際際滷

際際滷Share a Scribd company logo

High performance liquid chromatography as introduction

Download as PPT, PDF
B
bvocmithilesh

The document discusses high performance liquid chromatography (HPLC). It begins with introducing chromatography and its history. It then discusses why HPLC was developed to overcome limitations of column chromatography, noting that HPLC allows for more efficient separation, uses less solvent, and requires smaller sample sizes. The document goes on to describe key aspects of HPLC, including its instrumentation, components, separation mechanisms, and various modes like reversed phase, normal phase, and ion exchange chromatography. It emphasizes that HPLC is widely used in fields like pharmaceuticals, chemicals, food, and forensics.

1 of 32
Download to read offline
For Mixing your Video Broadcasting LOGO Dont write anything hereDont write anything here Dont write anything hereDont write anything here High Performance Liquid Chromatography Dr. Deepkumar Joshi Chemistry Department Sheth M. N. Science College, Patan
For Mixing your Video Broadcasting LOGO Dont write anything hereDont write anything here Dont write anything hereDont write anything here High Pressure Liquid Chromatography Dr. Deepkumar Joshi Chemistry Department Sheth M. N. Science College, Patan
For Mixing your Video Broadcasting LOGO Dont write anything hereDont write anything here Dont write anything hereDont write anything here Introduction & Basics Why need of HPLC ? Why Pressure ? Why Performance? Concept & Scope of HPLC Instrumentation & Working Separation Mechanism Todays Discussion
For Mixing your Video Broadcasting LOGO Dont write anything hereDont write anything here Dont write anything hereDont 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
For Mixing your Video Broadcasting LOGO Dont write anything hereDont write anything here Dont write anything hereDont write anything here History of Chromatography M. Tswett A Russian Botanist Introduction & Basics Petroleum ether CaCO3 Chlorophylls
For Mixing your Video Broadcasting LOGO Dont write anything hereDont write anything here Dont write anything hereDont write anything here Some Important Term(s) Stationary phase Mobile phase Chromatogram Retention Time Introduction & Basics
For Mixing your Video Broadcasting LOGO Dont write anything hereDont write anything here Dont write anything hereDont write anything here Column Chromatography Column : Diameter 5cm : Length 1 metre Packing Material : Silica Particle Size : 100 micrometer Introduction & Basics
For Mixing your Video Broadcasting LOGO Dont write anything hereDont write anything here Dont write anything hereDont write anything here Drawbacks of Column Chromatography Separation is not efficient More time consumption Wastage of Solvent Introduction & Basics
For Mixing your Video Broadcasting LOGO Dont write anything hereDont write anything here Dont write anything hereDont write anything here Overcoming the drawbacks Lesser time consumption Less solvent Efficient separation High sensitivity & resolution Small sample size (ppm / ppb) Why need HPLC ?
For Mixing your Video Broadcasting LOGO Dont write anything hereDont write anything here Dont write anything hereDont write anything here High Performance High Pressure Why P in HPLC
For Mixing your Video Broadcasting LOGO Dont write anything hereDont write anything here Dont write anything hereDont write anything here Pharmaceuticals Bio-chemicals Food products Industrial chemicals Forensic chemistry Environmental science Scope of HPLC
For Mixing your Video Broadcasting LOGO Dont write anything hereDont write anything here Dont write anything hereDont write anything here Separation of compounds take place as they move at different rates in the column Concept - Mechanism 2 1 column
For Mixing your Video Broadcasting LOGO Dont write anything hereDont write anything here Dont write anything hereDont 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
For Mixing your Video Broadcasting LOGO Dont write anything hereDont write anything here Dont write anything hereDont write anything here Instrumentation Pump Injector Column Oven Detector Mobile Phase Data processor
For Mixing your Video Broadcasting LOGO Dont write anything hereDont write anything here Dont write anything hereDont write anything here Components of HPLC instrument Solvent delivering reservoir High pressure pumps Sample injector Column oven Detectors Instrumentation
For Mixing your Video Broadcasting LOGO Dont write anything hereDont write anything here Dont write anything hereDont write anything here Possible configuration Isocratic system Low-pressure gradient system High-pressure gradient system Instrumentation
For Mixing your Video Broadcasting LOGO Dont write anything hereDont write anything here Dont write anything hereDont 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
For Mixing your Video Broadcasting LOGO Dont write anything hereDont write anything here Dont write anything hereDont 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
For Mixing your Video Broadcasting LOGO Dont write anything hereDont write anything here Dont write anything hereDont 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
For Mixing your Video Broadcasting LOGO Dont write anything hereDont write anything here Dont write anything hereDont write anything here Injector Manual Instrumentation
For Mixing your Video Broadcasting LOGO Dont write anything hereDont write anything here Dont write anything hereDont write anything here Detectors UV-Vis detector PDA detector Fluorescence detector Mass Spectrometer detector Instrumentation
For Mixing your Video Broadcasting LOGO Dont write anything hereDont write anything here Dont write anything hereDont write anything here Chromatogram tR : Retention time A : Area h : Height tR Signal Time Peak h A
For Mixing your Video Broadcasting LOGO Dont write anything hereDont write anything here Dont write anything hereDont write anything here Reverse phase chromatography Normal phase chromatography Ion exchange chromatography Size exclusion chromatography Affinity chromatography Working
For Mixing your Video Broadcasting LOGO Dont write anything hereDont write anything here Dont write anything hereDont write anything here Working Stationary phase: Non-polar property Mobile phase : Polar property This combination is defined as Reversed Phase Mode Reversed phase
For Mixing your Video Broadcasting LOGO Dont write anything hereDont write anything here Dont write anything hereDont write anything here Working Stationary phase: polar property Mobile phase : Non-Polar property This combination is defined as Normal Phase Mode Normal phase
For Mixing your Video Broadcasting LOGO Dont write anything hereDont write anything here Dont write anything hereDont 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
For Mixing your Video Broadcasting LOGO Dont write anything hereDont write anything here Dont write anything hereDont write anything here Mobile phase Water + Organic solvent Methanol Acetonitrile Tetrahydrofuran Working in Rev. Phase
For Mixing your Video Broadcasting LOGO Dont write anything hereDont write anything here Dont write anything hereDont 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
For Mixing your Video Broadcasting LOGO Dont write anything hereDont write anything here Dont write anything hereDont 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
For Mixing your Video Broadcasting LOGO Dont write anything hereDont write anything here Dont write anything hereDont write anything here Retention Time & Hydrophobicity Separation Mechanism OH OH C18 (ODS) Strong Weak 1 1 2 2
For Mixing your Video Broadcasting LOGO Dont write anything hereDont write anything here Dont write anything hereDont write anything here Effect of Stationary phase Separation Mechanism C18 (ODS) Strong C8 sample sample sample C4 Medium Weak
For Mixing your Video Broadcasting LOGO Dont write anything hereDont write anything here Dont write anything hereDont write anything here THANK YOU deepjoshi359@yahoo.co.in http://deepjeejoshi.wix.com/deepjoshi HPLC

More Related Content

High performance liquid chromatography as introduction

  • 1. For Mixing your Video Broadcasting LOGO Dont write anything hereDont write anything here Dont write anything hereDont 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 Dont write anything hereDont write anything here Dont write anything hereDont 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 Dont write anything hereDont write anything here Dont write anything hereDont write anything here Introduction & Basics Why need of HPLC ? Why Pressure ? Why Performance? Concept & Scope of HPLC Instrumentation & Working Separation Mechanism Todays Discussion
  • 4. For Mixing your Video Broadcasting LOGO Dont write anything hereDont write anything here Dont write anything hereDont 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 Dont write anything hereDont write anything here Dont write anything hereDont write anything here History of Chromatography M. Tswett A Russian Botanist Introduction & Basics Petroleum ether CaCO3 Chlorophylls
  • 6. For Mixing your Video Broadcasting LOGO Dont write anything hereDont write anything here Dont write anything hereDont write anything here Some Important Term(s) Stationary phase Mobile phase Chromatogram Retention Time Introduction & Basics
  • 7. For Mixing your Video Broadcasting LOGO Dont write anything hereDont write anything here Dont write anything hereDont 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 Dont write anything hereDont write anything here Dont write anything hereDont 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 Dont write anything hereDont write anything here Dont write anything hereDont 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 Dont write anything hereDont write anything here Dont write anything hereDont write anything here High Performance High Pressure Why P in HPLC
  • 11. For Mixing your Video Broadcasting LOGO Dont write anything hereDont write anything here Dont write anything hereDont write anything here Pharmaceuticals Bio-chemicals Food products Industrial chemicals Forensic chemistry Environmental science Scope of HPLC
  • 12. For Mixing your Video Broadcasting LOGO Dont write anything hereDont write anything here Dont write anything hereDont 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 Dont write anything hereDont write anything here Dont write anything hereDont 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 Dont write anything hereDont write anything here Dont write anything hereDont write anything here Instrumentation Pump Injector Column Oven Detector Mobile Phase Data processor
  • 15. For Mixing your Video Broadcasting LOGO Dont write anything hereDont write anything here Dont write anything hereDont 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 Dont write anything hereDont write anything here Dont write anything hereDont write anything here Possible configuration Isocratic system Low-pressure gradient system High-pressure gradient system Instrumentation
  • 17. For Mixing your Video Broadcasting LOGO Dont write anything hereDont write anything here Dont write anything hereDont 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 Dont write anything hereDont write anything here Dont write anything hereDont 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 Dont write anything hereDont write anything here Dont write anything hereDont 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 Dont write anything hereDont write anything here Dont write anything hereDont write anything here Injector Manual Instrumentation
  • 21. For Mixing your Video Broadcasting LOGO Dont write anything hereDont write anything here Dont write anything hereDont write anything here Detectors UV-Vis detector PDA detector Fluorescence detector Mass Spectrometer detector Instrumentation
  • 22. For Mixing your Video Broadcasting LOGO Dont write anything hereDont write anything here Dont write anything hereDont write anything here Chromatogram tR : Retention time A : Area h : Height tR Signal Time Peak h A
  • 23. For Mixing your Video Broadcasting LOGO Dont write anything hereDont write anything here Dont write anything hereDont 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 Dont write anything hereDont write anything here Dont write anything hereDont 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 Dont write anything hereDont write anything here Dont write anything hereDont 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 Dont write anything hereDont write anything here Dont write anything hereDont 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 Dont write anything hereDont write anything here Dont write anything hereDont write anything here Mobile phase Water + Organic solvent Methanol Acetonitrile Tetrahydrofuran Working in Rev. Phase
  • 28. For Mixing your Video Broadcasting LOGO Dont write anything hereDont write anything here Dont write anything hereDont 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 Dont write anything hereDont write anything here Dont write anything hereDont 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 Dont write anything hereDont write anything here Dont write anything hereDont 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 Dont write anything hereDont write anything here Dont write anything hereDont 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 Dont write anything hereDont write anything here Dont write anything hereDont write anything here THANK YOU deepjoshi359@yahoo.co.in http://deepjeejoshi.wix.com/deepjoshi HPLC