January 15, 1933
INDUSTRIAL AND ENGINEERING CHEMISTRY
LITERATURE CITED (I) Dickinson, J.Iron Steel Inst. (London), 113,177 (1926). (2) Egan, Crafts, and K i n d . Paper to be presented before Iron and Steel Division, American Institute of Mining and Metallurgioal Engineers, February, 1933. ( 3 ) Eggertz, Polytech. J.,188,119 (1868).
29
(4) Herty, Freeman, and Lightner, Bur. Mines, Repts. Investigations 3166 (April, 1932). ( 5 ) Kichline, J. IND. ENO.CHEM.,7, 806 (1915). (6) Westoott, Eckert, and Einert, Ibid., 19,1285 (1927). (7) willems, Arch. Eisenhattenw., 1, 665-8 (1928). R ~ C E I V EOotober D 29, 1932.
Estimation of Gossypol in Cottonseed Meal A Modified Method J. 0. HALVERSON AND F. H. SMITH, Agricultural Experiment Station, Raleigh, N. C. tion of the small a m o u n t s of OSSYPOL occurring in The Withers-Carruth method as modiJied by the ether-soluble gossypol rethe cotton seed is readily Schwartze and Alsberg for the estimation of maining in cottonseed meal, the soluble in ethyl ether. gossypol in cotton seed is not adequate for the results of which are presented in Carruth made use of this fact determination of gossypol in cottonseed meal. this paper. From this study in his method of estimation (a). The proposed method has overcome the objections the method was formulated for In the process of manufacture the estimation of gossypol. of cottonseed meal, the gossypol and consists in the removal of a part of the oil in The modifications introduced not expressed into the oil, but the meal by a preliminary extraction with are: a preliminary extraction of remaining in the meal, becomes petroleum ether, followed by thorough extraction the oil remaining in the meal; in a large part insoluble in ethyl of gossypol from the air-dried meal with U. S. P. the precipitation upon standing ether. I n order to estimate the ethyl ether and removal of the ether from the overnight and filtering off an small amounts of this remaining insoluble flocculent precipitate; ether-soluble g o s s y p o l in the extract by partial vacuum. The residual extract the use of ethylene glycol as an meal, there is need of a method is diluted with petroleum efher and allowed to which is expeditious and accuaid in precipitation of gossypol stand overnight. The material insoluble in rate, and which will avoid the as dianiline gossypol; and digespetroleum ether precipitates and is Jiltered off. tion on the steam bath in the difficulties of the present method Aniline and ethylene glycol are added, the latter of C a r r u t h as m o d i f i e d by presence of a high boiling point p e t r o l e u m ether, followed by Schwartze and Alsberg (6). aiding the precipitation on the steam bath of the constant shaking or s t a n d i n g But one p u b l i s h e d modifidianiline gossypol in the presence of a petroleum cation of the Carruth method has before the p r e c i p i t a t i o n of ether of high boiling point. After standing overbeen made, that of Schwartze dianiline gossypol is complete, night, precipitates of a satisfactory degree of and Alsberg. This consisted in when it is filtered off, dried, and purity are obtained. extracting the ground seed in weighed. a Soxhlet extraction t h i m b l e , METHOD IN DETAIL removing the ether, allowing the residue to stand overnight after the-addition of 8 to l0;olumes of petroleum ether, and PRELIMINARY EXTRACTION OF OIL' then filtering off the precipitated insoluble material that Transfer a charge of 75 grams of cottonseed meal, ground would otherwise contaminate the precipitate of dianiline gossypol. One cubic centimeter of aniline was then dis- to pass through a 40-mesh sieve, to a 500-cc. Erlenmeyer flask solved in the solution with shaking, and allowed to stand until with 100 cc. of petroleum ether (b. p. 30" to 80" C,). (Fracprecipitation occurred. After standing 3 to 7 days, this tions of ordinary gasoline distilling between 30' and 108" C. precipitate was filtered on a tared Gooch crucible, washed, have been used.) Shake vigorously for 5 minutes and dried a t 100" C., and weighed. The filtrate was further filter through a Biichner funnel. Remove thoroughly with a allowed to stand from a week to a month in order to small amount of petroleum ether any meal adhering to the determine whether precipitation was complete. If neces- Erlenmeyer flask. Carefully remove the petroleum ether sary, corrections were made for any subsequent precipitation. from the meal in the Biichner funnel. Transfer the meal The accuracy of this method is shown by a recovery of 90 to shallow pans, and allow to dry a t room temperature for per cent of 0.7 gram of gossypol from 25 cc. of oil. In order to several hours with occasional stirring in order to remove all obtain this degree of accuracy, the authors used a large the petroleum ether. After washing with petroleum ether and air-drying, examount of gossypol in comparison with the minute amounts tract the charge thoroughly (approximately 72 hours) with present in meal, The Carruth method, as modified by Schwartze and Als- U. S. P. ethyl ether to which 5 cc. of distilled water are added berg, is not sensitive enough to estimate the small amounts of in the receiving flask, using a Soxhlet extractor having an ether-soluble gossypol remaining in the meal because of inside diameter of 50 mm. (Anhydrous ether and moistureseveral factors. The excess oil in the oil-petroleum ether free samples of meal cannot be used as the gossypol does not mixture hinders precipitation of dianiline gossypol. There extract quantitatively.) After removing the thimbles, is not a complete elimination of the troublesome contaminat- recover the ether by distillation into the Soxhlet until 15 to ing insoluble precipitate (in petroleum ether) ; the length of 20 cc. of extract remain in the extraction flask. Remove the time required to precipitate aniline gossypol and the in1 Omit this step in the analysis of seed, t o prevent the precipitation of completeness of this precipitation are unsatisfactory. gossypol by the petroleum ether; however, the small amounts of gossypol A study has been made of the factors involved in the estima- present in the meal do not precipitate.
G
30
ANALYTICAL EDITION
flask and filter the extract through a Gooch crucible into a 250-cc. Erlenmeyer flask placed under a bell jar. Wash the residue with ethyl ether. The ether is distilled off rather completely under reduced pressure, and this is facilitated by placing the flask containing the filtrate in a beaker of warm water. (The ether extract is readily injured, detected by a burnt odor, if heated strongly on the steam bath.) Then add to the filtrate 75 cc. of petroleum ether of a high boiling point (65' to 110" C.), and let the flask stand overnight. The following morning filter the solution through a Gooch crucible in order to remove the material precipitated by the petroleum ether. Wash the precipitate with petroleum ether, combining the washings with the filtrate. Add 5 cc. of ethylene glycol (c. P. or practical, Eastman) and 2 cc. of aniline to the filtrate, thoroughly mix by shaking, and digest gently on the steam bath 1 to 2 hours. When the digestion is discontinued, replace the petroleum ether lost to give a total volume of 60 to 75 cc. Allow the flask to cool, stopper, and let stand overnight. (Longer standing is necessary for precipitates of gossypol smaller than the usual amounts.) Filter through a tared Gooch crucible and wash the precipitate of dianiline gossypol with petroleum ether (b. p. 30' to 80" C.). Follow by four washings with water and then with a few cubic centimeters of petroleum ether. Dry the precipitate to constant weight a t 100' C. In converting the dianiline gossypol to gossypol, multiply by the factor 0.775.
PREPARATION OF PURIFIED GOSSYPOL In order to study the factors involved in the proposed method for the estimation of gossypol, a supply of purified gossypol was necessary. This was prepared from the seed by the method of Clark (5), in which the ground seed is percolated for 3 hours with ether sufficient to yield 3 liters of extract per kilogram of seed. The ether is recovered by distillation, and the residue is filtered through activated carbon. The filtrate is dissolved in an equal volume of petroleum ether and then treated with the same volume of glacial acetic acid. Crystallization begins at once, and the process is complete within a few hours. Some difficulty was experienced in obtaining the gossypol acetate (gossypol combined with one molecule of acetic acid of crystallization) precipitate from the extracted oil from fresh seed. Removal of the ether from the extracted oil by distillation on a steam bath injurea the extract (it has a burnt odor) so that the gossypol will not readily precipitate upon the addition of glacial acetic acid. The ground seed was percolated with ether. The resulting oil-ether mixture was freed from ether by gently heating at a temperature which did not rise above 60' C. This was done by immersing in the solution an electric heater consisting of a coil of Nichrome wire, No. 20, 12.5 feet (31.8 cm.) long having a resistance of 25 ohms (suggested by E. P. Clark). The crude gossypol was recrystallized three times as the acetate from a mixture of ethyl ether and ethylene chloride (4 to 1) and a volume of glacial acetic acid equal to one-half the volume of the mixture. There is present a reddish impurity which is readily soluble in this mixture. The gossypol was then further purified free of any reddish color by the method of Clark (3,p. 731), and solvent-free gossypol was prepared by crystallizing the purified substance from ether (3, p. 733). The final product of purified gossypol was of a bright yellow color.
Vol. 5, No. 1
cottonseed oil upon the addition of 5 cc. of ethylene glycol in the presence of aniline. Comparisons were made by dissolving definite amounts of purified gossypol in 5 cc. of refined cottonseed oil and 50 cc. of petroleum ether, adding aniline to all determinations and 5 cc. of ethylene glycol. After thorough shaking and standing, those determinations containing the ethylene glycol began to precipitate in a short time. $Thosedeterminations not containing ethylene glycol, however, on further standing after filtering off the precipitate, gave an additional small amount of precipitate. The total yield of gossypol was not so great as where ethylene glycol was added. The results are given in Table I.
TABLEI. INFLUENCE OF ETHYLENE GLYCOL UPON RECOVERY RELATIVELY LARGEAMOUNTSOF GOSSYPOL WITHOUT HEAT OR CONSTANT AGITATION
OF
(Gossypol dissolved in 6 cc. of oil, 25 0 0 . of petroleum ether, and precipitated with 2 cc. of aniline) ANILINE
DETN.
GOSSYPOLGOSSYPOL DAYS TAKEN WEIGHED QOSSYPOL RECOVEREID STOOD R ~ M A R K S Mo. Me. Me. %
( a ) METHOD OF SCHWARTZE AND ALSBERQ WITHOUT U S E OF E T H Y L E N l GLYCOL
1 2 3 4 5 (b) 6 7 8 9 10
11
48.8 48.8 25.0 25.0 25.0
cc. 48.8 48.8 48.8 48.8 48.8 48.8
WITH 6
49.5 48.3 24.6 26.4 27.0
38.4 37.5 19.1 20.5 20.9 Av.
78.7 76.8 76.4 82.0 83.6 79.5
2 2 Slow and in2 complete 2 precipitation 2
OF E I T H Y L E N QLYCOL, ~ OTHER REAGENTS AS A B O V E
57.3 58.7 55.8 56.9 68.1 58.3
44.4 45.5 43.3 44.1 45.1 45.2 Av. 44.6
91.0 93.2 88.7 90.4 92.4 92.6 91.4
More
oomplete p r e cipitation
Ethylene glycol is not miscible with the oil-petroleum ether solution, but in the presence of this reagent the precipitation of gossypol is facilitated. It has been found that 10 mg. of gossypol dissolved in refined cottonseed oil can be recovered quantitatively from the oil by repeated extractions with ethylene glycol when approximately 40 mg. of anhydrous sodium carbonate are used together with 75 cc. of petroleum ether. If water is present, troublesome emulsions are encountered. The gossypol is also unstable in the presence of aqueous alkaline solutions. Aniline is readily soluble in ethylene glycol and much less soluble in the cottonseed oil-petroleum ether mixture, and it thus appears that gossypol and aniline are intimately brought together in the ethylene glycol layer where precipitation occurs. Therefore, agitation should hasten precipitation. This will be considered later. The favorable influence of ethylene glycol on precipitation resulted in an average of 91.4 per cent recovery of 48.8 mg. of gossypol. This is 11.9 per cent greater than by the method of Schwartze and Alsberg, in which the precipitation took twice as long.
PRECIPITATION OB SMALLAMOUNTS OF GOSSYPOL The amounts of gossypol in Table I are not comparable to the small amounts determined in cottonseed meal, so it ap-
peared desirable to determine whether such amounts as are estimated in meal could be precipitated in the presence of 2 cc. of refined cottonseed oil with the aid of ethylene glycol. The amount of the other reagents was the same as given in Table I. They were well mixed by shaking by hand, and then allowed to stand in a refrigerator a t about 10" C. until precipitation was complete. The dianiline gossypol began to precipitate on standing overnight. Details are given in Table 11. INFLUENCE OF ETHYLENE GLYCOLON PRECIPITATION The results show that 4 of the 5 mg. of gossypol dissolved OF DIANILINEGOSSYPOL in the oil were recovered after standing 8 days a t approxiWithout heat or agitation dianiline gossypol precipitated mately 10" C., and that 8.7 of 10 mg. were recovered after more completely from a petroleum ether solution of crude standing 13 days. This precipitation is complete enough for
January 15, 1933
INDUSTRIAL AND ENGINEERING CHEMISTRY
the estimation of gossypol in cottonseed meal. In a charge of 75 grams of meal, 1 mg. of gossypol not recovered is equivalent to 0.0013 per cent, which is not significant. TABLE11. RECOVERY OF SMALL AMOUNTSOF GOSSYPOL WITHOUT HEAT DETN.
ANILINE GOSSYPOL GOSSYPOL DAYS TAKEN WEIGHEDGOSSYPOI, RECOVERED STOODREMARKS M Q
.
%
MQ.
Mg.
( a ) W I T H O U T AGIT.ATIONa
1 2 3 4 5 6 7 8
5.0 5.0 5.0 5.0 5.0 5.0 5.0 5.0
5.3 5.6 5 8
4.9 4.8 4.7 5.1 5 5 Av. (b)
4.1 4.3 4.5 3.8 3.7 3.6 4.0 4 3
82.0 86.0 90.0 76.0 74.0 72.0
8 8 Stood in cooler 8 a t 19". C.; 8 precipita8 tion delaved
80 0 86 0
8 8
4.0
80.7
8
WITH A G I T A T I O B b
13 Stood in cooler 13 13 davs a t 13 10°C.;shaken 24 hours; preAv. 8.7 87.3 cipitation delayed Five mg. of gossypol were dissolved in 2 cc. of oil, 25 cc. of petroleum ether, 2 cc. of aniline, and 5 cc. of ethylene glycol. b With 10 mg. of gossypol in 2 cc. of oil and 75 cc. of petroleum ether, other reagents as given above. 9
11 lo
10.0 10.0 10.0
11.3 11.8 10.6
8.8 9.2 8.2
88.0 92.0 82.0
31
shown by Carruth @),who found that not more than 90 per cent of 0.5 gram of gossypol was recovered from 50 cc. of oil, thus leaving approximately 50 mg. in solution. Likewise, Schwartze and Alsberg (6) recovered approximately 90 per cent of 0.7 gram of gossypol in 25 cc. of refined oil, 70 mg. remaining in solution. Although the amount of oil and gossypol used by these investigators is more than is obtained from a charge of 75 grams of cottonseed meal, yet it is found that less gossypol remains in solution or is not precipitated by the reagents in the proposed method, when proportional amounts of oil are used. TABLEIV. EFFECTOF PRESENCE OF OILON PRECIPITATION OF GOSSYPOL (Two cc. of aniline and 5 cc. of ethylene glycol added t o each determination) GOSSY- PETRO-ANILINE POL LEUM GOSSYPOL D E T N .TAKEN ~ OIL ETHER WEIQHED GOSSYPOL RECOVERED REMARKS M a . Cc. Cc. Mo. Ma. % .1 5.0 2 25 5.1 3.95 79.0 S o m e precipi2 5.0 2 25 5.3 4.11 82.5 t a t e on first 3 5.0 2 5.8 4.50 25 90.0 andsecond 4 5.0 2 25 5.7 4.42 88.4 davs. considerable i n Av. 4.25 84.9 3 days 5 6
7
5.0 5.0 5.0 5.0
5 5 5 5
25 25 25 25
0.5 1.3 0.8 0.7
0.39 1.01 0.62 0.54
7.8 No p r e c i p i t a t e on first 20.2 14.4 day: s l i g h t 10.8 on t h i r d d a y
EFFECTOF HEATAND AGITATIONUPON PRECIPITATION 8 Av. 0.64 13.3 The results in Table I1 show good recovery of gossypol 9 10.0 2 75 11.8 9.16 91.5 Precipitatiopat upon long standing. I n order to hasten precipitation, 10 10.0 2 75 11.9 9.22 92.2 once.co 1ou8 11 10.0 2 75 11.0 8.53 85.3 bythfday the effect of heat was tried by digesting several determina12 10.0 2 75 12.1 9.38 93.8 tions of 10 and 25 mg. of gossypol 2.5 hours on the steam Av. 9.07 90.7 bath. The gossypol was dissolved in 2 cc. of oil to which 8.23 82.3 S l i g h t p r e 13 10.0 5 75 10.6 were added 5 cc. of ethylene glycol, 2 cc. of aniline, and 50 cc. 11.6 8.99 89.9 c i p i t a t e on 14 10.0 5 75 8.45 84.5 fir5t.da.y; 75 10.9 of petroleum ether of a high boiling point, as suggested by 15 10.0 5 7.98 79.8 precipitating 75 10.3 16 10.0 5 E. P. Clark. After the digestion, the cooled, stoppered flasks s l p w l y on Av. 8.41 84.1 thirdday were mechanically agitated continuously for 48 and 72 hours. a All determinations stood 8 days in an electric refrigerator at 10' C. The mechanical shaker was similar in type to the Camp shaking apparatus. It was driven by a water motor making 50 to 60 revolutions per minute. The beneficial effect of heat in The data presented in Table IV show the influence of 2 and hastening precipitation is shown in Table 111. 5 cc. of refined cottonseed oil, respectively, on the precipitation of 5 and 10 mg. of gossypol, other factors remaining the TABLE111. EFFECTOF DIQESTIOXAND AGITATION UPOX same. A n inspection of determinations 1 to 8, using 5 mg. of RECOVERY OF GOSSYPOL gossypol, shows that in the presence of the larger amount ( I n presence of 2 00. of oil, 50 cc. of petroleum ether,a 2 cc. of aniline, and of oil, the precipitation of dianiline gossypol is materially 5 ca. of ethylene glycol) slowed up, and that the recovery is much less, 13.3 per cent ANILINE GOSSYPOLGOSSYPOL HOURS DETN. TAKEN WEIQHEDGOSSYPOL RECOVERED SHAKENREMARKS compared to 84.9 per cent. Also, when 10 mg. of gossypol are used, recovery is quicker and better in the presence of 2 cc. Mg. Mg. MQ. % 1 10.0 10.8 8.4 84.0 48 Shaking and rather than 5 cc. of refined oil (determinations 9 to 16) with 2 10.0 10.8 84.0 48 heat aided 8.4 93.8 per cent recovered compared to 84.1 per cent. 3 10.0 10.8 84 0 48 precipita8.4 4 10.0 8.5 85.0 48 tion 11.0 Table V presents additional data in support of the effect of Av. 8.4 84.1 5 cc. of oil on the recovery of 5, 10, and 15 mg. of gossypol (determinations 1 to 10) compared to the recovery of 5, 10, 5 25.0 29.5 22.9 91.6 72b Shakin and 6 25.0 30.0 23.3 93.2 72 heattelpand 25 mg. of gossypol in the presence of 2 cc. of oil, obtained 7 25.0 29.1 90.4 72 ful as 22.6 8 25.0 30.3 23.5 94.0 72 above in previous results (Tables I1 and 111). I n every case the average results of the amounts of gossypol recovered are Av. 23.1 92.3 a Boiling point 6 G D t o llOo C. higher in the presence of less oil, 2 cc. compared to 5 cc. b After shaking 24 hours, precipitates appeared ready t o filter. Thus, in determinations 1 to 3, an average of 59.3 per cent of 5 mg. of gossypol is recovered in the presence of 5 cc. of oil, With 2.5 hours of digestion and 48 hours of shaking, 8.4 compared to 80.7 per cent in the presence of 2 cc. of oil. of 10 mg. of gossypol (Table 111) were recovered. Likewise Likewise, in determinations 4 to 7, an average of 78 per cent 23.1 of 25 mg. of gossypol were recovered by the same pro- of 10 mg. of gossypol is recovered in the presence of 5 cc. of cedure. But without the aid of heat, the precipitation of oil, in comparison with 84.1 per cent when 2 cc. of oil were gossypol was delayed so that a similar amount of 8.7 mg. of used. Also, in determinations 8 to 10, 83.1 per cent of 15 gossypol (Table 11,h) was recovered after standing 13 days mg. of gossypol was recovered with 5 cc. of oil, in comparia t 10" C. and with a final shaking of 24 hours. son to 92.3 per cent when 2 cc. of oil were used. The purpose in the proposed method of the preliminary exEFFECTSOF COTTONSEED OIL ON PRECIPITATION OF traction of a part of the oil from the cottonseed meal with GOSSYPOL AS DIANILINE GOSSYPOL petroleum ether is to eliminate this somewhat troublesome Small amounts of gossypol are not readily precipitated factor. Approximately 2 cc. of oil are left in the charge; resifrom large volumes of refined cottonseed oil. This has been dues yielding as high as 5 cc. of oil are to be avoided. This
ANALYTICAL EDITION
32
extraction did not remove a detectable quantity of gossypol, as shown by shaking the extract with ethylene glycol and anhydrous sodium carbonate in which the gossypol is more readily soluble. No gossypol could be precipitated from the petroleum ether extract as the dianiline compound.
Vol. 5, No. 1
order to prevent overheating of the extract, the ether is removed under reduced pressure. The results show that amounts of ether as small as 0.5 cc. do not interfere with the precipitation of dianiline gossypol.
SOLUBILITY OF DIAKILINE GOSSYPOL IN REAGENTS TABLE V. EFFECT OF OIL OR OIL AND DIGESTION ON PRECIPITAThe solubility of dianiline gossypol was determined in the TION OF GOSSYPOL IN PRESENCE OF 5 cc. OF ANILINEAND OF reagents used in this method of estimation. Weighed ETHYLENE GLYCOL amounts of dianiline gossypol which had been recrystallized GOSBY-ANILINE POL GOBSYPOL GOSSYPOL REDAYS DETN. TAKEN WEIQHED COVERED STOOD REMARKS from boiling benzene were agitated from 5 to 9 days a t laboraMe. (a)
Me.
Mg.
%
PRECIPITATED WITHOUT DIQESTION IN PRESENCE OF 6 16 CC. OF PETROLEUM ETHER
1 2 3
5.0 5.0 5.0
1-8, Table I1
(b)
3.4 4.7 3.5 Av. Av.
5.0
2.6 3.6 2.7
52.0 72.0 54.0
3.0 4.0
59.3 80.7
cc.
OF OIL AND
50 Precipitation 50 incomplete 40 8 2 cc. each of oil and aniline, and 25 cc. of petroleum ether used
tory temperature in the presence of the various reagents. The undissolved dianiline gossypol was filtered off, dried, and weighed. The results are given in Table VII.
TABLE VII. SOLUBILITY OF DIANILINE GOSSYPOL IN REAGENTS ANILINE EQUIV. GOBSYPOL ANILINEGOSSYPOL GOSSYPOLDAYS DETN. TAKENRecovered Dissolved DISSOLVED SHAKEN TEMP.
Me. TWO CC. OF
Me.
Mg.
OIL, 2
CC. OF
c.
Mg.
ANILINE,
6 CC. OF E T H Y L l N E PETROLEUM ETHER^
QLYCOL,AND
76 CC. OF
DIQEBTED WITH 60 CC. OF PETROLEUM ETHER, 6 CC. O F OIL, AND 2 CC. OF ANILINE; OTHER REAQENTS AS ABOVE
4 5 6
10.0 10.0 10.0 10.0
7
9.6 10.2 10.6 9.9 Av. Av.
1-4, Table I11 10.0 8 9 10
15.0 15.0 16.0
5-8, Table I11 25.0
7.7
74.0 79.0 82.0 77.0
7.8 8.4
78.0 84.1
12.2 12.6 12.6
81.3 84 0 84:0*
7.4 7.9 8.2
15.7 16.3 16.3
Av. 12.5 Av. 23.1
83.1 92.3
SHAKEN 8 6 6 6
Av. 2.8 TWO
6 7
10.0 10.0
cc.
OF OIL A N D T O
2 2 2
OF
PETROLEUM
1.6 1.2
1.2 0.9
AV. 1.4
1.1
8.4 8.8
2 2 eo. of oil used
2.2 cc.
ETHER
6 6
FIVE CC. OF OIL AND 76 CC. OF PETROLEUM ETHER
8 9 10
25.0 25.0 25.0
1.3 1.4 0.9
0.7
Av. 1.2
0.9
23.7 23.6 24.1
3 2 cc. of oil used
1.0 1.1
5 5 5
EFFECT OF ETHYL ETHERON PRECIPITATION It is desirable to know whether it is necessary to remove the small amount of residual ether in the ethyl ether extract of cottonseed meal in order to obtain complete precipitation of the gossypol. The effect was studied of the influence of 0.5 and of 1.0 cc. of ether upon precipitation of 10 mg. of gossypol dissolved in 2 cc. of refined oil, with the other reagenb present in the amounts given in the method. The results are presented in Table VI. TABLEVI. EFFECTOF ETHYLETHERUPON RECOVERY OF SMALL AMOUNTSOF GOSSYPOLWITHOUT DIGESTION (In 2 eo. of oil, 2 cc, of aniline, 75 00. of petroleum ether, and 5 eo. of ethylene glycol) ETHYLGOSBY-ANILINE ETHER POL GOSBYPOL DAYS DETN. USED TAKEN WEIQHEDGOSSYPOL RECOWRED STOOD REMARKS Cc. Mg. Me. Mg. % 92.0 15 All detns. 9.2 11.8 1 0.5 10.0 81.0 15 shaken 10 8.1 10.0 10.5 2 0.5 87.0 15 hours be8.7 11.2 3 0.5 10.0 fore filterAv. 8.7 86.7 ing
4 5 6
1.0 1.0 1.0
10.0 10.0 10.0
10.4 10.2 10.3
8.1 7.9 8.0 Av. 8.0
81.0 79.0 80.0
15 15 15
80.0
The results of determinations 1 to 3 show that 8.7 of 10 mg. can be recovered in the presence of 0.5 cc. of ethyl ether. This is the same amount which was recovered from 10 mg. of gossypol in determinations 9 to 11 (Table 11). One cubic centimeter of ethyl ether does appear to lower the per cent recovered slightly (80 versus 87 per cent without ethyl ether). However, when expressed in milligrams of gossypol, this difference is of doubtful significance. Because dianiline gossypol is slightly soluble in ether, the residual ether in the extract should not exceed one cc. I n
Av.
10.86
9.58
1.28
1.0
O N E HUNDRED CC. O F PETROLEUM ETHER
16 17 18 Av.
25.3 25.5 26.5
25.1 24.9 25.5
25.8
26.2
a Boiling point 30° to 80'
0.2 0.6 1.0
0.2 0.5 0.8
0.6
0.5
9
23 to 25
C.
The results of determinations 1 to 5 show that 2.8 mg. of dianiline gossypol (2.2 mg. of gossypol) are dissolved in the reagents used in the method. These results compare favorably with the amounts not recovered in studying the influence of the various factors in the method. The solubility of dianiline gossypol in the reagents, when some of the reagents are omitted, varies, but this is less than the solubility in all of the reagents. I n 100 cc. of petroleum ether, the dianiline gossypol is soluble to the extent of one-half of the amount soluble in the reagents prescribed by the method when using 75 cc. of petroleum ether, 2 cc. of oil, and 5 cc. of ethylene glycol. Small amounts of dianiline gossypol are soluble in aniline, especially with increase in temperature. The dissolved dianiline gossypol was calculated to its equivalent in gossypol in order to compare these results with the amounts not recovered in studying the effects of heat, agitation, and oil on precipitation (Tables I11 and IV). The amount which dissolved in the reagents agrees closely with the amounts not recovered (Table VIII). The small loss or incomplete recovery of gossypol (Table VIII) is largely accounted for by the solubility of dianiline gossypol in the reagents. The small discrepancies in the results may be due to slight changes in temperature a t which the determinations were made, although they are within the limits of the experimental error of the method.
January 15, 1933
INDUSTRIAL AND ENGINEERING
TABLEVIII. GOSSYPOL DISSOLVED AND SOT RECOVERED BY METHOD TABLE Av. OF No. DETNS. VI1 I11 I11
v V
1 to 5 1 to 4 5 to 8 4to7 8 to 10
GOBSYPOL DISSOLVED REMARKS Mg . 2.2 Dissolved 1.6 Not recovered, effect of heat and agitation 1.9 Not recovered, effect of heat and agitation 2.2 Not recovered, effect of oil and digestion 2.5 Not recovered, effect of oil and digestion
I n the recovery of small amounts of gossypol, an average of one mg. not recovered in determinations 1 to 8 agrees with the solubility figures shown in Table VI11 as well as could be expected when it is observed that only one-third as much petroleum ether was used in these determinations and that the refrigerator temperature where they stood was much lower than that in the laboratory. I n this case an average of one mg. was not recovered, whereas in determinations 9 to 11 (Table 11)with 75 cc. of petroleum ether, the amount was 1.3 mg.
PURITY OF DIANILINEGOSSYPOL PRECIPITATE The nitrogen content of the dianiline gossypol is an indication of the degree of its purity. Clark ( 4 ) calculated this to be 4.19 per cent. He reported duplicate results of 4.12 and 4.14 per cent on a purified sample of dianiline gossypol. The nitrogen content was determined on a sample of dianiline gossypol recrystallized from boiling benzene, and in precipitates obtained from cottonseed meal. Mercury was used as the catalyst in the micro-Kjeldahl method adapted from Pregl for the determination of nitrogen. Ammonium sulfate and urea served as controls on the accuracy of the method. The results obtained from dianiline gossypol are shown in Table IX. ANILINE
ESTIMATION OF GOSSYPOL IN COTTONSEED MEAL BY PROPOSED METHOD It was desirable to know what results could be obtained from meals with unlike gossypol content. These results are presented in Table X. One meal contained a comparatively high content of gossypol, whereas the other was relatively low, only 13.8 per cent as much as the former. TABLEX. RESULTS OF ESTIMATION OF GOSSYPOL IN COTTONSEED MEALBY PROPOSED METHOD DETN. SAMPLE^
Mg.
cc.
81.2 80.2 80.3 80.5 80.0
5.79 5.81 5.87 5.78 5.82
%
3.242 3.254 3.298 3.237 3.269 Av.
3.99 4.06 4.11 4.02 4.07 4.05"
FROM COTTONSEED MEAL
6 7
8
9 10 11 12 13 14 15 16 17 18 19 20
(I
32.9 98.0 102.5 87.3 39.5 41.4 21.7 26.5 87.4 49.2 93.2 112.4 32.9 62.7 53.6
2.25 6.71 6.83 6.24 3.02 3.14 1.60 1.99 6.28 3.76 6.79 7.35 2.42 4.22 3.89
GOSBYPOL Gram %
WITH 48 HOURS' EXTRACTION
1 2 3 4 5
1421 1421 1421 1421 1421
0.0971 0.0890 0.0954 0.0874 0.0932
0.0675 0.0690 0.0740 0.0678 0.0723
0.0450 0.0460 0.0493 0.0452 0.0482
Av. 0.0701
0.0467
WITH 72 HOURS' EXTRACTION
6 7
1421 1421 WITH 4s
8 9 10
1414 1414 1414
0.0980 0.1025
0.0760 0.0507 0.0795 0.0530
Av. 0.0777 HOURS'EXTRACTION 0.0114 0.0088 0.0126 0.0098 0.0137 0.0106 Av.
0.0097
0.0519 0.0059 0.0065 0.0071 0.0065
Charge, 150 grams,
NITROQEN
Mu.
RECRYBTALLIZED F R O M BOILING BENZENE
1 2 3 4 5
ANILINE GOE~YPOL Gram
a
DETN. TAKEN 0.04 N ACID
33
the greater amounts of nitrogen may have occluded free aniline. The variation in nitrogen content from the theoretical, as observed in the above precipitates (Table IX), is not great. These results show that the precipitates obtained from cottonseed meal by the proposed method consist of dianiline gossypol of a fair degree of purity. In fact, the method gives clean, uniform precipitates of definite crystalline structure.
TABLEIX. KITROOEN CONTENT OF DIANILINE GOSSYPOL GOSSYPOL
CHEMISTRY
1.260 3.758 3.825 3.494 1.691 1.753 0.896 1.114 3.517 2.106 3.802 4.116 1.355 2.363 2.178
3.83 3.83 3.73 4.00 4.28 4.23 4.13 4.20 4.02 4.28 4.08 3.66 4.12 3.77 4.06
The results of determinations 1 to 5 by this method are consistent, the maximum variation being 0.004 per cent. Determinations 6 to 8 of cottonseed meal No. 1414 gave an average of 0.0065 per cent. The results show that the method is applicable to meals containing very small amounts of gossypol. It is also seen that 72 hours of Soxhlet extraction of the meal gave 0.005 per cent more gossypol than 48 hours. It was found that air-dried samples of meal and U. S. P. grade of ethyl ether (from J. T. Baker Chemical Co.) should be used. Gossypol is extracted with great difficulty after the meal has been dried in vacuum with or without heat or over calcium chloride in a desiccator. Also anhydrous ether was not so efficient as U. S. P.ether in extracting gossypol from air-dried meal (0.0399 against 0.0701 gram) and gave even poorer results on moisture-free meals.
Av. 4 . 0 1 Theoretical amount calculated by Clark, 4.19%.
The nitrogen content of dianiline gossypol recrystallized from boiling benzene averaged 4.05 per cent (3.99 to 4.11), and thus agrees fairly closely with the theoretical nitrogen content of 4.19 per cent when it is considered that the dianiline gossypol was recrystallized but once. The precipitates of dianiline gossypol obtained from the determinations of ether-soluble gossypol in cottonseed meal showed more variation in nitrogen content, an average of 4.01 per cent (3.66 to 4.28). This variation, however, is only slightly greater than reported by Schwartze and Alsberg (3.75 to 4.04) in the aniline gossypol obtained from seed. Carruth ( I ) reported results varying between 3.97 and 4.84 per cent nitrogen. Some of his precipitates which contained
LITERATURE CITED (1) Carruth, F. E., J. Am. Chem. SOC.,40, 658 (1918). (2) Carruth, F. E., J. Biol. Chem., 32, 87-90 (1917). (3) Clark, E. P., Ibid., 7 5 , 7 3 1 , 7 3 3 (1927). (4) Clark, E. P., Ibid., 75, 725-39, especially 736 (1927); 76, 22935, especially 232 (1928). (5) Clark, E. P., J. Oil Fat Ind., 6 , 1-11 (1929). (6) Schwartze, E. W., and Alsberg, C. L., J. Agr. Res., 25, 285-95 (1923). RECEIYED July 23, 1932. Presented before the Division of Agricultural and Food Chemistry at the 82nd Meeting of the American Chemical Society, Buffalo, N. Y., August 31 to September 4, 1931. Published with the approval of the Director of the North Carolina Agricultural Experiment Station as Paper 48 of the Journal Series. From a thesis presented by the junior author to the Graduate School of the North Carolina State College of Agriculture and Engineering in partial fulfilment of the requirements for the degree of Master of Science.
