Figure 7.1 Thin Layer Chromatography Demonstration: Page No

Figure 7.
1 Thin layer chromatography demonstration
Page No:
Experiment No: 07 Date:
SEPARATION OF MIXTURES USING CHROMATOGRAPHY

METHODS
A) THIN LAYER CHROMATOGRAPHY
Aim
To separate components of a mixture using thin layer chromatography method.
Theory
Chromatography is the separation of two or more components or ions by the distribution between
two phases, a moving and a stationary phase. Differential polarities of the various components in
the analyte cause differential affinities to the stationary phase, leading to differential adhesion and
thereby separation. The two phases can be solid-liquid, liquid-liquid, or gas-liquid. Thin layer
chromatography (TLC) is an extremely useful technique for monitoring reactions. It is also used to
determine the proper solvent system for performing separations using column chromatography.
TLC uses a solid stationary phase, usually alumina or silica, and a liquid mobile phase, some
solvent whose polarity is chosen based on the analyte.
The stationary phase is chosen in such a way that the individual components of the unknown
mixture interact with the stationary phase in some way. One that interacts more strongly and prefer
to stay associated with the stationary phase will move slowly and will be held back. Species that
interacts weekly move more swiftly through the stream. In this manner the different components
of a mixture can be separated out by the amount of time it takes for them to elute through a
column of stationary phase.
Retention factor (Rf) is a measure of how strong or weak a compound is retained. It is the ratio of
the distance travelled by colored component to the distance travelled by the solvent front. The
value of Rf is unique for a given component, as it depends upon the structure and chemistry of that
compound. The distance travelled also depends on the solvent. In non-polar solvents like pentane
and hexane, most polar compounds will not move, while non-polar compounds will travel some
distance up the plate. In contrast, polar solvents will usually move non-polar compounds off the
baseline. A good solvent system is the one that moves all components of the mixture off the
baseline, but does not put anything on the solvent front. Here is a list of some standard solvents
and their polarity.
VERY POLAR ADDITIVES: Methanol > Ethanol > Iso-Propanol
MODERATELY POLAR ADDITIVES:
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Acetonitrile > Ethyl acetate > Chloroform > Dichloromethane > Diethyl ether > Toluene
NON-POLAR ADDITIVES:
Cyclo-hexane > Petroleum ether > Hexane > Pentane.
Apparatus required
An Oven, A weighing scale, A beaker, Pan or Porcelain plates for resting the plates on and for
putting in the oven, A mortar and pestle, A syringe (10 cc minimum), Glass slides (can also use
sheets of tin or plastic, basically anything stiff that won’t interact with water), Anhydrous
calcium sulfate, plaster of Paris, silica gel. The final materials that are needed for constructing
a developing chamber and developing slides of plant pigments are
● Eye dropper or Pasteur pipette

● Acetone 50 ml
● Hexane 50 ml
● A pencil
● A graduated cylinder
● 5 gm of silica gel
Procedure
1) Silica paste is prepared by adding a known amount of silica (2 g) to 20ml of distilled water.
2) The paste is coated over the glass plate and dried at 80°C for 2 minutes.
3) Baseline is marked by spotter at 1.5 cm from the bottom of the dried Chromatographic plates.
4) A pinch of each Amino acids is mixed with 10 ml of distilled water separately.
5) Finally, the mobile phase (amino acids) is placed in the baseline of the chromatographic plate and
it is kept undisturbed for 45 minutes. The colored spots are observed by adding a few drops of
ninhydrin on the chromatographic plate.
6) From the observed results, the retention factor can be calculated by taking the ratio of distances
between baseline and solvent front to distance between baseline and coloured spot.
Note:
• The eluting solution is prepared by Acetic acid, Acetone, and distilled water in the ratio of
2:1:1 respectively.
• The retention factor of three Amino acids such as L-Arginine hydrochlorine, DL- Phenyl alanine
and DL-valine is determined by measuring the distance travelled by the solvent and
component.
Result
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Observations
Table 7.1 Data for calculation of retention factor for the given protein sample
Distance travelled Distance Retention

Sl.No Sample Spot color by colored travelled by factor
component (cm) solvent (cm) Rf
L-Arginine
1 violet
hydrochlorine
2 DL-Valine Light Red
DL- Phenyl alanine
3 Purple
Model Calculations
Distancetravlled by component
Retention factor, Rf =
Distance travelled by solvent
1) Rf value of L-Arginine hydrochlorine =

2) Rf value of DL-valine =
3) Rf value of DL- Phenyl alanine =
Formula used
Rf =Distance travelled by component ¿ baseline ¿ baseline ¿

Distancetravelled by solvent ¿
Result
Inference
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Figure 7.3 Demonstration of Paper Chromatography
B) PAPER CHROMATOGRAPHY
Aim
To perform paper chromatography to separate the unknown compounds in red and blue dye and
to determine the Rf values of the dyes.
Theory
In paper chromatography, the stationary phase is a special quality paper called chromatography
paper. Mobile phase is a solvent or a mixture of solvents. A solution of the mixture is spotted
on a line about 2 cm above from the bottom of the paper, called original line or base line and
then suspended in a chromatography chamber containing suitable solvent. The solvent rises up
the paper by capillary action and flows over the spot. The paper selectively retains different
components according to their differing partition in the two phases. The paper strip so
developed is called chromatogram. The spots of the separated coloured compounds are visible
at different heights from the position of initial spot on the chromatogram. The spots of the
separated colourless components may be observed either under ultraviolet light or by the use of
an appropriate spray reagent. The distance travelled by the solvent from the original line is
called solvent front. The relative adsorption of each component of the mixture is expressed in
terms of its Retardation or Retention factor (Rf).
Rf =Distance travelled by component ¿ originalline ¿ original line ¿

Distance travelled by solvent ¿
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Ascending and Descending Paper Chromatography: The type of paper chromatography in
which the solvent rises up is called ascending paper chromatography. Alternatively, the solvent
may be taken on the top in a container and be allowed to come down, in which case it is termed
as descending paper chromatography. Figure 7.3 shows demonstration of the ascending paper
chromatography.
Apparatus required
Chromatography paper, toothpicks, binded paper, beaker, plastic cups, ceramic plate, heat
lamp.
Procedure
A cellulose fibre paper with dimensions of 8cm × 3cm is chosen as the chromatography paper.
The pigments are loaded on the baseline of stationary phase then the samples are kept in
mobile phase (pure acetone). The loaded ink pigments move along with mobile phase on
stationary phase and reach solvent front. Finally, the distance travelled by the loaded spot and
solvent front are noted.
Formula used
Rf =Distance travelled by component ¿ originalline ¿ original line ¿

Result
Inference
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Observations
Table 7.2 Data for calculation of retention factor of red and blue dye
Sample Dspot Dsolvent RF RF avg
Black ink
Black ink
Green ink
Green ink
Calculations
Rf =Distance travelled by component ¿ original line ¿ original line ¿

1) RF value of Black ink =

2) RF value of Black ink =
3) RF value of Green ink =
4) RF value of Green ink =
RF avg of Black ink =

RF avg of Green ink =
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Figure 7.

1 Thin layer chromatography demonstration

SEPARATION OF MIXTURES USING CHROMATOGRAPHY

To separate components of a mixture using thin layer chromatography method.

VERY POLAR ADDITIVES: Methanol > Ethanol > Iso-Propanol

MODERATELY POLAR ADDITIVES:

Cyclo-hexane > Petroleum ether > Hexane > Pentane.

● Eye dropper or Pasteur pipette

Distance travelled Distance Retention

1) Rf value of L-Arginine hydrochlorine =

Rf =Distance travelled by component ¿ baseline ¿ baseline ¿

Rf =Distance travelled by component ¿ originalline ¿ original line ¿

Rf =Distance travelled by component ¿ originalline ¿ original line ¿

Sample Dspot Dsolvent RF RF avg

Rf =Distance travelled by component ¿ original line ¿ original line ¿

1) RF value of Black ink =

RF avg of Black ink =

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