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HPLC
- 1. High Performance Liquid Chromatography FEROSEKHAN S FNB-41 CIFE MUMBAI
- 3. Elution chromatography Increasing polarity of pure solvents hexane ether acetone methanol water acetic acid Solvents mixed %hexane and % methanol miscible can be mixed continuously (solvent programming)
- 4. Outline What is HPLC? An Overview Types of HPLC Partition Chromatography Adsorption Chromatography Ion Chromatography Size-Exclusion Chromatography
- 5. What is HPLC? The most widely used analytical separations technique Utilizes a liquid mobile phase to separate components of mixture uses high pressure to push solvent through the column Popularity : sensitivity ready adaptability to accurate quantitative determination suitability for separating nonvolatile species or thermally fragile ones
- 6. HPLC is…. Popularity: widespread applicability to substances that are of prime interest to industry, to many fields of science, and to the public Ideally suited for separation and identification of, proteins, amino acids nucleic acids, hydrocarbons, carbohydrates, pharmaceuticals, pesticides, pigments, antibiotics, steroids, and a variety of other inorganic substances
- 7. History Early LC carried out in glass columns diameters: 1-5 cm lengths: 50-500 cm Size of solid stationary phase diameters: 150-200 m Flow rates still low! Separation times long! Decrease particle size of packing causes increase in column efficiency! diameters 3-10 m This technology required sophisticated instruments new method called HPLC
- 8. Advantages to HPLC Higher resolution and speed of analysis HPLC columns can be reused without repacking or regeneration Greater reproducibility due to close control of the parameters affecting the efficiency of separation Easy automation of instrument operation and data analysis Adaptability to large-scale, preparative procedures
- 9. Advantages to HPLC Advantages of HPLC are result of 2 major advances: stationary supports with very small particle sizes and large surface areas appliance of high pressure to solvent flow
- 10. Instrumentation Instruments required: Mobile phase reservoir Pump Injector Column Detector Data system
- 11. Schematic of liquid chromatograph
- 12. LC column LC injector
- 13. Partition Chromatography Most widely used Bonded-phase Chromatography Silica Stationary Phase: OH OH OH OH O O O Si Si Si Si Siloxanes: O CH 3 Si O Si R R= C 8 , C 18 O CH 3
- 14. Partition Chromatography Reverse Phase Chromatography Nonpolar Stationary Phase Polar Mobile Phase Normal Phase Chromatography Polar Stationary Phase Nonpolar Mobile Phase Column Selection Mobile-Phase Selection
- 15. Partition Chromatography Applications Parathion in Insecticides: O CH 3 CH 2 O P O NO 2 CH 3 CH 2 O
- 17. Adsorption Chromatography Classic Solvent Selection Non-polar Isomeric Mixtures Advantages/ Disadvantages Applications
- 18. What is Ion Chromatography? Modern methods of separating and determining ions based on ion-exchange resins Mid 1970s Anion or cation mixtures readily resolved on HPLC column Applied to a variety of organic & biochemical systems including drugs, their metabolites, serums, food preservatives, vitamin mixtures, sugars, pharmaceutical preparations
- 20. Size Exclusion Chromatography(SEC) Gel permeation(GPC), gel filtration(GFC) chromatography Technique applicable to separation of high-molecular weight species Rapid determination of the molecular weight or molecular-weight distribution of larger polymers or natural products Solute and solvent molecules can diffuse into pores -- trapped and removed from the flow of the mobile phase
- 21. Specific pore sizes.average residence time in the pores depends on the effective size of the analyte molecules larger molecules smaller molecules intermediate size molecules SEC(continued)
- 22. SEC Column Packing Small (~10 µm) silica or polymer particles containing a network of uniform pores Two types (diameters of 5 ~ 10 µm) Polymer beads silica-based particles
- 24. The Mobile Phases are... Aqueous solutions containing methanol, water-miscible organic solvents also contain ionic species, in the form of a buffer solvent strength & selectivity are determined by kind and concentration of added ingredients ions in this phase compete with analyte ions for the active site in the packing
- 25. Properties of the Mobile Phase Must dissolve the sample have a strong solvent strength leads to reasonable retention times interact with solutes in such a way as to lead to selectivity
- 26. Mobile phase reservoir Glass/stainless steel reservoir Removal of dissolved gases by degassers vacuum pumping system heating/stirring of solvents
- 27. Elution methods Isocratic elution single solvent of constant composition Gradient elution 2 or more solvents of differing polarity used
- 29. Sample Injection Systems For injecting the solvent through the column Minimize possible flow disturbances Volumes must be small .1-500 L Sampling loops interchangeable loops (5-500 L at pressures up to 7000 psi)
- 30. Types of Detector Refractive index UV/Visible Fluorescence Conductivity Evaporative light scattering Electrochemical
- 31. Detectors Refractive Index detector All solutes Measures change in RI of the mobile phase due to solutes Carbohydrates and lipids
- 32. Refractive Index Measure displacement of beam with respect to photosensitive surface of dectector
- 33. Refractive Index Advantages universal respond to nearly all solutes reliable unaffected by flow rate low sensitive to dirt and air bubbles in the flow cell
- 34. Refractive Index Disadvantages expensive highly temperature sensitive moderate sensitivity cannot be used with gradient elution
- 35. Detectors UV-VIS absorption detector (190-800 nm) Aromatics Conjugated molecules
- 36. UV/Visible Mercury lamp Photocell measures absorbance
- 37. UV/Visible Advantages high sensitivity small sample volume required linearity over wide concentration ranges can be used with gradient elution Disadvantage does not work with compounds that do not absorb light at this wavelength region
- 38. Fluorescence For compounds having natural fluorescing capability Fluorescence observed by photoelectric detector Mercury or Xenon source with grating monochromator to isolate fluorescent radiation
- 39. Fluorescence Advantages extremely high sensitivity high selectivity Disadvantage may not yield linear response over wide range of concentrations
- 40. Conductivity Measure conductivity of column effluent Sample indicated by change in conductivity Best in ion-exchange chromatography Cell instability
- 41. Data System For better accuracy and precision Routine analysis pre-programmed computing integrator Data station/computer needed for higher control levels add automation options complex data becomes more feasible software safeguard prevents misuse of data system