Chromatography

1. Chromatography Department of Pharmaceutical analysis CIPS, Guntur

2. Chromatography • Chromatography is used to separate pure substances from a mixture of substances, such as a cell extract. • It is based on different substances having different solubilities in different solvents. • A simple and common form of chromatography uses filter paper

3. Clip Direction of solvent movement Filter paper cylinder Concentrated spot of chemicals to be separated and identified Solvent Paper chromatography is a technique used for the separation & identification of relatively small chemical substances by a moving solvent on sheets or strips of filter paper Substances to be identified are ‘spotted’ near one end of the filter paper As the solvent moves up the paper, different molecules move at different rates with the smallestmolecules moving the fastest The technique is used for small molecules suchas amino acids, small peptides and sugars

4. Mark the solvent front & allow paper to dry Solvent front Purple spots develop located at different distances from the origin line Amino acid spots origin solvent

5. amino acid spots on the origin line Chromatography separates small molecules in a mixture on the basis of size As the solvent moves up the paper, molecules move at different rates When the spots are colourless (most amino acids), a locating agent is needed to visualise their positions on the chromatography paper

6. The chromatogram can be analysed by measuring the distance travelled by the solvent front, and the distance from the origin to the centre of each spot. • This is used to calculate the Rf (relative front) value for each spot: • An Rf value is characteristic of a particular solute in a particular solvent. It can be used to identify components of a mixture by comparing to tables of known Rf values.

8. X3 X1 X4 X2 X5 Y The Rf value is always a value less than one as the solvent front always moves further than the solute molecules

9. The mixture of unknown amino acids is seen to contain four different amino acids Of these four amino acids, two can be positively identified The mixture contains four amino acids; two unknown together with arginine & leucine

10. Sometimes chromatography with a single solvent is not enough to separate all the constituents of a mixture. • In this case the separation can be improved by two-dimensional chromatography, where the chromatography paper is turned through 90° and run a second time in a second solvent. • Solutes that didn't separate in one solvent will separate in another because they have different solubilities.

12. Solvent front Solvent front Paper dried and rotated clockwise through 90o Mixture of amino acidson origin line Second solvent First solvent Two-way chromatography provides better separation of substances that behave in a similar fashion in the first solvent. A second run in a different solvent resolves two very close spots more clearly

13. Different types of chromatography • Paper chromatography is the simplest, but does not always give very clean separation.

14. Different types of chromatography • Thin layer chromatography (tlc) uses a thin layer of cellulose or silica coated onto a plastic or glass sheet. This is more expensive, but gives much better and more reliable separation.

15. Different types of chromatography • Column chromatography uses a glass column filled with a cellulose slurry. Large samples can be pumped through the column and the separated fractions can be collected for further experiments, so this is preparative chromatography as opposed to analytical chromatography.

16. Different types of chromatography • High performance liquid chromatography (HPLC) is an improved form of column chromatography that delivers excellent separation very quickly.

17. Different types of chromatography • Electrophoresis uses an electric current to separate molecules on the basis of charge. It can also be used to separate on the basis of molecular size, and as such is used in DNA sequencing