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HPLC
HPLC
HPLC
HPLC
HPLC
HPLC
HPLC
HPLC
HPLC
HPLC
HPLC
HPLC
HPLC
HPLC
HPLC
HPLC
HPLC
HPLC
HPLC
HPLC
HPLC
HPLC
HPLC
HPLC
HPLC
HPLC
HPLC
HPLC
HPLC
HPLC
HPLC
HPLC
HPLC
HPLC
HPLC
HPLC
HPLC
HPLC
HPLC
HPLC
HPLC
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HPLC

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    • 1. High Performance Liquid Chromatography FEROSEKHAN S FNB-41 CIFE MUMBAI
    • 2. Liquid Chromatography
    • 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. 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. 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. 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. 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. 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. 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. 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. Schematic of liquid chromatograph
    • 12. LC column LC injector
    • 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. 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. 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. &nbsp;
    • 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>
    • 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 &amp; biochemical systems including drugs, their metabolites, serums, food preservatives, vitamin mixtures, sugars, pharmaceutical preparations </li></ul>
    • 19. &nbsp;
    • 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>
    • 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)
    • 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>
    • 23. &nbsp;
    • 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 &amp; 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>
    • 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>
    • 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>
    • 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>
    • 28. &nbsp;
    • 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>
    • 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>
    • 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>
    • 32. Refractive Index <ul><li>Measure displacement of beam with respect to photosensitive surface of dectector </li></ul>
    • 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>
    • 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>
    • 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>
    • 36. UV/Visible <ul><li>Mercury lamp </li></ul><ul><li>Photocell measures absorbance </li></ul>
    • 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>
    • 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>
    • 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>
    • 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>
    • 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>

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