Estimation of Gossypol in Crude Cottonseed Oil J. 0. HALVERSON
AND
F. H. SIMITH, North Carolina Agricultural Experiment Station, Raleigh, N. C.
wash oil adhering on the inside into the flask with petroleum ether (boiling point 30' to 60" C.). Into another Erlenmeyer flask containing 25 grams of refined cottonseed oil (Wesson) pipet 5 ml. of ether-extracted oil from cottonseed meats to determine the amount of gossypol added to the unknown charge. To each charge add 75 ml. of petroleum ether (boiling point 30' to 60' C.) and after allowing to stand overnight in the lightly stoppered flasks, add about 0.1 gram of Hyflo-Supercel to the mixture in each flask. Filter off the precipitated material with suction through dry Gooch crucibles (which contain a mat of HyfloSupercel over the asbestos pad) into a 250-ml. Erlenmeyer flask placed under a bell jar. Wash the flask and precipitate in each Gooch crucible with a small quantity of petroleum ether (10 to 15 ml,). Add the following reagents per determination: 4 ml. of aniline and 12ml.of fractionatedpyridine (boilingpoint 115"to 116.3'C.). Agitate 72 hours in a shaking constant-temperature water bath held at 43" to 46" C., using water-cooled condensers (Hopkins reflux type) attached to the Erlenmeyer flasks. In order to catch the few drops of water which form in the condenser tube, attach a small glass cup to the lower end of the cork stopper through which the condenser tube passes. The elimination of water prevents the precipitate from adhering to the glass vessel. After 72 hours' shaking at 43' C. and 1 hour's standing out of the bath, filter with suction through a tared Gooch crucible, first decanting off a portion of the mother liquor into a 150-ml. Erlenmeyer flask for use in washing out any precipitate which adheres to the flask. The precipitate is conveniently washed from the Erlenmeyer flask with the aid of a special wash bottle (4) containing petroleum ether and dry pyridine (100 3). Loosen any precipitate which adheres to the flask, with the aid of a rubbertipped glass rod and the mother liquor. After washing down the walls of the Gooch crucible, wash the precipitate five times by filling the crucible about half full of the petroleum ether-pyridine mixture, using suction (about 25.4 cm., 10 inches). Finally wash down the sides of the Gooch crucible carefully with the petroleum ether-pyridine mixture and wipe the outside surface clean with a cloth. Dry the precipitate in a hot air oven at 110' C. (by heating 18 to 24 hours to constant weight, depending upon the amount of precipitate) in order to drive off the two molecules of pyridine.
A clear-cut method for the estimation of gossypol in crude cottonseed oil is presented, with modifications which permit sets of ten determinations to be made fairly expeditiously. Precipitation is expedited by increased temperature, by the addition of gossypol in an ether-extracted oil prepared from cottonseed meats, and by constant agitation which precipitates the gossypol in a good crystalline condition for rapid filtration and washing without appreciable loss due to dissolving. Solubility is prevented by the use of pyridine in the wash solution. The gossypol compound is prevented from adhering to the glass container by the elimination of practically all water. Recovery of added gossypol and the reproducibility of results are good.
T
+
HE observation of Royce (8) that pyridine promotes the precipitation of gossypol from crude cottonseed oil has been confirmed. However, Royce's method of estimating gossypol in crude cottonseed oil (2, 3) can be improved in a number of respects. Gossypol in the presence of pyridine is not precipitated as dianilinogossypol but as dianilinogossypol dipyridine, a salt of gossypol (6). Driving off all the pyridine of crystallization by heating the precipitate at 100 O C. to constant weight, in the authors' hands, has not given consistent results and has not been practical. The factor 0.775 for converting dianilinogossypol to gossypol is not applicable unless all the pyridine has been removed. The process of precipitating gossypol from crude oil is slow and tedious and the precipitate a t times adheres tenaciously t o the glass vessel in which precipitation occurs. The dianilinogossypol containing pyridine of crystallization is appreciably soluble in the petroleum ether used in washing, in contrast to the relatively insoluble dianilinogossypol alone. A rapid clear-cut method is desirable. The precipitation of gossypol should be fairly rapid and complete. It should be readily removed from the flask in which precipitation occurred and washed without appreciable loss. I n attempting to overcome these objections the following factors were studied: the charge of crude oil necessary to expedite the precipitation of gossypol; the advantage of adding gossypol t o the charge of crude oil in order to expedite precipitation; the quantity of reagents necessary; the elimination of water from the reagents used in the precipitation of gossypol; the temperature and agitation necessary for a more rapid and complete precipitation; the prevention of loss of the precipitated gossypol by washing; and the length of time necessary for heating the precipitate to constant weight by driving off all the pyridine.
TABLE I. RECOVERY OF PURIFIED GOSSYPOL FROM REFINED OIL AND FROM ETHER-EXTRACTED OIL Gossypol Charge Mg.
100 100
100 100 100
... ... ...
...
...
.,.
Ether-Extracted Oil (Meats) Ml.
Total Dianilinogossypol
Gossypol Recovereda
Mu.
Mu.
Purified Gossypol in Refined Oil 128.1 128.1 Av. 128.1 127.3 127.3 .. 128.7 Av. 127.5
....
99.3
*. ..
Gossypol in Ether-Extracted Oil (Meats) 5 133.6 5 134.6 5 134.8 5 135.1 Av. 134.5 5 133.3 5 132.2 Av. 132.8
99.1
104.2
102.9
Purified Gossypol Added to Ether-Extracted Oil (Meats) 5 200.1 50 200.4 5 50 5 199.8 50 5 199.9 50 Av. 200.1 155.0 198.4 5 50 195.0 5 50 199.5 5 50 199.2 5 50 Av. 198.8 154.1
Method of Determination Filter the crude cottonseed oil by gravity through glass wool. Weigh a charge of 25 grams of crude oil into a 250-ml. Erlenmeyer flask and pipet 5 ml. of cottonseed oil containing considerable gossypol (extracted from cottonseed meats with ethyl ether) into the same Erlenmeyer flask, wiping off all the oil on the outside of the filled pipet with a clean cloth before adjust/ng the oil to the mark, After emptying the contents of the pipet lhto the flask,
5
46
0.775 X weight of disnilinogossypol.
ANALYTICAL EDITION
January 15, 1941
The total weight of dianilinogossypol in the charge, less that added in the 5 ml. of ether-extracted cottonseed oil, multiplied by 0.775, gives the equivalent amount of gossypol. Four times this amount of gossypol gives the per cent of gossypol present in crude oil. Reagents Used GOSSYPOL ADDEDTO CHARGE.An ether-extracted oil relatively high in gossypol was used, which was obtained by the extraction of prime cottonseed meats with peroxide-free ether. The meats consisted of the flattened decorticated seed passed through iron rolls set to within 0.025 mm. (0.001 inch), and were taken just before they entered the steam cookers (or the press in the expeller process). The ethyl ether was removed from the extracted oil by distillation, by placing the distilling flask in a water bath heated by a closed steam coil in which the temperature of the water was not allowed t o rise above 60" C. The hot oil was filtered through glass wool and any residual ether removed under reduced pressure while the flask was gently warmed in a vessel of warm water. The ether-extracted oil was stored at about 5" C. in an electric refrigerator. For the determination of its gossypol content, 5 ml. charges were taken after the oil had reached a temperature of 25' to 26" C. and had been carefully mixed by shaking. PETROLEUM ETHER(boiling point 30" to 60' C.), Skellysolve F, and aniline (from sulfate or practical), Eastman. PYRIDINE (boiling point 115" to 116.3' C.) was obtained by the fractionation of pyridine (practical, 112" to 117" e.),Eastman, in order t o eliminate water. All grades examined contained water, which, if present in excessive amounts, may interfere with the precipitation of gossypol. OF RESULTS ON CRUDE TABLE11. REPRODUCIBILITY COTTONSEED OIL(No. 2549)
EtherExtracted Oil Charge (Meats) Gram ME. 25 5 25 5 25 5
.. .. ..
5 5 5
25 25 25
5 5 5
.. ..
5 5 5
,
132.5 132.5 132.9 Av. 1 3 2 . 6 154.1 154.2 153.1
132.6 133.0 132.7 Av. 1 3 2 . 8 From ether-extracted oil (meats).
.. a
Dianilinogossypol Less amount added" Mg. M9. 22.2 154.8 22.7 155.3 154.3 21.7
Gossypol Mg % 17.2 0.069 17.6 0.070 16.8 0.067 Av. 0 . 0 6 9
.
*.
.. ..
21.3 21.4 20.3
16.5 16.6 15.7
..
.. .. ..
*.
..
*.
*.
..
... ... ...
0.066 0.066 0.063 Av. 0 . 0 6 5
... ... ...
Accuracy of Method The accuracy of the method is indicated by the quantity of gossypol recovered (99.3 and 99.1 mg.) from 100 mg. of purified gossypol dissolved in 25 grams of Wesson oil (Table I); from 50 mg. of purified gossypol added to 5 ml. of the ether-extracted oil mixed with 25 grams of refined oil, and from 5 ml. of the extracted oil mixed with 25 grams of refined oil. When the quantity of gossypol precipitated from 5 ml. of extracted oil is deducted from that precipitated in 5 ml. of extracted oil plus 50 mg. of added purified gossypol the result, 50.8 and 51.2 mg., shows that the added purified gossypol was recovered. The 50 mg. of gossypol added to the 5 ml. of etherextracted oil promotes the precipitation of gossypol from the latter, and i t is probable that at least a portion of the gossypol figured as recovered from the 50-mg. charge is in reality additional gossypol precipitated from the ether-extracted oil. The addition, in the ether-extracted oil of a relatively large amount of gossypol (approximately 100 mg.) also promotes the precipitation of additional gossypol from the crude oil. This slightly increased recovery is not accounted for by the occurrence of small amounts of a phosphorus-containing compound in the precipitated dianilinogossypol.
47
Compilation of previous results on dianilinogossypol obtained from 5 ml. of the ether-extracted oil gave a mean of 133.72 * 1.38 mg. which is equivalent to 103.63 * 1.07 mg. of gossypol (59 determinations). From refined cottonseed oil 99.3 and 99.1 per cent of 100 mg. of purified gossypol was recovered. Solution in the reagents probably accounts for the slightly low results.
Reproducibility of Results Representative results (Table 11)from a crude oil containing an average amount of gossypol show the usual variations obtained. The averages of 3 determinations may differ by 0.004 to 0.006 per cent. Discussion of Factors Studied The detailed data upon which the amounts of reagents recommended are based are not presented here. Tables I and 11, in which these amounts of reagents were used, show the percentage recovery and reproducibility of results. Gossypol is more readily recovered from refined cottonseed oil than from the commercial crude oil, and can be more readily precipitated from an oil extracted by ether from the decorticated cottonseed or meats. The commercial crude oils usually contain from 15 to 40 mg. of gossypol in a 25-gram charge. These small amounts are more readily and completely precipitated by the addition of about 100 mg. of purified gossypol or approximately that amount in 5 ml. of the ether-extracted oil from the meats. The gossypol content of crude oils, even though kept in cold storage, decreases over a period of months. With the amounts of reagents and solvent kept the same a charge of 25, instead of the 50 grams recommended by Royce, gave slightly higher yields of gossypol. The precipitation of gossypol was expedited by adding to the charge 5 ml. of an ether-extracted oil from cottonseed, which had a high gossypol content (approximately 100 mg. in 5 ml.). The use of the ether-extracted oil was more convenient than the longer process of purifying gossypol for this purpose, as the latter does not keep well a t ordinary laboratory temperature. Adding gossypol to the unknown charge gave a more complete and rapid precipitation, especially where the amount of gossypol in the oil was small (3). The procedure given above-filtering off a flocculent precipitate from the oil-petroleum ether mixture after it had stood overnight-probably does not remove all this material. Raymond Reiser found by the Fiske and Subbarow method that the weighed precipitate of dianilinogossypol contained a small amount of phosphorus (0.62 mg. or 0.4 per cent) which was probably due to the phosphatides lecithin and cephalin (1). The amount of this contaminant was not significant, as the nitrogen content of the dianilinogossypol checked theory. All grades of pyridine examined contained water, in some instances in rather large amounts and this had to be removed by fractional distillation, using the portion having a boiling point of 115' to 116.3" C. The reaction of aniline with gossypol also yields a small amount of mater. The few condensed drops of water were prevented from coming in contact with the dianilinogossypol dipyridine precipitate in the bottom of the Erlenmeyer flask by being caught in a small cup attached directly beneath the condenser tube. The cup was made by cutting off a 20-mm. test tube about 1 cm. from the bottom, and sealing to the bottom section a small glass rod about 3 cm. long, one end of which was drawn to a sharp point. This end was stuck into the lower surface of the cork stopper through which the condenser tube passes. A small amount of precipitate occasionally adhered to the glass container, indicating that a small quantity of moisture probably remained.
INDUSTRIAL AND ENGINEERING CHEMISTRY
48
Loss of precipitate due to solubility was prevented by using a decanted portion of the mother liquor in transferring the precipitate to the Gooch crucible and by washing with minimum amounts of petroleum ether containing 3 parts of the fractionated pyridine per 100 ml. of petroleum ether (boiling point 30" to 60" C.). Heating the precipitate in an air oven to constant weight a t 110" C. drove off all the pyridine. The nitrogen content of the heated residue showed it to be dianilinogossypol (6).
Need for Constant Temperature and Agitation Considerable difficulty was encountered in obtaining complete precipitation of the last 1 to 3 mg. of gossypol. In order to expedite and complete the precipitation, constant agitation with increased temperature was found necessary. The production of fair-sized crystals and the most complete precipitation of gossypol occurred with moderate shaking a t 43" to 46" C. The crystals of precipitated gossypol should be large enough so that they will not pass through the Gooch crucible or clog i t while filtering. Too quick precipitation of the gossypol compound results in small crystals, and too vigorous shaking causes a fine precipitate to form rapidly. If the solution is not sufficiently agitated, the gossypol precipitation may not be complete in 72 hours. When shaken 48 hours, 97 to 98 per cent of the gossypol was recovered. An additional 24 hours of shaking was necessary for a more complete precipitation. Precipitation also requires proper control of the temperature. Agitation was accomplished in a constant-temperature water bath suspended by 20.3-cm. (8-inch) arms from center to center from a wood frame by bearings. The bath holding 10 Erlenmeyer flasks is moved back and forth with a stroke of 1.27 cm. (0.5 inch), approximately 120 revolutions per minute, by means of an electric motor with the proper reducing gears (Eimer and Amend, ball mill, No. 21,481/1) and with an offset of 0.64 cm. (0.25 inch) off center for the connecting arm on the reducing gear to the water bath. The water bath had the following outside and inside dimensions: 40.6 X 87.7 X 24.1 cm. (16 X 34.5 X 9.5 inches)
Vol. 13, No. 1
and 33.0 X 81.3 X 20.3 cm. (13 X 32 X 8 inches), respectively. The bath was lined with 28.35-gram (16-ounce) soft sheet copper and was surrounded with a layer of plywood (Celotex) made from 24.1 X 19.1 cm. (9.5 X 0.75 inch) boards. Two heating units made from No. 25 Chromel A wire were drawn through two 2.54-cm. (1-inch) copper pipes lined with asbestos paper except at the top surface. They were placed 1.27 cm. (0.5 inch) from the bottom of the bath, running lengthwise through it. The Chromel A wire was 43.7 and 38.1 cm. (17.2 and 15 feet) long, having 25.3 and 30.5 ohms resistance, giving 3.16 and 3.65 amperes with a wattage of 347 and 402, respectively.
Summary A comparatively rapid method for the estimation of gossypol in crude cottonseed oil is presented. The gossypol was precipitated in a good crystalline condition a t 43" C. by constant agitation for 72 hours. Precipitation was in part expedited by the addition of gossypol in an ether-extracted oil made from cottonseed meats. The crystalline precipitate was readily washed and filtered. The gossypol compound was prevented from adhering to the glass container in which p r e cipitation occurred by the elimination of practically all water. Loss in washing the precipitate was also largely prevented. A rather high recovery of gossypol was obtained with good reproducibility of results. Acknowledgment The authors are indebted to the Buckeye Cotton Oil Company, subsidiary of the Procter & Gamble Company, for the cottonseed oil and the cottonseed meats used in this investigation. Literature Cited (1) Lishkevitch, M., Maslobolno Zhirovoe DeZo, No. 27, 6-8 (1939). (2) Royce, H. D., Oil & Soup, 10, 183-5 (1933). (3) Royce, H. D., and Kibler, M. C., Zbid., 11, 116, 118, 119 (1934). (4) Smith, F. H., IND.ENQ.CHEM.,Anal. Ed., 8, 400 (1936). (5) Smith, F. H., and Halverson, J. O., Ibid., 11, 475 (1939). PUBLISHED with the approval of the Acting Director of the North Carolina Agricultural Experiment Station as No. 116 of the Journal Series.
Rapid Method for Determination of Manganese in Feeds I
J. W. COOK', Agricultural Experiment Station, Pullman, Wash.
M
ETHODS for the determination of manganese have received much attention in the past due to the importance of manganese in both the steel industry and agriculture. Much more interest has been shown in the determination of manganese in biological materials since Wilgus, et al. (10) showed that this element is important in the prevention of perosis in chicks. Most methods that are used for the preparation of a sample previous to the determination of manganese in biological materials involve a lengthy dry-ash procedure which requires a number of steps of manipulation. Also, i t was shown by Bolin ( 2 ) , Davidson (S), and others that the acid-insoluble portion of ash contained appreciable quantities of manganese, but that it could be recovered by a sodium carbonate fusion or by volatilization of silica with hydrofluoric acid. Hundreds of fiamples of feeding materials have been wetashed in this laboratory by the procedure outlined by Gerritz (4, using a nitric acid oxidation followed by a perchloric acid 1 Present addresa, U. S. Department of Agriculture, Food and Drug Administration, Seattle, Wash.
oxidation previous to the determination of calcium and phosphorus. This procedure is rapid and lends itself well to the analysis of feed ingredients and plant materials. It seemed desirable to adapt it to the determination of manganese. Experimental The common methods for the final determination of manganese in minute quantities, as it is found in plant material, involve the oxidation of bivalent manganese to heptavalent manganese by some oxidizing agent stronger than the permanganic ion, such as periodic acid, with a subsequent colorimetric comparison of the permanganic ion. Chlorides interfere with this reaction and are commonly present in rather high concentration, especially in mixed feeds, but the use of perchloric acid readily eliminates this difficulty. The products of decomposition of perchloric acid (8) are chlorine and oxygen. Chlorides are volatilized either as hydrogen chloride or as chlorine after oxidation. Remaining small traces must be eliminated by boiling in the presence of sodium periodate before the color of permanganic acid will develop.
