creases in the tissue of flue-cured tobacco, the proporbion of oxalic acid in the aggregate of the acids becomes proportionally greater. On combustion tobacco ...
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INDUSTRIAL AND ENGINEERING CHEMISTRY
pleasing qualities and flavor and the smoker usually regards the smoke as being flat and insipid. The smoker’s desires are not satiated by such a product. If flue-cured tobaccos are smoked alone, those which contain from 1.70 to 2.30y0 total nitrogen usually give the greatest satisfaction t o the smoker. Midribs from flue-cured tobaccos suitable for cigarette manufacture usually contain less than these amounts of nitrogen. This is confirmed by the data in Table 111 for the tobacco from the bottom and middle sections of the plant. Tobaccos which are very low in nitrogenous materials usually are high in content of insoluble carbohydrates, organic acids, and ash constituents. The analyses in Table I11 show that the midrib portion of the veinous material contains more total acids and soluble ash than the laminar material. Unpublished work from this laboratory indicates that as the content of organic acids increases in the tissue of flue-cured tobacco, the proporbion of oxalic acid in the aggregate of the acids becomes proportionally greater. On combustion tobacco tissue which is high in content of oxalates and insoluble carbohydrates imparts to the smoke a bitter irritating taste. Such a smoke is not pleasing and may impart a lasting aftertaste which is undesirable. The difference in petroleum ether extractive between laminar and vein tissues is very striking; however, the significance of this difference is not clear. The data in Table I11 are believed to contain evidence of a chemical nature that supports the validity of the practice of eliminating the major portion of the midribs of flue-cured tobaccos in the manufacture of cigarettes.
Literature Cited Andreadis, T. B., andToole, E. J., Z . Untersuch. Lebenam., 68. 431 (1934).
Andreadis, T . B., Toole, E. J., and Binopoulos, J. T., Ibid., 77, 262 (1939).
Anon., Rev. Appl. Botan. Agr. Maurice, 2, 122 (1924). Askew, H. O.,Blick, R. T., Currie, K. E., and Joyce, W., New Zealand J . Sci., 30, 129 (1948). Bacon, C. W., J . Am. SOC.Agron., 21, 159 (1929). Cicerone, D., and Marocchi, G., Boll. tec. colt. tabacchi, 12, 119 (1935). Darkis, F. R., Dixon, L. F.,andGross, P. M., IND.ENQ.CHEM., 27, 1152 (1936). Darkis, F. R., Dixon, L. F., Wolf, F. A., and Gross, P. M., Ibid., 28, 1214 (1936). Zbid., 29, 1030 (1937). Darkis, F. R., Pederson, P. M., and Gross, P. M., Ibid.. 33, 1549 (1941). Dittmar, H., Pharm. Zentralhalle, 81, 169 (1940). Garner, W. W.,Bacon, C. W., Bowling, J. D., and Brown, D. E., U. 8. Dept. Agr., Tech. Bull. 414 (1934). Jehau, J. B., Mem. Manufactures etat (Pards), III, 21 (1898). Pantea, c., Bull. Facultat Stunte Agr. Chisinan. C m u n . Lab. Chim Agr., 3,194 (1940). Pyriki, C.,Z. Untermch. Lebensm., 83,221 (1942). Ridgway, C.W.,J . Agr. Research, 7 , 269 (1916). Vladescu. I. D.. Bull. Cult. Ferm. Tutunuli.. 29.. 307 (1940). . , ZbkZ., 30,’116(1941). Wenuech, A., Z . Untersuch. Lebensm., 79, 481 (1940). RECEIVED M a y 24, 1951. Papers 1, 2, 3, and 6 of this series appeared in IND. ENG.CEEM.,27, 1162 (1936): 28, 180 (1936); 29, 1030 (1937); 33, 1549 (1941). The fourth appeared in BUZZ. Torrey Boton. Club,64, 117 (1937).
Nitrogen Compounds in Fermented Cigar leaves A n essential step in the chain of industrial operations by which crude leaves of cigar tobacco are transformed into a good smoking product is the so-called fermentation. Substantial chemical changes occur during this phase of processing within the leaf tissues, including conversions of the nitrogenous leaf components. During the phases of processing thpt precede the fermentation, about half of the initial leaf protein is hydrolyzed by the leaf enzymes and yields considerable amounts of amino acids. A large part of these amino acids undergoes oxidative deamination with the formation of ammonia, which, after having reached a maximal concentration in the leaves, evaporates gradually during fermentation. The remain-
ing amino acids react with other constituents of the tobacco leaves, probably with polyphenolic substances, yielding Condensation products of increasing molecular size ond decreasing solubility in water. As a net result of these reactions, only very small amounts of amino ocids are left in the leaves after fermentation. The nitrates remain almost unaffected by the fermentation, but the contrary applies to nicotine and the related alkaloids of the leaves. These findings, in conjunction with studies of the catalytic and enzymic mechanism, have helped in replacing the traditional, purely empirical art of tobacco fermentation b y a catalytically controlled procedure which yields a mild and aromatic product of very low alkaloid content.
W. G. FRANKENBURG AND A. M. GOTTSCHO General Cigar Co., Inc., Lancasfer, Pa.
T H E end of the curing process the leaves of cigar tobacco are still unsuited for the manufacture of good smoking products. The cured leaves have t o undergo additional processes called sweat, fermentation, or resweat before they develop, on ignition, a mild and aromatic smoke. People, unfamiliar with the tobacco industry, are astonished t o learn t h a t these various processing operations take, as a rule, several years. We shall describe here briefly the processing operations as they are applied to the cured leaves of a typical cigar filler tobacco. For more detailed descriptions, see (7,18). Figure 1 illustratee the construction of a conventional cigar. As a rule, different types of tobacco are used for the filler, binder, and wrapper. Usually, the filler tobaccos undergo a more vigorous sweat and fermentation than the tobaccos used as binders, and partictdarly than tobaccos used as wrappem. I n the first phase called natural sweat the leaves, packed in tightly compressed units, undergo a prolonged storage in which
they are exposed t o the fluctuation of temperatures and humidities of at least one summer and one winter season. In the following process of fermentation, they are moistened with carefully controlled amounts of water, repacked, and kept in rooms at about 45’ C. and air humidity of about 60%. Periodically, the tobacco is withdrawn from these rooms, supplied with fresh air by unpacking and shaking the leaves, repacked, and returned to the heat rooms. These “cycles” are repeated four t o ten times, depending on the individual type and character of the tobacco. Concurrently, the periods in the heat room are increased from a few days t o many weeks. An aging process completes the treatment. Although it may sound ample enough, this processing is an art rather than a mechanical operation. No rigid schedule can be followed because of the extreme variability of the tobacco leaves which calls for a highly individualized treatment for every new crop and, within each crop, of every single unit of tobacco.
I N D U S T R 1.AL A N D E N G I N E E R I N G C H E M I S T R Y
The character and composition of the leaves depend, to an astonishing extent, on the weather conditions that prevailed during the growing and curing season, on local differences of soil and fertilization, and on a number of additional factors. An experienced tobacco processor will recognize the special nature of a given batch ottobacco from the appearance, feel, and smell of the leaves, and he will use these external signs for choosing the specific treatment t o be applied t o this batch. Much depends on his judgment since even slight deviations from the type of cycles t o be used, and from the correct amount of water incorporated into the leaves prior to fermentation, can lead to inadequate results. Leaf i) '7
Typical Construction of a Cigar
This intuitive method works well most of the time but can lead to occasional disappointments. Another difficulty encountered is that certain crops, or at least a part of them, fail to respond to even the most vigorous efforts to initiate their fermentation. To overcome these and related difficulties, i t appeared necessary to learn more about the chemical transformations that are caused in the tobacco leaves by natural sweat and fermentation. Any such information, and particularly any knowledge of the catalytic and enzymic factors involved, will serve for a better undersfanding of the fermentation process and possibly as a basis for its consistent controls. Investigation of Nitrogenous l e a f Components Analytical Methods. Several years ago, this laboratory started to study systematically the nitrogen compounds in cigar tobacco leaves and their fate during fermentation. By limiting this work to a thorough investigation of this specific group of leaf components, it was expected that more useful information could be obtained than would result from a cursory coverage of a wider scope of leaf constituents. The nitrogen compounds were chosen because preliminary observations had indicated a lively participation of these compounds in the fermentation process and also because Kjeldahl determinations permit a full accounting for all the nitrogen-containing compounds in the leaves, including any elusive and chemically ill-defined substances. Nitrogenous leaf components have been the subject of several earlier investigations (1-3, 14, 21, 24-26, 28-30, 34). However, most of the authors limited their work to a small number of tobacco samples and to the determination of a few typical compounds for which convenient analytical methods are available. Important progress was made in the analytical field by Vickery, Pucher, et al. (44, 48, 49, 50) who investigated thoroughly the chemistry of the curing process for a cigar wrapper tobacco (shade-grown Connecticut tobacco, U. S. Type No. 61). These authors improved considerably the existing analytical methods for a number of leaf components, including nitrogenous compounds, and developed new methods for the determination, i n tobacco samples, of proteins, and of various amino acids and amides, particularly of glutamine and asparagine (87, 45-47). The object of the studies a t General Cigar Co. was t o gain information on all the nitrogenous substances present in tobacco
Vol. 44, No. 2
leaves, including those which escaped previous investigations. This appeared desirable since some of the new unknown nitrogenous substances formed during the processing of the leaves may contribute to the improvement of the smoking properties. For this purpose, a special analytical scheme was developed that is based on a stepwise separation of individual fractions of nitrogenous substances from a given bulk sample and on the determination of the nitrogenous components in these successively withdrawn fractions. For a given sample, the sum of the individual increments of nitrogen found in the separate fractions is equal within the limits of analytical errors to its total nitrogen, a8 determined by direct kjeldahlization of the sample. Whereas most of the nitrogenous substances can be identified as specific compounds, the chemical nature of those contained in a few smaller fractions is, so far, only approximately known. The tobacco type selected for this work was Pennsylvania Seedleaf tobacco (U. S. Type No, 41). This tobacco, commonly used as a cigar filler, is subjected to a vigorous fermentation and undergoes correspondingly vigorous chemical changes. In the course of the program, samples of various crops and of different ease of fermentation were investigated. Table I gives a survey of the nitrogenous constituents found in tobacco samples, and of the analytical methods used for their determination.
Analysis of Nitrogenous Constituents in Tobacco Samples
Distillation, titration, Nessler (by optical scattering) Van Slyke (manometric), chromatography
a-Amino acids Nitrates
Re(;%lltion and distillation. titration. and Nessler Mild hydrolysis distillation of NHa (57 qS Stron hydrolysis. distillation of NHa '(34 Distil?ation, ultraviolet spectroscopy (@), titration Secondary alkaloids (nornico- Extraction with ether: ultraviolet spectrostine, possibly anabasine) copy, weights, and N content of silicotungstic acid precipitations Nitro enous condensation Coagulation in acid medium and kjeldahlipro%ucts (possibly with zation: N content of silicotungstic acid phenolic compounds) precipitations Transformed alkaloids (pyri- See (fd) dine compounds) Insoluble "protein" nitrogen Kjeldahlization Insolubilized alkaloid nitrogen Oxidation to nicotinic acid, extraction. ultraviolet spectroscopy Residual nitrogen, (probably Kjeldahlization bssic amino acids, purine and pyrimidine derivatives, etc.)
Glutamine Asparagine Nicotine
A detailed description of this stepwise scheme of nitrogen analysis and of the gravimetric, volumetric, colorimetric, and spectrophotometric methods used for determining the individual components will be given elsewhere, together with a detailed report on various analytical results. In this way hundreds of tobacco samples were analyzed a t different stages of their processing.
Conversions of Nitrogenous l e a f Components during Processing 'Instead of describing here in detail the chemical responses of samples of all kinds toward the various phases of natural sweat and fermentation, we shall attempt to outline briefly the predominant and characteristic chemical transformations in which the more important nitrogenous components of the leaves become involved in the course of processing. Ammonia. Ammonium compounds are present in the green tobacco leaf in negligible quantities only, but appear during the shed-curing in increasing amounts in the leaf tissues. They originate mainly from the oxidative deamination of those amino acids that ivere set free by enzymic hydrolysis of parts of the leaf protein. In Pennsylvania seedleaf tobacco, the formation of new ammonia continues throughout the phase of natural sweat until the quantities present in the leaf reach peak values as high aa
INDUSTRIAL A N D ENGINEERING CHEMISTRY
some 25% of the total leaf nitrogen. Thereafter, toward the end of the natural sweat or at the very beginning of fermentation, the ammonia in the leaves starts to decrease, as a result of its gradual evaporation as the free base. The timing of this effect is controlled by the shift, with progressing sweat and fermentation, of the pH of the leaf tissues from an initial value of about 5.4 for the green leaf to a final value of 6.8 to 7.0for the finished tobacco (7, 9.2,38). Essentially, this increased alkalinity of the leaf tissues is caused by the elimination of organic acids via total oxidation and -
although chemically quite different,product. Of the total amino acids which originated from leaf proteins and which represent some 35% of the total leaf nitrogen, only a very small amount, corresponding to roughly 1 to 2% of the total leaf nitrogen, is left as free amino acids in the finished tobacco. The detailed chemistry of the condensation reactions involving amino acids still has to be clarified. However, a number of independent observations make it likely that, in the tobacco types studied by the authors, the non-nitrogenous components of the condensation reaction are substances with phenolic hydroxyl groups belonging to the class of polyphenols and possibly of the The appearance of the Condensation products, their nitroge content, and their behavior toward precipitating agents are in accordance with this assumption. Polyphenols and flavonols have been found in the green tobacco leaf by various investigators (4-6,16-18, 23, 27, 31, 3.2, 36, 36, 41, 42). For green leaves of Pennsylvania seedleaf tobacco, Roberts (40) has established, by chromatography, the presence of at least five different components of polyphenolic character of which he identified two relatively abundant representatives as rutin and as chlorogenic acid. In the course of natural sweat and fermentation, the polyphenolic compounds in the tobacco leaves are known to change into products of oxidation and condensation. It has further been shown by British authors (19,90) that diphenolic compounds such as catechol form, in the presence of polyphenol oxidase, condensation products with amino acids-for example, with proline and glycine.
Figure 2. Chromatogram for Amino Acids (-) and Phenolic Compounds (- -) in Extract of Green Pennsylvania Seedleaf Tobacco (40)
decarboxylation. Only about one third of the ammonia that was formed in the leaves is left in the tissues after a proper fermentation. The loss of total nitrogen that occurs between the end of shed-curing and the end of fermentation corresponds to some 15% and is almost entirely due to these ammonia losses. To be exact, not all the ammonia formed in the leaves is derived from the or-amino group of amino acids; additional amounts originate from the chemical transforniatioii of other leaf components including the alkaloids. Amino acids. AB pointed out previously, most of the amino acids encountered in tobacco leaves at various stages of their processing were derived from the original leaf protein by enzymic hydrolysis in the curing phase. Glutamine and asparagine have been identified i n relatively large amounts by Vickery, Pucher, and associates in shade-grown tobacco. I n Pennsylvania seedleaf tobacco, no similarly large amounts of these two compounds were found a t corresponding stages of the processing. According to information from Roberts (40) who investigated the types of amino acids in green leaves of Pennsylvania tobacco by chromatographic analysis, the compounds listed in Table I1 were identified. Roughly, two thirds of the total or-amino nitrogen that originates from proteolysis in the leaves undergo oxidative deamination, leading to ammonia, as outlined. Of the remaining amino acids which represent some 10 to 12% of the total leaf nitrogen, a large percentage (about 80%) becomes involved in a different type of reaction. Qualitative and quantitative studies of certain fractions obtained in our stepwise scheme of analysis indicate that these amino acids form condensation products with non-nitrogenous lcaf constituents. These condensation products range from low molecular, easily watersoluble substances to high molecular water-insoluble materials with substances forming colloidal solutions as intermediates. As a net result most of the amino acid nitrogen that becomes involved in this condensation reaction is finally found in the fully fermented leaf in the form of water-insoluble nitrogenous products, whereas a smaller amount is present in the form of water-soluble colloidal substances. Thus, some of the nitrogen which initially was part of water-insoluble leaf proteins reverts into a water-insoluble,
The same authors found that these condensation products catalyze the oxitdation of excess amino acids to ammonia and keto acids. Should similar catalytically active complexes be formed in tobacco leaves, this would indicate an interesting mutual relationship between the two chemical pathways by which the amino acids are eliminated from the leaf tiasues. Nitrates and Minor Nitrogenous Leaf Constituents. Some 7 to 11% of the total leaf nitrogen is present in form of nitrates. I n these studies of Pennsylvania seedleaf samples of various crops, no major changes of the nitrates during natural sweat or fermentation were observed. Although this stability of the nitrates may be an apparent effect only, caused by a counterbalance of formation and consumption, the conclusion seems presently to be war-
Table II. Amino Acids identified in Pennsylvania Seedleaf Tobacco by Chromatography (40) 1 2 3 5 6 7
(The numbers refer to those in Figure 2) Aspartic acid Glutamic acid Serine Asparagine Lysine (?.)a Glutamine
Question marks are Roberts’ (40).
INDUSTRIAL A N D ENGINEERING CHEMISTRY
ranted that the nitrates do not essentially partake in the chemical transformations caused by the processing of the leaves. Little attention has been given so far to a number of other nitrogen-containing. substances that seem to be present in relatively small amountsin tobacco leaves. They include the non-aamino nitrogen of basic amino acids, pyrimidine and purine bases
Vol. 44, No. 2
treated with nascent hydrogen. A method was found for extracting the substance which, on reduction with hydrogen, yields nicotine. The extracted material represents a mixture of the reducible substance with other oxidation products of nicotine; this mixture resembles (8)a product obtained by Weil from nicotine via its photochemical oxidation in presence of methylene blue as optical sensitizer (51, 62). After the removal of the accompanying components and further purification, the reducible compound was identified as oxynicotine (11, 33). Another anomaly encountered in the analytical work was a foreign absorption band superimposed on the ultraviolet absorption spectrum of the secondary alkaloids contained in the fermented tobacco. This was the starting point for an extensive search for the substance or substances that cause the appearance of this foreign band. As a final result of this search, 2,3'-dipyridyl and methyl-3-pyridyl ketone were found and identified as minor alkaloid transformation products present in well-fermented tobacco leaves (12). At present, the following transformation products have been isolated and identified: oxynico, o! , tine, nicotinic acid (9),methyl-3-pyridyl ketone, and 2,3'-dipyridyl. Moreover, there are indiPARTS cations for the presence of myosmine (10) and 3 PARTS of one or more still unidentified, chloroform7 PARTS PS MIXTUR soluble pyridine derivatives. Besides, relatively large amounts of one or more water-insoluble 3-pyridine derivatives are contained in wellFigure 3. Quantitative Data on Transformation o f Nicotine in fermented leaves. These insoluble compounds Fermentation of Pennsylvania Cigar Tobacco can be determined by means of a permanganate oxidation of the residue left after thorough extracsuch as are contained in nucleic acids, and lecithin, betain, and tion of powdered fermented tobacco with boiling water, followed choline as well as transformation products of chlorophyll. More by extraction of nicotinic acid from the oxidation products. detailed work will be required before concise statements can be ma$e in regard to the quantities and chemical behavior of these and related compounds in tobacco leaves. Nicotine and Secondary Alkaloids. According to all previous investigations, the alkaloid content of cigar tobacco is diminished during the fermentation. Nicotine decreases have' been reported by various authors ( I , 11,SO) corresponding to some 10 to 6070 of the amount contained in the cured leaves. No detailed studies Nicotine Nornicotine were made on the fate of the disappearing nicotine except that its evaporation from the leaves was occasionally assumed. In the search for transformation products derived from the initially present nitrogenous leaf constituents, the authors paid particular attention to any reactions in which nicotine and the accompanying secondary alkaloids may be involved in the fermenting leaves. Transformation of these pyridine derivatives under the relatively mild conditions prevailing during the processing are of general scientific interest. Also, the removal of the Myosmine Anabasine alkaloids from the leaves has considerable practical importance since it results in a decisively enhanced mildness of the smoke. The elimination of nicotine also leads to an intensification of various, otherwise unrelated, oxidative processes in the leaf tissues, and thus to a faster and more complete fermentation. Obviously, the alkaloids act as inhibitors on these other reactions, and their disappearance from the leaves can be considered a key Oxynicotine Nicotinic acid process in the fermentation of cigar filler tobaccos (7'). Of the alkaloids contained in Pennsylvania tobacco, nicotine represents the main component. Nornicotine is contained in the 0 cured leaves in amounts of roughly 4 to lOyoof nicotine content and anabasine in ampunts that are probably still smaller (11, $3). The first indication for the presence of alkaloid transformation products in the fermented leaves was observed in the form of certain anomalies in the course of the analytical work. For instance, 2,3'-Dipyridyl Methyl-3-pyridyl ketone aqueous leaf extracts, after they had been carefully freed from Tobacco Alkaloids a n d Some of Their Conversion Products nicotine, yielded new amounts of this alkaloid when they were
INDUSTRIAL A N D ENGINEERING CHEMISTRY
Further work is required for elucidating the chemical nature of these insoluble pyridine derivatives. The quantitative distribution of all these alkaloid transformation products in well-fermented leaves is illustrated by Figure 3. Particularly interesting is the formation, during fermentation, of nicotinic acid in amounts which exceed by a factor of 10 to 20 the quantities of this compound t h a t were present in the leaves prior to t h e processing operations. No other natural product is known t o contain equally large amounts (-0.5% of dry weight) of nicotinic acid. After further work, it may well be possible tb clarify the specific paths of reaction leading from nicotine and from the secondary alkaloids t o their transformation products. Evidently, the pyridine ring of the alkaloid molecules survives the transformations in the leaf timues, whereas the pyrrolidine ring undergoes dehydrogenations, additions of oxygen, ring opening with formation of a straight side chain, and breakdown of the latter.
Catalytic Agents in l e a f Tissues Not less important than a detailed knowledge of the chemical reactions in the leaf tissues is more precise information on the catalytic agents which control these reactions. It seems t h a t at least three classes of such agents are operative in the leaf tissuesnamely, native enzymes of the tobacco leaf; enzymes of external sources such M microorganisms; and nonenzymic catalysts, including organic and inorganic leaf components (16). Whereas the leaf enymes appear to be the controlling agents in the curing process, bacterial enzymes seem to exert a decisive influence on the fermentation of cigar tobaccos (88, 39). Nonenzymic, heatstable catalysts may promote certain reactions in the leaves directly or function as activators of some of the enzymes. The previously mentioned complexes formed from amino acids may act as catalysts for the oxidative deamination of excess amino acids.
Practical Applications By way of systematic studies on the influence of a great number of catalytic agents on the fermentation of Pennsylvania seedleaf tobacco, we finally were led to a class of compounds which, if added in amounts of roughly 0.01% t o the leaves, promote the fermentation to a considerable extent. These catalysts have proved t o be useful, particularly for the processing of “sweatresistant” tobacco and have led in their practical application to a fermentation product of hitherto unobtainable low nicotine content (5 to 20% of the initial nicotine content) and of very satisfactory smoking quality. Furthermore, the correlation of chemical data with the practical experience of the tobacco experts has permitted the development of a more consistent and dependable control of the processing operations.
In spite of these encouraging signs, many questions remain unanswered. There are still some obscurities in the picture of the reactions involving nitrogen compounds, and only the rough outlines are known of the chemical transformations of the nonuitrogenous leaf components. The specific role played by leaf enzymes and by microorganisms a t the various stages of the processing needs further elucidation. The complications of tobacco chemistry are also illustrated by recently obtained indications that the chemical changes that occur during the fermentation of a cigar wrapper tobacco differ in many respects from those observed for filler tobaccos. Thus, there is no lack of problems for future investigations in the field of tobacco chemistry. l iCerat ure Ciled (1) Behrens, J., Landw. Vera. Stat., 43,291 (1894). (2) BodnAr, J., and Barta, L., Bioclterr,. Z., 247, 218 (1932). (3) Bodn&r,J., and Barta, L., Ergeb. Enz?Jmforsch.,4, 274 (1935).
(4) Couch, J. F., and Krewson, C. F., U. S. Dept. Agr., East. Regional Research Lab., Rept. AIC52 (1944). (5) Couch, J. F., Krewson, C. F., Naghski, J., and Copley, M. J., Ibid., AIC-115 (1946). (6) Couch, J. F., Naghski, J., and Krewson, C. F., Science, 103, 197 (1936). (7) Frankenburs. W. G . . Advances i n Enmmol., 6, 309-77 (1946); . . 247 (1949). (10) Ibid., 23, 333 (1949). (11) Frankenburg, W. G., and Gottscho, A. M., unpublished results. (12) Frankenburg, W. G., Gottsoho, A., Mayaud, E., and Tso, T. C . ,
(13) Garner, W. W., “Production of Tobacco,” Philadelphia, Toronto, Blakiston, 1946. (14) Garner, W. W., U. S’.Dept. Agr., Bull. 141 (1908). (15) Grob, K., Ber. Schweiz botan. Gea., 58, 172 (1948). (16) Hasegawa, H., Botun. Mag., Tokyo, 51,(306 (1937). (17) Hasegawa, H., J. Chem. SCC.Japan, 7 , 73, 1036 (1931). (18) Howard, W. L., Gage, T. B., and Wender, S. H., Arch. Biochem., 25, 74 (1950). (19) Jackson, H., and Kendall, L. P., Biochem. J., 44, 477 (1949). (20) James, 0. W., Roberts, E. A. H., Beevers, H., and DeKock, P. C., Biochem. J.,43, 626 (1948). (21) Jenkins, E. H., Conn. Agr. Expt. Sta. Rept. (1893, 1894, 1914). (22) Jensen, C. O., and Parmele, H. B., IND. ENQ.CHEM.,42, 519 (1950). (23) Johnson, E. F., Ann. J . Pharm., 118, 1 (1946). (24) Johnson, S. W., Conn. Agr. Expt. Sta. Rept. (1892). (25) Kissling, R., Chem. ZQ., 26, 672 (1902); 28,453 (1904). (26) Klein, L., Agr. Bot. Expt. Sta., Karlsruhe, Fifth Rept. (1896). (27) Koenig, P., and Dorr, W., Biochem. Z., 263, 295 (1933). (28) Kolenev, A. H., Pub. State Inst. Tobacco Invest. Krasnodar, U.S.S.R., Bull. 5 (1917); 6 (1917); 7 (1917). (29) K6sutany, T., “Chemical-Physiological Investigations of Characteristic Hungarian Tobacco Types,” Budapest, 1882. (30) Kraybill, H. R., J. IND. ENQ.CHEM.,8, 336 (1916). (31) Krewson, C. F., and Couch, J. F., J . Am. Chem. SOC.,70, 257 (1948). (32) Naghski, J., Beinhart, E. G., and Couch, J. F., IND.ENQ. CEEM.,36, 556 (1944). (33) Naghaki, J., Porter, W. L., and Eisner, A., Arch. Biochem. 24, 461 (1949); Naghski, J.,private communication to the authors. (34) Nessler, J., “Der Tabak,” Mannheim, 1867. (35) Neuberg, C., and Kobel, M., Biochem. Z . , 179, 459 (1926); 190, 232 (1927); Naturwiss.,.23, 800 (1935); Enzytologia, 1, 77 (1936). (36) Porter, W. L., Brice, B. A., Copley, M. J., and Couch, J. F., U. 9. Dept. Agr., East. Regional Research Lab., Rept. AIC-159 (1947). (37) Pucher, G. W., Vickery, H. B., and Leavenworth, C. S., IND. ENQ.CHEM.,ANAL.ED., 7, 152 (1935). (38) Pyriki, C., 2. Untermch. Lebemm., 77, 164 (1939). (39) Reid, J. J., McKinstry, D. W., and Haley, D. E., Penna. Agr. Expt. Sta., Bull. 356 (1938); 363 (1938); Science, 86, 404 (1937). (40) Roberts, E. A. H., privata communication to the authors (February 1951); see also Roberts, E. A. H., and Wood, D. F., Arch. Biochem.,33, 299 (1951). (41) Shmuk, A., and Kashirin, S., Pub. State Inst. Tobacco and Makhorka Ind., Krasnodar U.S.S.R., Bull. 69 (1930). (42) Shmuk, A., and Semonova, V., Ibid., 33 (1927). (43) Swain, L., Eisner, A., Woodward, C. F., and Brice, B. A., J. Am. Chem. SOC.,71, 1341 (1949). (44) Vickery, H. B., and Pucher, G. W., Conn. Agr. Expt. Sta., Bull. 324 (1931). (45) Vickery, H. B., and Pucher, G. W., IND.ENQ,CHEM.,ANAL. ED., 1, 121 (1929). (46) Zbid., 12, 27 (1940). (47) Vickery, H. B., and Pucher, G. W., J. BWZ. Chem., 83, 1 (1929). (48) Zbid., 128,703 (1939). (49) Vickery, H. B., Pucher, G. W., Wakeman, A. J., and Leavenworth, C. S., Carnegia Inst., Washington, D. C., Pub. 445 (1933). (50) Vickery, H. B., Pucher, G. W., Wakeman, A. J., and Leavenworth, C. s., Conn. Agr. Expt. Sta., Bull. 399 (1937); J . Bdol. C h m . , 119, 369 (1937). (51) Weil, L., Science, 107,426 (1948). (52) Weil, L., and Mahler, J., Arch. Biochem., 29, 241 (1950). RECEIVED Auguat 1, 1951.