1. Techniques of protein purification
chromatography

2. Chromatography
Based on the migration of the mobile phase against the stationary phase
The sample is dissolved in the mobile phase
As the mobile phase moves against the stationary phase the sample interacts with the matrix of the stationary phase
Stationary phases can be different and therefore can exploit different properties of the sample

3. Column chromatography

4. Ion exchange chromatography
Separates molecules according to charge
Ionizable groups are immobilized on support matrices such as cellulose
Commonly used ionizable groups are:
CM – cellulose
Carboxymethyl
DEAE – cellulose
Diethylamino ethyl

5. Ion exchange chromatography

6. Ion exchange chromatography

7. Ion exchange chromatography

8. Ion exchange resins

9. Size exclusion chromatography
Separates molecules according to size and shape
Stationary phase consists of beads with pores
Pores are of a certain size & can only allow only molecules of certain size to enter
Smallest molecular mass to enter the pores of the beads is called the exclusion limit
Molecules that are too large to enter are excluded first ie do not enter the pores

10. Size exclusion chromatography
The smaller molecules able to enter the beads will be excluded later
Fractions collected in different test tubes for further analysis
Affected by shape
Elongated molecules less likely to penetrate the pores compared to spherical ones

11. Size exclusion chromatography

12. Size exclusion chromatography

13. Size exclusion chromatography

14. Affinity chromatography
Exploits different affinities with which materials bind to different stationary phases
Can use antigen – antibody system

15. Affinity chromatography

17. Affinity chromatography

18. High Performance Liquid Chromatography
Faster than other column chromatography types
Requires more expensive equipment
Stainless steel columns – to withstand high pressures
Pressure generating pump – generates pressure to push mobile phase through stationary phase at higher speeds
Solvent degasser – to prevent formation of bubbles which would otherwise damage the column
Precolumn – column before separating column prevents any impurities reaching the separating column
Can use all types of stationary phases
Sample injection devises

19. HPLC device set up

20. Schematic HPLC diagram

21. HPLC Columns

22. Fraction collector

23. Fraction collector

24. Chromatogram

25. Thin layer chromatography

26. Thin layer chromatography

27. Paper chromatography