High performance(pressure

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High performance(pressure

  1. 1. HIGH PERFORMANCE(PRESSURE/PRICED) LIQUID CHROMATOGRAPHY ALTHOUGH GAS CHROMATOGRAPHY IS MOST WIDELY USED TECHNIQUE IN THE ANALYSIS OF OILS & FATS,BUT THE DESTRUCTION OF SAMPLE UNDER ANALYSIS & OTHER LIMITATION LIKE LOW MOL WT,HIGH VOLATILITY & HIGH STABILITY IN GAS PHASE NECESSITATED IN THE DEVELOPMENT OF NEWER TECHNIQUE AND HPLC IS THE RESULT OF THAT ONLY.
  2. 2. HPLC LIQUID CHROMATOGRAPHY INVOLVES SEPARATION OF COMPONENTS DUE TO DIFFERENCE IN EQUILIBRIUM DISTRIBUTION OF SAMPLE COMPONENTS BETWEEN A LIQUID MOBILE PHASE & STATIONARY PHASE (ONE WHICH DISTINGUISHES THIS FROM OTHER CHROMATOGRAPHIC TECHNIQUE).
  3. 3. INSTRUMENTATIONGradientController • Pump Column Detecor Injector Mobile Phases
  4. 4. MOBILE PHASE THERE ARE TWO TYPES OF MOBILE PHASES WIDELY USED BY ANALYST: ISOCRATIC ELUTION:BOTH MOBILE PHASE COMPOSITION & FLOW RATE IS FIXED. GRADIENT ELUTION: BOTH MOBILE PHSE COMPOSITION AND FLOW RATE IS PROGRAMMED TO GET BETTER ELUTION AND RESOLUTION
  5. 5. Types of HPLC Reverse-phase (polar mobile phase/non- polar stationary phase/somewhat polar analytes) Normal Phase (non-polar mobile phase/polar stationary phase/non-polar analytes) Adsorption (non-polar mobile phase/polar stationary phase/non-polar analytes); isomer separation Ion-Exchange (salts/ionic stationary phase) Size-exclusion (aqueous/gel for large MW solutes, >104)
  6. 6. STATIONARY PHASE IN THIS WE WILL BE TALKING ABOUT THE STATIONARY PHASES OF NORMAL PHASE AND REVERSE PHASE HPLC NORMAL PHASE:
  7. 7. STATIONARY PHASE REVERSE PHASE:
  8. 8. NORMAL PHASE NONPOLAR COMPONENTS ARE RETAINED WHILE NON POLAR COMPONENTS ARE ELUTED FIRST. INCREASE IN POLARITY OF MOBILE PHASE DECREASES RETENTION TIME & VICE-VERSA. FOR ANALYSIS OF OILS AND FATS THESE COLUMN IS GENERALLY NOT USED.
  9. 9. NORMAL PHASE
  10. 10. REVERSE PHASE HYDROPHILIC COMPOUNDS ELUTE MORE QUICLKY THAN HYDROPHOBIC COMPOUNDS. RETENTION TIME CAN BE INCREASED BY ADDITION OF NONPOLAR SOLVENTS. REVERSED PHASE TLC IS USED IN SEPERATION OF COMPOUNDS ACCORDING TO NO OF C ATOMS. RP GAVES BETTER RESOLUTUION BECAUSE NONPOLAR- NONPOLAR INTERACTION IS MUCH LARGER THAN POLAR-POLAR INTERACTION
  11. 11. REVERSE PHASE .

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