This document provides an in-depth overview of column chromatography, detailing its principles, methodologies, and practical applications. It discusses the necessity of stationary and mobile phases, the types of column and packing techniques, and various development techniques for component separation. Additionally, it addresses factors affecting efficiency, detection methods, and applications in purifying compounds and separating mixtures into individual components.

1. COLUMN
CHROMATOGRAPHY
Presented by:Anand Sagar Tiwari
M. Pharm (First Semester)
Dept. of Pharmacology
Guided by : Dr. S.M.Malipatil

4. Chromatography
Tswett (1906) stated that chromatography is a method in
which the components of a mixture are separated on
adsorbent column in a flowing system.
IUPAC definition (1993): Chromatography is a physical
method of separation in which the component to be
separated are distributed between two phases one of which
is stationary while the other moves in a definite direction.
Stationary phase may be a solid or a liquid
supported on a solid or gel , the mobile phase may be either a
gas or a liquid.

6. Column Chromatography
Introduction: Column chromatography is a separation
technique ion which a column of stationary phase is used.
The stationary phase used in column chromatography may
be a solid or liquid. Based on the type of stationary phase
used column chromatography may be divided into two
types:-
Adsorption Column Chromatography where solid is
stationary phase.
Partition Column Chromatography where liquid is stationary
phase.

12. PRACTICAL REQUIREMENTS
A. Stationary phase
B. Mobile phase
C. Column characteristics
D. Preparation of column
E. Introduction of sample
F. Development of technique
G. Detection of components
H. Recovery of components

14. Stationary Phase
An adsorbent used in column chromatography
should meet the following criteria :-
The particles should have uniform size distribution and have
spherical shape. Particle size: 60-200µ.
Should have high mechanical stability.
Should be inert and should not react with the solute or other
components.
Insoluble in the solvents or mobile phases used.
It should be colorless to facilitate observation of zones and
recovery of components.
Should allow free flow of mobile phase and should be capable of
separating for wide variety compounds . Most commonly used
adsorbent is 80-100 mesh or 100-200 mesh size.

17. Selection of Stationary phase
1) Removal of impurities
2) No. of components to be separated [ More
components to be separated a strong adsorbent is
used]
3) Affinity differences between components
4) Length of the column used
5) Quantity of adsorbent used[ Adsorbate : Adsorbent
ratio= 1:20 or 1:30]

18. Mobile phase
Mobile phase is very important and they
serve several functions. They act as a solvent,
developer and as a eluent. The functions of a mobile
phase are ;
To introduce the mixture into the column- As solvent
To develop the zones for separation – As developing
agent
To remove pure component out of the column- As
eluent
e.g. Petroleum ether, Carbon tetrachloride, Water etc.

19. Column characteristics
The material of the column is mostly good quality neutral
glass since it should not be affected by solvents, acids and
alkalies .
The column dimensions are important for effective column
separations.
The effective length : diameter ratio ranges from 10:1 to
30:1. The length of the column depends upon :-
1. Affinity of compounds towards the adsorbent used.
2. Number of compounds to be separated.
3. Types of adsorbents used.
4. Quantity of sample

20. Resolution
• The resolution of a quantitative measure of how well two
elution peaks can be differentiated in a chromatographic
separation.
• It is defined as the difference in retention times between
the two peaks, divided by the combined widths of the
elution peaks.
• A resolution value of 1.5 or greater between the two peaks
ensure that the sample components are well separated to a
degree where the area or height of each peak can/may be
accurately measured.
• The equation depends on selectivity(separation factor) ,
efficiency and retention(capacity factor).

24. Column efficiency
• It is expressed by the no. of theoretical plates.
• The no. of theoretical plates is a measure of the
goodness of the column.
• If the retention time is high and peak width is narrow
then it shows excellent chromatograms.
• It is expressed by the equation:
Where, tr= retention time measured from the instant of
injection and W= peak width

25. Contd……
oThe column is considered as being made of theoretical plates
which are parallel layers one above the other.
oSample being introduced into the column the components
distribute themselves in between the stationary phase and
mobile phase according to their partition coefficients.
oThe rate of movement of mobile phase is assumed to be in
dynamic equilibrium with stationary phase and the
components move down the column at a rate given by the
equation:-
R= Rate of movement of component
Rate of movement of mobile phase

26.  The thickness of the theoretical plate is called the
height equivalent to a theoretical plate(HETP).
HETP = Length of Column (L)
Number of theoretical plates (N)
The smaller the HETP the greater is the
number of theoretical plates and greater is the
efficiency of the column. Thus the efficiency of a
column is measured by its number of theoretical plates.

27. HETP value can also be determined by the Van Beemter
equation.
The equation of HETP is often used to describe the
efficiency of column.
An efficient column would have a minimum HETP value.

29. Factors affecting column efficiency
Nature of solvents: The rate of flow of a solvent is
inversely proportional to its viscosity. Hence the solvents
must have less viscosity for effective separation at faster rate
and also for proper elution strength.
Dimension of column: The efficiency can be increased by
means of increasing the length/width ratio . The range is
observed to be 20:1 to 100:1 depending upon the efficiency
required.
Particle size of the adsorbent : The adsorbent activity is
proportional to the surface area of the adsorbent i.e.
adsorbent activity increases with the increase in particle size.
Efficiency can be increased by decrease in particle size.

30. Contd…….
Temperature of the column: The rate of elution can be
increased by increasing the temperature of column , but, it
decreases the adsorbent power of the column . Hence a
compromise is made in between speed of elution and
adsorbent power. As a result difficult soluble samples are
separated at high temperature while others are separated at
room temperatures.
Pressure: Application of high pressure above the column
and lower pressure below the column, the efficiency of the
column can be increased. High pressure above the column is
achieved by maintaining a column of liquid on the top of the
column or by using pressure devices (pumps). Pressure
below the column is decreased by applying vacuum , using
vacuum pump.

31. Preparation of the column
• The bottom portion of the column is packed with cotton wool or
glass wool above which the column of adsorbent is packed.
• After packing the column with the adsorbent a similar paper disc is
kept on the top, so that the adsorbent layer is not disturbed during
the introduction of sample or mobile phase.
• Disturbance in the layer of adsorbent will lead to irregular bands in
separation.
• There are two types of preparing the column called as packing
techniques. They are:-
i. Dry packing technique
ii. Wet packing technique

32. •Dry Packing Technique: Adsorbent is packed in the
column in dry form. Solvent is filled till equilibrium is
reached.
Demerit : Air bubbles are entrapped in between M.P.
and S.P.----- cracks appear in the adsorbent layer.
•Wet Packing Technique: Ideal and common
technique. Adsorbent + M.P. in a beaker and poured
into the column.
 S.P. settles uniformly and no crack in the column of
adsorbent.

38. Introduction of sample
The sample which is usually a mixture of
components is dissolved in minimum quantity of the
mobile phase used for preparing the column or a
solvent of minimum polarity.
The entire sample is introduced into the column at
once and gets adsorbed on to the top portion of the
column.
From this zone the individual samples can be
separated by a process of elution.

39. Development technique
There are mainly three types of Development
technique:-
I. Frontal Analysis
II. Displacement Analysis
III.Elution Analysis

41. Frontal Analysis
• This technique was developed by Tisellius.
• Column is filled with a known amount of adsorbent and the
sample mixture is continuously added to the column.
• If any mixture is added the less adsorbed component leaves
the column in a pure form.
• Thus this method can be used for separation of readily
eluted component from others with greater affinity and is
also termed as preparative method.
• Used to separate relatively smaller qualities of unwanted
substances which are strongly adsorbed than that of desired
components from a given sample.

43. Displacement Analysis
Here the sample mixture is introduced first into the column.
The components in the mixture are separated by running a
solution which consists of a substance more strongly
adsorbed than any of the components in the sample
mixture.
The substance used to separate the components of mixture
is called as displacing agent.
Displacing agent is adsorbed strongly at the top of the
column and spreads downwards forcing out the components
of the sample mixture from adsorption sites . Components
leave the column based on their degree of adsorptivities.

45. Elution Analysis
In this method the sample components are separated by
means of eluting the column with fresh solvent.
A small quantity of mixture is introduced into the column .
Fresh solvent having lesser affinity for stationary phase than
sample components is allowed to run down towards the
column.
Partition coefficients of the components in the sample if
different from the mixture they separate out into bands and
migrate down towards the column at different rates and are
separated.
The components can be separated by means of elution
analysis by employing two different methods.

46. 1) Isocratic Elution Technique : (Iso means same or
similar) In this elution technique , the same solvent
composition or solvent of same polarity is used throughout
the process of separation. e.g. Chloroform only; Pet.ether :
Benzene = 1:1 only etc.
2) Gradient Elution Technique : (Gradient = gradually) In
this process solvents of gradually increasing polarity or
increasing elution strength are used during the process of
separation. Initially low polar solvent is used followed by
gradually increasing the polarity to a more polar solvent.
e.g. Initially benzene, then chloroform, then ethyl acetate,
then to methanol etc.

48. Detection of Components
• The detection of colored components can be done visually .
Different colored bands are seen moving down the column
which can be collected separately.
• But for colorless compounds the technique depends upon
the properties of the components.
• Different properties which can be used are:-
i. Absorption of light using (UV/Vis)- using UV/Vis detector
ii. Flame ionization detector
iii. Evaporation of solvent and weighing the residue
iv. By monitoring the fractions by TLC.

51. Recovery of components
Earlier recovery of the components were done by
cutting the column into several distinct zones.
Later extrusion of the column into zones were done
by using plunger.
The best technique is to recover the components by
a process called as elution.
The recovered fractions are detected by using the
techniques discussed earlier.
If a fraction still contains several components it can
be resolved by using another column.

52. Application
In the separation of the mixtures into the pure individual
components.
Removal of impurities and in the purification of the compounds.
Determination of the homogeneity of the charcoal substances.
Used in the separation of geo-isomers, diastereomers, racemates and
tautomers.
Identification of unknown compounds.
In the separation and identification of inorganic anions and cations.
The concentrated substance from dilute solutions such as those
obtained when neutral are products are extracted with large volumes
of the solvents from the leaves of plants, trees, roots and barks.

53. Contd….
Separation of Geometrical isomer: Winterstein
reported the first chromatographic separation of
cis/trans isomers of bixin and crocetin dimethyl ether .
Later Zechmeister separated cis/trans isomers
carotenoids on calcium carbonate, aluminum oxide
and other adsorbents.
The separation is based on steric factors .
Isomers whose functional group can approach the
surface of the adsorbent more easily and more strongly
adsorbed.

55. Contd…..
Separation of diastereomers : A derivative having an
optically active partner can’t be separated from the latter.
Such separations can be carried out by Column
Chromatography on various adsorbents . e.g. The separation
of diastereomeric 7-chloro-azibicyclo(4:1:0)- heptane has
been done on silica gel using pentane diethyl-ether as a
solvent.
Separation of tautomeric mixtures : Separation of
tautomeric mixtures can be carried at a high temperature by
column chromatography. e.g. The keto and enol forms of the
p-hydroxyl-phenyl pyruvic acid and indole pyruvic acid can
be separated in the liquid phase.

58. Column Partition Chromatography
Introduction: Partition chromatography is based on the
differences in partition coefficients of a mixture between a
liquid stationary phase and a gaseous or liquid mobile phase.
Principle : Some substance shaken with a mixture of two
immiscible liquids distribute themselves between the
immiscible liquids such that the concentration in each liquid
is proportional to the partition coefficient . Thus solvents
having larger difference in their partition coefficients can be
employed to separate the required components from a
mixture.

59. Theory
• In partition chromatography the liquid stationary
phase is supported in the column by means of a solid
material which is polar in character.
• Silica gel and cellulose powder are frequently used as
solid supports.
• The liquid phase should be chosen such that it is
immiscible with the liquid stationary phase.

61. Application
1) It is used for separation of wide range of biological
compounds.
2) Used for the separation of closely related substances
such as amino acids formed in the hydrolysis of
proteins.
3) Also used in separation of closely related aliphatic
alcohols, sugar derivatives and carboxylic acids.

62. Bonded Phase Chromatography

63. • In this technique the stationary phase may be bonded with
the support material.
• This type of partition chromatography in which both the
monomeric as well as polymeric phases have been bonded
to a large no of support materials is called as Bonded Phase
Chromatography.
• Polymeric bonded stationary phases can be produced by
using di- or tri- chlorosilanes in the presence of moisture as a
result of which a polymeric layer is formed at silica surface.
• The main advantage of this technique is to prevent loss of
stationary phase from the separation column and used for
the separation of bioactive molecules.