Column chromatography

i am hafiz m waseem from vehari
bsc in science college multan
msc in university of education lahore pakistan

1. HAFIZ MUHAMMAD WASEEM lahore Micrometry

2. Column Chromatography-Introduction Advanced Analytical Techniques

3. Introduction Column Chromatography? It is an analytical technique in which the substances to be separated are dissolved in a solvent The sample is introduced on the top of a column which is packed with an adsorbent like silica gel or alumina It passes through the column at different rates that depends on the affinities of the components for the adsorbent and for the solvent as they pass from the column at different times

4. Introduction Each component of a mixture is retained by the stationary phase differently and is thus separated from others Different components run at different speeds through the column depending on polarities of the stationary and mobile phases As a result they elute from the column one by one During the process the eluent is collected in a series of fractions for further analysis as a function of time

5. Introduction Stationary phase must have uniform distribution and free of air bubbles It is commonly used for the preparative applications Main advantage of column chromatography is that it is relatively low cost It is also preferred because stationary phase used in the process is disposable It also prevents cross contamination and degradation of stationary phase

6. Column Chromatography- Principle Advanced Analytical Techniques

7. Principle of CC Principle of CC depends on the type of Column adsorption Column partitioning Column adsorption . It is based on adsorption properties when a mixture of compounds is dissolved in a mobile phase Compounds travel according to their relative affinities for the stationary phase Compounds which have more affinity for stationary phase travel slower

8. Principle of CC Compounds with less affinity for the stationary phase travel faster Column partitioning. Principle involved in CC is the relative solubility When two immiscible liquids are present, a mixture of solutes will be distributed according to their partition co- efficients Component which is more soluble in the stationary phase travels slower as compared with others

9. Principle of CC While the component that is more soluble in mobile phase travels faster Stationary phase used cannot be a liquid A solid support is used on which a thin film or coating of a liquid is made which acts as a stationary phase for the separation Substances with large differences in their partition co- efficients are separated by the simple solvent extraction process

10. Column Chromatography -Components Advanced Analytical Techniques

11. Components of CC Stationary phase. A  number of compounds are used as adsorbents in the column chromatography (Starch , Alumina, Silica, etc.) Adsorbents may be selected depending on particle size and activity of the solid Activity grade is a measure of an adsorbent's attraction for solutes in the sample to be analyzed Complete anhydrous adsorbents have the highest activity grading

12. Mobile phase. It is very important to introduce the mixture into the column for the separation Its choice depends on polarity for the sample and also for the stationary phase It is the best strategy to start elution with a solvent of lower polarity for elution of the components that are weakly adsorbed to the stationary phase These also act as solvents, developers and as eluents Components of CC

13. Column. Dimensions of the column are very important for effective separation It consists of a glass tube with the bottom packed with glass wool, cotton wool, etc. After packing a paper disc is kept on the top so that the adsorbent layer is not disturbed during the introduction of sample Glass should be neutral to avoid the effects of the solvents, acids or alkalies on the process Components of CC

14. Sample injection. A graduated pipette is filled up with the sample mixture and introduced by touching the top of the adsorbent layer having a filter paper with a layer of sand Fraction collector. During the entire chromatography process the eluent is collected in a series of fractions from the column Fractions can be collected automatically by means of fraction collectors Components of CC

15. Column Chromatography-Applications Advanced Analytical Techniques

16. Forensic applications. Used in crime scene testing to determine levels of alcohol, drugs or poisonous substances in the body Arson may be detected which is leading cause of fires and related accidents Food industry. Used for quality control in the food industry by separating and analyzing additives, preservatives , etc. Proteomics. Components of complex mixture of proteins are identified Applications of CC

17. Quality control. It can also be used to separate and detect contaminants such as aflatoxins , heavy metals, etc. Used to test drinking water and to monitor air quality Monitoring of some contaminants in waste oil and pesticides such as DDT in groundwater Analytical chemistry. Used to separate desired compounds after organic synthesis Used to separate natural compounds like alkaloids, etc. Applications of CC

18. Pharmaceutical applications. It is possible to separate compounds during synthesis of drugs as part of its early stage development process Used to identify substances in blood stream for doping or performance enhancing drugs in athletes, etc. Analysis and study of kinetics of drugs in hepatic blood flow and other organs of the body Quality of drugs and screening and identification of drug metabolites Applications of CC