gHK DETERMINATION OP COUMARIN 301 TOBACCO AND ...

Coumarin standard solutions in dichloromethane (0.0015%i 0,0025%, 0,0050%,
0,0075%.)
GC CONDITIONS
Nitrogen flow : ^Omls/min.
Column.temperature : 220 isothermal
Attenuation : 50 x 1
Chart speed : 2,5 min,/cm, 9
PROCEDURE
1. Calibration of standard solutions
2ul of each of the standard solutions are injected into the gas
ohromatograph. Attenuation is 50 x 1, A graph of peak height vs
ug/ul of coumarin is plotted. This calibration graph is used to
obtain the approximate concentration of coumarin in the sample.
It is recommended that the calibration graphB are checked each day
and that the standard solutions are kept in a refrigerator and renewed
weekly*
2. Treatment of Sample
'. (a) The Cambridge filter containing the smoke from 25 cigarettes is
placed in a macerator flask, Celite (5g)» lead acetate (20ml,
Q% solution) and hot water (200ml, approx. 50 C) are added and
the mixture is macerated for 3 minutes. The macerator is then
turned off and the residue is washed off the lid and Bides of
the flask into the body of the solution with distilled water
(approx, 10ml) Maceration is continued for a further 2 minutes.
.The macerator is then switched off and the residue adhering to
the lid and sides of the flask is washed into the solution with
more distilled water (approx, 10ml),
http://legacy.library.ucsf.edu/tid/iyl97h00/pdf
(b) The mixture is filtered by suction through a Cambridge filter
pad into a Buchner flask. The residue on the filter pad is
washed with two 50ml portions of warm water.
(o) The aqueous filtrate (plus washings) 1B cooled to room temperature
by running cold water over the surface of the Buchner flask.
The contents of thG.-JBuchner flask are then transferred to a 50ml
separatory funnel |nd washed with four successive portions of
solvent mixture A (1 x 150, 2 x 100, 1 x 50ml),, the upper organic
layer being retained on each occasion. The organic extractions
are combined and washed with sodium bicarbonate (40ml, 4% solution)
in the same separatory funnel. The lower bicarbonate layer is
drawn off into a 50ml beaker. The upper organic layer is then
drawn off into a conical flask (11) containing anhydrous magnesium
sulphate (approx, 30-40g). The bicarbonate layer is washed with
an additional 40mle of solvent mixture A, The lower aqueous
bicarbonate layer is discarded and the organic layer is added to
the contents of the conical flask. The flask is stoppered and
the solution is left to dry for a minimum of 45 minutes.