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Kaumil Hplc

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HPLC basics

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Kaumil Hplc

  1. 1. Basics of - HPLC <ul><li>Kaumil Bhavsar </li></ul><ul><li>Date -21/08/07 </li></ul>
  2. 2. Index <ul><li>What is HPLC ? </li></ul><ul><li>Applications </li></ul><ul><li>Types of HPLC </li></ul><ul><li>HPLC Instrumentation </li></ul><ul><li>Solvent system </li></ul><ul><li>Pumping system </li></ul><ul><li>Sample injector system </li></ul><ul><li>Column </li></ul><ul><li>Detectors </li></ul><ul><li>P9 RP-HPLC salient features </li></ul>
  3. 3. What is HPLC ? <ul><li>Originally referred to as </li></ul><ul><li>H igh P ressure L iquid C hromatography </li></ul><ul><li>Now more commonly called </li></ul><ul><li>H igh P erformance L iquid C hromatography </li></ul><ul><li>HPLC is an analytical technique used to separate component of mixture by using variety of chemical interaction between the analyte and the chromatography column. </li></ul><ul><li>Why HPLC ? </li></ul><ul><li>Automated version of LC </li></ul><ul><li>Provides enhanced separations in short time duration. </li></ul><ul><li>It’s a most practiced quantitative, chromatographic technique which can separates wide range of molecule types and sizes. </li></ul>
  4. 4. Applications of HPLC <ul><li>Pharmaceutical / Biopharmaceutical </li></ul><ul><li>Pharmaceutical quality control. </li></ul><ul><li>Shelf-life determinations of pharmaceutical products. </li></ul><ul><li>Identification of counterfeit drug products. </li></ul><ul><li>Complex molecules separation. </li></ul><ul><li>Environmental </li></ul><ul><li>Biomonitoring of pollutents. </li></ul><ul><li>Water monitoring - Phenol content and toxic componants checking. </li></ul><ul><li>Clinical </li></ul><ul><li>Analysis of antibiotics and blood substances. </li></ul><ul><li>Detection of endogenous neuropeptides in brain extracellular fluids. </li></ul><ul><li>Food and Flavor </li></ul><ul><li>Sugar analysis in fruit juices. </li></ul><ul><li>Ensuring soft drink consistency and quality. </li></ul>
  5. 5. Types of HPLC <ul><li>Normal Phase : Separation of polar analytes by partitioning onto a polar, bonded stationary phase using non polar mobile phase. </li></ul><ul><li>Reversed Phase : Separation of non-polar analytes by partitioning onto a non-polar, bonded stationary phase using polar mobile phase. </li></ul><ul><li>Adsorption : In Between Normal and Reversed. Separation of moderately polar analytes using adsorption onto a pure stationary phase (e.g. alumina or silica) </li></ul><ul><li>Ion Exchange Chromatography : Separation of organic and inorganic ions by their partitioning onto ionic stationary phases bonded to a solid support. </li></ul><ul><li>Size Exclusion Chromatography : Also called as Gel filtration chromatography. Separation of molecules based on their size. </li></ul>
  6. 6. Selection of Chromatography
  7. 7. Separation based on selection of column material
  8. 8. Separation based on selection of column material
  9. 9. HPLC instrumentation <ul><li>HPLC components </li></ul><ul><li>Solvent system </li></ul><ul><li>Pumping system </li></ul><ul><li>Sample injection system </li></ul><ul><li>Column </li></ul><ul><li>Detector </li></ul>Detector Mobile phase Pump Injection Valve Separation column => => => =>
  10. 10. Solvent system <ul><li>Mobile phase reservoirs : Several reservoirs – Use to carry mobile phase </li></ul><ul><li>Degassing : Remove dissolved gas - interfering detection </li></ul><ul><li>Protect band spreading </li></ul><ul><li>Sparging: fine bubble of gas (He) </li></ul><ul><li>Vacuum pumping. </li></ul><ul><li>Dust removal: Dust interference with detection, column clogging, damage pumping system </li></ul><ul><li>Millipore filter under vacuum </li></ul><ul><li>Isocratic elution : constant composition </li></ul><ul><li>Gradient elution : different solvent systems during elution, continuous change or step wise, solvent proportion valve </li></ul>
  11. 11. Pumping system <ul><li>Requirements for HPLC pump. </li></ul><ul><li>High pressure (up to 6000 psi) </li></ul><ul><li>• pulse free, prevents remixing of solutes </li></ul><ul><li>• control flow rate from 0.1 to 10 mL/min </li></ul><ul><li>Types of HPLC pumps </li></ul><ul><li>1) Reciprocating pumps : Most commonly used. </li></ul><ul><li> Disadvantage: pulses from single piston. </li></ul><ul><li>2) Syringe pumps : Generates pulse free output. </li></ul><ul><ul><li>Disadvantage: Limited mobile phase capasity. </li></ul></ul>
  12. 12. Reciprocating pump : Single piston pump
  13. 13. Reciprocating pump : Double piston pump
  14. 14. Syringe pump
  15. 15. Sample injection system <ul><li>Sample Injection system: </li></ul><ul><li>Limit of precision of HPLC </li></ul><ul><li>Sample size: 0.5 ~ 500 μL </li></ul><ul><li>No interference with the pressure </li></ul><ul><li>Sample loop, 1 ~ 100 μL, </li></ul><ul><li>Auto sampler: inject continuously </li></ul><ul><li>Volume uptake: 1 μL – 1 mL </li></ul><ul><li>Controlled temperature environment </li></ul>
  16. 16. Sample loading & Injection.
  17. 17. Column <ul><li>Made of Stainless steel tubing for high pressure. </li></ul><ul><li>Analytical column: straight, L(5 ~ 25 cm), d (3 ~ 5 mm), dp(3~5 μm). N (40 k ~ 70 k plates/m) </li></ul><ul><li>Microcolumn: L (3 ~ 7.5 cm), d (1 ~ 5 mm), dp: (3 ~ 5 μm) , N: ~100k plates/m, high speed and minimum solvent consumption </li></ul><ul><li>Guard column: protects analytical column, similar packing, remove particulate matter and contamination. </li></ul><ul><li>Temperature control: 0.1 °C - 150 °C. </li></ul><ul><li>Column packing: silica, alumina, a polystyrene-divinylbenzene, synthetic or an ion-exchange resin </li></ul><ul><li>Pellicular particle: original, spherical,nonporous beads,proteins and large biomolecules separation (dp: 5 μm) </li></ul><ul><li>Porous particle: commonly used, dp: 3 ~ 10 μm. Narrow size distribution, porous microparticle coated with thin organic films. </li></ul>
  18. 18. Detectors <ul><li>Ideal Detector Properties: </li></ul><ul><li>High Sensitivity </li></ul><ul><li>Universality or predictable specificity </li></ul><ul><li>Large linear response range </li></ul><ul><li>Low dead volume </li></ul><ul><li>Non-Destructive </li></ul><ul><li>Insensitive to temperature & mobile phase </li></ul><ul><li>Continuous operation </li></ul><ul><li>Reliable and easy to use </li></ul><ul><li>No single detector fits all these criteria </li></ul>
  19. 19. Types of HPLC detectors Ability to ionize analyte Low LOD; analyte identification Mass Spec Non-specific; high LOD Commercially available Electrochemical Non-specific; interference from solvent Uniform response; 5ng/25  L LOD Scattering Temperature sensitive; high LOD Works w/ nearly all molecules Refractive Index Many solvents IR active Works w/all molecules IR Not everything fluoresces Very specific; low LOD Fluorescence High LOD Works for all wavelengths DAD Non-specific; complex samples; absorption wavelength Works w/all molecules UV-Vis Name Advantage Disadvantage
  20. 20. UV-VIS detector. <ul><li>Single Beam UV-VIS instrument with a flow-through flow cell (cuvette) </li></ul><ul><li>Usually typical UV-VIS lamps used having 254 nm default wavelenth </li></ul><ul><ul><li>Can be set to other wavelengths (most) </li></ul></ul><ul><ul><li>Simple filter detectors no longer widely used </li></ul></ul><ul><ul><li>Non-destructive, not-universal </li></ul></ul><ul><ul><li>not all compounds absorb light </li></ul></ul><ul><ul><li>can pass sample through several cells at several different wavelenghts </li></ul></ul><ul><li>Usually zeroed at the start of each run using an electronic software command. You can have real-time zeroing with a reference cell. </li></ul>
  21. 21. Diode array detector (DAD)
  22. 22. Refractive Index Detector.
  23. 23. ELSD (Evaporative Light Scattering Detector)
  24. 24. P9 RP- HPLC Salient features. <ul><li>Reverse phase HPLC. </li></ul><ul><li>15 min method, RT- Between 4 to 6 min. </li></ul><ul><li>Mobile phase : A - Water + TFA </li></ul><ul><li> B - ACN + TFA </li></ul><ul><li>Column: Brand Name - Discovery </li></ul><ul><li>Type of Column:Reverse phase C18 column </li></ul><ul><li>Specification of ColumnDiameter: 4.6mm Length: 50mm Porosity: 300•A, Particle size: 5 Micron </li></ul><ul><li>Guard column Brand Name - Zorbex 300 SB (Agilent) </li></ul><ul><li>Type of Column:Reverse phase Zorbax 300 SB-C18 </li></ul><ul><li>Specification of ColumnDiameter: 4.6mm, Length: 35 mmPorosity: 300•A, Particle size: 5 Micron </li></ul>
  25. 25. Typical P9 RP-HPLC peak (Agilent-1200).
  26. 26. Thank You

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