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.

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).

INSTRUMENTATION

Pump

Injector

ColumnDetecor

Mobile Phases

Gradient Controller

•

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

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)

STATIONARY PHASE

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

NORMAL PHASE:

STATIONARY PHASE

REVERSE PHASE:

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.

NORMAL PHASE

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

REVERSE PHASE

.