Column Chromatography

2. COLUMN
CHROMATOGRAPHY Faisal Shabir 2012-
ag-3612

3. CONTENTS
 HISTORY
 DEFINATION AND ITS TYPES
 PRINCIPLE
 PROCEDURE OF COLUMN CHROMATOGRAPHY
 FACTORS AFFECTING C .CHROMATOGRAPHY EFFICIENCY
 APPLICATIONS
 ADVANTAGES
 DISADVANTGES

4. Represented by:
Faisal Shabir
Department Of Agronomy
University Of Agriculture Faisalabad,
Pakistan.
Gmail_ Faisal.shabir3612@gmail.com

5. HISTORY
 This chromatography was first demonstrated by
TSWETT in 1906.
 In all the early applications, the procedure was limited
to the separation of coloured substances, hence the
name chromatography .

6. CHROMATOGRAPHY
It is a technique employed for separation of components of mixture by
continuous distribution of components between two phases i.e. one
phase moves (mobile )over the other(stationary) in a continuous
manner.
when chromatography is carried out in column it is called column
chromatography. It is otherwise known as gravity chromatography .
 Column chromatography is one of the most useful methods for the
separation and purification of both solids and liquids.
 This is a solid - liquid technique in which the stationary phase is a solid
& mobile phase is a liquid.

7. TYPES OF COLUMN CHROMATOGRAPHY
 ADSORPTION CHROMATOGRAPHY
 PARTITION CHROMATOGRAPHY
 ION EXCHANGE CHROMATOGRAPHY
 GEL CHROMATOGRAPHY

8. PRINCIPLE
 When a mixture of components dissolved in the mobile phase is
introduced into the column, the individual components move with
different rates depending upon their relative affinities.
 The compound with lesser affinity towards stationary phase moves
faster and it is eluted out of the column first. The one with greater
affinity towards stationary phase moves slower down the column and
hence it is eluted latter. Thus the compounds are separated.

9. Step 1: Prepare the column
o Plug a pipet with a small amount of cotton.
o Use a wood applicator stick to tamp it down lightly.
o Take care that you do not use either too much cotton or pack it too tightly.
You just need enough to prevent the adsorbent from leaking out.
o Add dry silica gel adsorbent, 230-400 mesh.
o Usually the jar is labeled "for flash chromatography."
o One way to fill the column is to invert it into the jar of silica gel and scoop
it out...
o Then tamp it down before scooping more out
o Another way to fill the column is to pour the gel into the column using a 10
mL beaker
PROCEDURE OF COLUMN CHROMATOGRAPHY

10. Step 1: Prepare the column

11. Step 1: Prepare the column

12. Step 1: Prepare the column
When properly packed, the silica gel
fills the column to just below the
indent on the pipet.
This leaves a space of 4-5 cm on
top of the adsorbent for the addition
of solvent.
Clamp the filled column securely to
a ring stand using a small three-
pronged clamp.

13. Step 2: Pre-elute the column
 Add solvent to the top of the silica gel
 The solvent flows slowly down the column
 Monitor the solvent level, both as it flows through the silica gel and the
level at the top
 But make sure it does not go below the top of the silica
 When the bottom solvent level is at the bottom of the column
 The pre-elution process is completed and the column is ready to load

14. Step 2: Pre-elute the column

15. Step 3:
Load the sample onto the silica gel column
 The sample to be purified is dissolved in a small amount of solvent,
such as hexanes, acetone, or other solvent.
 This solution is loaded onto the column
 In this case, if you use the wet method of column loading
 It is critical that you only use a few drops of solvent to load the sample

16. Step 3:
Load the sample onto the silica gel column

17. Step 4: Elute the column
 Force the solvent through the column by pressing on the top of the
Pasteur pipet with a pipet bulb
 The colored bands will travel down the column as the compound is
eluted
 Only force the solvent to the very top of the silica
 Do not let the silica go dry
 Add fresh solvent as necessary.
 The colored bands will travel down the column as the compound is
eluted.

18. Step 4: Elute the column

19. Step 5:
Elute the column with the second elution
solvent
 As soon as the colored compound begins to elute, the collection beaker
is changed
 The process is complicated if the compound is not colored
 In such experiments, equal sized fractions are collected sequentially
 carefully labelling is necessary for later analysis

20. Step 5:
Elute the column with the second elution
solvent

21. Step 6: DETECTION OF COMPONENTS
 If the compounds separated in a column chromatography procedure are
colored, the progress of the separation can simply be monitored
visually.
 If the compounds to be isolated from column chromatography are
colorless. In this case, small fractions of the eluent are collected
sequentially in labelled tubes and the composition of each fraction is
analyzed by TLC (Thin-layer chromatography).

23. FACTORS AFFECTING COLUMN EFFICIENCY
1. Dimension of the column: column efficiency has been improved by
increasing length/width ratio of the column.
2. Particle size of column packing: separation to be improved by
decreasing the particle size of the adsorbent.
3. Activity of the adsorbent
4. Temperature of the column: The speed of the elution increases at
higher temperatures.
5. Packing of the column
6. Quality of solvents: solvents having low viscosities is giving better
results.

24. APPLICATIONS
►Separation of mixture of compounds
►Purification process
►Isolation of active constituents
►Estimation of drugs in formulation
►Isolation of active constituents
►Determination of primary and secondary glycosides in
digitalis leaf.

25. Column Chromatography
 Advantages :
» Any type of mixture can be separated
» Any quantity of mixture can be separated
» Wider choice of Mobile Phase
» Automation is possible.
 Disadvantages :
» Time consuming
» more amount of Mobile Phase are required
» Automation makes the techniques more complicated & expensive.