DCTER.'.'IN.~TION OF 3:4 BE-NZO-(a )-PYRENE IN
CIGARErTE SMOKE
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BATCo document for Province of British Columbia 2
November 1999


IIIJI VBP/46C/46D nth August, 1961.
Experimental Method
1,13terials and Apparatus
All chromatographic eluants should have extinction
values less than 0.005
down to 360 m4 after purification.
Penzene - May R Baker Ltd. (pu- crystallisable)
purified by redistillati3n (three times)
through a fractionation column and rejection
of the first ICI;~ of distillate. The benzene
Petroleum ether -
r-Plexane
Ciethyl etl,er
Cyclohexane -
Methyl alcohol
Hydrochloric acid
podium Hydroxide
Acetylated Chromotoqraphy Paper
Alumina A -
is stored after drying with sodium wire.
Analar BP 400 - 600C - purified b~-
percolation through a 4.5 cm. diameter
15 cm. long fully activated alumina column
and redistilled as for benzene. Dry with
!;odiu-n wire.
Hopkin 9 'Nilliams Ltd. (Hexane fraction).
May & Baker Ltd. (Peroxide free). Ury with
sodium wire.
British Drug Houses Ltd. (Special for
spectroscopy.
flopkin & 4illiams Ltd.
2 N aqueous solution
2 N aqueous solution
Prepared as described by SpotsWO06.
J. Chromat., 3. 19601 101.
Woelm Ltd. - neutral grade - activity 1.
De-activated at 70A' relative humidity for
3 days, re-activatec' by heating at 2000C
for ?4 flours just before use.
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BATCo document for Province of BritiSh Columbia 2
November 1999


Alumina B
Silica qej
!,Iitroqen -
Solid Carbon dioxide -
14
7 :10 ~C benzpYrene -
Woelm Ltd. - neutral grade - activity 1.
De-activated by addition of ',% water and
mixed in closed rotary mixer for 3 hours.
British Drug Houses - de-activated hy
washing with methyl alcohol/water lil, dried
in air and activated by heating at llo'c
for 6 hours.
British Oxygen Co. Ltd. - (oxygen free).
Imperial Chemical Ineustries Ltd.
~;pecific activity 6 g C/mg. purified by
paper chromatography as in procedure
(200 vg/6" strip). Log E384 MV - 4.41
Annaratus
All glass apparatus is cleaned with chromic acid
and washed well with water
before use.
Soiral Glass traos - Drawing RR.100
Ca,,+ridqe filter - Drawing RR.70/1, 70/2, 70/4a.
~:lass separators - 500 ml.
Chrorratoqraphy tubes - 4.5 cm. diameter - 50 cm.
length.
A.-71a Micrometer Syringe
Spectrophotometer
Radiation counting
equipment
3.0 cm. diameter - 15 cm. length.
Fitted with a sintered glass disc to
support adsorbent.
Burroughs Wellcome Ltd.
Unicam Ltd. SP.500.
Labgear Ltd. - Scaler
G-M tube and manual sample changer
Probe quenching unit.
BATCo document for Province of British Columbia 2
November 1999


3 -
Spectroqraph Hilger & Watts Ltd. - medium quartz
E498
Cells Hilger & Watts Ltd. - Cat.11o. H857
Photooraphic Plates Ilford Ltd. - HPS (5" X 4")
Photooraphic developer Ilford Ltd. - Contrast FF
Photoqraphic fixer Ilford Ltd. - Hypam fixer with
Hypam hardener
1%72.
pectroqraph lens Hilger & Watts - Cat.No. FIM
U.V. illumination Mazda merciry vapour discharge
lamp (125w,
system fitted into a photographic enlarger
with objective lens removed. A 'Joods glass
filter (2 mm) is fitted between the
condensing lens and the lamp.
ON
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BATCo document for Province of BritiSh Columbia 2
November 1999


4
Procedure
jeneral Note3
All operations are carried out In subdued light to
reduce the risk of
photochemical decomposition of the benzpyrene,
columns and flasks are covered
with black paper.
All evaporations are carried out under reduced
pressure in an atmosphere of
nitrogen.. When solvents are changed. the
solutions are evaporated down with three
successive 10 ml. fractions of the new solvent.
Smoke Collection
The cold traps are connected to the smoking engine
via a Cambridge
filter; the latter is a precaution in case of
blocking or incomplete cold trap
collection. The smoking engine is then calibrated
to give a 35 ml puff, of 2
seconds duration, once a minute. The calibrator
for puff volume Is a soap
film burette with a glass capillary attached, the
resistance of which is
approximately equivalent to that of the cigarettes
to be smoked.
Smoke a sufficient number of cigarettes to produce
6 - 10 ~Lq of benzpyrene
(about 3 - 4g of condensate if dried at 1000C for
16 hours). Collect using
a smoking cycle of 35 ml. puff volume, once per
minute each puff of 2 seconds
duration. Collect the conHensate in the spiral
cold traps surrounded by solid
carbon dioxide (Note 1). Remove thD cambw e ItM
the traps ,and wash
out the condensate with 6 x 10 MI. portions of a
mixture of ether and
hy0rochloric acid (lil). Transfer the washings
into separators and each
separator should contain approximately equal
amounts Of 1g of condensate.
,'.,ash out any condensate -emaining in the traps
with methyl alcohol, evaporate
the alcohol and wash the residue into one of the
separators with 10 ml.
of warm ether/hydrochloric mixture. Extract the
filter pads of the Cambridge
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filterswith ether in a soxhlet apparatus for 1
hour and add the concentrated C--
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0-1
all
BATCo document for Province of BritiSh Columbia 2
November 1999


- 5 -
ether solution (20 ml.) to one of the separators.
To each of the separators
add equal volumes of 14 C-benzpyrene solution in
cyclohexane such that a total of
about 10 pg (accurately known) has been added in a
total volume of 5.J mi.
c yc lohexa ne.
Isnlation of the neutral tar fraction
Separate the aqueous layer and discard. Shake the
ether layer for I
minute with 5 successive 100 mi. fractions of
hydrochloric acid followed by
3 x 100 mi. fractions of water. Extract the ether
solution with 6 x 100 mi.
portions of sodium hydroxide and finally 100 ml.
portions of water until the
aqueous layer is neutral. Care should be taken to
avoid emulsion formation
especially with the sodium hydroxide solutions.
Run the ether layer into
a flask containing anhydrous sodium sulphate 5g)
and allow to stand overnight
First Chromatoqraphy
Prepare a column (4.5 cn. diameter) containing
160g of alumina A and using
n-hexane as the solvent (Note 2). Filter the
ethereal neutral tar solution and
wash the sodium 5Ulphate with 6 x 20 ml.
successive portions of dry ether,
transferring the washings to the bulk of the
solution. Change the solvent to
n-hexane and apply the neutral fraction In 10 mi.
of n-hexane to the column.
Llute with n-hexane until all the green
fluorescence has been removed from the
colunn (Note 3). Continue the elution with 2
litres of benzene and collect
the benzene eluate (Note 4). Evaporate the
benzene, and change the solvent to
petroleun ether and dissolve the residue in 10 mi.
of petroleum ether.
'econd Chromatoqraphy
Prepare the column (3.0 cm diameter) in petroleum
ether using 120 g of alumina
B. Apply the petroleum ether solution of the
active fractions obtained from the
first chromatographic separation to the column and
elute with 500 mi. of petroleum
ether. Collect 10 ml. fractions. Carry out a
linear gradient elution commencing <=
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November 1999


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with 100% petroleum other and finishing with "
petroleum other 2CK benzene
over 400 mi. of eluate (Note 6). Continue the
elution with 8Os2O petroleum
ether/benzene taking 50 mi. fractions from the
commencement of the gradient
elution. Evaporate the solvent from each fraction
and dissolve the residue
In 5 ml. of petroleum ether. Determine the
fluorescence spectra of the solutions Using
the spectrograph (Note 7 and Appendix 1). and
combine these fractions showing the
typical Lenzpyrene fluorescence spectral bands
(404 mp, 408 m11, 427 mg) together
with one fraction either side. Evaporate the
solution to near dryness and
t sfer -,uantitatively into a 10 ml. beaker using
petroleum ether. Concentrate
the solution to about 0.5 mi. on the steam bath
whilst blowing a current of
nitrogen over the surface.
T1,ird Chromatoqraphy
Apply the solution containing the benzpyrene onto
a base line drawn
approximately 3 cm. from the edge of a 15 cm. wide
strip of acetylated chromatography
paper (-*-'hatman UM) using an Agla micrometer
syringe. Wash the beaker and syrinqe
with small quantities' of petroleum ether until
all the fluorescent material has been
applied to the paper. Develop the paper in methyl
alcohol/ether/water (4:4il)
mixture for 6 hours. Withdraw the paper and allow
to dry overnight, then replace
ir 'he chromatography tank for a further 6 hours.
Examination of the paper under
the ultra violet lamp shows the intensely
fluorescent benzpyrene band 2 - 3 cm.
from the base line.
Determination of the benzpyrene
Cut out the strip of paper showing the benzpyrene
fluorescence and extract in
a small soxhlet apparatus with benzene for one
hour. Cool the benzene solution to
room temperature and run the solution through a
silica gel column
ri cm. diameter 2 cm. long (Note 8) which is then
washed with small portions of
CD
benzene (total volume 25 mi.). Changt the solvent
to cyclohexaneand dissolve %)
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BATCo document for Province of British Columbia 2
November 1999


the benzpyrene in 5.0 ml. of cyclohexans. Measure
the extinction of 1 cm.
thickness of this solution using cycloh*xane as a
blank at 374 aq1 (A), 384 mg (B)
394 m~L (C).
Prepare 12 planchets for radioactive counting as
described in Appendix 2;
6 containing benzpyrene solution from smoke and 6
planchets with the standard
14 C-benzpyrene solution. Determine the count rate
for each planchet over
IU,030 counts as soon as possible after
preparation and calculate the mean count
rate for the standard and sample (Note 9).
of Result
Benzpyrene found r 5 x D x F
Y
where D = B - (A + C) for the benzpyrene solution
Y = B - (A + C) for a standard benzpyrene solution
of concentration
F v9/ml.
Benzpyrene recovery -- count rate for the sample =
R
count rate for the standard
Total benzpyrene 5 x D x F Pg.
Y x R
Benzpyrene in smoke 5 x D x F Z
Y x R I -
where Z = ~ig of benzpyrene added as radioactive
tracer.
Notes on Experimental Procedure
1. Under these smoking conditions, (35 ml. puff,
of 2 secon(; duration,
1 per minute) each trap can be used to collect tar
from 30 cigarettes. If the
puff frecuency is increased there is a tendency
for the trap to block and the puff
volume will decrease. The efficiency of the smoke
collection was estimated as
98% as determined by measurement of the smoke
deposited on the Cambridge filter
placed at the suction end of the trap. The draw
resistance of these traps was
less than C.2 cm. water cauae at a flow rate of
1C50 ml./min.
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BATCo document for Province of Brit!Sh Columbia 2
November 1999


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2. The activity of this alumina was found to be
approximately No. 2 as
determined by the Brackmann methodl^4&,. Ju.
h&J5,ke.,, , C4-1- - ~-"4XJ - q4t, ~4 '13
3. Fluorescence measurements showed that there is
no loss of benzpyrene
in the n-hexane eluate. About I - 1.4 litre of
n-hexane Is required to elute
the green fluorescing substance.
4. Although most of the benzpyrene is eluted in
the first litre of benzene,
2 litres are used to ensure the complete elution.
5. The activity of this alumina was approximately
No. 3 as determined by
the Prockmann mcthod.
6. The gradient elution is conveniently carried
Out Using the apparatus
described by Bock and Nan-Sing Linglr-(A-~,t- 6 ,
IS45- f-j (-. )
7. The spectra d.,Xrecorded on Ilford HPS
photographic plates 4eveloped for
3 minutes in Ilford Contrast FF developer.
S. This column removes paper fibres and suspended
material which will
interfere with the U.V. measurement.
9. 10,003 counts are taken because this is the
minimum number of counts
requircd to even out the effect of the natural
random emission of the radiation.
PTOVided the geometry of the counting system is
reproducible the count rate is
repro6ucible to within Li& with a theoretical
confidence limit of 6a.
The uniformity of distribution of the sample on
the dimple of the planchet
was checked by screening of various areas, when
the count rate was proportional
to the exposed area of the dimple.
From 0 - 0.5 pq of 14 C-benzpyrene the count rate
was proportional to the
amount of radioactive subs tance on the planchet
showing that self absorption of
radiation was negligible.
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BATCo document for Province of BritiSh Columbia 2
November 1999


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The count rate fell by A per day when the prepared
planchet was kept in
the dark, this decrease doubled on exposure to
daylight. Care must be taken
to avoid counting in polluted atmospheres as
certain substances cause a rapid
decrease In count rate. Chloroform was found to
cause a 2C% reduction in 30
minutes when the planchet was exposed to the
vapour.
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BATCo document for Province of BritiSh Columbia 2
November 1999


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APPENDIX 1
Procedure for use of the Spectroqraph
The spectrograph is set up using a slit width of
0.15 mm and slit height
if emm. A half plate (5" x 4") is placed in the
plate holder to photograph
the 28Qm4 - 1,UOO mp spectral range; the vacant
space in the holder (5" x 4")
is occupied by a piece of plain glass. A cell
containing the solution is
placed 48 cm. from the slit with one side facing
the slit. A lens in placed
10 cm. from the slit to focus light from the cell
onto the slit face. The
cell Is illuminated by a beam of light projected
at 600 to the cell face.
The spectrum is photographed using an exposure
time of 3 minutes and a spectrum
can be obtained on each plate. The plate is
developed for exactly 3 minutes,
washed in water for approximately 2 seconds and
placed in the fixer for 20
minutes. The plate development is carried out In
complete darkness and only
after the plate has been placed in a covered
fixing bath is light admitted
in the darkroom.
I
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BATCo document for Province of British Columbia 2
November 1999


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APPENDIX 2
Preparation of samples for countinq 14C-bEn7pyrene
The planchets are cleaned by boiling with
cyclohexane and wiped with a
soft paper tissue to remove last traces of grease.
The temperature of the
heating apparatus is adjusted to 1000C and the
planchet then placed in position.
Using the agla syringe 0.04 ml. of solution is
transferred into the dimple of
the planchet and allowed to evaporate. This is
followed by 2 x 0.08 ml.
portions of sample. Immediately the last trace of
solvent has evaporated,
the planchet is removed and stored in a dark place
before counting.
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BATCo document for Province of British Columbia 2
November 1999


OPTICAL ARRANGEMENT FOR
FLUORESCENCE SPECIROGRAPHY.
125 W BLACK GLASS LAMP
GEC MlaW/U
2 -V.. CHA.NCE
OXI FILTER
OPTIC
AXIS
(OPTICAL SEWICH)
10 QUARTZ CELL
LENS F1172 to C-
8-3c.m FOCA
LENGTH L)
As CM.
SCAITTERED U.V.
SLIT
MEDIUM QUARTZ
SPECTROGRAPIA
RTO 11.(=
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BATCo document for Province of British Columbia 2
November 1999


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BATCo document for Province of British Columbia 2
November 1999


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BATCo document for Province of British Columbia 2
November 1999


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BATCo document for Province of British Columbia 2
November 1999


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BATCo document for Province of British Columbia 2
November 1999


REF. REVISON DATE
6 L.Al.. WOOL. CILTIER
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FLEXASLE QU
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MATFPIAL PYP.F_,A. Z 0 D. 3Z 0. APPPox.
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C.D. RESEARCH A DEVELOPMENT
SCALE DRAWING No.
4-/IC~- FULL. R 12. 100
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