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

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 VolatilitySample Polarity HPLC No volatility requirement Sample must be soluble in mobile phase GC Sample must be volatile HPLC GC Separates both polar and non polar compounds PAH - inorganic ions Samples are nonpolar and polar

6 Comparison of HPLC and GC

7 Sample Thermal LabilitySample Molecular Weight HPLC Analysis can take place at or below room temperature GC Sample must be able to survive high temperature injection port and column HPLC GC No theoretical upper limit In practicality, solubility is limit. Typically < 500 amu

8 Comparison of HPLC and GC Sample PreparationSample Size HPLC Sample must be filtered Sample should be in same solvent as mobile phase GC Solvent must be volatile and generally lower boiling than analytes HPLC GC Sample size based upon column i.d. Typically  L

9 Comparison of HPLC and GC Separation MechanismDetectors HPLC Both stationary phase and mobile phase take part GC Mobile phase is a sample carrier only HPLC GC Most common UV-Vis Wide range of non- destructive detectors 3-dimensional detectors Sensitivity to fg (detector dependent) Most common FID, universal to organic compounds

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

11 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.

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

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

30 Modes of High Performance Liquid Chromatography Types of CompoundsModeStationary Phase Mobile Phase Neutrals Weak Acids Weak Bases Reversed Phase C18, C8, C4 cyano, amino Water/Organic Modifiers Ionics, Bases, AcidsIon Pair C-18, C-8Water/Organic Ion-Pair Reagent Compounds not soluble in water Normal Phase Silica, Amino, Cyano, Diol Organics Ionics Inorganic IonsIon 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

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