Product Design
Automated Determination of Nicotine in Total Particulate Matter of Cigarette Smoke
Abstract
Reports on development of an automated procedure to determine nicotine in 2-propanol extract of cigarette smoke condensate. States that this is partly in response to the Federal Trade Commission's requirements of analysis of nicotine and water in smoke particulate matter. Describes the procedure as using an AutoAnalyzer based on the reaction of nicotine, cyanogen bromide and amiline to produce a color indicating nicotine concentration. Compares this procedure with steam distillation method. Finds that water and nicotine determination can be done from the same Cambridge filter pad with the cyanogen bromide method, that the procedure is more efficient and that the baseline is stable. Reports best agreement between the methods on 83 and 80mm cigarettes with cellulose acetate filters. Averages the nicotine delivery to be 1.33mg/cigarette with a standard deviation of 0.058, as determined on 610 replicates.
Fields
- Author
- Charles, James L., Ph.D. (PM, R&D VP, Pharmacologist, Industry Expert)Vice President of Research and a scientist for Philip Morris, Inc. Vice President of Research for Philip Morris, Inc. in 1986 and then again from 1992 to 1993.
- Ikeda, Robert M. Dr. (PM R&D)
Defense- STAHR, HM
- Ikeda, Robert M. Dr. (PM R&D)
- Hypothesis
- Use of filters, paper, and ventilationModification of tobacco products through use of filters, paper, and ventilation, and measuring effects on dependence, behavior, and toxicity.
- Nicotine transport, transfer, and uptake
Design changes which alter nicotine delivery or effect how the product causes and maintains dependence, including transfer of nicotine from tobacco to smoke, and uptake into the body. - Nicotine transport, transfer, and uptake
- Keyword
- Invention disclosure
- Nicotine delivery (Smoke nicotine or nicotine yield)
- Total particulate matter (TPM or Tar)
- Nicotine delivery (Smoke nicotine or nicotine yield)
- Smoke Constituent
- Nicotine
- Total particulate matter
- Design Component
- Cellulose acetate filter (CA filter, Conventional filter)
- Nicotine content (Tobacco nicotine content)
Total nicotine in the unburnt tobacco rod - Nicotine content (Tobacco nicotine content)
- Named Organization
- Eastman Kodak Co. (Kodak) (Cigarette filter mfg from 1950s to 1994.)Manufacturers of quality control equipment for cigarette packaging
- Federal Trade Commission (Enforcement agency for laws against deceptive advertising)
Enforces laws against false and deceptive advertising, including ads for tobacco products. Ensures proper display of health warnings in ads and on tobacco products;collects and reports to Congress information concerning cigarette and smokeless tobacco advertising, sales expenditures, and the tar, nicotine, and carbon monoxide content of cigarettes.- M R B
- Federal Trade Commission (Enforcement agency for laws against deceptive advertising)
- Technology/Method
- Technicon AutoAnalyzer
- Subject
- nicotine technology
- Smoke Constituents
- Smoke Nicotine (Measures)
- Test/Smoke Condensate (Testing)
- Smoke Constituents
Document Images
AUTOMATED DETERMINATION OF NICOTINE
INITOTAL PARTICULATE MATTER OF CIGARETTE SMOKE
J.L. Ch.a~ries, H.M. Stalh-, and R.M. Ikeda
P-1:1ILIP MORRIS, INCORPORATED
RESEARCH CENTER
RICHMOND, VIRGINIA, U.S'._4.
INTRODUCTION
An automated procedure has been developed for the deter-
mination of nicotine in a 2-propanol extract of the particulate
mattei of cigarette smoke collected on a Cambridge filter pad.
This work was und'ertaken in part because the Federal Trade
Commission's tar determination as set forth in the Commission's
announcement of July 21, 1967 requires the analysis of nicotine
and water in smoke particulate matter.
The `ne_'~od uses an AutoAnalyzer with room temperature
dialysis and is based on the reaction of nicotine, cyanogEn
bromide and~aniline to produce a color which is proportional to
the nicotine conce-r:t---ation. Sadler et al (1) have ad'apted the
reaction for use with an AutoAnalyzer to determine the nicotine
conterxt of steam distillates. Ha~wvey et al (2) have modified
Sadlle_'s procedu~fe to determine simultaneously the nicotine and
reduci-ag sugar co-nteM*` in an acetic acid-methanol extract of
ground tobacco leaf.
In our labo-=atory we have routi.ely determined nicotine in
particulate matter of cigarette smoke by a modification of the
methods of Griffith (3) and Pleasants et al (4~). A Cambridge
filter pad co-_^tain i_-°g the particulate matter from four cigarettes
smoked under standard conditions is introduced into a Griffith
still containing,hydrochloric acid. A clean up distillation is
performed. The contents of the still are made basic with sodium
hydroxide,, and the distillate is collected~ir_ a flask containing
sulfuric acid < kn ult-_-aviolet absorptio x spEctr;3rn~ is obtained on
(2)
the distillate, and the nicotine content determined by the method
of Willits (5). Standards are prepared from
nicotinea Hereafter I will refer to this as
tion method.
The automated cyanogen bromide method offers
tages over the steam distillation method. First,
is more efficienb for large numbers of
extraction procedure permits water and
on the same Cambridge filter pad, thus
machine smoking and sample preparation
Eastman White Label
the steam distilla-
analyses.
several advan-
the AutoAna:lyzer
Second, our
nicotine to be determined
reducing the amount of
required per tar deter-
mination. Third, the AutoAnalyzer baseline is stable, and absorb-
ances may be obtained directly from the recorder by using absorb-
ance paper. This simple peak height to concentration relation-
ship represents a co_r._siderable savings in time over the more
laborious background corrections required by the ultraviolet read-
_out of Willits. Furthermore, by means of an automated readout
device it is possible to obtain a direc.t readout with mg, of
nicotine per cigasette printed on paper tape thus completely
eliminating calculatio~~~~s.
MATERIALS AND METHODS
Appar.atus
Technicor AutoAnalyzer with Sampler II, 15 mm flow
cuvette, 460 mu interference filters, half-length
delay coil and dialyzer with Type C membrane.
Arthur H. Thomas Mod'el 4 Concentration Readout, C-8
Digital Converter and Printer
(3)
Reagents
2-Propanol, reagent grade
Ethanol, anhydrous, reagent grade
Molecular Sieve, Type 4A, 1/16" pellets
Cyanogen Bromide, Eastman White Label
Aniline, Eastman White Label
Citric Acid, reagent grade
Disodium Phosphate, reagent grade
Nicotine, Eastman White Label
BRIJ-35, 20°la
Preparation of Solutions The extraction solvent is prepared
by pipett--:ng 2.0 ml of ethanol into 1 liter of 2-propanol. This
solution is protected from the atmosphere with a drying tube
filled'. with Type 4A molecular sieve. In the event the analysis
for water does not precede the nicotine determination the internal
standard ethanol and the drying tube may be omitted.
The cyanoger bromide solution is prepared by dissolving,
20 g of cyanogen bromid'e in 1 liter of distilled water which
contains 0.5 ml of BRIJ-35 surfactant.
The buffered aniline solution is prepared by dissolving
11.24 g of Na2HP04 and 8.2 g of citric acid monohydrate in
about 900 ml of distilled water and 0.5 ml of BRIJ-35 in a
1 liter volumetric flask. Three ml of aniline is added, and
the solution is diluted to volume with distilled water.
The stock standard nicotine in particulate matter solution
is prepared by adding 60 ml of 2-propanol to 12 Cambridge
(4)
pads containing the particulate matter from 4$ monitor cigarettes.
The extraction is performed by mechanical shaking for 15 minutes.
The extract is thenfiltered through~Whatman #1 filter paper
while expressing all possible extract from the pads. Three 5 ml
aliquots are pipetted into separate Griffith stills containing
0.5 ml of 2 N HC1. The first 100 ml of distillate is discarded.
Five ml of 20% HaOH is added to each still, and the distillate
is collected to volume in a 250 ml volumetric flask containing
10 ml of 3 N H2SO4. The nicotine content of the distillate is
determined by the method of Willits using Eastman White Label
Nicotine as the stand'ard. The nicotine content of the stock
standard nicotine in particulate matter solution can thenbe
calculated.
Dilute standards of nicotine in particulate matter are pre-
pared by pipetting 1.5, 4.5, 7.5, 12.0, and 15.0 ml of the stock
standard solution into separate 25 ml volumetric flasks and
diluting to volume with 2-propanol.
In practice the above stock and dilute standards are pre-
pared by multiplying all volumes and Cambridge pads by a factor
of 10 to obtain an~a:dlequate quantity of standards for routine use.
The monitor solution is prepared by combining the extracts
from completed'samples. Four liters of combined extracts will
last approximately three months.
Sample Preparation Two Cambridge filter pads containing
the particulate matter from eight cigarettes smoked under standard
(5)
conditions are extracted with 23 ml of extracting solution on a
mechanical shaker for 1 hour. The sample is centrifuged for 1
minute, and gas chromatographic water is determined (6). After
standing overnight a portion of the extract is analyzedifor
nicotine on the AutoAnalyzer.
Instrumental Figure 1 is a flow schematic of the Auto-
Analyzer channel. The samples are placed on the Sampler II
turntable in a known counter-clockwise order and are separated by
alternate water washes. Sampling occurs at a rate of 70 per
hour with a 2 to 1 sampling to wash ratio. This yields an effective
analysis rate of 35 per hour. The air segmented sample stream
is diluted with 2-propanol, mixed, and a portion of the stream
cycled through the proportioning pump. The sample stream:is
pumped to a dialyzer where the nicotine dialyzes across a Type C
membrane into an air segmented stream of buffered aniline. The
dialyzer bath is at room temperature. Cyanogen bromide is added
to the nicotine-buffered aniline stream, andithe reagents are
mixed in a double mixing coil. The color is developed in a one-
half length delay coil. The stream is pumped into a colrimeter
with a 15 mm tubular flow cell and 460 mu interference filters.
The signal from the colorimeter is fed to a strip chart recorder F-~
O
O
u
~
~
where the samples appear as a series of symmetrical peaks. A t1:
retransmitting potentiometer sends a signal to an Arthur H. Thomas
Model 4 Concentration Readout where either absorbance or concen-
tration is displayed. The voltage proportional to the cor_centration
(6)
is passed through a digital converter and'~mg of nicotine per
cigarette is printedIon paper tape with an appropriate sample
identification number.
The data in this paper was obtained with a manual 1 to 4
dilution of sample extracts prior to analysis on the AutoAnalyzer.
Subsequently, the on-line dilution of extracts was incorporated
into the manifold, and'.it was demonstrated that there was no
significant difference between the two methods of sample treat-
ment.
Figure 2 is a typical recorder tracing of standards, monitor,
and unknowns. The purpose of the monitor solution is to check
the operation of the AutoAnalyzer channel on a daily and~long
termbasis. The preferred sequence is standards-monitorUsamples-
monitor-standards.
EXPERIMENTAL
Standardization Nicotine values obtained by the cyanogen
bromide method are approximately 10%Q higher than values obtained
by the steam distillation method~when Eastman White Label nicotine
is used as the primary standard for both~procedLres. Secondary
standards prepared as a 2-propanol extract of particulate matter
are standardized by the steam distillation method prior to use
as standards for the cyanogen bromide procedure.
Figure 3 demonstrates the difference in slopes
obtained with
Eastman nicotine standards in 2-propanol and secondary
stand'a-rds
prepared as a 2-propanol extract of particulate matter and
standardized by steam distillation.
(7)
Table 1 shows the effect of the method of standardization.
Ten replicates of a sample were analyzed by the cyanogen bromide
method with Eastman r:icotine in 2®propanol as standards, and the
average was 1.46 mg/cigarette. Ten replicates of the same sample
were analyzed with secondary nicotine in particulate matter
stand3rds, and the average was 1.34 mg/cigarette. Eighty deter-
miTa;:ions of the nicotine delivery of this sample by steam
distillation aver3ged~1.33 mg/cigarette with a standard deviation
of 0.068. Thirty-seven determinations on this same sample by
the cyanogen bromide procedure with secondary standardization
averaged 1.33' mg/cigarette with a standard deviation of 0.067.
Secondary standardization results in good agreement between
the two methods for this sample.
Effec±` of Dialysis The flow system in Figure 1 was altered
so that the dialyzer could be bypassed. Five standards of nicotine
in particulate matter and seven aliquots of a monitor solution
were pumped through the system with the reagent lines cor_ts.ining
only water and BRIju35. A definite turbidity formed when the
sample staeam entered the "reagent" stream when the dialyzer was
bypassed as in&icated in Table 2. The dia:lyzer was reco nnected
so that the system was in its standard configuration. The same
samples were pumped through the AutoAnalyzer with insignificant
N
O
absorbances. 0
Room temperature dialysis was chosen because of significant
~
r.~
background f rom samples dialyzed at 37 °C . Reduction to room
(Q
temperature gave insignificant background. While the temperature O
(8)
may vary slightly over long periods of time the variation within
a day is insignificant in our laboratory. Throughout the course
of the day the sensitivity of the method will increase slightly
as indicated by steeper slopes on the Beer's law plots. Frequent
standardizatio_n_ compensates for this effect and also guards
against undetected temperature fluctuations in the dialyzer.
Precision The precision of the method was determined'by
smoking two separate samples on four days over a seven day period.
The samples were analyzed for water by gas chromatography and
then analyzed for nicotine with the results as indicated in
Table 3. Sample number 2 is routinely analyzed by steam distil-
lation. Over a one month period the average nicotine delivery
was 1.33 mg/cigarette with a standard deviation of 0.058'as
deter,nined on 610 -replicates,
RESULTS AND DISCUSSION
A methods comparison study of the cyanogen bromide method
versus the steam distillation method was conducted over a
three month pe~riod. All major cigarette types were tested,
and the results are demonstrated in Figures 4 thrrough 8. For
the first two months each data point represents the averages
of 9 replicates by steam distillation and 4 replicates by
the cyanogen bromide method. Data points for the third :nocrth
represent the ave-r.ages of 5 replicates by the cyanogen bromide
procedure and 4 replicates by the steam distillation method. No
attempt was made at monthly distinctions on the figures. The
best agzeemerit between the two methods was obtained on 85 and 80 nun
cigarettes with cellulose acetate filte-rs. This is to be expected
(9)
since the standards for the cyanogen bromide procedure are
prepared from a particulate matter extract of a 85 tan monitor
cigarette with a cellulose acetate filter. Carbon filtered
and mentholated cigarettes also showed excellent agreement.
The 100 mm and non-filtered cigarettes gave the largest degree
of data scattering. This was due to the fact that these
cigarettes are departi.ng from~the standard matrix established
by the 85 mrn monitor cigarette.
The individual results for the first two months were
subjected to statistical analyses of variances. Pertinent
conclusions drawn were: 1. The average nicotine deliveries
obtained by both methods were not significantly different.
2. The variances of nicotine measurements by both procedures
were not significantly different.
SUMMAkY
An automated method for the determination of nicotine in
a 2-propanol extract of the particulate matter of cigarette smoke
has been developed. The method is particularly applicable to the
detei*.ninaticn of wate.: and nicotine on the same Cambridge filter ~
pad thus red::cing the amount of machine smoking and sample prep-
O
N
aratior. required per Federal Trade C'ommission tar detetmi:natiot? . ~
~
The Technicon AutoAnalyzer with room temperature dialysis is usedl N
Cd
to obtain analyses at the rate of 35 per hour. The method utilizes W'
.'L7 I
the reaction of r:icotir_e, cyanogen bromide andl buffered anilir_e
to produce a color which is proportional to the nicotir_e cor_cer_tration.
