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© © Chemical Analysis il. a x F mg. nicotin8 in aliquot. a = weight gain of funnel by precipitate in g. = proportion of nicotine to reagent in a molecule of precipitate = O.01012 From here. data can be extrapolated to the number of milligrams of nicotine per cigarette or to the percentagef of nicotine in dry "tar". The far most advanced method of quantitative determination of nicotine utilizes the isotope dilution technique with cl4-1abelcd nicotine as internal standard and involves gas chromatography in the final step. ~nis method, however, requlres special equipment and is, therefore, applied in a limited number of laboratorieS. With a liquid scintillation counzer, unquenched cl4-samples in toluene with 0.4% FPO (2.5-diphenyloxazole) and 0.005~ POPOP (p-bis~(5-phenyloxazolyl~enzene) ,as scintillators will give an efficiency of about 75~. A quench correction curve is established by counting a number of aliquot samplas before and after addition of small volumes of chloroform in ascending quantity. Nicotine-N-methyl-C14 is available from the Nuclear Chicago Corporation. A sm~ll amount of the isotopic nico- tSne is added to the wet PM from four cigarettes in 500 ml. Kjel~abl. •Between 2 and 5 x l05 DPM is a suitable

© \ O Chemical Analysis 12. range" of radioactivity fob the added "tracer". Nicotine is enriched by water steam distillation as described above. Fifty milliliter portions from 500 ml. distillate in the volumetric flask are combined with 10.5 ml. of 5n NaOH and 30 ml. of ether and shaken. ~he ether portion is removed and the alkaline layer is extracted two more times with 30 ml. portions of ether. The combined ether extracts are dried over sodium sulfate for at least two / hours; the ether is filtered and carefully evapora£ed under nitrogen. 0.3 ml of toluene are added to the residual oil and aliquots of 0.05 ml of this sample solution are prepared for liquid scintillation counting whereas aliquots of 0.005 ml. are analyzed by gas chromatography• Data for gas chromatography are: Column inside diameter 32 mm., length 2 m., stationary phase 20~Apiezon L on Gas Chrom P(60-80 mesh), column temperature 170° C., injection port and detector 200° C., helium with a flow rate o~ 55 ml. per minute, and an inlet pressure of 2.4, atm. serves as carrier• (~ne Perkin Elmer Model 800 with flame ionization detector with a 1 mv. recorder is used for this analysis in our laboratory)~ The described set-up clearly separates nicotine from nornicotine.

13. © Chemical Analysis The original nicotine content in the smoke of o~e cigarette is calculated with this equation: a(e - 0.1 b1 X 5 x 0.1 b) ~g. nicotine in the smoke of 1 clgarette a added nicotine - C14 b = isolated nicotine - C14 in 0.05 ml. aliquot (average from 3 units) c isolated nicotine in 0.005 ml. alignot determined by gas chromatography (average from 3 samples) When one determines X as an average from ~wo complete analyses for each test, the experimental deviation remains below 5~.

Chemical Analysls 14. © REFERENCES PERTAINING TO ~XTRACT FROM "S~LECTE~ LABORATORY METHODS IN CARCiNOGE~ESISY, E.L.Wynder, and D. Hoffmann from: "Methods in C~neer Research" Vol. II, in p~int. Editor: H.'Buscb. crowell, E.P., 19~i, Tobacco Sci.5:19-23 Holmes, J.C, and Cridlin, W.B., Jr., 1960. J. Assoc. Offic. Agr. Chemists 43, 1515 / Jarrell, J.E. and Wickhm~., J.E., 1962 Tobacco Sci. 6, 154-157. Quin L.D. and Pappas, N.A., 1962~ J. Agrlc. Food Chemists i0, 79-82 Sloan, C.H., and Sublett, N.J., 1965, Tobacco Sci. 9, o-74. ©

Chemical Analysis, 15. ADDENDUM~ © Since the submission of "Selected Methods in Tobacco Carcinogenesis," two new papers were published on the moisture determination in particulate matter. ~nese are 1. © 20 Thome, F.A. "Gas Chromatographic Determination of the Water in Cigarette Malnstream Smoke and Total Particu- later Matter." Tobacco SCI. i0, 29-33, 1966. Schultz, F.J., and Spears, A.W. "Determlnatlon of Moisture in Total Particulate Matter.' Tobacco Sci. i0, 32-33, 1966. The second method by Schultz and Spears is an improv- ment in the method previously reported by Sloan and Sublett (1965) of determinlng moisture con~en~. Furthermore, since only three samples of 0.01 ml. are taken from i0 ml• dioxane- isopzopanol solution, more than 999 remains for the nicotine determination after evaporation of the solvent• using cl4-1abeled nicotine as internal standard, mental deviation for Also, when the experi- nicotine stays~ below 5~ with this method. *September i, 1966. Addendu~ will appear in the final article.

O CIGARETTE SMOKE ANALYSIS '~- Date: II E xDerimental Details (a) Name of Cigarette (b) Type of Cigarette (c) Length of filter plus overlap (mm.) (d) Date and place of purchase (e) Laboratory temperature (o C.) (f) Laboratory humidity (~) (g) Moisture content of cigarette (~) (h) Average weight of cigarette (mg.) (i) Smoking machine (j) Butt length Particulate Matter(PM) (a) Average number of puffs taken i. .2. 3. 4. Average number of puffs resulting from 20 clgarettes smoked (b) Wet PM from 4 cigarettes I. 2. 3. Average weight wet PM per cigarette (mg.) (e) Method for wa~er determination (d) Water content of PM from 4 cigarettes (mg.) (e) I. i. 2. 3. 4. Average water content per cigarette (mg.) PM minus water; average from 20 cigarettes (mg.) Ill Nicotine Determination (a) Method used (b) Nicotine per cigarette (rag.) l, 2. 3. 4.

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