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Introduction to High Performance Liquid Chromatography

Introduction to High Performance Liquid Chromatography. In This Section, We Will Discuss:. The differences between High Performance Liquid Chromatography and Gas Chromatography. The components of the high performance liquid chromatograph (HPLC). The separation process. The chromatogram.

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Introduction to High Performance Liquid Chromatography

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  1. Introduction to High Performance Liquid Chromatography

  2. In This Section, We Will Discuss: • The differences between High Performance Liquid Chromatography and Gas Chromatography. • The components of the high performance liquid chromatograph (HPLC). • The separation process. • The chromatogram. • The most common modes of HPLC.

  3. I need a quantitative separation of carbohydrates in some of our products as soon as possible. I’ll get on it! I’ll need a separation technique. You’ve Got a Problem to Solve

  4. Separation Techniques I have two separation techniques in my lab, High Performance Liquid Chromatography and Gas Chromatography. Which should I use?

  5. Comparison of HPLC and GC Sample Volatility Sample Polarity HPLC HPLC • Separates both polar andnon polar compounds • PAH - inorganic ions • No volatility requirement • Sample must be solublein mobile phase GC GC • Samples are nonpolarand polar • Sample must be volatile

  6. Comparison of HPLC and GC

  7. Comparison of HPLC and GC Sample Thermal Lability Sample Molecular Weight HPLC HPLC • Analysis can take placeat or below roomtemperature • No theoretical upper limit • In practicality, solubility islimit. GC GC • Sample must be able to survive high temperature injection port and column • Typically < 500 amu

  8. Comparison of HPLC and GC Sample Preparation Sample Size HPLC HPLC • Sample must be filtered • Sample should be insame solvent as mobilephase • Sample size based uponcolumn i.d. GC GC • Typically 1 - 5 L • Solvent must be volatileand generally lower boiling than analytes

  9. Separation Mechanism Detectors HPLC HPLC • Both stationary phase and mobile phase take part • Most common UV-Vis • Wide range of non-destructive detectors • 3-dimensional detectors • Sensitivity to fg (detectordependent) GC GC • Most common FID,universal to organiccompounds • Mobile phase is a sample carrier only Comparison of HPLC and GC

  10. 5 2 3 mAU 4 1 6 time How can We Analyze the Sample? Carbohydrates 1. fructose 2. Glucose 3. Saccharose 4. Palatinose 5. Trehalulose 6. isomaltose Zorbax NH2 (4.6 x 250 mm) 70/30 Acetonitrile/Water 1 mL/min Detect=Refractive Index

  11. Injector Mixer Pumps Column Detector Waste Solvents High Performance Liquid Chromatograph Separations Separation in based upon differential migration between the stationary and mobile phases. Stationary Phase - the phase which remains fixed in the column, e.g. C18, Silica Mobile Phase - carries the sample through the stationary phase as it moves through the column.

  12. Injector Chromatogram mAU Mixer Pumps time Start Injection Column Detector Solvents High Performance Liquid Chromatograph Separations

  13. Injector Chromatogram mAU Mixer Pumps time Start Injection Column Detector Solvents Separations

  14. Injector Chromatogram mAU Mixer Pumps time Start Injection Column Detector Solvents Separations

  15. Injector Chromatogram mAU Mixer Pumps time Start Injection Column Detector Solvents Separations

  16. Injector Chromatogram mAU Mixer Pumps time Start Injection Column Detector Solvents Separations

  17. Injector Chromatogram mAU Mixer Pumps time Start Injection Column Detector Solvents Separations

  18. Injector Chromatogram mAU Mixer Pumps time Start Injection Column Detector Solvents Separations

  19. Injector Chromatogram mAU Mixer Pumps time Start Injection Column Detector Solvents Separations

  20. Injector Chromatogram mAU Mixer Pumps time Start Injection Column Detector Solvents Separations

  21. Injector Chromatogram mAU Mixer Pumps time Start Injection Column Detector Solvents Separations

  22. Injector Chromatogram mAU Mixer Pumps time Start Injection Column Detector Solvents Separations

  23. Injector Chromatogram mAU Mixer Pumps time Start Injection Column Detector Solvents Separations

  24. Injector Chromatogram mAU Mixer Pumps time Start Injection Column Detector Solvents Separations

  25. Injector Chromatogram mAU Mixer Pumps time Start Injection Column Detector Solvents Separations

  26. Injector Chromatogram mAU Mixer Pumps time Start Injection Column Detector Solvents Separations

  27. Injector Chromatogram mAU Mixer Pumps time Start Injection Column Detector Solvents Separations

  28. to - elution time of unretained peak tR- retention time - determines sample identity tR tR mAU Area or height is proportional to the quantity of analyte. to time Injection The Chromatogram

  29. HPLC Analysis Parameters Mobile Phases Flow Rate Composition Injection Volume Column Oven Temperature Wavelength Time Constant

  30. Types of Compounds Mode Stationary Phase Mobile Phase Neutrals Weak Acids Weak Bases Reversed Phase C18, C8, C4 cyano, amino Water/Organic Modifiers Ionics, Bases, Acids Ion Pair C-18, C-8 Water/Organic Ion-Pair Reagent Compounds not soluble in water Normal Phase Silica, Amino, Cyano, Diol Organics Ionics Inorganic Ions Ion Exchange Anion or Cation Exchange Resin Aqueous/Buffer Counter Ion High Molecular Weight Compounds Polymers Size Exclusion Polystyrene Silica Gel Filtration- Aqueous Gel Permeation- Organic Modes of High Performance Liquid Chromatography

  31. Chemical Bioscience proteins peptides nucleotides polystyrenes dyes phthalates tetracyclines corticosteroids antidepressants barbiturates Pharmaceuticals Consumer Products lipids antioxidants sugars Environmental Clinical polyaromatic hydrocarbons Inorganic ions herbicides amino acids vitamins homocysteine HPLC Applications

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