Fig X.

1 Thin Layer Chromatography

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Experiment No: X Date:xx-yy-zzzz

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 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 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 base line. 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

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Observations and Calculations

Table X.1 Retention factor for the given protein sample

Distance
Distance Retention
travelled by
Sl.No Sample Spot color travlled by factor
colored
solvent (cm) Rf
component (cm)

1 DL-Serine Purple,brown

2 DL-Valine Red,maroon
L-ornithine
3 Purple,red
monohydrochloride

Model Calculations

Distance travlled by component

Retention factor, Rf = Distance travelled by solvent

1) Rf value of DL-serine =
2) Rf value of DL-valine =
3) Rf value of L-ornithine monohydrochloride =

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MODERATELY POLAR ADDITIVES:

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 50ml

• Hexane 50ml

• A pencil

• A graduate 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 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 ratio of distances between baseline
and solvent front to distance between base line 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-ornithine monohydrochloride, DL-sarine and DL-valine is
determined by measuring the distance travelled by the solvent and component.
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Fig X.2 TLC Slides

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Formula used

𝐷𝑖𝑠𝑡𝑎𝑛𝑐𝑒 𝑡𝑟𝑎𝑣𝑒𝑙𝑙𝑒𝑑 𝑏𝑦 𝑐𝑜𝑚𝑝𝑜𝑛𝑒𝑛𝑡 𝑓𝑟𝑜𝑚 𝑏𝑎𝑠𝑒𝑙𝑖𝑛𝑒

𝑅𝑓 =
𝐷𝑖𝑠𝑡𝑎𝑛𝑐𝑒 𝑡𝑟𝑎𝑣𝑒𝑙𝑙𝑒𝑑 𝑏𝑦 𝑠𝑜𝑙𝑣𝑒𝑛𝑡 𝑓𝑟𝑜𝑚 𝑏𝑎𝑠𝑒𝑙𝑖𝑛𝑒

Result

Rf value for DL-serine=

Rf value for DL-valine =

Rf value for L-ornithine monohydrochloride =

Inference

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Fig X.3 Demonstration of Paper Chromatography

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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).

𝐷𝑖𝑠𝑡𝑎𝑛𝑐𝑒 𝑡𝑟𝑎𝑣𝑒𝑙𝑙𝑒𝑑 𝑏𝑦 𝑐𝑜𝑚𝑝𝑜𝑛𝑒𝑛𝑡 𝑓𝑟𝑜𝑚 𝑜𝑟𝑖𝑔𝑖𝑛𝑎𝑙 𝑙𝑖𝑛𝑒

𝑅𝑓 =
𝐷𝑖𝑠𝑡𝑎𝑛𝑐𝑒 𝑡𝑟𝑎𝑣𝑒𝑙𝑙𝑒𝑑 𝑏𝑦 𝑠𝑜𝑙𝑣𝑒𝑛𝑡 𝑓𝑟𝑜𝑚 𝑜𝑟𝑖𝑔𝑖𝑛𝑎𝑙 𝑙𝑖𝑛𝑒

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. Fig X.3 shows
demonstration of the ascending paper chromatography.

Apparatus required

Chromatography paper, tooth picks, binded paper, beaker, plastic cups, ceramic plate, heat lamp.

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Observations
Table X.2 Retention factor of red and blue dye

Sample Dspot Dsolvent RF RF avg

Blue ink
Blue ink
Red ink
Red ink

Calculations
𝐷𝑖𝑠𝑡𝑎𝑛𝑐𝑒 𝑡𝑟𝑎𝑣𝑒𝑙𝑙𝑒𝑑 𝑏𝑦 𝑐𝑜𝑚𝑝𝑜𝑛𝑒𝑛𝑡 𝑓𝑟𝑜𝑚 𝑜𝑟𝑖𝑔𝑖𝑛𝑎𝑙 𝑙𝑖𝑛𝑒
𝑅𝑓 =
𝐷𝑖𝑠𝑡𝑎𝑛𝑐𝑒 𝑡𝑟𝑎𝑣𝑒𝑙𝑙𝑒𝑑 𝑏𝑦 𝑠𝑜𝑙𝑣𝑒𝑛𝑡 𝑓𝑟𝑜𝑚 𝑜𝑟𝑖𝑔𝑖𝑛𝑎𝑙 𝑙𝑖𝑛𝑒

1) RF value of blue ink =

2) RF value of blue ink =
3) RF value of red ink =
4) RF value of red ink =
RF avg of blue ink = 0.9825
RF avg of red ink = 0.841

Fig X.4 Paper Chromatography

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Procedure

A cellulose fibre paper with dimensions of 8cm × 3cm is chosen as the chromatography paper. The pigments are
loaded on the base line 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

𝐷𝑖𝑠𝑡𝑎𝑛𝑐𝑒 𝑡𝑟𝑎𝑣𝑒𝑙𝑙𝑒𝑑 𝑏𝑦 𝑐𝑜𝑚𝑝𝑜𝑛𝑒𝑛𝑡 𝑓𝑟𝑜𝑚 𝑜𝑟𝑖𝑔𝑖𝑛𝑎𝑙 𝑙𝑖𝑛𝑒

𝑅𝑓 =
𝐷𝑖𝑠𝑡𝑎𝑛𝑐𝑒 𝑡𝑟𝑎𝑣𝑒𝑙𝑙𝑒𝑑 𝑏𝑦 𝑠𝑜𝑙𝑣𝑒𝑛𝑡 𝑓𝑟𝑜𝑚 𝑜𝑟𝑖𝑔𝑖𝑛𝑎𝑙 𝑙𝑖𝑛𝑒

Result

Rf value of blue ink =

Rf value of red ink =

Inference

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