column chromatography details about column preparation

column chromatography

1. COLUMN CHROMATOGRAPHY Presented by - Amartya Nandi M.Pharm (Pharmaceutics)

2. Introduction Chromatography may be defined as a method of separating a mixture of components into individual components through equilibrium distribution between two phases. When a column of stationary phase is used, the technique is called as column chromatography. When the mixture move through a porous medium (called stationary phase) under the influence of some solvent or gas (called moving phase). Elution : Passing of mobile phase through a stationary phase for separation. Eluent : It is the solvent used to separate mixture of components (mobile phase). Elute : Desired solute taken out from the chromatographic column using elution.

3. Principles A solid stationary phase and a liquid mobile phase is used, and the principle of separation is adsorption. The mixture to be separated is dissolved in a suitable solvent and allowed to pass through a tube containing the adsorbent The component which have greater absorbing power is adsorb in the upper part of the column. The next component is adsorbed in lower portion of the column which have less adsorbing power than the first component. The process is continued. As a result, the materials are partially separated and adsorbed in the various part of the column.

4. Types of Adsorbents Use Weak adsorbent ( Sucrose , Starch, talc ) Few components. Different affinities. Longer column. Use Strong adsorbent ( Silica Gel, Activated Alumina, Activated Charcoal) More components. Similar affinities. Shorter column. The most commonly used adsorbent is silica gel of 80-100 mesh or 100-200 mesh size which has a particle size of 60-200 μ.

5. Selection of Stationary phase Stationary phases used in column adsorption chromatography are also known as adsorbents . Requirements of an ideal stationary phase: They should be insoluble in solvents or mobile phases. chemically inert. colorless to facilitate observation of zones. The particle should have uniform size distribution and have spherical particle size :60-200μ

6. Mobile Phase They act as solvent, developer & eluent. The function of a mobile phase are: As developing agent To introduce the mixture into the column - as solvent To remove pure components out of the column - as eluent The choice of the solvent is dependent on the solubility characteristics of the mixture. The solvents should also have sufficiently low boiling points to permit ready recovery of eluted material. Different mobile phases used: Petroleum ether, carbon tetrachloride, cyclohexane, ether, acetone, benzene, toluene, esters, water It can be used in either pure form or as mixture of solvents

7. Preparation of Column Bottom portion of the column - packed with glass wool/cotton wool or may contain asbestos pad. Above which adsorbent is packed After packing a paper disc kept on the top, so that the adsorbent layer do not get disturbed on introduction of sample or mobile phase. PACKING CAN BE: Dry packing Wet packing

8. Dry Packing

9. Dry packing Wet packing

10. Wet packing

11. Introducion 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 the top portion of the column. From this zone, the individual sample can be separated by a process of elution.

12. Devolopment Technique Isocratic elution technique In this elution technique, the same solvent amposition or solvent of the same polarity is used throughout the process of separation. Chloroform only, petroleum ether: Benzene 1: 1 Gradient elution technique In this elution technique, solvents 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. Benzene , Chloroform , Ethyl Acetate , Methanol

13. Application Separation of inorganic ions like copper, cobalt, nickel etc. Separation of tautomers  and racemates. Isolation of metabolites from important components Used for determination of phytomenadione in tablets and injections Determination of flucinolone , acetonide, betamethasone in formulations.

14. 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

15. Thank you