HPLC AND ITS APPLICATIONS

USE OF HPLC IN MEDICAL FIELD ,SPECIALLY IN PHARMACOLOGY .

1. HIGH PERFORMANCE LIQUID chromatography DR. AKHTAR ALI FIRST YEAR RESIDENT(PG) DEPARTMENT OF PHARMACOLOGY DR. S.N. MEDICAL COLLEGE JODHPUR ( RAJ.)

2. Basic principal of chromatography

3. Concept of chromatography Chromatography is an analytical method in which compound are physically separated prior to measurement. The main purpose of chromatography is to separate and quantify the target sample in the matrix. chromatography Liquid chromatography Gas chromatography Supercritical-fluid chromatography

4. H istory of chromatography

5. Why use HPLC? High resolution High sensitivity Good repeatability Small sample size Easy to fractionate the sample and purify Non destructive

6. Scope of HPLC

7. Flow Diagram of HPLC

8. Chromatogram

9. Separation mechanism Compound are separated because the molecules move at different rates in the column. Due to different interaction between stationary phase and different sample , the molecules move at different rate, therefore separation can be done.

10. Separation modes Normal phase chromatography Reversed phase chromatography Ion chromatography Size exclusion chromatography

11. Normal Phase chromatography First developer used - CaCO3 as separation column - Petroleum ether as solvent Stationary phase: Polar property Mobil phase: Non-polar property This combination is defined as Normal phase chromatography

12. Normal Phase chromatography Mobil phase: Non-polar Organic solvent: Hexane, Benzene, Methylene chloride, Chloroform, Diethyl ether etc. Stationary phase:

13. Reversed Phase chromatography Stationary phase: Non-polar property Mobil phase: Polar property This combination is defined as reversed phase chromatography

14. Staionary phase C18 (ODS) type C8 ( octyl ) type C4 (butyl) type Cyno type TMS type Mobile phase Water/buffer + Organic solvent Organic solvent: Methanol Acetonitrile etc. Buffer: Phosphate buffer Acetate buffer etc. Reversed phase chromatography

15. What is the interaction

16. Hydrophobicity

17. Retention Time and Hydrophobicity

18. Increase of solvent polarity

19. Effect of stationary phase

20. Peaks: Methyl benzoate Ethyl benzoate n- Propyl benzoate n- Butyl benzoate Effect of Stationary phase

21. Reversed phase Ion-Pair Chromatography

22. Ion-Pair Reagent Anion compounds Tetra-n- butylammonium hydroxide (TBA) Cation compounds Butanesulfonic acid sodium salt (C4) Pentaesulfonic acid sodium salt (C5) Hexanesulfonic acid sodium salt (C6) Heptanesulfonic acid sodium salt (C7) Octanesulfonic acid sodium salt (C8) Decanesulfonic acid sodium salt (C10)

23. Reversed Phase Ion Pair

24. Reversed Phase Ion Pair Advantage: Share several features with reversed phase HPLC- column and mobile phase. By selecting ion pair reagent, concentration or pH, selectivity can be improved. Ion and natural compound can usually be done in the same run. Disadvantage: Equilibration time is long. Dedicated columns are often recommended. Beware of ion pair reagent precipitation in organic solvent eg . Methanol, acetonitrile , etc.

25. Polarity of common organic functional groups and solvents Organic solvents: Hexane Carbon tetrachloride Ether Benzene Methylene chloride THF Iso-propanol Chloroform Ethyl acetate Acetonitrile Methanol Water Functional groups: Alkane Alkyl halide Alkene Dienes Ethers Ketones Aldehydes Amines Alcohol Phenols Carboxylic acid Sulfonic acid Non-polar Polar

26. Ion Exchange chromatography Anion exchange Cation exchange

27. Example of anionic and cationic ion chromatography Cationic Anionic

28. Size Exclusion Chromatography Purpose of SEC GPC (Gel Permeation chromatography) - Molecular weight measurement of polymer GFC (Gel filtration chromatography) - Separation of protein

29. Affinity chromatography

30. Choice of liquid chromatography

31. Instrumentation of HPLC Column Detector Mobil phase Pump Injection Port Auto-injector

32. Isocratic System

33. High-pressure gradient system

34. Low-pressure gradient system

35. Selection of Detector

36. PDA Detector

37. Thank you