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High performance liquid chromatography as introduction
- 1. For Mixing your Video Broadcasting LOGO Don’t write anything hereDon’t write anything here Don’t write anything hereDon’t write anything here High Performance Liquid Chromatography Dr. Deepkumar Joshi Chemistry Department Sheth M. N. Science College, Patan
- 2. For Mixing your Video Broadcasting LOGO Don’t write anything hereDon’t write anything here Don’t write anything hereDon’t write anything here High Pressure Liquid Chromatography Dr. Deepkumar Joshi Chemistry Department Sheth M. N. Science College, Patan
- 3. For Mixing your Video Broadcasting LOGO Don’t write anything hereDon’t write anything here Don’t write anything hereDon’t write anything here • Introduction & Basics • Why need of HPLC ? • Why Pressure ? Why Performance? • Concept & Scope of HPLC • Instrumentation & Working • Separation Mechanism Today’s Discussion
- 4. For Mixing your Video Broadcasting LOGO Don’t write anything hereDon’t write anything here Don’t write anything hereDon’t write anything here • Chromatography is an analytical method used to separate compounds physically prior to measurement • Satisfies the purpose of separation and quantification of target Introduction & Basics
- 5. For Mixing your Video Broadcasting LOGO Don’t write anything hereDon’t write anything here Don’t write anything hereDon’t write anything here History of Chromatography M. Tswett – A Russian Botanist Introduction & Basics Petroleum ether CaCO3 Chlorophylls
- 6. For Mixing your Video Broadcasting LOGO Don’t write anything hereDon’t write anything here Don’t write anything hereDon’t write anything here Some Important Term(s) • Stationary phase • Mobile phase • Chromatogram • Retention Time Introduction & Basics
- 7. For Mixing your Video Broadcasting LOGO Don’t write anything hereDon’t write anything here Don’t write anything hereDon’t write anything here Column Chromatography Column : Diameter – 5cm : Length – 1 metre Packing Material : Silica Particle Size : 100 micrometer Introduction & Basics
- 8. For Mixing your Video Broadcasting LOGO Don’t write anything hereDon’t write anything here Don’t write anything hereDon’t write anything here Drawbacks of Column Chromatography • Separation is not efficient • More time consumption • Wastage of Solvent Introduction & Basics
- 9. For Mixing your Video Broadcasting LOGO Don’t write anything hereDon’t write anything here Don’t write anything hereDon’t write anything here Overcoming the drawbacks • Lesser time consumption • Less solvent • Efficient separation • High sensitivity & resolution • Small sample size (ppm / ppb) Why need HPLC ?
- 10. For Mixing your Video Broadcasting LOGO Don’t write anything hereDon’t write anything here Don’t write anything hereDon’t write anything here High Performance High Pressure Why ‘P’ in HPLC
- 11. For Mixing your Video Broadcasting LOGO Don’t write anything hereDon’t write anything here Don’t write anything hereDon’t write anything here • Pharmaceuticals • Bio-chemicals • Food products • Industrial chemicals • Forensic chemistry • Environmental science Scope of HPLC
- 12. For Mixing your Video Broadcasting LOGO Don’t write anything hereDon’t write anything here Don’t write anything hereDon’t write anything here • Separation of compounds take place as they move at different rates in the column Concept - Mechanism 2 1 column
- 13. For Mixing your Video Broadcasting LOGO Don’t write anything hereDon’t write anything here Don’t write anything hereDon’t write anything here • Due to different interactions between sample and the stationary phase, molecules move at different rate Concept - Mechanism Stationary Phase Stronger interaction Weaker interaction Mobile Phase 2 1
- 14. For Mixing your Video Broadcasting LOGO Don’t write anything hereDon’t write anything here Don’t write anything hereDon’t write anything here Instrumentation Pump Injector Column Oven Detector Mobile Phase Data processor
- 15. For Mixing your Video Broadcasting LOGO Don’t write anything hereDon’t write anything here Don’t write anything hereDon’t write anything here Components of HPLC instrument • Solvent delivering reservoir • High pressure pumps • Sample injector • Column oven • Detectors Instrumentation
- 16. For Mixing your Video Broadcasting LOGO Don’t write anything hereDon’t write anything here Don’t write anything hereDon’t write anything here Possible configuration • Isocratic system • Low-pressure gradient system • High-pressure gradient system Instrumentation
- 17. For Mixing your Video Broadcasting LOGO Don’t write anything hereDon’t write anything here Don’t write anything hereDon’t write anything here Isocratic System Instrumentation Simple system with one pump and one solvent reservoir. If more than one solvent is used, solvents should be premixed. Data processor Pump Injector Column Oven Detector Mobile Phase
- 18. For Mixing your Video Broadcasting LOGO Don’t write anything hereDon’t write anything here Don’t write anything hereDon’t write anything here Low-pressure Gradient System Instrumentation •One pump used to control 4 reservoirs •Mixing is done before pump. . low pressure gradientvalve Data processor A B DC Pump Injector Column Oven Detector
- 19. For Mixing your Video Broadcasting LOGO Don’t write anything hereDon’t write anything here Don’t write anything hereDon’t write anything here • Excellent gradient accuracy. • 2-3 pumps required - one pump per solvent used. Data processor pump pump pump A B C Injector Column Oven Detector Mixer High-pressure Gradient System Instrumentation
- 20. For Mixing your Video Broadcasting LOGO Don’t write anything hereDon’t write anything here Don’t write anything hereDon’t write anything here Injector – Manual Instrumentation
- 21. For Mixing your Video Broadcasting LOGO Don’t write anything hereDon’t write anything here Don’t write anything hereDon’t write anything here Detectors • UV-Vis detector • PDA detector • Fluorescence detector • Mass Spectrometer detector Instrumentation
- 22. For Mixing your Video Broadcasting LOGO Don’t write anything hereDon’t write anything here Don’t write anything hereDon’t write anything here Chromatogram tR : Retention time A : Area h : Height tR Signal Time Peak h A
- 23. For Mixing your Video Broadcasting LOGO Don’t write anything hereDon’t write anything here Don’t write anything hereDon’t write anything here • Reverse phase chromatography • Normal phase chromatography • Ion exchange chromatography • Size exclusion chromatography • Affinity chromatography Working
- 24. For Mixing your Video Broadcasting LOGO Don’t write anything hereDon’t write anything here Don’t write anything hereDon’t write anything here Working Stationary phase: Non-polar property Mobile phase : Polar property This combination is defined as Reversed Phase Mode Reversed phase
- 25. For Mixing your Video Broadcasting LOGO Don’t write anything hereDon’t write anything here Don’t write anything hereDon’t write anything here Working Stationary phase: polar property Mobile phase : Non-Polar property This combination is defined as Normal Phase Mode Normal phase
- 26. For Mixing your Video Broadcasting LOGO Don’t write anything hereDon’t write anything here Don’t write anything hereDon’t write anything here Working in Rev. Phase • C18 (ODS) type • C8 (octyl) type • C4 (butyl) type • Phenyl type • TMS type • Cyano type C18H37Si O Si CH3 CH3 Non-polar Reversed phase HPLC • Stationary phase: Non-polar property • Mobile phase: Polar property Stationary Phase
- 27. For Mixing your Video Broadcasting LOGO Don’t write anything hereDon’t write anything here Don’t write anything hereDon’t write anything here Mobile phase • Water + Organic solvent –Methanol –Acetonitrile –Tetrahydrofuran Working in Rev. Phase
- 28. For Mixing your Video Broadcasting LOGO Don’t write anything hereDon’t write anything here Don’t write anything hereDon’t write anything here Hydrophobic Interaction Separation Mechanism A B B B B B A A A A A B Support particle Nonpolar bonded phase Interstitial area (mobile phase) Less polar analyte More polar analyteB A
- 29. For Mixing your Video Broadcasting LOGO Don’t write anything hereDon’t write anything here Don’t write anything hereDon’t write anything here • Sample having carbon (alkyl) chain(s) or aromatic ring(s) are more hydrophobic • Sample having –COOH, -NH2, -OH are less hydrophobic Separation Mechanism
- 30. For Mixing your Video Broadcasting LOGO Don’t write anything hereDon’t write anything here Don’t write anything hereDon’t write anything here Retention Time & Hydrophobicity Separation Mechanism OH OH C18 (ODS) Strong Weak 1 1 2 2
- 31. For Mixing your Video Broadcasting LOGO Don’t write anything hereDon’t write anything here Don’t write anything hereDon’t write anything here Effect of Stationary phase Separation Mechanism C18 (ODS) Strong C8 sample sample sample C4 Medium Weak
- 32. For Mixing your Video Broadcasting LOGO Don’t write anything hereDon’t write anything here Don’t write anything hereDon’t write anything here THANK YOU deepjoshi359@yahoo.co.in http://deepjeejoshi.wix.com/deepjoshi HPLC