Chromatographic techniques – I

OBJECTIVE:
After this class, student should be able to understand:
i. the basic principle of chromatography
ii. different types of chromatography
iii. the procedure of paper and thin layer chromatography
iv. applications of paper and thin layer chromatography

INTRODUCTION:
Chromatography is a separation process where a mixture of solute is allowed
to interact with two physically distinct entities, that is, the stationary phase
and the mobile phase. The mixture of solutes is dissolved in a common
solvent and is separated from one another by a differential distribution of
solutes between the stationary and the mobile phase. The stationary phase
can be the solid/gel/liquid/solid-liquid mixture that is immobilised and it
may have the ability to bind some type of solutes on to it. And the mobile
phase can be liquid or gas which passes over the stationary phase.

GENERAL PRINCIPLE OF CHROMATOGRAPHY:

All forms of chromatography work on the same principle. They have a

stationary phase (a solid or liquid supported on a solid) and a mobile phase
(a liquid or a gas).
During the process, the sample containing many molecular components
comes into contact with the stationary phase and the components distribute
themselves between the stationary and the mobile phase. If some
components in the sample are bound to the stationary phase then they will
spend more time in stationary phase and hence their movement in the
chromatographic system will be retarded. On the other hand, the molecules
that show weak affinity/interaction with the stationary phase spends more
time with the mobile phase and are rapidly removed and eluted from the
system.
Thus the rate of migration of the solute depends on the rate of interaction of
it with the stationary and mobile phase.
Distribution or Partition coefficient (Kd) describes the way an analyte
distributes between the two immiscible phases.

Concentration of solute in Stationary phase

Kd =
Concentration of solute in Mobile phase

Thus the difference in Kd value of the components results in their

separation.
And the general process of moving a solute mixture through a
chromatographic system is called development.

CLASSIFICATION OF CHROMATOGRAPHY:

1) Based on mechanism of separation:

i) Adsorption chromatography
ii) Partition chromatography

2) Based on phases:
I) Solid phase chromatography
i) solid- liquid chromatography
ii) solid-gas chromatography
II) Liquid phase chromatography
i) Liquid- liquid chromatography
ii) Liquid-gas chromatography

3) Based on shape of chromatographic bed:

I) Planner chromatography
i) Paper chromatography
ii) Thin layer chromatography
II) Column chromatography
i) Packed column chromatography
 ii) Open tubular column chromatography

So, the concerned topic, that is, the Paper chromatography and thin layer
chromatography are examples of Partition chromatography.

PRINCIPLE OF PAPER CHROMATOGRAPHY AND THIN LAYER

CHROMATOGRAPHY:

For partition chromatography, the stationary phase consists of inert solid

particles coated with liquid adsorbent. The mobile phase is the developing
solution that travels up the stationary phase, carrying samples with it. So, the
substances are distributed between the stationary and mobile phase.
Therefore, the components of the sample will separate readily according to
how strongly they adsorb onto the stationary phase as compared to how
readily they dissolve in the mobile phase. The distribution of the solutes
between two phases is based primarily on solubility differences. The
distribution may be quantified by using the partition coefficient, Kd.

In paper chromatography, the stationary phase is the water molecules

trapped between the cellulose fibres of the paper. Cellulose chromatography
paper is used as the stationary support. The mobile phase travels up the
stationary phase due to capillary action. The mobile phase is generally an
alcohol solvent mixture.
In TLC, the stationary phase is a layer which is as thin as 0.25-2.0 mm and
it is coated on a glass or metal foil plate. The glass or plate is commonly
square or rectangle of 5-20 cm size. The test sample is applied as spot near
the end of the plate. This spot is called the origin.
This plate is placed in a reservoir of mobile phase. Due to capillary action,
the mobile phase moves rapidly across the layer. The silica gel (or the
alumina) is the stationary phase. A pencil line is drawn near the bottom of
the plate and a small drop of a solution of the dye mixture is placed on it.

In both TLC and paper chromatography, the fundamental measurement is

that of the retardation factor (Rf). The retardation factor is defined as the
ratio of the distance travelled by the solute from the origin to the distance
travelled by the solvent from the origin.

Distance travelled by the solute from the origin

Rf =
Distance travelled by the solvent from the origin

If Rf value is Zero, it means that the distance travelled by solute is nil and
the solute remains in the stationary phase and thus it is immobile.
If Rf value is 1, it means that the solute has no affinity with the stationary
phase and travels with the solvent front.
For example,
Rf values depend on the temperature and solvent used in the experiment, so
same compound can have different Rf values in different solvents.

TYPES OF PAPER CHROMATOGRAPHY:

1) Ascending paper chromatography: In this case, the separation of the
sample is against gravity. This is why this is termed as ascending
technique. The mobile phase is at the bottom of the tank. The point of
application of sample is just above the solvent. The mobile phase will
gradually rise upwards and carry the components of the mixture. The
most polar substance will be at the bottom with respect to the tank
whereas the least polar will be on top end of the tank. This is
relatively a slow process as compared to descending technique.
 Fig: Ascending paper chromatography
2) Descending paper chromatography: Here, the development of
chromatogram is done by allowing the solvent to travel down the
paper under the influence of gravity. The mobile phase is placed in
solvent holder at the top. The sample application spot is kept at the top
and the solvent flow down the paper from above.

Fig: Descending paper chromatography

Both descending and ascending chromatography are used for the separation
of organic and inorganic substances. For the detection of the separated
compounds on the chromatogram, the compounds should be coloured.
Several methods can be used to locate the spots, including fluorescence,
radioactivity and treatment with chemicals that develop colours. Universal
reagents like concentrated sulphuric acid or Iodine can be used to colour the
organic molecules black or brown respectively. Specific reagents react with a
particular class of compounds, for example, Rhodamine B is often used for
visualization of lipids; Ninhydrin for visualizing amino acids and Aniline
phthalate for carbohydrates.
The position of each component of a mixture is quantified by calculation the
Rf value. For example in this figure, purple colour component travels 6 cm
and the solvent front travels 10 cm, then Rf value is calculated as 6/10 = 0.6.

Fig: Separated components after paper chromatography

3) Radial paper chromatography: It is also called circular

chromatography. Here a circular filter paper is taken and the sample is
deposited at the centre of the paper. After drying the spot, the filter
paper is tied horizontally on a Petri dish containing solvent, so that the
wick of the paper is dipped in the solvent. The solvent rises through
the wick and the components are separated into concentric circles.

Fig: Radial paper chromatography

4) Two dimensional paper chromatography: The sample is applied to
one of the corners and after the development is performed in the first
run, the filter paper is turned 90o clockwise; another solvent is used
and then performed a second chromatographic run. This will lead to a
satisfactory separation.
 Fig: Two dimensional paper chromatography

PROCEDURE OF THIN LAYER CHROMATOGRAPHY (TLC):

1) Preparation of plates: The stationary phase is applied as slurry with a
uniform layer of thickness. The coated plates are allowed to dry at
room temperature before use. The plate can be dried by heating the
plate at 100-200oC and also the temperature serves to activate the
adsorbent.
2) Application of sample: The sample is dissolved in an organic solvent
and it is applied at about 2 cm from the edge of the plate by using a
micropipette. The solvent can be removed from the spot by gentle
heating with an air dryer. Many different samples can be applied as
discrete spots on a single plate. The sample can also be applied as a
band across the plate for preparative purpose.
3) Plate development: Before the separation process, the tank is allowed
to equilibrate with the mobile phase for about 1 hour with a glass lid
on top. After drying the spot of mixtures, the plate is allowed to stand
in a beaker containing solvent. The solvent layer will be just below
the point of application of the sample. And then the beaker is covered.
The plate is then vertically placed in the tank with the edge near the
origin standing in the liquid. When the mobile phase front approaches
the distant edge of the plate, the plate is removed from the tank and
the separated analytes will be detected.
 Fig: Thin layer chromatography set up

When the solvent travels up the plate, it carries along with it the different
components of the sample. The components travel at different speed and
thus the mixture is separated at different spots. The plate can be removed
from the beaker when the solvent front reaches almost the top of the plate.
And the position of the solvent front is marked with another line before the
solvent evaporates.

Fig: Separation of a mixture of compounds by TLC.

4) The separated components can be identified by calculating the Rf

value and then comparing with the standards.

DETECTION OF ANALYTES:
If the compounds are colourless, then the detection can be done in a number
of ways.
1) by spraying the plate with a specific reagent that converts the analyte
to a coloured product. For example, during the separation of a mixture
of amino acids, ninhydrin is sprayed on the dried chromatogram.
 Ninhydrin reacts with amino acids to give coloured compounds,
mainly brown or purple. So after spraying ninhydrin the spots of
amino acids will be visible.

Fig:

2) by incorporating a fluorescent dye into the stationary phase so that

when the plate is examined under ultraviolet light the analytes show as
blue, green or black spots against a fluorescent background and thus
the separated components can be visually detected.

Fig:

3) by using radiolabelled analytes and subjected the developed plate to

autoradiography using an X-ray film or by scanning the plate with a
radio-chromatograph scanner.

APPLICATION OF PAPER CHROMATOGRAPHY AND THIN

LAYER CHROMATOGRAPHY:
1) Paper chromatography is used to estimate free amino acid in tissues.
A quantitative estimation of these fractions was made possible by
spectrophotometric analysis of the colored solution produced by
ninhydrin.
 Suppose we want to find out which particular amino acids are
contained in a given amino acid mixture. In the diagram, the mixture
is represented by M, and the known amino acids are labelled 1 to 5.
Thus after comparing the Rf values of the separated spots with that of
the known amino acids, it is found that the mixture contains amino
acids labelled as 1, 4 and 5.

Fig: identifiation of amino acid using paper chromatography

2) Detection of purity of sample can be carried out by TLC. If any
impurity is detected, then it shows extra spots and thus can be
detected easily.
3) Thin layer chromatography can be employed in purification, isolation
and identification of natural products like volatile oil or essential oil,
fixed oil, waxes, terpenes, alkaloids, glycosides, steroids etc.
4) Reaction mixture can be examined by Thin layer chromatography to
access whether the reaction is complete or not. This method is also
used in checking other separational processes and purification
processes like distillation, molecular distillation etc.

CONCLUSION:
Chromatography is used to separate mixtures of substances into their
components. TLC and Paper chromatography are practically simple, low
cost and ability to separate several test samples simultaneously. It requires a
minute sample size and detection is straightforward. Unknown samples are
applied to a plate with appropriate standards and the chromatography is
developed as a single experiment.

SUMMARY:
Chromatography is a separation process where a mixture of solute is allowed
to interact with two physically distinct entities, that is, the stationary phase
and the mobile phase. All forms of chromatography work on the same
principle. The molecules that show weak affinity/interaction with the
stationary phase spends more time with the mobile phase and are rapidly
removed and eluted from the system. Thus the rate of migration of the solute
depends on the rate of interaction of it with the stationary and mobile phase.
Distribution or Partition coefficient (Kd) describes the way an analyte
distributes between the two immiscible phases. In both TLC and paper
chromatography, the fundamental measurement is that of the retardation
factor (Rf). Colourless compounds can be detection in a number of ways.
Some of them include use of ninhydrin, rhodamine B, Iodine, etc. Due to
their low cost, simple procedure, they have been used to separate and
identify compounds like amino acid or carbohydrate mixtures.

GLOSSARY:
1) Stationary phase: It is the solid/gel/liquid/solid-liquid mixture that is
immobilised and it may have the ability to bind some type of solutes on to it.
2) Mobile phase: The mobile phase can be liquid or gas which passes over
the stationary phase.
3) Retardation factor (Rf): The retardation factor is defined as the ratio of the
distance travelled by the solute from the origin to the distance travelled by
the solvent from the origin.
4) Analytes: It is the sample mixture which is to be separated.

FAQs:

1) What information you get from the Retardation factor value?

Answer – Retardation factor (Rf) is a measure of the separation of a
particular component. It is expressed as
Rf = distance travelled by the solute from the origin /distance travelled by
the solvent from the origin.
Rf is a unit less quantity and lies between 0and 1.A value of 0 indicates no
separation has taken place and 1 represents that the component has moved
entire length along with the solvent front.
2) Why paper chromatography has retained its applicability in the face of
an emergence of advanced instrumental techniques?
Answer- Chromatographic technique of analysis has seen an impressive
growth over time. Such advances have increased laboratory throughputs,
lowered limits of detection and have made forays into new areas of
applications. Paper chromatography has retained its ground till date and is
popular in laboratories across the world. Some of the reasons for this are:
Low cost of analysis and freedom from maintenance. Separated spots are
visible for coloured compounds and colourless compounds can be viewed by
using alternate techniques. Minimum operation and training required.
Solvent consumption is much less as compared to more sophisticated
techniques. Paper chromatography serves as a good demonstration of basic
concepts of separation for school and undergraduate students.
3) I got a long smear on the plate, rather than a discrete spot. Why?
Answer- This happens when the plate is overloaded. Another plate can be
run after diluting the sample by about fivefold.
4) What are the limitations of paper chromatography technique?

Answer- Paper chromatography has some limitations such as: Semi-

quantitative in nature. Overlapping of spots of components having close Rf
values. Higher concentration of components often leads to streaking
instead of well-defined spots. Errors in Rf calculations can result from
uneven flow of solvent front. This can be caused by running out of solvent
at the bottom of the chamber, uneven cutting of the filter paper or
unevenness of the bottom of the development chamber. Improper sample
spotting, spotting below the marked line resulting in dipping into the
solvent or accidental dipping of spot into solvent while inserting the paper
into the solvent chamber.