740
INDUSTRIAL AND ENGINEERING CHEMISTRY
of the Michigan State College of Agriculture and Applied Science for studies on the industrial utilization of agricultural products.
Literature Cited (1)
Abrams, A., and Brabender, G. J . , Paper Trade J . , 102,No. 15,
32 (1936). (2) Brabender, G. J., Proc. f a t Food Conf. Inst. Food Tech., 1, 227 (1940). (3) Carson, F.T.,Natl. Bur. Standards, Misc. Pub. M127 (1937). (4) Charch, W.H., and 'Scroggie,A. G., Paper Trade J . , 101,N o . 14, 31 (1935). (5) Edwards, J. D.,IND. ENO.CHEM., ANAL.ED.,25,846 (1933). (6) Gardner, H.A., "Physical and Chemical Examination of Paints,
Vol. 15, No. 12
Varnishes, Lacquers, and Colors", 9th ed., Washington, Institute of Paint and Varnish Research, 1939. (7) Gardner, H.A.,and Sward, G. G., IND.ENQ.CIIEM.,19, 972 (1927). (8) Kline, G. M., J . Research Natl. Bur. StandardP, 18,235 (1937). (9) Lishmund, R. E., and Siddle, F. J., J . Oil Colour Chem. Assoc., 24, 122 (1941). (10) Payne, H. F.,IND. ENQ.CHEM.,ANAL.ED.,11,453(1939). (11) Taylor, R. L.,Hermann, D. B., and Kemp, A. R., IND.ENO. CHEM.,28, 1255 (1936). (12) Wing, H.J., Ibid., 28, 786 (1936). (13) Wray, R.I., and Van Vorst, A.R., Ibid., 25, 842 (1933). P U E L I R Hwith ~ D the permission of the Director of the Experiment Station 8s Journal Article 639 (n. E.).
Determination of Nicotine and Nornicotine in Tobaccos C. V. BOWEN AND W. F. BARTHEL United States Department of Agriculture Bureau of Entomology and Plant Quarantine, Beltsville, Md.
To determine nicotine and nornicotine in tobacco the total steam-volatile alkaloids are isolated by steam distillation from a strongly alkaline solution containing sodium chloride in excess. One aliquant of the distillate is treated with silicotungstic acid precipitating nicotine and nornicotine, and another is treated with nitrous acid, steam-distilled from an alkaline solution, and the nicotine
A
METHOD of analysis for nicotine and nornicotine when present together in solution has been presented by Markwood (6). It WES developed for cases in which the alkaloids are already separated from interfering materials, and no procedure is given for obtaining the alkaloids from tobacco or tobacco products. To separate nicotine and nornicotine from the plant material, a steam distillation or continuous extraction with an organic solvent such as benzene is necessary. In view of the difficulty experienced in completely removing nornicotine from alkaline plant material by repeated extraction with benzene, ether, or gasoline, the method presented here resorts to a steam distillation, which completely separates both the nicotine and nornicotine from the sample, This method is based on the assumption that anabasine or other steam-volatile secondary-amine alkaloids aside from nornicotine are absent; such alkaloids have been found only in traces in Nicotiana tabacum. The steam-volatile secondary-amine alkaloid has been identified as nornicotine by methylation in most of the samples analyzed, The nornicotine is determined as the difference between the total steam-volatile alkaloid and the nicotine; consequently the analysis takes place in two parts. The first is an application to nornicotine of Burkat's method (4) for determining anabasine; it depends upon the complete steam distillation of nicotine and nornicotine from strong alkali solutions saturated with sodium chloride. The second part, which has been proved satisfactory by Markwood (6), ihvolves treating the solution containing both alkaloids with nitrous acid and steam-distilling from an alkaline solution, whereby the secondary amines, including nornicotine,
precipitated as a silicotungstate. The nornicotine is determined by difference. Analysis of tobaccos differing widely in alkaloid content shows the rather common occurrence of nornicotine. Comparison with nicotine determinations by the conventional methods shows the unreliability of the heretofore accepted method of analysis when nornicotine is present.
remain behind as nitrosoamines and the nicotine is obtained in the distillate.
Analysis of Standard Solutions The standard solution of nicotine used for proving the method was repared from a sample of nicotine that had been vacuumdistifed, treated with nitrous acid to remove any secondary amine, and then steamdistilled from an alkaline aqueous solution. The nornicotine used in preparing the standard nornicotine solution was identical with that used by Markwood (6),had been obtained from Robinson Medium Broadleaf, a strain of Maryland tobacco, and formed a picrate melting at ~9O-l9IoC. The absence of nicotine was established by treating a sample with nitrous acid and steam-distilling the a ueous solution after making it basic to phenolphthalein. No Jkaloid was present in
OF NICOTINEAND NORNICOTINE IN TABLEI. RECOVERY MIXTURESBY STEAM DISTILLATION FROM STRONQ
ALKALIAND SODIUMCHLORIDE
Composition of Mixture Nicotine Nornicotine Mg. Ma. 18.2 2.6 14.5 4.5 10.9 6.2 7.7 7.2 4.5 8.2 a
b
Direct pptn. YO.
Siliootungstate Residue" Pptn. of distillate Reoov ery
Yo.
%
97.2 98.6 101.8 98.7 99.4 Av. 99.1 Average of three determinations except where otherwise noted. Average of two determinations. 190.1 172.7 152.2 138.95 118.6b
184.86 170.3 155.0 137.1 117.9
December 15, 1943
ANALYTICAL EDITION
TABLE11. ANALYSIS OF KNOWNSOLUTIONSOF NICOTINENORNICOTINE MIXTURES
------Nicotine-------Calcd. Found Recovered
MU.
Mg.
%
23 51,
23.47 16.52 14.43 7.71
99.9 98.3 96.7 98.5
16.80
14.93 7.83
...
...
...
Av.
-NornicotineCalcd. Found .ug.
...
2.58 3.65 7.32 10.90
Recovered
Ma.
%
2.62 3.67 7.23 10.40
100.6
...
ioi:6 98.8 95.4 99.I
98.4
the distillate. This nornicotine was steam-distilled and the distillate used in preparin the, standard solution. Both solutions were standardized by t f e silicotungstic acid method.
74 1
opalescence when tested with silicotun stic acid solution. The distillate is concentrated by boiling (bohng stones added to prevent bum ing) until the volume is less than 25 ml., and then transferred' to a 25-ml. volumetric flask. Washings are added to the flask, and the liquid is brought to the mark. A 10-mI. aliquant is taken for precipitation of the nicotine and nornicotine with silicotungstic acid solution (12 per cent) according to the A. 0. A . . C p d u r e for nicotine (1). The silicotungstate re cipitate IS tered through a tared Gooch-type crucible ancfi, nited, giving residue A. Another 10-ml. aliquant is made neutral to phenolphthalein, and 2 ml. of acetic acid (30 per cent) and 0.5 gram of solid sodium nitrite are added (6). After standing at room temperature for 20 minutes, this solution is made slight1 alkaline with sodium hydroxide and steam-distilled into 3 m l of hydrochloric acid from the improved distillation apparatus as used above. The distillate contains the nicotine, which is precipitated, filtered, and ignited aa for the first aliquant, giving residue B. The nicotine is calculated from residue B and the nornicotine from the difference between residues A and B. The factor 0.1042 has been assigned to the nornicotine calculation. This factor was obtained as the theoretical value and was substantiated by use in the analysis of standard solutions of nornicotine.
The reliability of the silicotungstate precipitation for the determination of nornicotine is evidenced by the data presented in Table I. The conventional method ( 1 ) for the steam distillation of nicoWeight of residue B X 0.1140 = tine specifies the use of a slight excess of alkali. When nornicotine weight of nicotine present in aliqumt is present, the time for complete distillation of the alkaloids is (Weight of residue A weight of residue B) x 0.1042 = long and the volume of distillate consequently large, because the weight of nornicotine in aliquant nornicotine distills only slowly from a weakly alkaline solution. When the distillation is made from a strongly TABLE 111. STEAM-VOLATILE ALKALOIDSI N ANALYZEDSAMPLES O F TOBACCO AND alkaline solution, the speed of removal COMMERCIAL NICOTINESULFATE of alkaloids is greatly increased, and the addition of sodium chloride further Calculated 8 8 Nicotine and NornicoCalculated BE Nicotine tine, Proposed Method Proposed A. 0. A. C. hastens t h e process. Avens and Sample Nicotine Nornicotine Total method methods Pearce (8) report that in their nicotine % % % % % procedure excessively large amounts of Nicoliana labarum Robinson's Maryland Medium sodium hydroxide give abnormally high Broadleaf, Sayple 1 b 0.34 1.71 2.05 2.16 0.76 results, but that they are due to someCash, Bue-cured 0.70 2.40 3.10 .,. Robinson's Maryland Medium thing besides nicotine. This material is Broadleaf, Sample 2 C 0.98 2.16 3.14 3.32 1.77 Burley, Halleye 1.23 1.41 2.64 2.78 2,1? undoubtedly nornicotine, which did not Turkish, Xanthi, American distill readily a t the lower concengrown e 6.59 1.28 7.87 .., ... Maryland-Connecticut Broadtration. A comparison of the weights leaf 5 2.22 0.49 2.71 2.75 2.78 N. rustica of the alkaloid-silicotungstate ignition Sample 1 4.57 0.99 5.56 ... ... residues of nicotine-nornicotine mixSample 2 (Brasjlia) 6.71 0.66 7.37 ... ... Commercial nicotine sulfate tures obtained by direct precipitation Sample 1 c 34.75 4.54 39.29 39.70 39.70 Sample 2 38.82 1.10 39.92 40.02 39.75 and by precipitating after steam disa Determined elsewhere. tillation from strong alkali and sodium b Source of nornicotine used to pre are standard solution. chloride (Table I) shows that an average c Steam-volatile necondary-amine a?kaloid identified a8 nornicotine by methylation. recovery of 99.1 per cent may be expected. The alkali should be 30 per cent by- weight - and the sodium chloride The results of analyses of several samples of tobacco (Nicoin excess in order to have a saturated solution at all times during tiana tubacum), N . rualica, and commercial nicotine sulfate by the distillation. When the fresh distillate and the residue from this method are given in Table 111. For some of the samples the some of the samples were tested, the absence of alkaloid was table gives the total steam-volatile alkaloid calculated as nicotine indicated by no precipitation with silicotungstic acid. both by the proposed method and by the A. 0. A. C. method. When mixtures of the standard nicotine and nornicotine soluUpon calculation of the alkdoids as nicotine and nornicotine, tions were analyzed, one aliquot was steamdistilled directly to an unexpectedly widespread occurrence of nornicotine is indiget all the steam-volatile alkaloid in the distillate, while another cated. It is also'evident that when nornicotine is present the aliquot was treated with nitrous acid and then alkali before steam accepted method of analysis cannot be depended upon to give distillation. The alkaloids in the distillates were precipitated as reliable results. silicotungstates and the precipitates ignited. The nornicotine Although the improved steam-distillation apparatus was used waa calculated from the difference in weights of the residues. The in the proposed procedure, the conventional steam-distillation results shown in Table I1 indicate that such a procedure is satisapparatus may be substituted with a resulting increase in time factory. required for distillation.
-
,
Analysis of Tobacco for Nicotine and Nornicotine A 2.5-gram sample of powdered tobacco, a small piece of paraffin, 10 ml. of sodium hydroxide (30 per cent by weight), and 10 grams of sodium chloride are placed in the inner chamber of an improved steamdistillation apparatus (3) and steamdistilled into 3 ml. of dilute hydrochloric acid (1 4). Distillation is continued until a few drops of fresh distillate fail to give an
+
I
,
Literature Cited (1) Assoc. Official Agr. Chem., Official and Tentative Methoda of A n a l y s i a , 5 t h e d . , p . 6 4 (1940). (2) Avens, A . W., and Pearce, G . W., IND. ENQ.CHEM.,ANAL.ED., 11,505-8 (1939). (3) Bowen, C . V., and Barthel, W. F., Ibid., 15, 596 (1943). (4) Burkat, S.,Farm. Zhur., No.41,228-34 (1937). (5) Markwood, L.N., J . Asaoc. OfiCiaZ Agr. Chem., 26,283-9 (1943).
