Column Chromatography

1. COLUMN
CHROMATOGRAPHY
PRESENTED BY
VED PRAKASH PANDA
M. Pharm (Pharmacology)

2. CONTENT
▶ History and introduction to chromatography
▶ Principle of column chromatography
▶ Requirements for column chromatography
▶ Factors affecting column chromatography
▶ Applications

3. History & introduction to
chromatography
▶ M. Tswett (1906) defined chromatography as the method
in which the components of a mixture are separated on an
adsorbent column in a flowing system.
▶ The international union of pure & applied
chemistry(IUPAC) definition of chromatography:
▶ “Chromatography is a physical method of separation in
which the components to be separated are distributed
between two phases, one of which is stationary, while the
other(mobile phase)moves in a definite direction”

4. Column chromatography
▶ Introduction:
▶ Column chromatography is a separation technique in
which components of mixture is separated by using a glass
column packed with stationary phase and the liquid
mobile phase flowing continuously through the column.
▶ Principle:
▶ Column adsorption chromatography

5. Principle
▶ A solid stationary phase and a liquid mobile phase is used and the
principle of separation is adsorption.
▶ When a mixture of component dissolved in the mobile phase, & is
introduced in to the column, the individual component move with
different rate depending upon the relative affinities.
▶ The compound with less affinity toward the stationary phase
(adsorbent) moves faster and hence eluted out of the column first.
The one with greater affinity towards the stationary phase moves
slower down the column & hence it is eluted later. Thus the
compounds are separated.
▶ The type of interaction between the stationary phase and the solute is
reversible in nature.
▶ The rate of movement of a component(R) is given by:
▶ R = rate of movement of component rate of movement of mobile
phase

6. Practical requirements
▶ 1. Column characteristics & selection
▶ 2. Stationary phases
▶ 3. Mobile phases
▶ 4. Preparation of column
▶ 5. Introduction of sample
▶ 6. Development of column
▶ 7. Detection of components
▶ 8. Recovery of components

7. COLUMN CHARACTERISTICS
▶ The material of the column is mostly good quality neutral glass since it should not
be affected by solvent, acid or alkali.
▶ The column dimension are important for effective column separations.
▶ The length : diameter ratio ranges from 10:1 to 30:1, for more efficiency the
length : diameter ratio ranges from 100:1
▶ The length of the column depends upon:
▶ Number of compounds to be separated
▶ Type of adsorbent used
▶ Quantity of the sample
▶ Affinity of compounds towards the adsorbent used
▶ Better separation will be obtained with a long narrow column than short thick
column because number of theoretical plates will be more.

8. Stationary phase

10. MOBILE PHASE OR ELUENTS OR SOLVENTS
USED
▶ Mobile phase act as a solvent to introduce the mixture into the column as
developer to develop the zones for separation and as an eluent to remove the
pure component out to the column.
▶ Strain(1942) has arranged the solvents in order of eluting power. A grouping
of solvents in order of polarity index is known as eluotrophic series.
▶ Increasing eluting power: Light petroleum ether (petroleum ether, hexane,
heptane), Carbon tetra chloride, Cyclohexane, Carbon disulphide ,Benzene
Toluene, Chloroform, Ether Ethyl acetate, Acetone, Alcohols ,Water, Pyridine
Organic acids, Inorganic acids & bases.

11. Preparation (packing) of 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 dics is
kept on the top, so that the adsorbent layer is not disturbed, during
the introduction of the sample or mobile phase.
▶ There are two types of packing techniques:
▶ 1. Dry packing technique
▶ 2. wet packing technique

12. Dry packing technique
▶ In this technique, the required quantity of adsorbent is
packed in the column in dry form and the solvent allowed
to flow through the column till equilibrium is reached.
▶ Disadvantage:
▶ 1. air bubbles are entrapped in the column
▶ 2. cracks appear in the adsorbent present in the column
▶ 3. hence the uniformity flow character & clear band of
the separated component may not be obtained.

15. Wet packing technique:
▶ This is the ideal technique. The required amount of adsorbent is
mixed with the mobile phase in a beaker and poured in to the column.
▶ The stationary phase settles uniformly in the column.
▶ Advantages:
▶ 1. there is no entrapment of air bubbles.
▶ 2. there will not be any crack in the column of the adsorbent.
▶ 3. the band eluted from the column will be uniform and ideal for
separation.

16. Introduction of sample
▶ The sample which is usually a mixture of component is
dissolved in minimum quantity of the mobile phase.
▶ The entire sample is introduced into the column at once
and get adsorbed on the top portion of the column.
▶ From this zone, the individual samples can be separated
by a process of elution.

17. Development technique (elution)
▶ Elution:
▶ It is a common method used in column chromatography.
In this method a small volume of mixture to be separated
is added on the top of column & mobile phase is allowed
to flow through the column.
▶ There are two elution technique:
▶ 1. Isocratic elution technique
▶ 2. Gradient elution technique

19. Isocratic & Gradient elution technique:
▶ Isocratic elution technique :
▶ In this elution technique, the same solvent composition or solvent of same
polarity is used through out the process of separation.
▶ Ex. Chloroform only
▶ Gradient elution technique :
▶ In this elution technique, 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
of the solvent.
▶ Ex. Initially benzene, then chloroform, then Ethyl acetate etc.

20. Detection of component
▶ 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, different techniques are used;
▶ Using UV/visible detector.
▶ Using fluoresence detector.
▶ Refractive index detector.
▶ By monitoring the fraction by thin layer chromatography.

21. Recovery of components:
▶ The best technique to recover the components by process of elution.
▶ The components are called as eluate, the solvent are called as eleunt.
▶ Recovery is done by collecting different fraction of mobile phase of equal
volume like 10ml, 20ml, with fraction of 10 or 20 min.
▶ Eluting the sample: Components a, b, and c separate as column progresses,
Fractions can be collected in test tubes, vials, beakers.
▶ Analyze the fractions by thin-layer chromatography or by detectors.

22. Factor 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: Dry or wet packing
▶ 6. Quality of solvents: solvents having low viscosities is giving better results.

23. Application of column chromatography
▶ ►Separation of mixture of compounds
▶ ►Purification process
▶ ►Isolation of active constituents
▶ ►Estimation of drugs in formulation
▶ ►Determination of primary and secondary glycosides in
digitalis leaf.
▶ ► separation of diastereomers & separation of
geometrical isomers.

24. Advantages & disadvantages:
▶ Advantages of C.C:
▶ Any type of mixture can be separated & Any quantity of mixture can be
separated
▶ Used as qualitative as well as quantitative analysis
▶ Wider choice of Mobile Phase
▶ Automation is possible
▶ Disadvantages of C.C:
▶ Time consuming
▶ more amount of Mobile Phase are required
▶ To over come this, HPLC is developed with a pump.