EXPERIMENT 4: COLUMN CHROMATOGRAPHY AND THIN …

1 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.

2 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.

3 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 - AND -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.

4 THIN LAYER CHROMATOGRAPHY : PREPARATION ~ 1cmbaseline drawnin pencil PREPARE THREE DEVELOPING CHAMBERS CONTAINING: - HEXANE - ETHYL ACETATE - 10% ETHYL ACETATE IN HEXANE SPOTTING A TLC PLATE THIN LAYER CHROMATOGRAPHY : RUNNING solvent~ ~ 1cmTHE 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 ABCdistance travelled by solvensolvent fr ontbaselinedista nce tr avelledby spot:ABCRf =distance travelled by the compounddistance travelled by the solventdecreasing polarity of compoundincreasing Rf valueTHIN 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.

5 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.