Column chromatography is a separation technique that uses a column packed with a stationary phase and a liquid mobile phase. Components of a mixture are separated as they travel through the column at different rates depending on how strongly they interact with the stationary phase. Factors that affect column chromatography include the type of stationary phase, particle size, flow rate of the mobile phase, column temperature, and concentration of the mixture. Column chromatography is commonly used to purify compounds in chemistry and biochemistry.

1. COLUMN CHROMATOGRAPHY
Bilawal Shaikh
Lecturer, IPS
PUMHSW

2. CONTENTS
 Introduction
 Types of column chromatography
 Principle of column chromatography
 Instrumentation techniques of column
chromatography
 Factors affecting column efficiency
2

3. INTRODUCTION
Chromatography is a powerful and advanced techniques for
separating mixtures. Many types of chromatographic
techniques are known, such as paper, thin layer, column
chromatography, each with its own strength and weakness.
 Chromatography system in general have a stationary phase
and a mobile phase.
 In column chromatography both phases are placed in a
column container, i.e. all the chromatographic operations are
carried out using column.
 Column chromatography in chemistry is a method using for
the Identification, separation and purify individual chemical
compounds from mixtures of compounds in the large amount.
3

4.  Column Chromatography is a separation technique in
which components of mixture is separated by using a
glass column packed with stationary phase and liquid
mobile phase flowing continuously through the column.
 It is suitable for the physical separation of gram quantities
of material. A solvent acts as the mobile phase while a
finely divided solid surface acts as the stationary phase.
 Usually a glass tube with a diameter from 1cm to 10cm
and a height of 20 cm to 50cm with a tap at the
bottom, is used for this purpose.
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5. TYPES OF COLUMN CHROMATOGRAPHY
 Depending upon the flow of solvent down, column
chromatography may be separated into two categories.
Gravity column chromatography
 If the solvent is allowed to flow down the column by
Gravity, or downward process, it is known as gravity
column chromatography .
Flash chromatography
 If the solvent is forced down the column by positive air
pressure , it is called flash chromatography.
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6. 6

7. TYPES OF COLUMN CHROMATOGRAPHY
 1. Based on the difference of the flow rates of the mobile phase,
column chromatography can be of several types:
1. Gravity column chromatography
2. Flash column chromatography
2.1 Low and medium pressure column chromatography
2.2 Vacuum Liquid Chromatography (VLC)
2.3 High Performance/Pressure Liquid Chromatography (HPLC)
7

8.  Low and medium pressure column chromatography:
The movement of mobile phase is accelerated by using
pumps that generate low or medium pressure. The
increase in the flow rate shortens the time of
separation.
8

9.  Vacuum Liquid Chromatography (VLC): The movement
of the mobile phase is accelerated by creating vacuum.
The adsorbent is applied dry into a sintered glass
funnel. The sample is applied by dry method or as
solution. Then the mobile phase is added portion by
portion and vacuum is applied after each portion to
collect each fraction.
9

10.  High Performance/Pressure Liquid Chromatography
(HPLC): In this column very fine silica gel is used, so
separation power is greatly increased. However, the
flow rate of the mobile phase is severely decreased.
High pressure pumps are used to push the solvent
through the column which in this case must be made of
stainless steel.
10

11.  2. Based on types of stationary phase and mobile phase:
 Liquid-solid chromatography: also known as column
adsorption chromatography or classical column
chromatography. Here the stationary phase is solid and the
mobile phase is liquid.
 Liquid-liquid chromatography: also known as column
partition chromatography or partition chromatography. Here
both the stationary and mobile phase is liquid.
11

12. PRINCIPLES OF COLUMN CHROMATOGRAPHY
 Column Adsorption Chromatography
 Partition Chromatography
 Ion-Exchange Chromatography
 Size exclusion chromatography or Gel permeation
chromatography
 Affinity Chromatography
12

13. PRINCIPLE OF COLUMN CHROMATOGRAPHY
 Column adsorption chromatography:
 A solid stationary phase and a liquid mobile phase and is
used and the principle of separation is adsorption.
 It is known that the rate of adsorption varies with a given
adsorbent for different materials.
 Generally , in this techniques , the mixture to be separated
is taken in suitable solvent and small amount of solution is
added to the uniformly packed column with adsorbent. The
sample is allowed to the enter.
 A suitable developing solvent is added on the top of the
column and solvent allowed to run down. 13

14.  Elution development procedure of two solute drug A
and B, here A is more strongly adsorbed than B , and E
is the eluent . B is less absorbing power than A. 14

15.  In this principle, the mixture to be separated is dissolved
in a suitable solvent and allowed to pass through a tube
containing adsorbent.
 The components which has greater absorbing power is
adsorbed in the upper part of the column.
 The next component is adsorbed in the lower portion of
the column which has less adsorbing power than the first
components.
 The process is continued. As a result the materials are
partially separated and adsorbed in the various parts of
the column. The type of interaction between the
stationary phase and the solute is reversible in nature.
15

16. INSTRUMENTATION TECHNIQUES OF
COLUMN CHROMATOGRAPHY
A) Column characteristics
B) Adsorbents
C) Preparation of the adsorption column
D) Packing of the columns
E) Mobile phase used in cc
F) Introduction of sample
G) Separation of compounds
H) Detection of the compounds
I) Recovery of components
16

17. INSTRUMENTATION TECHNIQUES OF COLUMN
CHROMATOGRAPHY
17

18. (A) COLUMN CHARACTERISTICS
The materials of the column is mostly good quality neutral glass since it
should not be affected by solvents ,acids or alkalis.
 It is obtained commercially with spring loaded stopcocks with a
sintered glass disc at the bottom. An ordinary burette can also be
used as column for separation
 The length of the columns depends upon
 Affinity of compounds towards the adsorbent used.
 Number of the compound to be separated .
 Type of adsorbent used .
 Quantity of the sample.
 A stopcock fitted to a column is allow to control the flow rate of solvent
and a sintered glass plate or small plug of glass wool is used at the
bottom to support the adsorbent used in columns.

19. (B)ADSORBENTS
Based upon their adsorbent activity and good filtration property they
can be classified into three parts :-
 Weak adsorbents
 Intermediate adsorbents
 Strong adsorbents
 These adsorbents are sold in different mesh sizes, as indicated by a
number on the bottle label such as silica gel 60 or silica gel 230-
400. The larger the mesh size, the smaller the adsorbent particles.
 Silica gel 70–230 is used in gravity column chromatography
 Silica gel 230–400 is used in flash chromatography.
 The most commonly used adsorbents for column chromatography
are Silica gel (SiO2) and Alumina (Al2O3).

20. (C) PREPARATION OF THE ADSORPTION COLUMN
 At first a plug of cotton wool or glass wool is
placed in the bottom of the column and pressed
down evenly. Then some cleaned, washed sand
can be poured on the top of the plug to form a thin
layer.
 The sand serves to give a flat base to the column
of adsorbent when cotton wool is used instead of
sinter glass.
20

21. 21

22. (D) PACKING OF THE COLUMNS
 The adsorbent is applied to the column in two ways:
 1. Dry packing: In this method the dry adsorbent is poured
to the column directly. Vibration/vacuum pump is the applied
to get rid of air bubbles then the mobile phase is passed
through the adsorbent.
 Demerits:
 Air bubbles are entrapped between mobile phase and
stationary phase
 Column may not be uniformly packed
 Cracks appear in the adsorbent present in the column.
22

23.  2. Slurry packing (Wet method):
 This is the ideal technique.
 Slurry is prepared by taking at first small amount silica (10-20 gm)
& least polar solvent (PE) & mixed well in a beaker & then poured
into the column.
 This process is continued for several times until the stationary phase
is firmly packed in the column.
 Sand may be added after the slurry.
23

24.  PRECAUTION: After setting the adsorbent the excess solvent is
allowed to drain out by the outlet duct but a “head” or layer of
solvent should always cover the adsorbent. Otherwise cracks
will develop in the column and becomes useless for
chromatography because the solvent runs through the cracks
rather than between the particles of adsorbent.
24

25. (E) MOBILE PHASE USED IN CC
 Different solvents are used for this purpose such as:
 1. Petroleum ether
 2. Carbon tetra chloride
 3. Chloroform
 4. Acetone
 5. Benzene
 7. Toluene
 8. Water
 9. Pyridine etc.
 Choice of an eluting solvent is very important for the successful
separation of a mixture of chemical compounds.
 Polar solvents elute polar molecules more rapidly and vice-
versa. 25

26. (F) INTRODUCTION OF SAMPLE
 The sample is placed at the top of the
column (stationary phase) as a band either
by two ways; i.e. Wet application and Dry loading
 1. Wet application: Dissolve the sample in
the initial mobile phase and apply by pipette
to the top of the column. This is very good
method but in most of cases the samples are
not soluble in the initial mobile phase.
 2. Dry loading: Dissolve sample in any
volatile solvent. The sample solution is then
adsorbed on small weight of adsorbent
(stationary phase) and the solvent is allowed
to evaporate. The dry adsorbent loaded with
the sample is then applied to the column.
26

27. ELUTING THE SAMPLE:
 Once the sample solution is added onto the silica, eluent is
added to the top of the column at a rate sufficient to ensure
a “Head” of liquid on the top layer of sand at any point of
chromatogram development.
 The composition of the eluent can be changed as the
column progresses.
 The constituents of the sample will pass down the column
at varying rates.
27

28. (G) SEPARATION OF COMPOUNDS
(a) isocratic elution (same)
(b) Gradient elution ( gradually)
 Isocratic elution technique: Iso means same/similar. In this
elution technique, the same solvent or solvent system of
same polarity is used throughout the process of separation.
E.g.: chloroform, petroleum ether etc.
 Gradient/Stepwise elution technique: The 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. E.g.: Initially benzene then chloroform,
ethyl acetate, methanol etc. 28

29. (H) RECOVERY OF COMPONENTS:
 The eluate is collected as it is released from the
column in small volumes of 5, 10 or 15ml test tubes.
 Analyzing the fractions:
1. Then the fractions are analyzed by thin-layer
chromatography.
2. Similar fractions are combined.
3. If the fraction contains a single compound the solvent
is evaporated to obtain the pure compound.
4. But if the fraction contains more than one component
they are separated using another suitable technique.
29

30. 30

31. (I) DETECTION OF THE COMPOUNDS
 By visual examination of the column: After developing the
chromatogram by running the solvent, different bands of
components are formed. If the bands are colored then we can
detect the component visually if color of the component is
known.
 Eluting the various components with solvents: It is
however more convenient to complete the chromatogram by
eluting the various components with solvents. The eluate is
collected as a large number of fractions, each of small volume.
The large number of fractions assists in obtaining better
separation of components. Each fraction is examined
appropriately for the presence of compounds by:
 By simple spot tests by paper or TLC
 By passing UV light compounds can be detected visually.
 By addition of reagents
31

32. OTHER METHODS OF VISUALIZATION:
1. Measuring changes in pH,
2. Measuring changes in conductivity,
3. Measuring changes in refractive index,
4. Measuring absorption of UV light,
5. Measuring fluorescence (for quinine).
6. Adding fluorescein (0.04%) to the adsorbent and then
viewing the column under UV light. The background will
fluoresce except in those areas where chemicals are present
and they will appear as dark bands.
7. Adding dimethyl amino azobenzene with silica gel to identify
alkaloids. The silica will appear red colored except where the
alkaloids are present.
8. Adding ethereal solution of ferric chloride with alumina to identify
organic compounds which react with ferric chloride to give colored
compounds. 32

33. FACTORS AFFECTING SEPARATIONS
 Factors due to stationary phase:
 Particle size of the stationary phase: Reducing the particle size
increases the surface area and improves separation. However,
reduction of the particle size will decrease the flow rate of the mobile
phase.
In HPLC we use very fine particles to get very good separation. The
flow rate problem is solved by the use high pressure pumps to push
the mobile phase through the stationary phase. Columns are made
of stainless steel to withstand the high pressure.
 Adsorbent activity: The choice of the suitable adsorbent is very
important.
 Uniformity of packing of the column: If the stationary phase is not
packed uniformly then the bands will be irregular and resulting in
poor separation.
 Concentration of the mixture: the proper ratio between sample to
be separated and the amount of stationary phase is very important.
Too much samples resulted in poor separation. 33

34.  Factors due to mobile phase:
 Selection of the proper mobile phase: Very polar
mobile phase will wash out all components without any
separation. On the other hand very non polar mobile
phase will result in broad band and poor separation.
 Rate of flow: Slower flow rate usually resulted in a better
separation and narrower bands.
 Consistency of flow: The continuous flow of the mobile
phase during the whole experiment gives better
separation.
34

35.  Factors due to column:
 Column dimensions: Increasing the length of the
column improve separation. However, that usually
leads to slower flow rate.
 Column temperature: Increasing the temperature
usually reduces the adsorption power of the stationary
phase and increase elution speed. This may leads to
decrease in the separation efficiency.
35

36. COLUMN PARTITION CHROMATOGRAPHY
 The separation of a component in partition
chromatography is based on the partition co-efficient of the
individual component of a mixture between a liquid
stationary phase and gaseous or liquid mobile phase. But
in case of column partition chromatography, the mobile
phase is a liquid.
36

37. PC PROCESS
37

38.  Partition chromatography may be conducted in
either of two ways:
Parameter Normal phase Reversed phase
Stationary phase Polar Non-polar
Mobile phase Non-polar Polar
Compound eluted
first
Non-polar Polar
Compound eluted
last
Polar Non-polar
Example of
stationary phase
Silica gel C18, C8-bonded
phase
Frequently used in CC, TLC HPLC
38

39.  Stationary phase
 In partition column chromatography, solid materials are used solely
as a thin layer to support the liquid stationary phase. Usually the
liquids used as stationary phase is polar in nature. For example,
water or aqueous buffer solution may be used as stationary phase.
Silica gel is the most convenient supporting material for the liquid
stationary phase because it is capable of retaining considerable
volume of liquid phase. Cellulose powder can also be used as
supporting material.
 Organic liquids or solvents may also be used as stationary phase. In
this case, kieselguhr is commonly used as supporting material.
39

40.  Mobile phase
 The solvent system which is used for column adsorption
chromatography may also be used for partition column
chromatography.
 Reversed-phase C18 columns (ODS): octadecyl
carbon chain (C18) is bonded with. Because of steric
effects, not all of the hydroxyl groups of the silica are
derivatized by the ODS reagent, so the remainder are
reacted with trimethylsilane in a process called capping
to reduce adsorption effects.
O
Si
O
O
Si
O
OH
O
Si
O
O
O
Si
O
O
O
R
R
Normal phase
R = H
Reversed phase
R = C18, C8, C4 and more
R
40

41.  Advantages:
 CC is advantageous over most other chromatographic
techniques because it can be used to separate and
purify substantial quantities of components from a
mixture.
 Disadvantages:
 1. Packing of the column requires some technical skill.
 2. The technique is time-consuming and tedious,
especially for larger samples utilizing gravity column
chromatographic technique.
 3. Requires large amount of solvents.
 4. It requires constant attention while the experiment is
being performed.
 5. Collection vessels must be frequently changed and
solvent should be added continuously to the top of the
column at a rate sufficient to cover the adsorbent. 41

42. 42