.1, No.2, No.3, No.4,
No.20, No.40, No.42 etc are used. In general this paper contains 98-99%
of -cellulose, 0.3 1% -cellulose
Factors that governs the choice of paper:
Nature of Sample and solvents used.
 Based on Quantitative or Qualitative analysis.
Based on thickness of the paper.
Modified Papers acid or base washed filter paper, glass fiber type paper.
Hydrophilic Papers Papers modified with methanol, formamide, glycol,
glycerol etc.
Hydrophobic papers acetylation of OH groups leads to hydrophobic
nature, hence can be used for reverse phase chromatography.
Paper impregnated with silica, alumina, or ion exchange resins can also be
made.

Types/modes of Paper chromatography

Based upon the techniques employed for the development of paper
chromatograms, its been divided into various categories:

Ascending technique (Ascending chromatography):

In this type, 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.
After equilibration of the chamber is achieved, the development of the
chromatogram may be started.
If the development is to be performed by ascending technique, the paper is
allowed to hang in or is suspended in a manner that the base of the paper is
in contact with the solvent at the base of the chamber.
The sample spot should be in a position just above the surface of the solvent
so that as the solvent moves vertically up the paper by capillary action,
separation of the sample is achieved.

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.

Advantage of Ascending technique over descending:

Firstly, the set up required for it is very simple.

Secondly, the resolution of sample by ascending technique is somewhat
better as compared to descending technique. This is so because in
ascending chromatography, two forces are acting on the solute:
1. Capillary force which makes the solute move up, &
2. Gravitational force which opposes this movement.

Under the influence of these two forces, the sample components are
resolved better than in the descending technique.

Disadvantage:

Ascending technique is very slow while descending technique on the

other hand is much faster than ascending.

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.

Two- Dimensional Chromatography

This type of chromatography involves the sequential development in two
directions using two different solvents as mobile phase.
The sample is applied as a spot close to one of the corner of the paper and
it is developed in the normal fashion by either ascending or descending
procedure.
The development is continued until the faster moving component or
solvent front approaches the end of the paper.
The paper is then removed and solvent is allowed to evaporate.
This paper is then turned 90 0 and developed for a second time with
another solvent having totally different eluting properties.
Since the two solvents used have different eluting properties, the
distribution coefficients of individual components in them will also
differ.
Thus, components which could not be separated using one solvent alone
can be easily separated by this procedure.
Fig 1 Two-dimensional chromatography. (A) First development in the direction indicated by the arrow
doesnt resolve B and C completely. (B) Second development in the direction at right angles to the first
using a different solvent system resolves all components completely.

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.

Desirable characteristics that a solvent should possess are listed below.

(i) Partition coefficient of substances to be analysed should range from 1-

100 in favor of the aqueous phase.
(ii) The solvent should be removed from the paper and to this effect its
boiling point should ideally be less than 200 0C.
(iii) The solvent should be stable. It should not oxidize when spread over the
paper.

Solvent system (v/v) Ratio Compounds

Butan-1-ol/Acetic 40/10/50 Amino acids
acid/water
Butan-1- 33/33/33 Amino acids
ol/pyridine/water
Methanol/pyridine/water 25/12/63 Amino acids
Propan-1-ol/petroleum 4/96 Plant pigment
ether
Chloroform/petroleum 30/70 Plant pigment
ether
Table 1. Examples of solvent system used in paper chromatography.

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.

Physical method: In this method observation of spots are done under UV

light (usually in the range of 200-400 nm). Other physical method includes
measurement of fluorescence and radioactivity. Usually these methods are
non-destructive method of detection.

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.