2. Types of Chromatography
Stationary Phase
• Thin Layer Chromatography
• Paper Chromatography
• Column Chromatography
Mobile Phase
• Liquid chromatography
• Gas Chromatography
Classification according to the force of separation
• Adsorption chromatography.
• Partition chromatography.
• Ion exchange chromatography.
• Gel filtration chromatography.
• Affinity chromatography.
4. Thin layer chromatography
Use of thin layer chromatography was first reported by two Russian
scientists, N.A Izmailov and M.S Schreiber. Later this technique
was developed further by other scientist. Thin layer
chromatography (TLC) depends on the separation principle. The
separation relies on the relative affinity of compounds towards
both the phases. The compounds in the mobile phase move over
the surface of the stationary phase.
The movement occurs in such a way that the compounds which
have a higher affinity to the stationary phase move slowly while
the other compounds travel fast. Therefore, the separation of the
mixture is attained. On completion of the separation process, the
individual components from the mixture appear as spots at
respective levels on the plates. Their character and nature are
identified by suitable detection techniques.
5. • The TLC plate is a thin piece of aluminium coated on one side
with the stationary phase (this phase does not move), an inert
material that does not react chemically with the sample
components. A mobile phase, which consists of a solvent that
moves through the stationary phase, is used to move the
components of the sample up through the stationary phase. A
drop of the sample is placed near the bottom end of the TLC plate.
The bottom end of the TLC plate is then dipped into the solvent
(mobile phase). The solvent creeps slowly up the TLC plate.
• The various components of the sample move along with the
solvent at a rate that depends on how strongly they are bound to
the stationary phase. Strongly bound substances hardly move at
all, a very weakly bound substance may move at almost the same
speed as the solvent. Thus the different components (or fractions)
of the sample are separated into bands (or spots) along the length
of the TLC plate. This separation process is the basis of the
chromatography method
6. Component of TLC
• TLC plates, preferably ready made with a stationary phase: These are stable
and chemically inert plates, where a thin layer of stationary phase is applied on
its whole surface layer. The stationary phase on the plates is of uniform
thickness and is in a fine particle size.
• TLC chamber- This is used for the development of TLC plate. The chamber
maintains a uniform environment inside for proper development of spots. It
also prevents the evaporation of solvents, and keeps the process dust free.
• Mobile phase- This comprises of a solvent or solvent mixture The mobile
phase used should be particulate-free and of the highest purity for proper
development of TLC spots. The solvents recommended are chemically inert
with the sample, a stationary phase.
• A filter paper- This is moistened in the mobile phase, to be placed inside the
chamber. This helps develop a uniform rise in a mobile phase over the length of
the stationary phase.
7. Applications of Thin Layer
Chromatography (TLC)
• In monitoring the progress of reactions
• Identify compounds present in a given mixture
• Determine the purity of a substance.
• Analyzing ceramides and fatty acids
• Detection of pesticides or insecticides in food and water
• Analyzing the dye composition of fibers in forensics
• Assaying the radiochemical purity of
radiopharmaceuticals
• Identification of medicinal plants and their constituents
8. Paper Chromatography
• Invented by Archer John Porter Martin and Richard Laurence
Millington Synge.
• Its development successfully solved the problem of separating
amino acids which are very similar to each other.
• Special papers are usually used for this technique. These
papers should be highly purified.
• The papers used contain sufficient adsorbed water.
• Other liquids as silicone, paraffin oil are used
• Choice paper is dependent on the type of analysis under
investigation
• Whatman chromatographic papers of different types are used.
9. • The principle of separation is mainly partition rather than
adsorption.
• Substances are distributed between a stationary phase and
mobile phase.
• Cellulose layers in filter paper contain moisture which acts as
stationary phase.
• Organic solvents/buffers are used as mobile phase.
• The developing solution travels up the stationary phase
carrying the sample with it.
• Components of the sample will separate readily according to
how strongly they adsorb onto the stationary phase versus how
readily they dissolve in the mobile phase.
10. Steps in Paper Chromatography
Selection of Solid Support
Selection of Mobile Phase
Saturation of Tank
Sample Preparation and Loading
Development of the Chromatogram
Drying of Chromatogram
Detection
11. Application of Paper
Chromatography
• To check the control of purity of pharmaceuticals,
• For detection of adulterants,
• Detect the contaminants in foods and drinks,
• In the study of ripening and fermentation,
• For the detection of drugs and dopes in animals &
humans
• In analysis of cosmetics
• Analysis of the reaction mixtures in biochemical labs
12. Column Chromatography
• Stationary phase is held in a narrow tube through
which the mobile phase is forced under pressure
or under the effect of gravity.
• Column chromatography is a technique which is
used to separate a single chemical compound
from a mixture dissolved in a fluid. It separates
substances based on differential adsorption of
compounds to the adsorbent as the compounds
move through the column at different rates which
allow them to get separated in fractions. This
technique can be used on small scale as well as
large scale to purify materials that can be used in
future experiments. This method is a type of
adsorption chromatography technique.
13. When the mobile phase along with the mixture that needs to be
separated is introduced from the top of the column, the
movement of the individual components of the mixture is at
different rates. The components with lower adsorption and
affinity to stationary phase travel faster when compared to the
greater adsorption and affinity with the stationary phase. The
components that move fast are removed first whereas the
components that move slow are eluted out last. The adsorption
of solute molecules to the column occurs in a reversible
manner.
The rate of the movement of the components is expressed as:
Rf = the distance travelled by solute/ the distance travelled by
solvent
Rf is the retardation factor
14. Column Chromatography Procedure
Before starting with the Column Chromatography Experiment let us understand the different
phases involved.
Mobile phase – This phase is made up of solvents and it performs the following functions:
1. It acts as a solvent – sample mixture can be introduced in the column.
2. It acts as a developing agent – helps in the separation of components in the sample to
form bands.
3. It acts as an eluting agent – the components that are separated during the experiment are
removed from the column
4. Some examples of solvents used as mobile phase based on their polarity are – ethanol,
acetone, water, acetic acid, pyridine, etc.
Stationary phase – It is a solid material which should have good adsorption property and meet
the conditions given below:
1. Shape and size of particle: Particles should have uniform shape and size in the range of 60 –
200μ in diameter.
2. Stability and inertness of particles: high mechanical stability and chemically inert. Also, no
reaction with acids or bases or any other solvents used during the experiment.
3. It should be colourless, inexpensive and readily available.
4. Should allow free flow of mobile phase
5. It should be suitable for the separation of mixtures of various compounds.
15. Column Chromatography Experiment
• The stationary phase is made wet with the help of solvent as the upper level of the
mobile phase and the stationary phase should match. The mobile phase or eluent is
either solvent or mixture of solvents. In the first step the compound mixture that needs
to be separated, is added from the top of the column without disturbing the top level.
The tap is turned on and the adsorption process on the surface of silica begins.
• Without disturbing the stationary phase solvent mixture is added slowly by touching the
sides of the glass column. The solvent is added throughout the experiment as per the
requirement.
• The tap is turned on to initiate the movement of compounds in the mixture. The
movement is based on the polarity of molecules in the sample. The nonpolar
components move at a greater speed when compared to the polar components.
• For example, a compound mixture consists of three different compounds viz red, blue,
green then their order based on polarity will be as follows blue>red>green
• As the polarity of the green compound is less, it will move first. When it arrives at the
end of the column it is collected in a clean test tube. After this, the red compound is
collected and at last blue compound is collected. All these are collected in separate test
tubes.
16. Column Chromatography
Applications
• Column Chromatography is used to isolate
active ingredients.
• It is very helpful in Separating compound
mixtures.
• It is used to determine drug estimation from
drug formulations
• It is used to remove impurities.
• Used to isolation metabolites from biological
fluids.
17. Types of Column Chromatography
• Adsorption column chromatography – Adsorption
chromatography is a technique of separation, in which the
components of the mixture are adsorbed on the surface of the
adsorbent.
• Partition column chromatography – The stationary phase, as
well as mobile phase, are liquid in partition chromatography.
• Gel column chromatography – In this method of
chromatography, the separation takes place through a column
packed with gel. The stationary phase is a solvent held in the
gap of a solvent.
• Ion exchange column chromatography – A chromatography
technique in which the stationary phase is always ion exchange
resin.
