experiment 4: column chromatography and … · experiment 4: column . chromatography and thin layer...
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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.
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