This document provides information about chromatography techniques. It discusses paper chromatography, thin layer chromatography (TLC), and column chromatography. Paper chromatography uses a paper strip as the stationary phase to separate mixture components through selective adsorption. TLC uses a thin layer of adsorbent like silica or alumina coated on a plate. Column chromatography uses a column packed with an adsorbent like silica gel or alumina to separate gram quantities of materials.

2. Group Members
Masoom Shani 41
Abdul karim 12
Mohammad Munawer 23
Mubasher 46
Mohammad Anas 47

4. Introduction
 The word “chromatography” is derived from the two
Greek words,”chroma” and “graphein”.
 "Chroma" meaning “colour ” and "graphein "meaning
“to write”. So, the word “chromatography” means,
“colour writing”. This technique was first discovered by
Tswett in 1903.

5. Definition
 “Chromatography is a physical separation technique
in which the components of a mixture is separated by
differences in their distribution between two phases:
stationary und mobile phase.”
 So, in simple words, we can say that “chromatography
is the physical separation of a mixture into its
individual components”.

6. Purpose
Chromatography is used to:
 Analyze
 Identify
 Purify and
 Quantify the compounds.

7. Phases
There are two phases of chromatography:
 Stationary phase.
 Mobile phase.

8. Stationary phase
 “A phase, which becomes adsorbed on the filter media
or a surface, is called “stationary phase”.
 It may be a liquid or a solid.
 It may be packed in in a column.

9. Examples
 Silica gel
 Alumina
 Filter paper, etc.
are some important stationary phases.

10. Mobile phase
 “The solvent or mixture of solvents used for the
separation of components, in chromatography, is
called “mobile phase”.
OR
 “The phase, which passes over stationary phase, is
called “mobile phase”.
 It may be a liquid or a gas.
 It is also called “eluent”.

11. Examples
 Alcohol
 Water
 Ethanol
 Acetic acid
 Acetone or gas etc.
are some important mobile phases

12. Principle
 This is a separation technique based upon the
difference of adhesive forces (relative affinities) of
solute with stationary and mobile phases.
 The distribution of the components of a mixture
between the phases is governed by distribution co-
efficient KD.
 It can be calculated as:
 KD = Conc. of components in mobile phase
Conc. of components in stationary phase

14. Types
On the basis of stationary phase, chromatography has
two types:
1. Partition chromatography.
2. Adsorption chromatography.

15. Partition Chromatography
 A chromatography, having liquid stationary phase, is
called “partition chromatography”.

16. Example
 “Paper chromatography” is the common example of
partition chromatography.

17. Paper Chromatography
Definition:
 “A chromatography, that uses paper strips or sheets as
the adsorbent stationary phase through which a
solution flows.”
OR
 “ A type of partition chromatography, that uses
selective adsorption on a strip of paper”.

18. Different ways of Paper
Chromatography
 There are three common ways of carrying out this
technique:
 Ascending paper chromatography.
 Descending paper chromatography.
 Radial / circular paper chromatography.
 But we only discuss ascending paper chromatography.

19. Ascending paper
chromatography
Material :
 Paper, pencil, eraser, filter paper, test tube, rubber
stopper, paper clip, metric ruler etc.

20. Procedure
 Take some solvent in a chromatographic tank and
cover it, so that the vapours of solvent homogenize
with the walls of container or tank.
 Take proper cut filter paper and draw a line of 2.5 cm
from the bottom with the help of lead pencil. At the
midpoint, put a drop of sample mixture with the help
of capillary tube. Dry it for about 15-30 minutes.

22. After that suspend the strip in a tank, in such a way that
its bottom should be dipped 1-2 cm in a solvent.

23.  . When the solvent front rises to about 3/4 of the
length of the strip or paper, then remove the strip.
Mark the solvent front with the lead pencil and dry the
strip. When the strip or paper is dried, the pattern on
the paper is called “chromatogram”.
 Each component has a Rf value and it can be
calculated as:
Rf =Distance travelled by component from spot
Distance travelled by solvent from original spot

26. Uses
 Among all the chromatography methods, paper
chromatography is an inexpensive and rapid method that
provides graphic and clear results.
 It is used as a qualitative method for identifying the
components in a mixture.
 The separated spots on the finished and dried chromatogram
can be cut out and re-dissolved to obtain a pure sample of
component of the sample mixtures.
 It is used in several scientific studies in identification of
unknown organic and inorganic compounds from a mixture.

27.  It is used as an analytical chemistry technique for
identifying and separating colored mixtures like
pigments.
 Paper chromatography can be reproduced easily as
long as the conditions are controlled and maintained.

28. Limitations
There are some disadvantages of using paper
chromatography:
1. It cannot be used as a preparative technique because
we can't apply a large sample quantity.
2. It can't be used in quantitative analysis.
3. It doesn't allow the separation of complex mixtures.
4.This is one of oldest method.

29. Adsorption
Chromatography
 “A chromatography, having solid stationary phase,is
called “adsorption chromatography.

30. Examples
 “TLC (thin layer chromatography)” and “column
chromatography” are the common examples of
adsorption chromatography.

31. TLC(Thin layer
chromatography)
“Thin layer chromatography (TLC) is a method for
identifying substances and testing the purity of
compounds. TLC is a chromatography technique to
separate mixtures”. The technique of TLC closely
resembles those of column and paper chromatography

32. Definition
 “A chromatographic technique in which a thin layer of
solid adsorbent, supported on a glass or plastic plate is
used as a stationary phase”.
 Adsorbent used in it are silica and alumina. TLC plates
are commercially available in ready form.

33. Procedure
 In thin layer chromatography, a sheet of glass of plastic
plate is coated with a thin layer of adsorbent (cellulose,
powder alumina, silica). This is done by mixing the
adsorbent with a suitable liquid, usually water, to form a
slurry. This is applied to the sheet of glass or plastic plate by
spreading or dipping. After drying the plate a drop of
mixture to be separated is placed just above one edge which
is then placed in the air tight pool of solvent for
development. After development, the developed spot can
be located by holding the plate, under the UV lamp and
their Rf value can be determined similar to paper
chromatography.

36. Applications
1. Thin layer chromatography is being increasingly used for
qualitative, quantitative and preparative analysis.
 2.The technique of TLC is extremely suited for analysis of trace
components.
 3.In TLC a large number of organic and inorganic compounds
have been separated and identified and whenever possible
quantitatively analysed.
 4.The technique of TLC is very sensitive and gives sharper zones
hence better resolution.
 5. The application of TLC include the detection of by-products in
synthetic processes, determination of the presence of impurity,
peptides, carbohydrates, lipids, steroids, hormones, sterols,
vitamins, pigments and inorganic anions and cations, etc.

37. Limitations
 Although TLC is very simple and convenient technique
it can not tells the difference between enantiomers
and isomers.
 Another disadvantage of TLC is that in order to
identify specific compounds the Rf value for the
compounds of interest must be known beforehand.
 The components of the mixture must be soluble. It is
just qualitative and not quantitative.
 Development of the spot takes less time, so it does not
provide better resolution than paper chromatography.

38. Column Chromatography
Definition:
 “Column chromatography is suitable for the physical separation of gram
quantities of material”.
OR
 The form of liquid chromatography in which a column is used to hold
the stationery phase, whether the stationary phase is solid or liquid”

39. Principle
 When a mixture of mobile phase and sample to be
separated are introduced from top of the column, the
individual components of mixture move with different
rates. Those with lower affinity and adsorption to
stationary phase move faster and eluted out first while
those with greater adsorption affinity move or travel slower
and get eluted out last.
 The solute molecules adsorb to the column in a reversible
manner. The rate of the movement of the components is
given as follows:
 R= Rate of movement of a component / Rate of movement
of mobile phase. i.e; it is the ratio of distance moved by
solute to the distance moved by solvent.

40. Apparatus
 Solvent acts as mobile phase.
 A finely divided solid surface acts as the stationary
phase.
 The stationary phase will adsorb the components of
the mixture to varying degree and adsorbent surface.
This process may be described by a three-way
equilibrium between the sample, the solvent and the
adsorbent.

41. Adsorbents
 The most common solid adsorbents are alumina
(aluminum oxide) and silica gel (silicon dioxide).
Silica gel is slightly acidic while alumina may be acidic,
neutral or basic.

42. Solvent
 The mobile phase or eluent is either a pure solvent or a
mixture of different solvents. It is chosen so that
the retention factor value of the compound of interest
is roughly around 0.2 - 0.3 in order to minimize the
time and the amount of eluent to run the
chromatography.
 For most separations, the solvent should be less polar
than the compounds. The compounds must also be
soluble in the solvent so they are not permanently
adsorbed.

43. Columns
 The columns available in the department are simple
glass tubes, varying in length and diameter.

44. Procedure
 Add the sample to the top of the column, either as a neat liquid,
or dissolved in a minimum amount of the solvent used to pack
the column. The sample should be added directly to the sand
layer.
 Solvent is drawn from the bottom of the column until the level of
the liquid is just above the level of the sand
 The identity of the fractions may be determined by one of several
methods. If the compounds are coloured, they can be seen to
separate and visible spectroscopy can confirm the degree of
separation. For colourless compounds, either TLC or GC may be
used to identify the compounds present in the different
fractions. The preferred method is usually TLC. Once the
desired fractions are identified the solvent may be removed by
rotary evaporation and the compound isolated.

46. Uses
 1. Column chromatography is best suited to separate
active principle from plant materials.
 2. In separation of compounds after organic synthesis
to obtain desired molecule.
 3.To separate or purify natural compound mixtures
like alkaloids, glycosides.

47. Limitations
 1. Properly setting up the column requires some
technical skill and manual dexterity.
 2. It is very time consuming and tedious, especially for
large samples.
 3. Collecting vessels must be frequently switched
and solvent levels need to be topped up