1. The document discusses column chromatography, which is a technique used to separate mixtures based on differential adsorption of substances onto a stationary phase. 2. Key aspects covered include the principle of column chromatography, types of adsorption (e.g. normal phase, reversed phase, ion exchange), factors that influence separation like nature of adsorbent and mobile phase, and applications like purification and analysis of mixtures. 3. Column chromatography works by flowing a mobile liquid phase over a packed stationary solid phase, allowing different substances in a mixture to migrate through the column at different rates based on their relative affinities for the mobile vs. stationary phases.

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Contents
Sl.No Content Page
number
1 Introduction
2 Adsorption phenomenon
3 Principle
4 Migration of solutes
5 Nature of adsorbent forces
6 Types of adsorbent
7 Adsorption Column
8 Packing Technique
9 Retention time and
chromatogram
10 Applications of column
chromatography
11 Conclusion
12 References

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COLUMN CHROMATOGRAPHY
OBJECTIVE
The aim of this assignment is to know how to separate two substances
using column chromatography. As an example, methylene blue and methyl
orange will be separated using an alumina packed column. The separated
substances will then be analyzed spectrophotometrically using a visible
spectrophotometer.
INTRODUCTION
Chromatography is a non destructive process for resolving a
multicomponent mixture into its individual fractions.

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It was discovered by Dr.M.Tswett in 1906.
We already came across paper chromatography, thin layer chromatography
by using chromatography we can separate the mixture of amino acids,
which resembles one another in chemical properties ,can be separated
fairly and rapidly. There are different kinds of different types of
chromatography which differ in the mobile phase and stationary phase.
Column chromatography is type of chromatographic technique was
developed by the American botanist D.T. Day in1900 and M. Tswett in
1906 used absorption columns in their investigations of plant pigments.
Column chromatography is one of the useful methods for separation and
purification of both solids and liquids. Column chromatography is also
known as adsorption chromatography. In this chromatography is of solid-
liquid technique in which the stationary phase is a solid and mobile phase
is liquid.
ADSORPTION PHENOMENON
The column chromatography involves adsorption, partition and ion
exchange phenomenon
1. Adsorption Column Chromatography
Here in this type of chromatography the substances are absorbed by
adsorbent packed in the column
2. Partition Column Chromatography
Here in this type of chromatography the separation of components of
a mixture distribute themselves in different ratios between two
different solvents. The column is packed with silica gel or cellulose
which contain some amount of water.
3. Ion-exchange Column Chromatography

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Here in this type of chromatography the usual solvent is water and
selective desorption of ions is carried out by altering the pH or
concentration of ions in the eluting solvent.
PRINCIPLE
The principle involved in the column chromatography is based on
differential adsorption of substance by the usual adsorbents used in
column chromatography are silica, alumina, calcium carbonate ,calcium
phosphate, magnesia, starch etc.
Selection of solvent is based on the nature of both the solvent and
adsorbent. The rate at which the components of a mixture are separated
depends on the adsorbent and polarity of a solvent. If the activity of the
adsorbent is high and polarity of the solvent very low but gives a good
separation, on the other hand, If the activity adsorbent is low and polarity
of the solvent is high the separation is rapid but gives only poor separation
i.e., the components separated are not 100% pure.
Column chromatography involves a mobile phase flowing over a
stationary phase.
Migration Rates of Solutes
The Partition Coefficient

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An analyte is in equilibrium between the two phases;
Amobile ↔ Astationary MS CCK 
Where the equilibrium constant K is called the partition coefficient.
CS: Molar concentration of analyte in stationary phase
CM: Molar concentration of analyte in mobile phase
The above diagram showing the separation of a mixture of components A
and B by column chromatography
NATURE OF ADSORBENT FORCES1
The adsorbent provides a very large surface area and has the ability to
absorb chemical substances on its surface through some physical and
chemical interactions and they are Van der Waals forces, inductive
forces, hydrogen bonding, charge transfer and covalent bonding.
i. Van der Waals forces
Van der Waals forces hold neutral molecule together in the liquid or
solid state. Adsorption based on this is purely physical in nature
characterized by low adsorption energies and rapid equilibrium takes
place and results in good separation .Adsorption of non-polar solutes
on non polar adsorbents occurs by Van der Waals forces as, for
example, in case of hydrocarbons and graphite.

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ii. Inductive forces
Inductive forces or dipole-dipole attractions arise when a chemical
bond has a permanent electrical field with it (e.g.,C-NO2 , C-Cl etc).
The electrons of an adjacent atom or group or molecule get polarized
under the influence of this field.
This in turn gives rise to an induced dipole-dipole attraction between
the adsorbent and solute. Many adsorption on alumina illustrate
operation of these inductive forces.
iii. Hydrogen bonding
Hydrogen bonding becomes important when the solutes have a
proton donor group which can undergo hydrogen bonding with the
polar groups present at the surface of adsorbent (e.g., the surface
hydroxyl groups possessed by silica or alumina).
These surface hydroxyl groups will themselves act as proton-donor
groups, thus giving rise to hydrogen bonding on coming in contact
with, for example, ethers, nitriles or aromatic hydrocarbons.
iv. Charge transfer
The contribution of charge transfer to adsorption energy is reported
to be very little in this case of most compounds. An adsorbed
complex of the type, (Solute)+ (Adsorbent site) - results by the
transfer of an electron from the solute to a surface site.
v. Covalent bonding
Covalent bonding results to the operation of relatively strong
chemical forces between the solute and the adsorbent. Components
of the mixture obtained by chromatographic separation may not
possess high degree of purity in cases where these strong forces are
operating.

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Types of adsorbents2
Adsorbent is generally an active solid with a large surface area.
Weak adsorbents include talc, sucrose, starch, insulin etc. Intermediate
adsorbents are slaked lime, magnesia, CaCO3, Ca3(PO4)2 , Na2SO4 etc
Strong adsorbents are alumina, bauxite, charcoal etc.
Highly active adsorbents may give rise to irreversible solute adsorption.
Silica gel (acidic) may strongly retain basic compounds whereas alumina
(basic) should not be used for base-sensitive compounds. Equilibrium is
attained as the adsorbed layer consists of a monolayer covering the entire
adsorbent volume (Va) given by,
Va=3.5 ×10-8cm × surface area in cm2/ g-0.01% (H2O)
SOLVENT SYSTEM2
As we know the separation in a column involves adsorption, partition and
ion exchange phenomenon so the choice of solvent depends on these
properties and also depends on polarity and solubility.
For placing the solute on column, developing the chromatogram and
eluting the adsorbed materials, different solvents will be used. Generally a
single solvent is used but in certain cases simultaneous use of two or more
solvents is better. The purity of the solvents is very important because
impurities may slightly affect the column performance.
The role of solvents is very important because mobile phase molecules
compete with solute molecules for polar adsorption sites.

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The stronger the interaction between mobile phase and stationary phase,
the weaker the solute adsorption. Solvent also elute the components of
each separated zones.
The classification of solvents according to their strength of adsorption is
known as elutropic series. The eluting power of solvents is practically
proportional to their dielectric constants.
Eluting power of solvents2
Solvent ϵ293
Petroleum ether 1.90
Benzene 2.28
n-Propanol 21.80
Water 80.40
Chloroform 4.81
Carbon tetrachloride 2.24
Ethanol 25.80
Pyridine 12.40
Formamide 84.0
Absolute alcohol 4.34
Acetone 21.40
Methanol 33.60
Increasing order of polarity of common solvents
Increasing order of polarity of common solvents is: Petroleum ether <
carbon tetrachloride < Cyclohexane <Carbon disulphide <Ether <acetone
<Benzene <Chloroform <Alcohols <Water <Pyridine <Organic acids.
ADSORPTION COLUMN
Adsorption column can be of any size, shape, length or design. Generally
the size of the column is determined by the quantity of the mixture being

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fractioned. The geometry on the column depends on the form and size of
the zones to be separated. The column is commonly made of pyrex glass.
The smaller the diameter of the column, the more effective will be the
separation and the bands will be more distinct.
Chromatographic adsorption apparatus shown below.
PACKING TECHNIQUES2
Packing of the column can be done in two ways
1) Wet packing
For wet packing the column is clamped in a vertical position
and thick slurry of the adsorbent in a suitable medium is
poured through the open end.
It is allowed to settle under gravity until a column of a desired
height is obtained. The tap at the lower end is opened to allow
the liquid to run out until it just covers the top medium. Wet

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packing is common with adsorbents like alumina and
magnesia etc.
2) Dry packing
In dry packing the dry powdered adsorbent is poured though
the open end. Vacuum is created at the bottom and column is
tapped with a light object until no more settling takes place.
Ensure that the top is solid and unbroken.
Retention Time (tR )
The time it takes after sample injection for the analyte peak to reach
the detector is called retention time and its symbol is Rt .
RtLv  MtLu 
where v : average linear rate of analyte migration
L: Length of column packing
u : Average linear rate of movement of molecules of mobile phase
Mt : Time required for an average, molecule of mobile phase to pass
through the column, dead time.
Chromatograms
If a detector that responds to the presence of an analyte is placed at
the end of the column and its signal is plotted as a function of time
(volume of added mobile phase), a series of peaks is obtained. Such a plot,
called a chromatogram, is useful for both qualitative and quantitative
analysis.

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APPLICATIONS OF COLUMN CHROMATOGRAPHY
The applications of column chromatography are:
 In the separation of mixtures into pure individual components.
 Removal impurities and in the purification of compounds.
 The the concentration of substance from dilute solutions such as
those obtained when natural products are extracted with large
volumes of solvents from the leaves, trees, roots and barks.
 Determination of homogeneity of chemical substances.
 Identification of unknown compounds.
 In the separation of geometrical isomers, diastereomers, recemates
and tautomers.

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REFERENCES
 Dr.H.Kaur.”Instrumental methods of chemical
analysis”, 9th
Edition , Pragathi Prakashana
Publication :1020-1026
 R.P. BUDHIRAJA. Copy right 2OO4”Separation
Chemistry”, New Age International (P) Ltd. :79-80