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. 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. INSTRUMENTATION
Gradient
Controller
•
Pump Column
Detecor
Injector
Mobile
Phases

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. 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. STATIONARY PHASE
 IN THIS WE WILL BE TALKING ABOUT THE STATIONARY
PHASES OF NORMAL PHASE AND REVERSE PHASE HPLC
 NORMAL PHASE:

7. STATIONARY PHASE
 REVERSE PHASE:

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. NORMAL PHASE

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. REVERSE PHASE
 .