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  • HPLC

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