chromatography, types, column chromatography, working principle, applications

2. COLUMN CHROMATOGRAPHY
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Submitted by ;
A.Suruthi I M.sc Biotechnology
Under the guidance
Dr. R.Kalaivani
Assistant Professor of Biotechnology
Bon Secours College for Women, Thanjavur

3. • COLUMN CHROMATOGRAPHY
INTRODUCTION :
• A chromatography column is a device Used in chromatography For
the separation of chemical compounds.
• A chromatography column The stationary phase, allowing the Mobile
phase to pass though it.
• chromatography columns of different types are Used in both Gas and
liquid chromatography.

4. • COLUMN CHROMATOGRAPHY
• Column chromatography Was developed by The American chemist D.T day
In 1990, M.S tswett, the polish botanist, in 1906 used adsorption Columns In
his investigations of Plant Pigments.

5. • COLUMN CHROMATOGRAPHY
• olumn chromatography is one of most useful Methods for The
seperation and purification of both solids and liquids.
• This is a solid-liquid technique in which the Stationary phase Is a solid
and mobile phase is a liquid.

6. • COLUMN CHROMATOGRAPHY
. PRINCIPLE
• Adsorption
• Mixture of Components Dissolved In the M.P is Introduced In to the
column. Components moves depends upon their relatives affinities.
• Adsorbtion Column Chromatography, The Adsorbent, Packed in a
glass column, and a solvent, The mobile phase, That movies Slowly
Through The Packed Column A Solvent Used as a Mobile phase Is
called an elunt.

7. COLUMN CHROMATOGRAPHY

8. • COLUMN CHROMATOGRAPHY
STATIONARY PHASE
• Success Of chromatography depends Upon Proper Selection of S.P, it
depends following :
• 1.Removal Of Impurities.
• 2. No.of components to be seperated.
• 3.Length of the column used.
• 4.Affinities difference between Components.
• 5.Quentity Of adsorption Used.

9. • COLUMN CHROMATOGRAPHY
MOBILE PHASE
• The act as Solvent, develaper and eluent. The function of a mobile
phase are :
• 1.As developing agent
• 2.To introduce the mixture into the column-as Solvent.
• 3. To developing agent.
• 4.To remove pure components Out of the column – as eluent.

10. COLUMN CHROMATOGRAPHY
• Packing technique in Column Chromatography
• there are two types of preparing The column They are:
• 1. Dry packing / dry filling
• 2. Wet packing / wet filling
. The column Should be free form impurity before using Column it shoud
be washed properly and dry it.
• Before Filling Column with Stationary Phase, cotton / glass Wool Is
kept.
• it should be Uniformly filled.

11. COLUMN CHROMATOGRAPHY
Dry packing Technique:
• Adsorbent is backed in the column in dry form
• Fill the solvent, Till Equilibrium Is reached.
• Ofter filling Tapping Can be tone to Remove void Spaces.

12. DRY PACKING TECHNIQUE

13. COLUMN CHROMATOGRAPHY
Wet packing technique :
• Ideal and common technique:
• the meterial is Slurried With solvent and generally added to the
Column in portions.
• S.P Settles Uniformly And no crack In the Column of Adsorbent .
• Solid Settles down while the solvent remain Upward.
• This solvent is removed then again cotton plug is placed.

14. COLUMN CHROMATOGRAPHY
• Application:
• Separation of mixture of Components.
• Purification Process.
• Isolation of active Constituent.
• Estimation of drugs in formulation.
• Determination of Primary and secondary glycosides in digitalis Leaf.
• Sepration of diasteromers.

15. COLUMN CHROMATOGRAPHY
• Advantages:
• Any type of Mixture can be separated.
• Any quantity mixture can be seperated.
• Wider choice of mobile phase.
• Automation is possible.

16. COLUMN CHROMATOGRAPHY
• Disadvantages :
• time Consuming.
• More amount of Mobile phase as requested.
• Automation Makes the technique More complicated And
expensive.