EXPERIMENT 4: COLUMN CHROMATOGRAPHY AND THIN LAYER

CHROMATOGRAPHY THE SEPARATION, ISOLATION AND ANALYSIS OF THE DIFFERENT COMPONENTS IN A MIXTURE.

GRAVITY COLUMN CHROMATOGRAPHY

USED TO SEPARATE (AND THUS PURIFY) THE COMPONENTS OF A MIXTURE (PAPRIKA PIGMENTS). SEPARATION IS ACHIEVED BY PARTITIONING THE COMPONENTS BETWEEN A STATIONARY PHASE AND A MOBILE PHASE.

STATIONARY PHASE

MOBILE PHASE

SILICA GEL

ORGANIC SOLVENTS

COLUMN CHROMATOGRAPHY: PREPARATION

HALF FILL THE COLUMN WITH HEXANE, THEN ADD ~ 4g OF SILICA GEL WHILST GENTLY TAPPING THE COLUMN.

COLUMN CHROMATOGRAPHY: TROUBLESHOOTING

IF THE SILICA GEL GETS STUCK WHEN LOADING THE COLUMN, REMOVE IT FROM THE CLAMP, COVER THE OPEN END, SHAKE.

RETURN TO UPRIGHT POSITION, ALLOW TO SETTLE.

COLUMN CHROMATOGRAPHY: PREPARATION

DRAIN THE EXCESS HEXANE FROM THE COLUMN UNTIL THE TOP SURACE OF THE SILICA GEL JUST BEGINS TO DRY.

IMMEDIATELY CLOSE THE TAP.

COLUMN CHROMATOGRAPHY: LOADING SAMPLE

DISSOLVE THE PIGMENTS IN 10 DROPS OF HEXANE. APPLY SIX DROPS OF THIS MIXTURE TO THE TOP OF THE COLUMN USING A LONG PASTEUR PIPETTE. USE A CLEAN PASTEUR PIPETTE TO RINSE THE SIDES OF THE COLUMN WITH THE MINIMUM AMOUNT OF HEXANE. OPEN THE TAP AND ALLOW THE SOLUTION TO DRAIN INTO THE TOP OF THE ADSORBENT. CLOSE THE TAP, STOPPER THE COLUMN AND LET STAND FOR TWO MINS.

COLUMN CHROMATOGRAPHY: LOADING SOLVENT

KEEPING THE TAP CLOSED, FILL THE COLUMN WITH HEXANE. DO NOT DISTURB THE SILICA GEL. ADD HEXANE USING A PASTUER PIPETTE INITIALLY, THEN BY POURING. THE COLUMN IS NOW READY TO BE RUN.

COLUMN CHROMATOGRAPHY: ELUTING

BEGIN ELUTION WITH HEXANE, THEN MOVE TO 2% ETHYL ACETATE IN HEXANE. NEVER LET THE LEVEL OF THE

SOLVENT FALL BELOW THE TOP SURFACE OF THE SILICA GEL.

THE YELLOW BAND IS α- AND β-CAROTENE.

THIN LAYER CHROMATOGRAPHY

CLOSELY RELATED TO COLUMN CHROMATOGRAPHY: INSTEAD OF A COLUMN THE ADSORBENT IS COATED ON A STRIP OR PLATE OF GLASS, PLASTIC OR ALUMINIUM. INSTEAD OF TRAVELLING DOWN THE ADSORBENT, AS IN COLUMN CHROMATOGRAPHY, THE SOLVENT AND THE COMPOUNDS TRAVEL UP THE PLATE BY CAPILLARY ACTION.

THIN LAYER CHROMATOGRAPHY: PREPARATION

~ 1cm

baseline drawnin pencil

PREPARE THREE DEVELOPING CHAMBERS CONTAINING:

- HEXANE - ETHYL ACETATE - 10% ETHYL ACETATE IN HEXANE

SPOTTING A TLC PLATE

THIN LAYER CHROMATOGRAPHY: RUNNING

solvent~ 0.5cm ~ 1cm

THE SOLVENT LEVEL MUST BE BELOW THAT OF

THE SPOTTED SAMPLE

REMOVE THE PLATE WHEN THE SOLVENT REACHES ABOUT 1cm FROM THE TOP OF THE PLATE. QUICKLY MARK ON THE SOLVENT FRONT BEFORE THE SOLVENT EVAPORATES FROM THE PLATE.

THIN LAYER CHROMATOGRAPHY: SOLVENTS

HEXANE TOO NON-POLAR

ETHYL ACETATE TOO POLAR

10% ETHYL ACETATE IN HEXANE

JUST RIGHT

A

B

C

distance t ra v el led by s o lv en

solvent front

baseline

distance travelledby spot: A

BC

Rf =distance travelled by the compound

distance travelled by the solvent

decreasing polarity of com

pound

increasing Rf value

THIN LAYER CHROMATOGRAPHY: Rf VALUE

MORE POLAR COMPOUNDS ARE FOUND CLOSER TO THE

BASELINE

COMPLETING EXPERIMENT 4

REMEMBER TO REWEIGH THE PIGMENTS BEFORE DISSOLVING THEM IN HEXANE. PERFORM COLUMN CHROMATOGRAPHY IN THE FUME HOOD, TLC ON THE BENCH. TO SAVE TIME, RUN THE TLC EXPERIMENT WHILE THE COLUMN CHROMATOGRAPHY IS UNDERWAY. KEEP AN EYE ON SOLVENT LABELS AND DISPOSE OF ALL WASTE IN THE APPROPRIATE CONTAINERS. NO LAB REPORT IS REQUIRED FOR EXPERIMENT 4. THERE WILL BE A QUIZ ON EXPERIMENTS 3 & 4 NEXT WEEK.