HPLC AND ITS APPLICATIONS
- 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
- 5. Why use HPLC? High resolution High sensitivity Good repeatability Small sample size Easy to fractionate the sample and purify Non destructive
- 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: 1. Methyl benzoate 2. Ethyl benzoate 3. n- Propyl benzoate 4. n- Butyl benzoate Effect of Stationary phase
- 21. Reversed phase Ion-Pair Chromatogr
- 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
- 30. Choice of liquid chromatography
- 31. Instrumentation of HPLC Column Detector Mobil phase Pump Injection Port Auto-injector
- 32. Isocratic System
- 36. PDA Detector
- 47. Thank you