1. Dr. BASAVARAJAIAH S. M.
Assistant Professor and Coordinator
P.G. Department of Chemistry
Vijaya College
Bangalore-560 004
CHROMATOGRAPHY-II
(TLC, Paper, and Column)

2. Thin LayerChromatography
Paper chromatography
Column chromatography
CONTENTS:

3. ThinLayerChromatography
Thin-Layer Chromatography (TLC) is a solid-liquid
chromatography and is based on the principle of adsorption.
In this technique of chromatography, the moving liquid phase is
allowed to ascend a thin layer of adsorbent coated onto backing
support like a glass or aluminum strip, or plastic strip.
Thin Layer Chromatography (TLC) is a quick and easy method
for analyzing mixtures of organic compounds.
Introduction:

4. Thin Layer Chromatography Principle
The separation principle of the TLC procedure is based on the
given compound’s relative affinity towards the mobile and the
stationary phase.
The process begins here by moving the mobile phase over the
stationary phase’s surface. During this movement, the higher
affinity compounds gain less speed as compared to the lower
affinity compounds. This results in their separation.
The compounds that are more attracted towards the stationary
phase secure their position at lower levels while others move
towards the higher levels of the surface. So their spots can be seen
accordingly.

5. ThinLayerChromatographyCOMPONENTS
TLC Plates or Stationary Phase: These are used for applying the
thin layer of stationary phase. They are inert or stable in nature. The
layer of stationary phase is kept even throughout these plates for
better analysis. Usually, ready-to-use plates are preferred by the
people conducting experiments.
Mobile Phase: This comprises a solvent (or solvent mixture). The
taken solvent needs to be chemically inert, of the highest possible
purity, and particulate-free. Only then can the TLC spots be able to
develop.

6. TLC Chamber: This is where the thin layer chromatography procedure
takes place. It keeps the dust particles away from the process and does not
let the solvent evaporate. In order to develop the spots appropriately, a
uniform environment is maintained inside this chamber.
Filter Paper: This gets placed inside the chamber after being moistened
with the mobile phase solution. It ensures that the mobile phase rises
uniformly throughout the TLC plate’s length.
Detection of the spots or Visualizing agents: If the components
separated on the TLC are colored, then these can be visualized easily. But if
the components are colorless, then these are made visible by the use of
some reagent called visualization reagents.

7. ThinLayerChromatographyProcedure
Developing the chromatogram: It involves three steps
1. Spotting: First the sample to be analyzed is dissolved in a volatile
(easily evaporated) solvent to produce a very dilute (about 1%) solution.
Spotting consists of using a micropipette to transfer a small amount of
this dilute solution to one end of a TLC plate, in this case, a thin layer of
powdered silica gel that has been coated onto a plastic sheet. The
spotting solvent quickly evaporates and leaves behind a small spot of the
material.

8. Development: Development consists of placing the bottom of the TLC
plate into a shallow pool of a development solvent, which then travels up
the plate by capillary action. As the solvent travels up the plate, it moves
over the original spot. Different components in the original spot, having
different polarities, will move different distances from the original spot
location and show up as separate spots. When the solvent has traveled
almost to the top of the plate, the plate is removed, the solvent front
marked with a pencil, and the solvent is allowed to evaporate (Drying).

9. 3. Visualization: The spots can be directly observed after
development. Because most compounds are colorless, however,
a visualization method is needed. The silica gel on the TLC plate
is impregnated with a fluorescent material that glows under
ultraviolet (UV) light. While under the UV light, the spots can be
outlined with a pencil to mark their locations. The second method
of visualization is accomplished by placing the plate into iodine
vapors for a few minutes.
A= Distance travelled by solvent front.
B= Distance travelled by solute (Analyte).
Rf =B / A

10. Reagents Substances
Iodine vapor General organic and unsaturated compounds
Phosphomolybdic acid General organic compounds
Fluorescein/Bromine General organic compounds
UV light General organic compounds
Sulphuric acid General organic compounds
2,4-Dintrophenylhydrazine Ketones and aldehydes
Bromocresol green Acids
Potassium hydrogen platinate Basic nitrogen or sulfur compounds
Antimony pentachloride in CCl4 Terpeniods
Ninhydrin Amino acids
Dragendroff’s reagent Alkaloids
Aniline phthalate Sugars
Bromthymol blue Lipids
List of some common visualizing agents used in TLC

11. The checking of purity of samples.
The identification of organic compounds.
The separation of inorganic ions.
Monitoring the progress of a reaction.
Monitoring a column chromatographic separation.
Determining the appropriate solvent for a column
chromatographic separation.
Estimation of biomolecules.
Applications of Thin Layer Chromatography:

12. PAPER CHROMATOGRAPHY
In paper chromatography, the stationary phase is a sheet of paper
of suitable texture and thickness, which may sometimes be
impregnated with a liquid phase that is immiscible with the mobile
phase.
Chromatographic separations on paper are usually considerably
slower than on thin-layer plates and the method is, in general, not as
versatile as thin-layer chromatography since the degree of variation
of the stationary phase is much more restricted.
The concept of Rf value referred to in the discussion on thin-layer
chromatography applies equally well to paper chromatography.

13. There are three types of paper chromatography:
(i). Ascending Paper Chromatography
(ii). Descending Paper Chromatography and
(iii). Circular or Horizontal Paper Chromatography.
(i). Ascending Paper Chromatography: Here the solvent travels
in an upward direction of the chromatographic paper. It is used for
the separation of organic and inorganic substances.

14. (ii). Descending Paper Chromatography: The development of
the chromatogram is downwards by allowing the solvent to travel
down the paper. This requires the solvent to be kept at the upper
portion in a trough and the end of paper close to the spotting line is
dipped into a solvent. The solvent flows downwards.

15. (iii). Circular or Horizontal Paper Chromatography: Here a
circular filter paper is taken and the sample is given at the center of
the paper. After drying the spot, the filter paper is tied horizontally
on a Petri dish containing solvent. The solvent rises through the
wick and the component gets separated in form of a concentrate
circular zone.

16. Rf =
Distance travelled by solute
Distance travelled by solvent front
Applications of Paper Chromatography:
The separation of amino acids.
Structural analysis.
Determination of purity of the compounds.
Separation of inorganic cations or complexes.

17. COLUMN CHROMATOGRAPHY
Column chromatography is one of the most useful methods for the
separation and purification of both solids and liquids when carrying
out small-scale experiments.
The separation can be liquid/solid (adsorption) or liquid/liquid
(partition) in column chromatography.
The stationary phase, a solid adsorbent, is usually placed in a
vertical glass column and the mobile phase, is added from the top
and let flow down through the column by either gravity or external
pressure.

18. Column chromatography is advantageous over most other
chromatographic techniques because it can be used in both
analytical and preparative applications.
It can be used to determine the number of components of a
mixture and as well as the separation and purification of those
components.

19. Column Chromatography principle
The main principle involved in column chromatography is the
adsorption of the solutes of the solution with the help of a stationary phase
and afterward separates the mixture into independent components.
At the point when the mobile phase together with the mixture that
requires to be isolated is brought in from the top of the column, the
movement of the individual components of the mixture is at various rates.
The components with lower adsorption and affinity to the stationary
phase head out quicker when contrasted with the greater adsorption and
affinity with the stationary phase. The components that move rapidly are
taken out first through the components that move slowly are eluted out
last.

20. Rf = The distance traveled by solute/ The distance traveled by the solvent

21. Steps involved in the column chromatography:
The steps included in the column chromatography are:
1. Preparation of the column
Mostly the column is comprised of a glass tube with an appropriate
stationary phase.
The bottom end of the column is packed with a glass wool/cotton wool
or an asbestos pad after which the stationary phase is packed.
After packing the column, a paper disc is placed on the top to avoid
the disturbance of the stationary phase during the introduction of the
sample or mobile phase.
The disturbance in the stationary phase (adsorbent layer) leads to the
irregular bands of separation.

22. Two types of preparing the column, known as packing techniques
namely:
Dry packing technique – The amount of absorbent needed is
added as a fine dry powder in the column and the solvent flows
freely through the column until equilibrium is achieved.
Wet packing technique – The slurry of adsorbent is prepared
along with the mobile phase and is poured into the column.
It is regarded as the ideal technique for packaging.
The column should be properly washed and completely dried
before in-use.

23. 2. Introduction of the sample
The sample (a mixture of components) is dissolved in the
minimum amount of the mobile phase.
At one instant, the sample is introduced into the column and on
the top portion of the column, it is absorbed.
Through the elution process, the individual sample can be
isolated from this zone.
3. Elution technique
Through this technique, the individual components are
separated completely from the column.
The process of elution can be carried out by employing two
techniques: Isocratic elution and Gradient elution .

24. Isocratic elution technique – Throughout the procedure, a
solvent of the same polarity or same solvent composition is
utilized. Example: Use of chloroform alone
Gradient elution technique – Throughout the separation
procedure, solvents of gradually increased polarity or increased
elution strength are utilized.
Example: Benzene → Chloroform → Ethyl acetate → Methanol
4. Detection of Components
In case the mixture separated in a column chromatography
procedure are colored compounds, then monitoring the separation
progress is simple.

25. In case the compounds undergoing separation are colorless, then
small fractions of the eluent are sequentially collected in tubes that
are labeled. Thorugh TLC, the composition of each fraction is
determined.

26. Types of Column chromatography:
 Adsorption column chromatography
 Partition column chromatography
 Gel column chromatography
 Ion exchange column chromatography
 Gas Chromatography (GC)
 High-Performance Liquid Chromatography (HPLC)

27. Column chromatography uses:
To isolate active constituents.
To separate compound mixtures.
To remove impurities or carry purification process.
To isolate metabolites from biological fluids.
To estimate drugs in drug formulations or crude extracts.

28. Thank you