.
INTRODUCE 15 ML OF ETHYL ACETATE , CONTAINING 2 % OF ASCETIC ACID , INTO THE CENTRIFUGE TUBE . CLOSE THE TUBE WITH ITS GROUND-GLASS STOPPER AND SHAKE VIGOROUSLY FOR 30 SECONDS . CENTRIFUGE FOR FIVE MINUTES AT 1 000 TO 1 200 R / MIN .
PIPETTE 10 ML OF THE ETHYL ACETATE EXTRACT INTO A CONICAL FLASK FITTED WITH A GROUND-GLASS STOPPER .
INTRODUCE A FURTHER 5 ML OF ETHYL ACETATE CONTAINING 2 % OF ACETIC ACID INTO THE CENTRIFUGE TUBE ; AGAIN SHAKE FOR 30 SECONDS AND CENTRIFUGE FOR FIVE MINUTES AT 1 000 TO 1 200 R / MIN . PIPETTE 5 ML OF THE ETHYL ACETATE EXTRACT INTO THE CONICAL FLASK CONTAINING THE 10 ML FROM THE FIRST EXTRACTION . MIX .
SEPARATION OF THE BIS ( 2,4-DINITROPHENYLHYDRAZONE ) BY CHROMATOGRAPHY
APPLY 0 * 2 ML OF THE ETHYL ACETATE EXTRACT TO THE WHOLE OF A STARTING LINE SITUATED 2 CM FROM THE EDGE OF THE PLATE BUT LEAVE A MARGIN OF 2 CM AT THE SIDES OF THE PLATE . INTRODUCE SOLVENT TO A DEPTH OF APPROXIMATELY 1 CM INTO A CHROMATOGRAPHY TANK AND ALLOW THE ATMOSPHERE TO BECOME SATURATED WITH SOLVENT VAPOUR BEFORE INTRODUCING THE PLATE . ALLOW THE SOLVENT TO RUN TO THE TOP EDGE OF THE PLATE . ( THIS CHROMATOGRAPHIC SEPARATION SHOULD BE CARRIED OUT WITHIN TWO HOURS AFTER CARRYING OUT THE EXTRACTION . )
DRY THE PLATES FOR ONE HOUR UNDER A VENTILATED HOOD . HOLD THE PLATE IN A VERTICAL POSITION ON A SHEET OF GLAZED PAPER AND WITH A SPATULA SCRAPE OFF ( PERPENDICULARLY TO THE DIRECTION OF THE RUN ) THE REDCOLOURED ZONE , WHICH IS CHARACTERISTIC OF THE BIS ( 2,4-DINITROPHENYLHYDRAZONE ) DERIVATIVE . ENSURE THAT ANY CURRENT OF AIR IS EXCLUDED DURING THIS OPERATION . TRANSFER THE POWDERED PRODUCT OBTAINED , WITHOUT LOSS , INTO A SMALL GROUND-GLASS STOPPERED WEIGHING BOTTLE CONTAINING 4 ML OF GLACIAL ACETIC ACID . ALLOW TO STAND FOR 30 MINUTES STIRRING FREQUENTLY DURING THIS TIME . FILTER THROUGH A SMALL FLUTED FILTER PAPER DIRECTLY INTO A 10 MM THICK SPECTROPHOTOMETRIC CELL BUT RETURN THE FIRST 25 TO 30 DROPS TO THE FILTER PAPER IN ORDER TO OBTAIN A PERFECTLY CLEAR FILTRATE .
MEASURE THE ABSORBANCE OF THIS SOLUTION AT 500 NM USING ACETIC ACID IN THE REFERENCE CELL AND DETERMINE THE CONCENTRATION BY REFERENCE TO THE CALIBRATION CURVE .
PREPARATION OF THE CALIBRATION CURVE
PREPARE A SOLUTION OF L-ASCORBIC ACID OF CONCENTRATION 1 G PER 100 ML USING THE 1 % METAPHOSPHORIC ACID SOLUTION SOLVENT .
DILUTE 5 , 10 AND 15 ML OF THIS SOLUTION TO 100 ML USING THE SAME SOLUTION OF METAPHOSPHORIC ACID AS THE DILUENT .
CARRY OUT THE REACTIONS DESCRIBED ABOVE WITH 50 ML OF EACH OF THESE DILUTIONS WHICH CONTAIN 50 , 100 , 150 MG OF ASCORBIC ACID PER LITRE TO PREPARE THE CALIBRATION CURVE .
A GRAPH OF THE ABSORBANCE AS A FUNCTION OF THE CONCENTRATION OF THE SOLUTION SHOULD