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7 TABLE 2 Leaf Analysis of Tobacco after Sequential Increments of Ammonium Hydroxide Tobacco NH40H (ml) mmo l ) NH3 (g) % Nicotine * 1. Burley Grade HH - 3.25% 2. 200.Og 0 0 0 2.70% 3. 200.Og 10 150 2.55g 1.50% 4. 200.Og 18.8 280 4.76g 1.30% 5. 200.Og 25.0 375 6. 37g 1.26% 6. 200.Og 31.6 475 8.08g 1.40% 7. 200.0g 40.0 600 10.20g 1.40% * Starting Material - untreated TABLE 3 Gas Chromatographic Analysis of Extracts NH3 Added (mmol) g nicotine/100g tobacco 1. 0 0.56 2. 150 1.61 3. 280 1.61 4. 375 1.91 5. 475 1.86 6. 600 1.71

8 mately 18 fold molar excess based upon the relative amount of nicotine in the tobacco. Much of this ammonia is lost upon drying of the tobacco material since the total nitrogen increases by only 0.5% while the total volatile bases increases by about 0.3%'. These experiments have determined that the approximate amount of ammonia required to effectively alkalinize the tobacco for the maximum efficiency of extraction of the nicotine alkaloids is 2.8 - 3% of the tobacco weight as free ammonia at a 25%moisture level. Before any large scale freon extraction was attempted more information was required'to determine the optimum ratio of freon to tobacco in order to extract the most nicotine using the least quantity of freon. In eachexperiment 100.Og of cut HL '75 Burley tobacco was mixed with a solution of 10 ml of concentrated ammonium hydroxid'e and 15 ml of distilled water. This mixture was allowed to stand in a covered beaker for 20 minutes then the desired amount of freon was added. The suspension was allowed to stand at room temperature (21°C) for 20 minutes then suction filtered. The tobacco was washed with an additional 100 ml of fresh freon and rapidly pressed as dry as possible. The tobacco was spread out in a hood and dried under an air stream for 24 hours before leaf analysis. The following Table 4 shows the results of these experi- ments: TABLE 4 Leaf Analysis and Recovery volumes - Sequential Addition of Freon liters FC-11 liters FC-11 Leaf Analysis Added * Recovered * %nicotine recovery %total nit. @ - - 3.39 @' 4.21 @ 1) 0.40 0.21 (53%) 1.18 (34%) 4.45 2) 0.60 0.39 (65%') 1.39 (41%) 4.40 3) 1.10 0.88 (80%) 1.05 (31%) 4.50 4) 1.60 1.36 (85%) 0.80 (24%) 4.27 5) 2.10 1.89 (90%) 0.85 (25%) - * Includes the 100 ml wash @ Starting Tobacco analysis without ammonia treatment As expected the freon recovery efficiency percentage increases with increasing amounts of added freon. In~all cases ti 3 times the tobacco weight in freon remains absorbed on the leaf particles.

9 Although~ this large amount of freon was absorbed it could be efficiently recovered by technology currently in use for our tobacco puffing process. The minimum freon to tobacco ratio required for maximum extraction efficiency on one extraction is a 22.5 to 1 by weight or a 15 to 1 freon volume to tobacco weight. The maximum leaf reduction we obtained'on any freon-11/ ammonia extraction system was 85%. These experiments were all performed on laboratory size equipment which might not accurately reflect either the total extraction and recovery efficiency or difficulties sometimes encountered upon in- creasing the size of the process. A larger scale extraction was performed to determine "scale up" efficiency, to supply Ms. S. Ireland with approxi- mately 1/2 lb. of nicotine extracted from our tobacco and to identify any associated problems. A 25.5 lb. (11.58 kg) portion of cut burley tobacco, grade 7, was placed into a 30 gal. polyethylene settling tank which had the tap covered with wire screen. A mixture of 1 Q of concentrated ammonium hydroxide and 1.5 Q of distilled water was added. The tobacco was mixed with~a large paddle until it appeared'to be evenly moistened. The tank was covered and allowed to stand for 15 minutes. A 25 gal. (313 lbs.) portion~of freon was added in 5 gal. lots and the tobacco-freon slurry was allowed to stand at ambient temperature (45°F, 7°C) for 2.5 hours without agi- tation. The freon was drained from the tank into another 30 gal. polyethylene settling tank and the tobacco washed with one 5 gal. portion of fresh freon. The freon extracts and wash were combined to yield a recovered 20 gal. (2501lbs.) The tobacco was removed from the tank and a small representative portion was spread out and allowed~to air dry in a hood for 24 hours. The tobacco was then sent for leaf analysis. The results of this analysis are listed in Table 5. TABLE 5 Leaf Analysis of Tobacco from Large Scale Extraction I Tobacco % nicotine % total nitrogen Burley Grade 7 2.85 3.15 Burley 7 Extracted 1.58 3.59 This analysis indicates that only 45% of the nicotine present in the original tobacco was removed. A laboratory scale reaction with a freon/tobacco ratio of 12 to 1, as in the large scale extraction, effected a 68% reduction of the leaf nicotine.

One major difference was in the amount of mixing. The smaller scale was easily stirred allowing a more uniform distribution~of the ammonia solution. The temperature was also about 30°F (15°C) higher. This combination of higher temperature and more efficient mixing could allow better absorption of the ammonia by the tobacco and consequently better release of the nicotine. Two separate isolation procedures were attempted to determine which would give the greatest recovery of the extracted nicotine alkaloids. The isolation procedures attempted were (1) direct distillation and~ (2) aqueous acid extraction. (1) A 3 gal. portion of the freon extract was rotary evaporated to yield 65g of black semi-solid residue. This residue was vacuum distilled and 17.8g!of pale yellow liquid was col- lected', b.p. = 110°-112°C/7 mm, leaving a 46.7g pot residue. The residue solidified upon cooling. Capillary GC analysis indicated the distillate was 94% pure nicotine. (2) The remainder of the freon (17 gal.) was extracted portion- wise using a total of 700 ml of 50% aqueous sulfuric acid. The nicotine sulfate solution was made strongly alkaline with technical sodium hydroxide and vacuum distilled. As shown in a later experiment the isolation of the pure alka- loid by this method'was successful when applied to "Black Leaf 40", i.e. 40% nicotine sulfate solutions. The distillate (104g) had a boiling range of 50°- 70°C and was found to contain only 16% nicotine or 16.6g by capillary G.C. analysis. The 104g of aq,ueous nicotine was fractionally vacuum distilled to afford 20.8g of pure alkaloids, b.p. = 117°- 118°C/10 mm. The direct distillation technique gave by far a more efficient recovery of the nicotine alkaloids. The low efficiency of recovery in the second procedure could possibly be attributed to the concentration~differences between Black Leaf 40, 40% nicotine sulfate, and our extracts, calculated to be approximately 16% nicotine sulfate with 200g of unreacted sulfuric acid. Upon treatment with sodium hydroxide the large amount of sodium sulfate which preci- pitated could prevent transfer of heat during the vacuum distillation or strongly occlude the nicotine. In order to supply Ms. Ireland with 1/2 lb. of nicotine the extraction was repeated with certain modifications. A mixture of 15.5 lbs. of cut Burley Grade 7, 3.5 lbs. of cut Burley Grade HL'75 and 8.5 lbs. of cut Burley Grade OH'73 was mixed portionwise with a solution of 1.3 Q con- centrated ammonium hydroxide and 1.7 liters of water.

The ammoniated tobacco was allowed to stand at ambient temperature (0°C) for 24 hours. To this was added 27 gal. of FC-11 and the slurry was allowed to stand for 3 days. The freon was drained from the tobacco and the tobacco washed with 5 gal. of fresh~ freon. A small amount of the freon was spilled during the transfer from the extraction tank to the freon holding~tank. The recovered extract and wash totaled 26 gallons. The entire extract was treated by the first procedtiire to more efficiently recover the nicotine alkaloids. The 26 gallons were distilled'.from two 12 Q flasks portionwise until ti 3 liters remained in each flask. This was further concentrated in a 2 k flask, by distillation, until 1.5 liter remained. The dark residue was vacuum dis- tilled~to afford two fractions, both containing!substantial amounts of nicotine. The first fraction, b.p. = 111°-119°C/ 10 mm, amounted to 219.2g. This was given to S. Ireland for respraying onto tobacco along with the 20.8g from the pre- vious extraction. The second fraction, b.p. = 120°-128°C/ 10 mm, amounted to 41.4g. Upon standing the first fraction became pale yellow whereas the second fraction turned~light yellow/orange. Capillary G.C. analysis indicates the second fraction was contaminated with about 5% unknown volatiles. A small representative sample of the tobacco was spread out and air dried in a hood overnight. The results of the leaf analysis are listed in Table 6. TABLE 6 Leaf Analysis of Tobacco from Large Scale Extraction II Tobacco Weight (lbs.) % nicotine %total ni trogen Burley Grade 7 15.5 2.84 3.15 3.32% 3.71% Burley Grade HL'75 3.5 3.39 Ave. * 4.21 Ave. * Burley Grade OH'73 8.5 4.17 4.54 Mixed and Extracted 27.5 0.65 4.72 * These average values are of the entire mixed sample based upon the relative amounts of each tobacco in the mixture. The leaf analysis indicated that 80% of the contained nicotine was removed. The total nicotine recovered amounts to 258.5g nicotine or 62% of the total leaf nicotine available before extraction. This gives a recovery efficiency of 78% from the freon.

The freon 11 ammonia extraction technique appears to be generally applicable to a large scale process. The long reaction time required for the 80% extraction observed' here could possibly be shortened by agitation and some- what higher temperatures. The extreme volatility of the freon made it undesirable to operate at higher temperatures usingthe present equipment. The aqueous acid~workup would still seem like a viable concentration route. Possibly a series of scrubbers could be used to bring the nicotine sulfate concentration to 40% before the scrubbing efficiency dropped below acceptable limits. One problem encountered in the large scale freon extraction of'tobacco was the large amounts of non-alkaloid~ freon solubles. From the 27.5 lbs. of tobacco processed the residue weighed slightly over 850g (7$' by weight). Upon workup only 29% of this material was found to be nicotine. This means that 5% of the tobacco material by weight which is not nicotine is soluble in the freon. It might be desirable to readd this 5% by weight of non-alkaloidfreon solubles since they might contribute a number of valuable natural flavoring materials. A possible solution to this residue problem would'be a pre-extraction of non-alka:linized tobacco to remove most of the freon solubles, then ammonia treatment and re-extraction to remove the released nicotine. It was shown previously that freon 11 extraction of tobacco would not remove any substantial amount of the entrained nicotine. The following experiment was performed in order to determine the relative amounts of non-alkaloid materials which could be extracted with freon 11. By pre-treating the tobacco with freon the tobacco should~ remain saturated and~allow use of a smaller volume of freon for nicotine extraction. A 100.Og portion of cut Burley, Grade 7, was mixed with 1 Q of freon 11 and allowed to stand for 30 minutes. The freon was suction filtered off the tobacco. The 800 ml of recovered freon (80%) was rotary evaporated to yield 3.3g of viscous semi-solid residue. The tobacco was then treated with a mixture of 10 ml of concentrated ammonium hydroxide and 15 ml of water and allowed to stand for 20 minutes. To this mixture was added 1k of fresh freon 11 and the slurry was allowed to stand for an additional 20 minutes. The freon was suction filtered off the tobacco. The tobacco was pressed dry and washed with 100 ml of freon. The 900 ml of recovered freon (82$) was rotary evaporated to yield 3.60g of dark viscous oil.

The tobacco was air dried in a hood for 24 hours then sent for leaf analysis. The following Table 7 summarizes the results found in this experiment: TABLE 7 Leaf Analysis of Tobacco from Two-Stage Extraction Tobacco % nicotine % nitrogen Burley Grade 7 2.85 3.15 Grade 7 Extracted 0.45 4.44 The leaf analysis results show that 84% of the nicotine was extracted from the tobacco. The capillary gas chromato- graphic analyses of the two extracts agreed with the previous freon extraction experiments and indicated that the majority of the nicotine (>95g of the total extracted) was extracted only after treatment with ammonia. The pre-extraction technique can be used successfully to lessen the amount of non-nicotine extractables in the final nicotine recovery process. A large scale technique could involve two distillation steps, requiring additional heating and cooling costs. The advantage would be in the purer fraction of nicotine, possibly not requiring a final distillation to be suitable for augmenting low tar low nicotine tobacco. The procedure may more readily allow for the isolation of valuable natural flavorants from waste or scrap tobacco as well as from reusable smoking tobacco. Waste stems that are not used in the reconstituted leaf process, are a large source of waste tobacco material for use as a nicotine source. The following experiment was performed'in order to roughly determine the applicability of the freon 11-ammonia extraction procedure to the recovery of nicotine from these stems. The dry stems were received from the Louisville stemming operation. A 1000g portion of coarsely chopped stems was treated with a solution of 150 ml of water and 100 ml of concen- trated ammonium hydroxide and stirred until the mixture appeared evenly moistened. The mixture was covered and allowed to stand at room temperature for 30 minutes. A 5.5 Q portion of freon 11 was added and~the suspension was allowed to stand without agitation at 21°C for 1 hour covered with a glass plate. The freon was drained off through a wire screen and upon evaporation afforded 15.8g of residue. (4.9 liters recovered)

The stems were spread~out and air dried in a hood for 24 hours. TABLE 8 Leaf Analysis of Stems from F'reon Extraction Tobacco % nicotine o' nitrogen TVB Waste Stems 1.05 2.94 0.76 Stems - Extracted 0.55 2.90 0.91 It can be seen that there was a 48% reduction in nicotine in the stems using the freon extraction technique. The stems were very dry and very compact. The 5.5 k of freon 11 used covered the stems, although~, they had a tendency to float and the top portion was not under the freon surface. The large fraction of the freon recovered (89%) suggests that the compactness of the stems prevented good absorption and therefore prevented good dissolution of the entrained nicotine. With the use of stems it can be seen that most of the 2.6% added ammonia was not retained by the tobacco material. Only about 0.15% by weight was absorbed com- pared to ti 0.7% by weight retained on~whole or cut burley leaf. The fact that there was no apparent change in total nitrogen can be explained. The 0.50% of nicotine extracted accounts for a decrease in total N by 0.09%. If the 0.15% TVB increase were all added ammonia the total N increase would be 0.12%. The overall change in total N would therefore be 0.03%. This is within the experimental error of the determination. Relative to the 80% reduction of total nicotine in leaf or cut tobacco the stem extraction is less efficient (48%). The parameters of this FC-11-ammonia technique could be changed to determine if a higher efficiency can be obtained. Possibly a higher moisture would allow better transfer of the nicotine. More agitatim, high temperature and pressure for better solvent penetration or a finer cut stem material might also effect a more efficient extraction of the nicotine from stems.

In order for the freon/ammonia extraction technique to be economically feasible for the isolation of nicotine, from!leaf material purchased specifically as a nicotine source, the tobacco should remain a usable smoking material. Expert evaluation of tobacco from which 85% of the nicotine had been removed was not encouraging. The following Table 9 lists the taste profile for tobacco which had been extracted with freon. The nicotine was extracted from the freon by washing with two 50 ml portions of 10% sulfuric acid solution and~the non-alkaloid freon solubles were diluted in ethanol. Half of this solution was replaced on half of the extracted tobacco. TABLE 9 Taste Profile of Freon-Ammonia Extracted Tobacco Tobacco Nicotine ($) Taste Evaluation 29 (a) Burley OH'73 3.95 Good character, strength and body (b) Burley Extracted 0.47 No character, no strength, no body, burnt, smoky bitter off-taste (c) Burley Extracted 0.57 No character, no body, and Resprayed slightly more strength with "Flavor same off-taste as (b) Fraction" but less intense Without further processing the tobacco is not suitable as a smoking material. It was theorized that an alternate nicotine releasing agent might impart a suitable flavor and/or allow easier removal of the alkalinizing reagent. This experiment was performed using both a minimal amount of freon for partial nicotine extraction and an equal weight percentage of amine to nicotine to lessen the off'taste. A 100.Og portion of cut Burley, Grade 7, was mixed with 5 ml of 40% aqueous methylamine and 20 ml of water. This was covered and allowed to stand for 20 minutes. A 300 ml portion of freon was added and the slurry allowed to stand for an additional 20 minutes. The freon was suction filtered from the tobacco. The 90 ml of recovered freon (30%) was rotary evaporated to yield 2.80g of viscous residue. The residue was taken up in 100 ml of 95% ethanol and analyzed by capillary G.C. to indicate 1.34g (47%) of nicotine had been extracted.

The tobacco was air dried overnight in a hood and sent for leaf analysis. Table 10 lists the results of the leaf analys is . TABLE 10 Leaf Analysis of Methylamine - Freon Extracted Tobacco Tobacco % nicotine % nitrogen Burley Grade 7 2.85 3.15 Grade 7 Extracted 1.50 4.61 No formal evaluation of this tobacco has been completed'. Preliminary smoking results indicated the tobacco still retained the burnt, bitter off-taste however was less objection- able. The methylamine seemed to impart a cigar/pipe taste to the tobacco. Since less methylamine was used'than ammonia only 47% of the nicotine was extracted. It is not known whether the altered taste was due to less amine added and less nicotine removed or rather to the alternate amine added for alkalinization. The methylamine seems to bind somewhat more to the tobacco than does ammonia since the total nitrogen on analysis was over 0.5% larger than when ammonia is used. Conclusions to Freon Work 1. The freon 11-ammonia extraction technique is a viable method for the efficient (80-85%) removal and recovery of nicotine from tobacco materials. Maximum extraction efficiency can be obtained by using 2.8-3$ by weight of free ammonia and a 15 to 1 ratio of freon to tobacco 2. The use of a sequential or "two-fold" extraction tech- nique is desirable, in which the first extraction removes the "Flavor fraction" and the second'extraction, after ammonia treatment, removes the nicotine. This "flavor fraction", as well as the nicotine to be used for augmentation, could be recovered from currently discarded tobacco waste affording potential natural flavor additives as byproducts. 3. The direct distillation of the freon extracts, either from the one extraction technique or from the sequential extraction process, affords the most efficient recovery of the pure nicotine alkaloids. 4. The dramatic change in the flavor characteristics of the tobacco when methylamine was used as the alkalinizing agent suggests that R & D operations might solve the off-taste problem associated with the ammonia treatment by the selection of an alternate reagent or3$dditional processing of the ammonia treated tobacco.