March, 1946
INDUSTRIAL AND ENGINEERING CHEMISTRY
Bailey, and cc-workers ( 7 , 8 )and plotted in Figure 2 for comparison, appear to show the same tendency. The values reported in Table I1 seem to indicate that the drying and semidrying oils polymerized to some extent as a result of the heat treatment received during a determination, since their iodine numbers decreased and their viscosities increased. These properties changed in the opposite direction or remained the same for the nondrying castor oil and hydrogenated cottonseed oil. Tung oil began to body noticeably while being heated at about 200” C. after the determination had progressed for about 8 hours, and gelled soon afterward with continued heating. The specific heat values of castor oil are in good agreement with the two values reported by Deaglio and Montu (9)but are consistently a little higher than those determined by Delaplace (3) as shown in Figure 2. The specific heat values reported by Long and co-workers (6) for soybean and tung oils are consistently higher than those given in this paper. They reported values for alkali-refined linseed oil which agree fairly well with those in this paper except that their values are considerably higher in the range 190” to 270’ C. (Figure 2).
353
ACJLNOWLEDGM ENT
The authors take pleasure in expressing their indebtedness to the Armstrong Cork Company for financial assistance which made this work possible, for permission to publish this paper, and for furnishing the oils and gasket materials. Appreciation is also expressed for the assistance of Harold E. Adams of Armstrong Cork Company for furnishing the values in Table I1 and for advice and many helpful suggestions. LITERATURE CITED
(1) Bailey, Todd, Singleton,and Oliver, Oil & Soup, 21,293 (1944). (2) Deaglio and Montu, 2. tech. Physik., 10, 460 (1929). (3) Delaplace, Reni, Contpt. rend., 208, 515 (1939). (4) Gudheim, A. R., Oil & Soup, 21, 129 (1944). (6) Long, Reynolds, and Napravnik, IND. ENG.CHSM., 26, 864 (1934). (6) Newton, Kaura, and De Vries, Ibid., 23, 35 (1931). . (7) Oliver and Bailey, Oil & Soap, 22, 39 (1945). (8) Oliver, Singleton, Todd, and Bailey, Ibid., 21,297 (1944).
LINSEED PROTEINS.
..
Alkali Dispersion and Acid Precipitation Information and data are given for the isolation of linseed protein by alkali extraction and acid precipitation. The flaxseed hulls contain a mucilage which interferes with the settling of acid-precipitated protein. Methods for the decortication of the linseed meal and analytical information on the composition of the hulls and embryo fractions are given. The amount of nitrogenous matter which can be dispersed within a wide range of p H values from the decorticated and undecorticated linseed meal is presented. The pH value of maximum nitrogen precipitation is determined, and protein yields are estimated.
F
LAXSEED has been an important crop (11) in our country since pioneering days, and in recent years the United States has ranked fourth among the nations of the world in flaxseed production. The peak year for United States flaxseed production, over an extended period prior to the war, was in 1924 with a record of 31 million bushels; the trend then became downward with a low of 5 million bu+els in 1936. The war economy revised this trend so that more than 50 million bushels were pr’oduced for 1943. Furthermore, the domestic supply of flaxseed is normally supplemented by importations. While the composition of flaxseed varies considerably with climatic conditions (S),the oil content on a moisture-free basis is.about 40 to 43%. The commercial linseed meal containing several per cent of oil is sold at Minneapolis on a 34% protein basis; however, the solventextracted meal, when moisture-free, contains 40 to 49% protein (1, 6). Although t h e oil has sold at 8 to 12 cents per pound at Minneapolis, the oil meal has brought only 1.25 to 2.25 cents per pound. Thus, on the basis of their abundance’and low cost, the linseed proteins are entitled to serious consideration in a research program directed toward the development of vegetable proteins for industrial utilization. Of the two principal linseed products, the oil has always held the major share of scientific attention because of its great importance as a drying oil. I n the field-of protein research the‘only investigation of importance concerned with the isolation and identification of linseed protein is the classical work of Osborne (7) in 1892. Osborne,
A. K. SMITH, V. L. JOHNSEN, AND A. C. BECKEL Northern Regional Research Laboratory, of Agriculture, Peoria, I l l .
U. S . Department
working principally with his salt-extraction technique, showed that “the extracts of the flaxseed contain a globulin precipitated by dialysis, a proteid, resembling both globulin and albumin, precipitated by long-continued heating at 100 ” C., as well as by sodium chloride in the presence of acid; proteose and peptonelike bodies, and a proteid not extracted by sodium chloride solution, but soluble in dilute potash-water”. He described the dialyzed globulin as forming octahedral crystals. I n regard to the peptones, Osborne concluded that “they are wholly formed during the extraction and separation”; this suggests the presence of a proteolytic enzyme in the linseed dispersions. The globulin was found to contain 18.6% nitrogen; the albuminlike body, 17.7% nitrogen. Ohe sample of the proteose contained 18.78% nitrogen; another contained 18.33%. From these values and estimated quantities of protein for each fraction, he arrived at a nitrogen-protein conversion factor of 5.5. The work of the few investigators (8,9, 10) who have worked on linseed proteins since the time of Osborne has been limited to studies on salt peptization. The salt extragtion and dialysis methods heretofore used for isolating flaxseed proteins are not well suited for large-scale operation, since the dialysis of the protein dispersions and recovery of the salts are time consuming. The present investigation explores the possibility of isolating the protein by the method of alkali extraction and acid precipitation similar to that described for the isolation of soybean protein (a, 8). REMOVAL OF HULL AND MUCILAGE
Preliminary studies on protein isolation showed that the mucilage, which occurs in the flaxseed hull in relative abundance, seriously interferes with the settling of the precipitated protein.
.
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I N D U S T R I A L AND E N G I N E E R I N G CHEMISTRY
Vol. 38, No. 3
TABLE I. P E R C E N T I C E COMPOSITION O F HULL AND EXBRYO FRACTIONS IN HAND-DECORTICATED FLAXSEED Whole Seed Moisture (5 hr. in vacuum oven a t 105’ C.) Nitrogen Oil Ash (4 hr. a t 550’ C.) Weight %. -
7.13 4.01 38.7
70 of
...
., ,
---EmbryoFull Fatfat freea 4.31 4.64 53.20 3.38 58.60
10:92 ,..
7.95 40.04
-__-
-HullFull fat 7.89 3.18 1.84 2.99 41.40
Fatfreea
... ...
3.52 3.31 59.96
total oil in: 96 7 3.3 The oil- and moisture-free data which are calculated from the full f a t analysis. a
TABLE 11. THE
EFFECT O F LIOISTURE ON GRINDING AND SEPARATING HULLSF R O M UNDEF.4TTED FLAXSEED BY SCREENING
Moisture Content,
70 yield of Compn. (Moisture-Free Basis) When When Total Seed, 3 ground analyzed % Kitrogen Oil Ash 1-4 12 8.94 56.1 3.54 34.9 3.46 50 5 3.23 1B 12 7 22 20.4 4.82 1c 12 6.02 23.5 5.09 51.1 3.16 3.19 29.3 .. 10 9.98 43.2 2A 19.8 4.75 49.8 2B 10 6.06 51.4 .. 37.0 5.10 2c 10 6.06 3.5. 8 9.22 35.1 3.12 27.1 .. 8 6.63 16.8 4.31 44.1 3B 3C 8 5.50 48.1 5.01 51.5 .. 2 3 . 3 3 . 64 16.8 2.94 4A 2 7.12 3.64 2.74 24.8 8.24 11.4 4B 2 3 . 2 4 4 . 8 1 4 9 . 7 4 . 1 5 7 1 . 8 4c 2 a A, hull fraction, retainedon R 38-grit gauze; R , mixed hulls and embryo fraction, passes through 38- and remains on 50-grit gauze; C , embryo fraction passes through 50-grit gauze. Tyler Standard equivalents for 38and’5O-grit gauze are 42 and 54, respectively. ~
pH VALUES
Figure 1. Total Nitrogen Dispersed from Oil-Free Linseed Jleal by Acids and Bases Whole linseed meal B . Hexane-extracted and decorticated linseed meal A.
Sample N0.a
I
.
..
While i t has not been experimentally demonstrated that all of the mucilage occurs in the hull, it is apparent from available information that the hull is its principal location. The hull also contains dark-colorcd pigments which, if not eliminated, are extracted with the protein and impart to it a dark and objectionable color. Therefore an efficient method for removing the hull is a preliminary step to protein refining. TWOmethods were investigated-the simple process of fractionating the ground seed with graded sieves, and an air separation method with the Raymond Whizzer. For either process the flaxseed was ground between smoot,h rolls to avoid breaking the hull as much as possible. Fortunately the hull tends to remain whole Yhile the embryo breaks up readily. The term “embryo” is used here to include all of the nonhull part of the flaxseed. The method of grinding and the moisture content of the flaxseed have a marked effect on the fineness of grinding of the embryo and the breaking up of the hull. As there is no fundamental method suitable for measuring the completeness of flaxseed decortication, visual observations of the various fractions and their chemical analyses were made to serve the purpose reasonably well. To obtain information which would serve as a guide in the later study of mechanical decortication, a sample of Bison-type flaxseed was dehulled by hand, and the fractions were analyzed (Table I). The greatest difference between the composition of the hull and of the embryo fractions for a full-fat hand-separated sample is in oil content, but this same difference does not occur for mechanically separated hulls and embryos. When the seeds are crushed and separated mechanically, oil is smeared on the hull; or broken embryo particles cling to the hulls to raise their oil content. For solventextracted meal the largest difference in composition between hull and embryo is in protein content. Table I shows that, on a full-fat basis, the hulls comprise 41.4% of the seed, are higher in moisture than embryos, are lower in nitrogen, and contain only 1.84% oil. On an oil- and moisture-free basis some of the values are reversed; the hull constitutes essentially 60% of the linseed meal and has a nitrogen value approximately one third that of the embryo fraction. The most striking fact revealed by Table I is that 96.7% of the total oil is in the embryos. Table I1 presents data for a mechanical separation of fullfat flaxseed, having various moisture levels at the time of grinding, and the composition of the separated fract,ion. The seeds were adjusted to the moistare levels indicated, ground by being passed twice between smooth rolls, and sifted on grit gauze. Visual examination of the embryo fractions and consideration of the
data in Tables I and I1 indicated that the best results from the standpoint of both purity and yield were samples 2C and 3C which had been ground in the moisture range 8 to 10%. The hull and embryo fractions were next separated after solvent extraction of the oil with hexane. Three different moisture levels were used in grinding the seed prior to extraction, and both the screen method and Raymond Whizzer were used for the separation. The data are given in Table 111. An intermediate fraction, which amounted to 100 minus the sum of fractions A and C, is n o t shown. T h i s intermediate fraction would be added to fraction A to be used for stock feed. I n evaluating these data, it must be kept in mind that on an oilfree basis the hulls constitute about 60% of the meal; therefore the yield of embryo is small. The embryo fraction of samples 2C and 3C have the highest I nitrogen values, but I p H V4LUE OF DISPERSION the yields were only 10 6 7 4 5 12.6% and 4.6%, Figure 2. Precipitation of Lintoo small to be seed Protein from Water Disperof practical value. sion a t Various pH Values Samples 8C and Curve A is based on the total nitrogrn in 9C, ground a t normal the dispersion B is based on the total nitrogen in th.e linseed meal, and C is a and above-normal nitrogen dispersion curve for the same moisture content and linseed meal used for A and B .
March, 1946
,
INDUSTRIAL AND ENGINEERING CHEMISTRY
355
was removed in an International centrifuge, and an aliquot of the supernatant solution was measured into a Kjeldahl flask for nitrogen determination. Nitrogen Moisture in Figure 1 shows two pH-nitrogen dispersion curves for hexaneSample Flaxseed When Yield, Moisture, (Moisture-Free No.'" Ground, % % % Basis), % extracted linseed meal. These curves indicate that the isoelectric point of the protein is at p H 3.8. However, this must Screened without Further Treatment 1A 4 64.6 7.58 5.25 not be accepted as the'correct isoelectric point for maximum 1c 4 18.0 7.24 7.92 protein precipitation; data obtained by precipitation methods 5.52 2A 8 77.6 9.50 2c 8 12.6 8.34 8.89 show a higher value. The curves show further that 21 to 23% 5.40 3A 12 91.8 8.32 of the total nitrogen of the meal is soluble a t the point of minimum 3c 12 4.6 8.40 8.82 nitrogen solubility and, therefore, will not be recoverable by a Ground in Ball Mill 30 Minutes protein isolation process of alkali extraction and acid precipita4A 4 23.0 8.02 3.59 4c 4 62.0 7.02 7.42 tion. There was some indication that complete elimination of 5A 8 37.5 10.22 3.85 the hulls would reduce ,the water-soluble nitrogen fraction to a 53.0 7.84 8.13 5c 8 6A 12 , 36.0 8.90 3.53 little lower value than the 21% shown in curve B. 6C 12 56.5 7.24 7.76 It is apparent that several extractions of the meal would reFractionated in Raymond Whizzer (Blades No. 6 and 24) cover more protein than a single extraction. Therefore single 7A 4 68.0 7.17 4.64 and double extractions were carried out at various ratios of 7c 4 32.0 7.89 8.10. 8A 8 75.0 8.60 4.90 water to meal between 10 to I and 50 to 1. Table IV gives these 8C 8 25.0 8.94 8.57 SA 12 81.6 8.06 4.85 data and the amount of total nitrogen which can be recovered 9C 12 18.4 8.68 8.43 by centrifuging. Distilled water was the dispersing agent, and A (hull fraction) remains on 38-grit gause; C (embryo fraction) passes the p~ ofthe dispersion was 6.8. through 50-grit gauze. Columns 2, 3, and 4 show that, in making two successive extractions of the linseed meal with water, the amount of nitrogen TABLEIV. EFFECTOF DOUBLEEXTRACTION OF NITROGEN" BY WATER FROM DECORTICATED HEXANE-EXTRACTED dispersed is independent of the water-to-meal ratio, provided the ratio is greater than 10 to 1, and that water readily disperses LINSEED MEAL 95% of the total nitrogen. Columns 5 and 6 are the quantities 6 J 2 3 4 5 % N Recovered, of total nitrogen that can be recovered from the protein disperyo Nitrogen F.utv=c+ed m-."_ -" _-Ratio Erulll 181 sions after removal of the insoluble residue, and conversely they First Secon Frpm 1st + 2nd Sum of Water Bo extn. extn. 2 extns. Meal extn. extnl.d indicate the amount of dispersion the insoluble residue holds. 10 94.4 75.5 48.8 The nitrogen yielda are definitely best a t the higher water-to97.2 15 80.2 57.3 meal ratios. The maximum protein yield which could be antici93.9 78.6 60.1 20 95.2 83.1 25 66.6 pated by the water extraction procedures can be estimated by 95.4 83.4 68.1 30 94.5 35 84.4 70.7 subtracting 23% from the values in columns 5 and 6. It is also 94.4 84.1 71.1 40 evident from these data that the most efficient method of ex94.2 87.2 74.9 50 tracting protein from decorticated linseed meal would be a con0 All values are based on the total nitrogen in the meal. Columns 3 and 4 are based on the total dispersing agents,added. Columns 5 and 6 @re tinuous countercurrent process. based on the amount of dispersing agent which could be separated from the
TABLE111. SEPARATION OF HULL AND EMBRYO FRACTIONS OF SOLVENT-EXTRACTED LINSEED MEAL
*
-
../
meal by centrifuging. b In the supernatant solution after centrifuging.
fractionated in the Raymond Whiszer, give the most practical results from the standpoint of yield and purity of product. While the described methods of hull removal have not been perfected, they are sufficiently effective for the preparation of an embryo product which may be used for protein isolation. Continued investigation on decortication methods is expected to improve the results. PROTEIN PEPTIZATION
In the decorticating process the embryo fraction of the flaxseed appeared to be ground finely enough for satisfactory protein extraction. A screen analysis of an embryo fraction, which had been solvent-extracted and separated in a Raymond Whizzer, gave 77% through a 54-mesh screen and 50% through a 100mesh. I n an experiment to determine the approximate time required for complete dispersion of the soluble protein, the water and meal were shaken together at a ratio of 40 to 1 for 30 minutes, and the dispersion was analyzed for nitrogen. Repetition of the same procedure for longer periods did not increase the amount of nitrogen dispersed, which indicated that 30 minutes was sufficienttime for dispersion of the protein. To carry out a protein peptization or extraction experiment, 2.5 grams of the hexane-extracted and dehulled meal were weighed into a 250-ml. centrifuge bottle, and ,100 ml. of water, which had been adjusted to the desired p H value with hydrochloric acid or sodium hydroxide, were added; then the bottle was shaken for 1 hour a t room temperature (25' C.). The insoluble residue
PRECIPITATION OF LINSEED PROTEIN
The precipitation characteristics of a protein cannot be safely predicted from dispersion data alone; therefore, the precipitation properties of the linseed protein were investigated. A linseed protein dispersion was prepared from hexane-extracted flaxseed by extracting the decorticated meal twice with water-the first extraction with a water-to-meal ratio of 20 to 1, and the second with a ratio of 10 to 1. To precipi80 tate the protein, 100 ml. of the dis70 persion were measured into a 250-ml. centrifuge bottle, 60 the desired amount of sulfuric acid was 50 added, and the bottle was shaken 40 mechanically to establish equilibrium conditions. The 30 protein curd which formedwasremoved by centrifuging, and an aliquot of the supernatant solu10 P 3 4 5 6 7 tion was taken for analysis. The Figure 3. Precipitation Curves for amount of precipiLinseed Protein, Showing Isoelectric Point with Hydrochloric tated nitrogen was Acid ( A ) and Sulfuric Acid ( B ) calculated a8 the
356
INDUSTRIAL AND ENGINEERING CHEMISTRY
Vol. 38, No. 3
results; therefore, it may be safely concluded that the flaxseed contains no albumin, &borne found peptones in his protein dispersions prepared by salt extraction techniques which he 1 2 3 4 5 6 7 8 9 thought mere derived as cleavage products of the globulins; N in N in N Whey this suggested the possible presence of a proteolytic enzyme Rewhich might cause a loss in recoverable protein. There is covery Protein N in P p t d . P.T.A. Tannic Acid no evidence of a rapid or substantial loss of acid-precipit,able Extn. pH of of N, by Pptn., Acidb, Pptn., Yield, Whey protein when the water extract of the embryo fraction is kept at No. 70 Ext. "j, 70 ' P . T A a 70 70 70 9, 67, 39, 1 5 ,8 14, 0.9 14.2 1.6 room temperature for 7 hours, as the following experiment shows: 2 9.5 6.8 4.0 2.3 1.9 0.4 1.7 0.6 A water extraction of linseed meal was divided into two equal 3 9.5 1.1 0.7 0.9 .. ... .. ... parts. To the first part, acid was added and the protein imTotal 73.3 43.7 lg.O 16.' '.a l'.' 2.2 mediately removed by centrifuging; to the second part, the acid a Nitrogen precipitated from whey with phosphotungstic acid. was added 7 hours after the protein extraction, the b Kitrogen precipitated from whey. protein was removed, and its yield was compared with the first precipitate. The first precipitation gave a PROTEIX YIELD AND SITROGEN CONTENT OF TABLE VI. APPROXIMATE protein yield of 11.9 grams with 0.85 gram of nitroLINSEEDPROTEIN PREPARED BY VARIOUS METHODS gen remaining in the whey; the second precipitaappro^.^ Prep. Dispersing Pptn. Times Yield, Analysis, % %N NFaction gave a protein yield of 12.2 grams with 0.65 No. Reagent Reagent Repptd. % Ash AIoisture N (Cor') tor' % gram nitrogen in the whey. This experiment 1 38 0.31 7.62 12.88 13.99 7.15 1 NaOH HzSOl 2 NaOH H&OI 2 34 0.08 5.31 13.10 13.85 7.22 shows there is no loss of protein through the 3 NaOH Hi301 ::'?: formation of cleavage products, by enzyme action 4 NaOH Hi304 5 NaCl Dialysis 1 18 0.28 5.46 16.17 17.37 5.76 or otherwise, which would cause a serious loss in 6 Ha0 (NHI)%SOI 1 6 0.14 9.38 16.35 18.07 6.53 yield.
TABLE V.
-4XD S I T R O C E N FROM SOLVENTEXTRACTED DECORTICATED LIYSEEDMEAL YIELD O F P R O T E I N
'{id. ,",',"a,
_ _ _ _ - - -
:;
a
t:!: ::::! i;:;;
Based on weight of embryo meal used.
NITROGEN CONTENT OF LINSEED PROTEIN
difference between the nitrogen in the original dispersion and the amount left in solution after protein precipitation. Curves A and B of Figure 2 give the precipitation results. C is a dispersion curve made a t the water-to-meal ratio of 40 to 1 from the same decorticated linseed meal used for curves A and B , and is shown to compare the minimum solubility points on the dispersion and precipitation curves. Sulfuric acid was the acidifying agent for both experiments. For the precipitation curve the minimum solubility point is a t about pH 5.1, in contrast to 3.8 for the dispersion curves. The two different minimum values may cause some confusion in regard to the isoelectric point of the protein, but from experience gained with soybean protein, the higher value is more nearly correct. Figure 3 presents additional precipitation data. The protein extractions for curve A were made at a water-to-meal ratio of 40 to 1, using hydrochloric acid as the precipitating agent; for curve B the extractions were made at a 20 to 1 ratio with sulfuric acid as the precipitant. These data confirm the pH value of 5.1 for the isoelectric point in Figure 2 and demonstrate that 20 to 23% of the total nitrogen is not precipitated by acids. The hydrochloric acid gives a narrower precipitation range than sulfuric acid; in this respect it is similar to the precipitation behavior of soybean protein. PROTEIN YIELDS
Table V, column 4, shows yields for linseed protein prepared by alkali extraction and acid precipitation. Based on the weight of solvent-extracted decorticated meal, the yields are 39.0, 4.0, and 0.7%, respectively, for three successive extractions at water-to-meal ratios of 20 to 1, 10 to 1, and 10 to 1. The nitrogen values of column 3 are taken from the analysis of the protein values in column 4. The solution remaining after removal of the acid-precipitated protein contains sugars and soluble nitrogen compounds. This solution is called "whey" for convenience. The nitrogen in the whey varied from 19 to 23%. This variation may be due partly to the different water-to-meal ratios used, but the completeness of decortication is also a factor. The lower nitrogen values in the whey correspond to the purest embryo fractions. The mhey fraction contains the soluble nitrogen compounds such as albumin, proteoses, peptones, aiid nonprotein nitrogen. There was no evidence, however, of a water-soluble heat-coagulable protein in the whey. This finding agrees with Osborne's
By the use of salt extraction and precipitation methods, Osborne estimated a nitrogen-protein conversion factor of 5.5 (18.18% nitrogen) for the whole of the linseed protein. More than fifty years have elapsed since Osborne's work was published, and the lack of recent work on linseed protein made desirable a comparison of the nitrogen content of his proteins with those prepared in our laboratory by the salt extraction procedures and by the method of alkali extraction-acid precipitation. The analytical data for protein samples prepared by different methods are given in Table VI. Preparations 1, 2, 3, aiid 4 are successive reprecipitations of the same protein-that is, by alkali dispersion and acid precipitation with each precipitate receiving two washings; 0.27, alkali solution was used in the original extraction, and for redispersion the pH was 8-9.5. The number of reprecipitations are indicated, and there is no increase in nitrogen content (13.99%) of protein purified by this method. Another publication will show that this method of purification increases the nitrogen content of soybean protein several tenths per cent. Preparation 5, with a nitrogen content of 17.37%, was made by salt extraction of the meal and precipitation by dialysis; preparation 6, with a nitrogen content of 18.07%, was made by water extraction, ammonium sulfate precipitation, and dialysis. The separated proteins were washed twice with water and dried in an air oven at low temperature. The nitrogen content of preparation 6 is the same as for Osborne's average value but not so high as for the "globulin" or "albuminlilte" products which he isolated. LITERATURE CITED
(1) Anderson, J., and Eva, W. J., Special Bull. Grain Research (2)
Lab., Board of Grain Commissioners, Winnipeg, 1941. Belter, P. A., Beckel, A. C., and Smith, A. K., ILD. E ~ GCHEM., .
36,799 (1944). (3) Hopper, T. H., and Johnson, Muriel, N. Dak. Agr. Exp. Sta., H i i l l . 298 (19411. (4) N ~ ~ i I l ~ ~ , AJ.'Agr. l l k n , Sci., 5, 113-28 (1913). (5) IT.Dak. h g r . Exp. Sta., Bull. 286. (6) O'Hara, L. P., and Saunders, Felix, J . Am. Chem. Soc., 59, 352-4 (1937). (7) Osborne,T. B.,Ibid., 14,629-61 (1892). (8) Smith, A. K., and Circle, S. J., IND.ESG. CHEM.,30, 1414 (1938). (9) Smith, A. K., Circle, S. J., and Brother, G. H., J . Am. Chem. Soc., 60, 1316 (1938). (10) Staker, E. V., and Gortner, R. A., J . Phys. Chem., 35, 1565 (1931). (11) U. S. Dept. of Agr., Agriculture Statistics, 1914.
