5 86-914-10m TEXAS AGRICULTURAL EXPERIMENT STATION BULLETIN NO. 169 SEPTEMBER, 1914 DIVISION OF CHEMISTRY The Total Fatty Acids and Other Ether-Soluble Constituents 0f Feedstuffs BY J. B. RATHER Assistant Chemist n: _' Witwifi " i: POSTOFFICE COLLEGE STATION, BRAZOS COUNTY, TEXAS “O VON BOECKMANN-JONES 00., PRINTERS, AUSTIN, TEXAS 1914 AGRICULTURAL AND MECHANICAL COLLEGE OF TEXAS CIIAnLIzs PURYEAR, President Pro Tem. TEXAS AGRICULTURAL EXPERIMENT STATION BOARD OF DIRECTORS 3111 . H. . J. HART, San Antonio ................... .. . L. BENNETT, Paris ........................ .. . E. BATTLE, lVIarlin..... . . WILLIAMS, Paris .................... .. . LLEN KvLE, Houston.. PIZJPFI 86-4 >0) é . B. CUSHING, President, Houston. ..................... .. HN I. GUioN, Vice-President, Ballinger ............. .. AsTIN, Bryan .................................................. .. ALT0N PETEET, Waco ............................................. .. ................................................... .. Term expires 1915 .................... .. Term expires 1919 .................... .. Term expires 1919 Term expires 1919 Term expires 1917 Term expires 1917 Term expires 1917 Term expires 1915 ............................ .. Term expires 1915 GOVERNING BOARD, STATE SUBSTATIONS P. L. DowNs, President, Temple........... CHARLES HOGAN, Austin ....... .. . V. A. C0LLINs, Beaumont ................................................... .. Term expires 1915 ................................................... .. Term expires 1919 Term expires 1917 Term‘ expires 1915 W. A. TYNES, Cooper .................................... .. STATION STAFF ADMINISTRATION B. YOUNGBLOOD, M. S., Director A. B. CONNER, B. S., Assistant Director CHAS. A. FELKER, Chief Clerk A. S. WARE, Secretary DIVISION OF VETERINARY SCIENCE M. FRANcIs, D. V. S., Veterinarian in harge H. SCHMIDT, D. V. M., Assistant Veter- inarian DIVISION OF CHEMISTRY G. S. FRAPS, Ph. D., Chemist in Charge J. B. RATHER, M. S., Assistant Chemist WILLIAM LEVIN, A; B., Assistant Chemist J. W. CHEWNING, B. S., Assistant Chemist DIVISION OF HORTICULTURE H. NESS, M. S., Horticulturist in Charge W. S. HOTCHKISS, Horticulturist DIVISION OF ANIMAL HUSBANDRY J. _C. BURNs, B. S., Animal Husbandman in Charge J. M. JONES, B. S., Animal Husbandman. DIVISION OF ENTOMOLOGY WILMON NEWELL, M. S., Entomologist in harge F. B. PADDOCK, B. S. E., Entomologist’ DIVISION OF AGRONOMY A. B. CONNER, B. S., Agronomist in Charge A. H. LEIDIGH, B. S., Agronomist in Charge of Soil Improvement H. H.- JoissoN, B. S., Assistant Agronomist E. W. HARRIsoN, B. S., Assistant Agronomist DIVISION OF PLANT PATHOLOGY AND PHYSIOLOGY F. H. BLODCETT, P_h. D., Plant Pathologist and Physiologist in Charge ‘DIVISION OF FARM MANAGEMENT Bax E. WILLARD, M. S., Farm Manage- ment Expert in Charge DIVISION OF FEED CONTROL W. L. BOYETI‘, Feed Inspector in Charge J. H. RooERs, Feed Inspector . H. W001), Feed Inspector . H. WQLTERs, Feed Inspector . M. SCHAEDEL, Feed Inspector . D. PEARCE, Feed Inspector JAivIizs SULLIVAN, Feed Inspector SUBSTATION NO. 1: Beeville, Bee County E. E. BINFORD, B. S., Superintendent _ O. K. COURTNEY, B. S.,Scientific Assistant SUBSTATION NO. 2: Troup, Smith County W. S. HOTCHKISS, Superintendent J. W. JAcxsoN, B. S., Assistant Supt. SUBSTATION NO. 3: Angleton, County N. E. WINTERS, B. S., Superintendent *SUBSTATION NO. 4: County H. H. LAUDE, B S., Superintendenr SUBSTATION NO. 5: Temple, Bell County A. K. SHoRT, B. S., Superintendent G. F. JonnAN, B. S., Scientific Assistant SUBSTATION NO.6: Denton, Denton County T. W. BUELL, B. S., Superintendent SUBSTATION NO. 7: Spur, Dickens County R. E. DICKSON, B. S., Superintendent SUBSTATION N0. 8: lLubbock, Lubbock County' V. L. CORY, B. S., Superintendent SUBSTATION N0. 9: Pecos, Reeves County H. C. STEWART, B. S.. Superintendent SUBSTATION'NO.‘l0: Feeding and Breeding Substation, College Station, Brazos County J. M. THOMSON, B. S., Foreman SUBSTATION NO. ll: Nacogdoches, Nacog- doches‘ County G. T. McNIzss, Superintendent muqg Brazoria Beaumont, Jefllerson CLERICAL ASSISTANTS STATION C. A. CAsE, Stenographer IVIATFIE THOMAS, Stenographer FEED CONTROL DAISY LEE,- Registration Clerk T. C. STROETER, Stenographer C. L. DURsT, Mailing Clerk and Shipping Clerk *In Cooperation with the Bureau of Plant Industry, United States Department of Agriculture. CONTENTS. PAGE. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 5 Historical . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 5 An Improved Method for the Determination of Total Fatty Acids and Other Constituents of Ether Extracts . . . . . . . . . . . . . . . . . 7 A New Method for the Extraction of Total Fatty Acids and Other Constituents of Feed Stuffs . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 13 A Study of Feed Stuffs with the New Methods . . . . . . . . . . . . . . . .. 15 The Total Fatty Acids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16 The Unsaponifiable Matter . . . . . . . . . . . . . . . . . . . . . . . . : . . . . . . 18 The Saponified Residue . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 20 The Nature of the Acids Soluble in Petroleum Ether and Not Extracted from the Samples by Ethyl Ether . . . . . . . . . . . . . . 22 The Digestibility of the Various Ether-Soluble Fractions . . . . . . 24 Ether-Soluble Matter in the Nitrogen-free Extract of Feed ‘Stuffs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 26 Tables of Calculations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 29 Summary and Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 30 BLANK PAGE IN ORIGINAL THE TOTAL FATTY ACIDS AND OTHER ETHER-‘SOLUBLE CONSTITUENTS OF FEEDSTUFFS. BY J. B. RATHER, ASSISTANT CI~IEMIST.* In previous publications of this Experiment Station (Fraps and Rather, Bulletins Nos. 150 and 162) it has been shown that the un- saponifiable matter in the ether extract of hays and fodders averages about 58 per cent. of the total extract, and is of much lower digesti- bility than the saponifiablo matter. It has also been shown that chloro- form extracts comparatively large percentages of material from hays and fodders previously extracted with ether, and that this extract con- tains fatty acids. A method was devised by means of which it was pos- sible to separate the constituents of the extract into- three fractions: unsaponified (largely wax alcohols), uncolored saponified (fatty acids) and colored saponified (chlorophyll and related compounds). The above method will be hereinafter designated the Digestion Method. The low digestibility of the ether extract of hays and fodders was shown to be due to the presence of wax alcohols, waxes, chlorophyll, and other sub- stances not as easily digested as fats or as fatty acids. In the hays and fodders examined the unsaponified matter of the ether extract varied from 36 to 72 per cent. and averaged 58 per cent. It had already been shown by Stellwaag (Land. V ersuch. Stat. 37, 148) that the ether extract of seeds and of concentrated feeding stuffs contain unsaponifiable matter. This varied from 34.5 per cent. for malt germ to 0.5 per cent. for cocoanut cake. In most of the feeds examined, however, from 2 to 8 per cent. of the ether extract consisted of unsaponifiable matter. ' In view of the above results, it appears that a rapid and reasonably accurate method is desirable for the systematic removal of unsaponified matter in the determination of fats. HISTORICAL. The writer has been unable to find any mention of an analytical method for the determination of fats in feeding stuffs, suitable for routttine work, that makes any provision for the removal of unsaponifiable ma er. Attempts have been made to produce a purer ether extract by filter- ing through animal charcoal. Hayward states (Ann. Rpt. Md. Exp. Sta, 1890, 90-103) that the use of animal charcoal gives lower results than the oflicial method, varying from 0.1 per cent. in corn meal to 3.6 per cent. in dried tomatoes, and averaging 1.5 per cent. In the light of our present knowledge of the nature of the constituents of the ether extract of feeding stuffs, it is evident that the possibility of get- ting a pure fat by such means is slight. *Under the general direction of G. S. Fraps, Chemist. 6 TEXAS AGRICULTURAL EXPERIMENT STATION. Poole (J, Am, Chem, Soc. 19, 877-881) recommends a method for the determination of fat in feces in which the ether extract is saponified with alcoholic potash, the chloresterol removed from the soap with ether, and fatty acids determined in the residue. The method of Liebermann and Szekely (Pflugefs Arch/iv. 72 (1898) 360-366), for the determination of fat, consists of saponifying the fat in the sample without extracting the sample with ether, by _means of aqueous potassium hydroxide, dissolving the liberated fatty acids in petroleum ether, titrating an aliquot with 0.1/N alcoholic potash and phenolphthalein, and Weighing the resulting soap. The amount of fat which corresponds to the soap is then calculated. This method is rec- ommended by Haas and Hill (Chemistry of Plant Products, London, 1913, p. 26) as a reliable and convenient method applicable to the de- termination of fat in fodders, meat, feces, and physiological work in general. The method is, however, open to several serious objections. It makes no provision for the elimination of unsaponifiable matter, or for the elimination of chlorophyll products, both of which are present in large amounts in the petroleum-ether soluble matter of‘ hays and animal excrement-s therefrom. The calculation of the fat depends on the assumption that the fatty acid is stearic or some other fatty acid, that the fats are present as triglycerides, and that there are no free fatty acids present. The mean molecular weight of the fats of plant prod- ucts vary with the nature of the sample under examination, the fats are not necessarily present as triglycerides, and free fatty acids, always present to a considerable extent, may make up 8'7 per cent. (rStellwaag, Zoc. cit.) of the so-called fat. Kumagawa and Suto (Biochcm. Zeit. 8, 212) determine fat in plant products by saponifying the sample direct with 5/N aqueous sodium hydroxide, acidifying and shaking the fatty acids out with ether in a separatory funnel. The ethereal solution is filtered, evaporated, dried and taken up with light petroleum. The solution is filtered, evaporated, and dried to constant weight. The unsaponifiable constituents of the ether extract of plants are readily soluble in petroleum ether, and that solvent undoubtedly dissolves chlorophyll when that substance is present. The method, therefore, cannot be considered to give accurate results. It has long been known that ether does not extract all of the ether- soluble constituents of plant and animal products, but the extraction is generally considered to be complete enough for all practical purposes. However, in the case of human feces, fatty acids may be present as calcium soaps, which are not soluble in ether. A number of methods have been devised for the extraction of such fatty acids in feces, most of them depending upon the solubility of the fatty acids in the ether , after treatment of the soap with hydrochloric acid. Among methods of this type may be mentioned that of Folin and Went-worth (J. Biol. Chem. 7, 421-6), in which ether saturated with HCl gas is used to extract the feces. Dormeyer (Pflugeris Arch. Physiol. 61, 341-343) states that only '75 to 85 per cent. of the fat of meat can be obtained by extracting 100 hours with ether. He proposes digestion of the meat "with pepsin- hydrochloric acid and shaking the fat out with ether. Voit, on the _ . -____ --=¢_€.-< i_p__ _ ‘_Z y __. .___ -_ I" FATTY Acrns AND ErrrER-SOLUBLE CONSTITUENTS or Fl-IEDSTUFFS. 4 other hand (Ztschr. Biol. 35, 555-582) claims that when the ether ex- traction is properly carried out, with small amounts of substance as free from water as possible, 2-1 hours’ extraction will remove 95 per cent. of the fat. Tangl and Weiser (Pflugofs zlrclzi. Physiol. 72, 360-366) state that Liebermann and Szeklefs method gives as good results as Dormeyer’s method on meat and feces. Bogdanoiv (Pflugefs Arch. Physiol. 68, 431-433) states that meat- ext-racted with alcohol after previous extraction with ether yields almost as much ether-soluble mat- ter as was extracted by ether from the sample direct. He regards this excess over the original ether extract as fat intimately mixed with something insoluble in ether but soluble in alcohol. Browne (Proc. 20th Coho. A. O. A. U.) states that pepsin digestion of residues from ether extraction renders part of some substances solu- ble in ether. He cites steer feces and mixed feeds containing molasses as examples. Methods for the determination of unsaponifiable matter of fats and oils fall into two divisions: (1) extraction of the soap solution with solvents, either hot or cold, and (2) extraction of the dried soap with solvents. A number of the methods are described by Lewkowitsch ,( Chem. Technology and Analysis of Oils, Fats and Waxes, Vol. I, 5th Ed _, pp. 456-460) and will not be described here. The method proposed in this bulletin differs from both these general processes in that the fatty acids, after saponification, acidification and solution in ether, are precipitated from that solvent with alkali. This process, so far as the writer is aware, has never been used before. . We have been unable to find any mention of the use of alcoholic soda or potash, the basis of another method pro-posed in this bulletin, as a solvent for fats. Alcoholic soda probably dissolves less non-fats than aqueous soda; soap solutions in alcohol are more easily manipulated, and fats are soluble in alcohol alone. The advantages of alcohol over water thus appear to be considerable. The use of aqueous alkali would not prevent the contamination of the soap with unsaponified material, because the latter is both soluble in soap solutions, and emulsifiable with aqueous alkali. AN IMPROVED METHOD FOR ‘THE DETERMINATION OF TOTAL FATTY ACIDS AND OTHER CONSTITUENTS OF THE ETHER EXTRACT‘. As mentioned above, a new process was devised for the separation of the unsaponified matter from the fatty acids. This method for the separation of the constituents of ether extracts into three fractions is essentially as follows: Saponify the ether extract, acidifv and dissolve in ether, precipitate the fatty acids from ethereal solution with aqueous alkali and remove by washing with water. Acidity the soap with acetic acid and shake with petroleum ether to dissolve fatty acids and then with ethyl ether to dissolve the residue. The novel feature of the above process consists in the method of sepa- rating the unsaponified from the saponified material. The method of fractionating the saponified matter into colored and uncolored portions 8 Texas AGRICULTURAL EXPERIMENT STATION. is essentially the same as that already reported from this Station (Fraps and Rather, Bulletin No. 162). This method, designated the Precipitation Method, is given in detail below. Extraction of Ether-Soluble; Illa-tier: Dry 10 grams of the sample, or 5 grams if it contains more than 4 per cent. ether extract, and ex- tract with redistilled ether in a suitable continuous extractor for 16 hours. The extraction flask should have a capacity of '75 to 150 c.c. and should be of such a type that the extract may be readily and com- pletely poured out. Knorr flasks should not be used. If it is desired to determine the total ether extract, the flask should be dried and weighed before the extraction. Evaporate off the ether after the com- pletion of the extraction, and dry and weigh if the percentage o-f ether extract is desired. Deter~rrz.irirzii01i. of U/nsagaonifiable illaitter: Add 20 c.c. of approxi- mately 2/N alcoholic sodium hydroxide, and boil under a reflux con- denser for one hour. Evaporate nearly to dryness and add 3% c.c. glacial acetic acid, or its equivalent in weaker acetic acid. Add 50f c.c. of redistilled ether and warm to dissolve the extract. Add 25 c.c. water and warm a minute more. Transfer to a 500 c.c. pear-shaped sepa- ratory funnel with a short neck and wash out the flask with 5 successive 20 c.c. portions of ether, pouring the washings in the funnel. Turn funnel on its ide and shake gently. The two layers should now be clear and the aqueous layer nearly colorless. Draw off the lower layer into a. 500 c.c. Erlenmeyer flash. Add to the ethereal solution 1O c.c. of a warm 1:2 aqueous sodium hydroxide solution, turn the funnel on its side and shake gently. Allow the precipitate to settle, add 25 c.c.- warm water, hold funnel in vertical position and give rotary motion. Allow the two layers to separate and draw off the clear aqueous solution into the Erlenmeyer flask, leaving any emulsion in the funnel. Repeat and then shake gently as above with 5 successive 30 c.c. portions of cold water, allowing a short time for the two solutions to separate, and add the washings to the soap solution in the Erlenmeyer flash. Transfer the ethereal solution to a tared 200 c.c. flask, evaporate or distil the other and dry to constant weight in a. steam oven at 100° C. _ Correction for Fatty Acids inf L7fl/86Lp0flifi6d (for material high in fatty acids and low in unsaponified only) : Add 2O c.c. of 0.2/N hydro- chloric acid to the ethereal solution of the unsaponified matter in the separatory funnel before evaporation, stopper and» shake vigorously. Draw off the aqueous layer and discard. Evaporate ethereal solution and weigh. Heat to boiling with 20 c.c. alcohol, titrate with N/lO sodium hydroxide and phenolphthalein, running a blank on the alcohol. Multiply the corrected reading by .28 (or .56 if 5 gm. is taken). The result is percentage fatty acids in the unsaponified matter. Subtract this from the percentage of unsaponified matter and add it to the fatty acids. This amount of fatty acids dissolved in the ethereal solution of unsaponifled matter is on an average 16 milligrams for concentrated- feeding stuffs, and this figure may be used instead of determining the correction if very accurate figures on the percentage of unsaponifiable matter are not desired. FATTY Aorns AND ETHER-SOLUBLE CONSTITUENTS or Funnsrtrrs. 9 - Determinaltion of the Fatty Acids: Heat the soap solution on a steam bath to remove dissolved ether, shaking gently from time to time. This must be done carefully to avoid frothing of the soap. Cool the soap solution nearly to room temperature under the tap, add 8 c.c. of glacial acetic acid or its equivalent in Weaker acetic acid and extract in the separatory funnel with 4O c.c. of redistilled petroleum ether, dis- tilling below 75° C, shaking violently. Difaw off aqueous layer, hold funnel in a vertical position, giving it a rotary motion, and let stand a minute. This will cause the suspended matter to settle in a compact mass at the bottom of the funnel. Draw off this portion into the flask containing the aqueous mixture. Extract the aqueous mixture I)’ times more in a similanmanner. By using a pear-shaped funnel, and follow- ing the above procedure, filtration to remove suspended matter may gen- erally be avoided. Shake well with two 5O c.c. portions of water to remove suspended matter and traces of inorganic substances, allowing any emulsion to g0 into aqueous mixture. Extract the latter a fifth time with petroleum ether, and after ivashing this extract twice with small portions of water, add to the other extracts. Evaporate and dry to constant weight. If the evaporation is carried to completeness on a steam bath, the fiask being turned on its side to facilitate removal of gasoline fumes, the product may be dried to constant weight in 3 or at hours. This fraction is fatty acids. Determination of Saponified Residue: Acidify further the aqueous residue from the extraction of the fatty acids, with hydrochloric acid, warm and extract 5 times as above with 40 c.c. portions of ethyl ether. Wash the combined extracts twice with 5O c.c. portions of water, evap- orate and dry to constant weight. Include any suspended matter in this fraction. Tests of the method were made by examining the groups of ether~ soluble material obtained with it, by the Digestion Method already developed in this laboratory (I00. cit.) and by other means. The vari- ous factors affecting the accuracy of the process are discussed below. The results are expressed in percentage of the original sample. Uompleteness of Sapcnification: Incomplete saponification will cause low results in the saponifiable material and high results in the nnsaponifiable. That the saponification is complete at the end of 1 hour under the conditions of the method is shown by the following tests: 1. Samples of ether extract of wheat bran, corn chops, and milo maize chops were saponified for 1 hour with 2O c.c. l/N alcoholic soda and with 20 c.c. 2/N alcoholic soda. The unsaponified was determined by the Precipitation Method. The results follow: Percent Unsaponified § 1N Alkali 2N Alkali l 1299s Wheat bran .............................................................................. .. .21 .24 12999 Corn (chops) ................................................................................... .. .13 .12 13030 Milo Maize (chops).......... .20 .18 Average ............................................................................ .. . 18 . 18 l 1O TEXAS AGRICULTURAL EXPERIMENT STATION. Concentration of the alkali did not decrease the percentage of un- saponified. 2. The unsaponified matter was determined by the Precipitation Method in three hays and three exerements, the product was then re- saponified and the unsaponified determined by the Digestion Method of Fraps and Rather. The results follow: Unsaponified Matter, Per cent. A. By Precipitation B. In (A) by Diges- Method. tion Method. 7980 ................................................................... .. .47 .43 7991 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. .43 . 7999 ................................................................ .. 1.02 1.02 8002 ................................................................... .. .68 .68 8011 ............................................................. .. 1.17 1.13 8013 ....................................................................... .. 2. 14 1 , 99 Average ............................................. .. 0. 98 0.95 The results are practically the same in all cases. It is to be expected that the results would be slightly lower by (B) because of the large amount of manipulation required. . U nsapoirtifiecl JlIatc/rial in Fatty zlcids: This would be due to incom- plete separation of the two groups. Determinations by the Digestion Method showed it to be negligible in amount. In percentage of the original sample the results were as follows: Maximum Minimum Average 12 Concentrated feeding stuffs ........... .. _ . . . . . . . . . . . . . . . . . . . . . . . . . .. .04 6 Hays and excrements, 20 determinations. .. .05 i .00%' .o2% I .o1% 03% Fatty Acids in [Tnsapmzified Material: This could be due to incom- plete washing of the ethereal solution with water to remove soap, or to hydrolysis of the soap to form free fatty acids. The acids were set free and determined by the process described in the Precipitation Method, .it being found that no hydrochloric acid remained upon evaporation and drying after such treatment. The results are as follows: Maximum Minimum Average 12 Concentrates, 42 determinations: . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. .50% 04% .16%. 12 Hays and excrements, 27 determinations ....................... .. .09% .00% .05% The fatty acids in the unsaponified material of the concentrates varied greatly, even in duliilicate (lQlQ1W11l1IEItlOI1S 011 the same samples. Increas- ing the number of washings of the ethereal solution of the unsaponified to eight did not decrease the amount of fatty acids in the unsaponified fraction. We believe the presence of the acids is due to- hydrolysis of the soap and not to incomplete removal of the latter. We think that the correction given in the Precipitation Method should always be‘ ap- plied in the examination of plant and animal products rich in fat and low in unsaponifiable material. FATTY Aorns AND ErHER-SoLUBLE Consrrrunnrs or Fnnnsrurrs. 11 The agreement of the determinations of fatty acids in the unsaponi- fied matter is much more satisfactory in the case of products poor in _fat and high in unsaponified matter. The unsaponified in the fatty acids, and the fatty acids in the unsaponified, very nearly balance each other in determinations on these Products. The difference is only 0.02 per cent, well within the limit of error of determination. - Colnzpletcncss 0f the Eaftractton. of the Fatty Acids by Petroleum Lltltcr: The fifth extraction of the fatty acids with petroleum ether was dried and Weighed separately as a test. of the completeness of ex- traction. The results follow: Maximum Minimum} Average 12 Concentrates, 18 determinations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. .06% .Ol% .027 12 Hays and excrements, 12 determinations . . . . . . . . . . . . . . . . . , .. .02% .01 % o .O1% Five extractions with petroleum ether seem to be sufficient to extract practically all of the material soluble ‘in that medium, under the condi- tions of the method. _ The method as given difiers slightly from that 0n which the above tests were made. The change was made for convenience in manipula- tion and does not affect the accuracy of the results. The Sctyaon/ified Residue: Under this heading we include ether-solu- ble saponifiable matter, which is not dissolved by petroleum ether after acidifying with acetic acid. The reason acetic acid is used instead of" hydrochloric acid, is that the latter renders chlorophyll more reatlily" soluble in petroleum ether than the former. Petroleum ether is recog- nized as a solvent for fats, and is said to be more desirable than ether- under certain conditions. All fatty acids are more or less readily solu- ble in petroleum ether, and since the ratio of solvent to solute in our’ work is about 1000 to 1, and the weight of the fifth extraction usually less than 2 milligrams, it does not seem likely that‘ any fatty acids re- mained UHCllSSOlVGCl after five successive extractions with petroleum ether. The petroleum ether extracts a. fraction of the saponifiable mat- ter which is relatively free from chlorophyll, and, for that reason, We~ know that, in the case of the samples containing that substance, the fraction of-the ether-soluble plant material designated by us as “saponi- fied residue” consists, in part, of a substance which is not a fatty acid- This does not make up a very great percentage of the fraction, however, and we find the latter in considerable amount in samples free from chlorophyll. V A sample of lard compound‘ weighing 30 milligrams yielded only "4 milligrams of the “saponified residue,” which would be equivalent t0- 0.04 per cent. on a 10-gram sample. Since the sample contained com- mercial fats of only average purity, it is possible that pure fats would be entirely free from the fraction designated “unsaponified residue.” Completeness of Est-traction of Sapovzilfied Residue: The fourth ex- traction of this fraction was dried and weighed separately. The re--- sults follow : 12 TEXAS AGRICULTURAL EXPERIMENT STATION. Maximum Minimum} Average 12 Concentrates, 24 determinations ................................... .. .04% .00% ,O2% h 12 Hays and excrements ..................................................... .. . 02% W.101% .02% . ‘ " .\ gr- Four extractions appear to be sufficient. iSYzpcnifietl Residue in Zflatty Acid Fraction." The fatty acids may be contaminated with petroleum-ether soluble non-fatty acids. The only data we have on this point is qualitative. The fatty acids were all tinged with green in samples where chlorophyll was present, and the depth of color was apparently related to the amount of chlorophyll present. Compared with the saponified residue fraction, however, they were very lightly colored. Chlorophyll constitutes a part only of the latter fraction, and its coloring power is very great. We believe the amount of chlorophyll, at least, in the fatty acid fraction, is relatively small. Sciponified Residue in the Unsaponified Fraction: This is indicated by green color and by quantitative tests. We have already discussed the amount of acids in the unsaponified. We have assumed these to be fatty acids. Our reason for this is that, in every case Where chlorophyll is present in the sample, the unsaponified fraction was entirely free from green color. Washing Required t0 Remove flydrochloric Acid After Liberating Acids in [Tnsapo-nified: The method of liberating the acids in the un saponified as used by Fraps and Rather is somewhat long; we substi- tuted a process in which the acids were liberated in ethereal solution be- fore evaporation, and the excess of mineral acid washed out with water. Later it was found that the washing was unnecessary when the 11n- saponifiedmatter is evaporated to dryness. Samples of unsaponified matter equivalent to one or more per cent. in the sample yielded an acid value of zero by the process as finally adopted in the method. Fatty acids are, however, always found in the unsaponified matter of concen- trates. Comparative determinations are given below: Per cent Acid in Unsaponified. ‘ 2O c.c. 2N HCl 2O c.c. 2N HCl washed 5 times not washed 1 .............................................................................................. .. .06 .04 2 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. .04 .04 1 3 ..................... ...................................................................... .. .03 .06 Average ...................................................................... .. .04 .05 There is practically no diiference in the results by the two processes, and as the one in which no washing is done is much shorter, it is to be recommended. This process reduces the time required for the correc- tion to a negligible amount. The process has additional confirmation in a large number of determinations not reported here. (Viecking 0f Duplicate Deteivninatimis: All determinations were made at least twice. The results given in the tables in this bulletin are FATTY Aoins AND ETI-IER-SOLUBLE OoXsTITUENTs on FEEnsTUFrs. 13 averages. A few duplicate determinations, none run at the same time, are given here to show the accuracy possible with the method. Unsaponified Fat Acids Non-fat Or- ganic Acids 12996 Corn Chops ...................................................... .. .17 2.96 .43 .24 3.05 44 .23 3.14 29 .31 3.17 .... .. 21 . . . . . . . . . . . . .. 13030 Milo Maize Chops .............................................. .. .22 .. .59 .23 . . . . .. 69 .22 . . . . .. .18 . . . . . .. .20 . . . . .. 13045 Cold Pressed Cottonseed..... ............................ .. 6.66 6.47 . . . . . .33 13023 Rice Bran................. ' .77 Results on the hays and on both concentrates and roughages by the Alcoholic Soda Method checked equally as Well. A NEW METHOD FOR THE EXTRACTION OF "TOTAL FATTY ACIDS AND OTHER EITHER-SOLUBLE OONSTITUENTS OF FEEDING STUFFS AND EXOREMENTS. This process was devised with the intention of securing a substitute for the method of determining fats as fatty acids in feed stuffs. The results obtained are in every casemuch higher than the fatty acids of the ether extract, and do not justify its use as a substitute for the latter. The method is valuable in another direction, which will be discussed later. The method consists in the digestion‘ of the sample for 1 hour with Z/N alcoholic soda, removal of the alcohol, addition of water and shak- ing the acidified mixture, of ethyl ether. The ether solution is frac- tionated, as described in the Precipitation Method above. The method in detail is given below. Digest 10‘ gm. of the sample, or 5 gm. if it is bulky, or contains more than 4 per cent, ether extract, with 50 c.c. of approximately 2/N alco- holic soda, for 1 hour under a reflux condenser. Filter with the aid 0f suction through asbestos in a carbon funnel of suitable size and wash 1O times with the boiling alcohol. Transfer to a dish with water and evaporate to about 1O c.c. Transfer with hot water to a pear-shaped separatory funnel. Acidify with 10 c.c. glacial acetic acid and extract the warm solution 5 times with 5O c.c. portions of ethyl ether (redis- tilled), removing the bulk of the suspended matter in the manner de- scribed under “Determination of Fatty Acids” in the Precipitation ‘Method. Separate the constituents of this- extract as described under the Precipitation Method, laeginning with the addition of the 1:2 sodium hydroxide solution. Discard the insoluble matter remaining after the determination of the saponified residue. 14 TEXAS AGRICULTURAL EXPERIMENT STATION. 110st of the factors affecting the accuracy of this method are the same as those. discussed under the Precipitation Method. Two additional factors require notice. They are discussed below. Uonzplctcncss of Saponi/ictltio-rz: The percentage of fatty acids ob- tained under different conditions is an indication of the completeness of saponification. Six samples of hays and excrements were used. The fatty acids were (letermined (A) in an extract prepared by digesting 10 grams of the sample 1 hour with 20 c.c. N alcoholic soda and 100 c.c. alcohol, (B) in an extract prepared as above except 40 c.c. Q/N alco- holic soda and 60 c.c. alcohol were used, (C) in an extract prepared by digesting 5 grams of the sample 1 hour with 50 c.c. Q/N alcoholic soda. The results follow: Percentage Fatty Acids. (A) (B) (C) 6288 .............................................................................. .. . 19 .67 .54 6290 ........................................................................... .. .26 .89 .91 6724 ............................................................................. .. .32 1.15 1.06 7763 ................................................................................. .. 87 1.05 1.00 7799... 21 1.49 1.48 7970 ............................................................................... .. 85 1.49 1.72 Average ..................................................................... .37 1.12 1.12 There is a great difference between the results by methods (A) and , (B), but very little between those by (B) and We have included the process (C) in the method as finally used. Completcncsis of Extraction. anitlz. Ethyl Ether: In extracting the aqueous mixture of fatty acids, etc., with ethyl ether to separate it from ether insoluble matter, the fifth extract was dried and weighed separately. The results follow: Maximum Minimum Average 12 Concentrates... .. .O6% .O1% .O2% 12 Hays and excrements._... .. .O2% .O1% .01% Five extractions seem to be sufiicient. The number of ivashings of the sample, aft-er digestion with the alco- holic soda, was _ten. This number was entirely arbitrary. The entire amount of chlorophyll is probably not removed by this number of wash- ings, but we believe all fatty acids are removed. , The Solubility of Calct/zuiz Salts in Alcoholic Soclrlt: _ A number of methods for the determination of fat in human feces have been pro- posed which aim to extract, in addition to glyrcerides and free fatty acids, the fatty acids present in the sample as lime soap. Acids are usually used for this purpose, either in connection with or before the use of ether. In order to see what effect our alcoholic soda process would have on lime soaps, we prepared lime salts of mixed fatty acids. and determined the fatty acids soluble and insoluble in alcoholic soda under the conditions of the method, with and without an excess of lime (CaCO3). The results follow: FATTY Aorns AND ETHEn-SoLUBLE OoNsTrrUENTs or Fnnnstrtlrrs. 15 nissolvod Insoluble Solubility 1 Without lime . . . . . . . . . . . . . . . . . .................................... .. 121 mg. 12 mg. 91% 2 With lime ................................ .. ................................. .. 85 mg. 4 mg. 90% The amount dissolved is equivalent to 1.7 per cent. to 2.1 per ce11t. on a 5-gram sample, which is alaout the amount of fatty acids present in the alcoholic soda extract in excess of those of the ether extract. We believe that lilne soap would be dissolved nearly completely from excrements under the conditions of the Alcoholic Soda method. A STUDY OF FEED STUFFS WITH THE NEW METHODS. Twelve samples of concentrated feeding stuffs, salnples of hays and six samples of sheep excrements from the hays were used. The concentrates consisted of some of the more common ilnmixed feeds on the Texas market, and were products or by-products of the seeds of corn, cotton, rice, Wheat, milo maize, and kafir corn. Thev were fresh and in good condition. With the exception of one sample of “wheat shorts,” No. 12996. which appeared to be ground wheat bran, the ma- terials were true to- name. The hays were prairie hay, and tabosa grass, both native Texas hays, sorghum, Sudan grass, and nloth-laean hay, and Sudan straw. FThe Sudan straw was made from fully matured Sudan grass with the heads removed. All of these samples were in good condition. Determinations of the ether-soluble constituents of the samples were made by the methods given above. In addition, determinations were made as follows: Ether Extract, by the A. O. A. C. Method; Con- stituents of the Ether Extract, by the Digestion Method of Fraps and Rather; and Fatty Acids in the Chloroforln and Alcohol Extract Fol- lowing Ether Extract-ion. The Digestion Method is given in detail in Bulletin No. 162 of the Texas Experiment Station. It consists essentially in digesting the aqueous solution of the saponified ether extract in the warm, with suc- cessive portions of petroleum ether followed lry ethyl ether, to remove the ilnsaponifiable matter, and treatment of the residue as described in the Precipitation Method given above. Fatty acids were determined in the chloroform and alcohol extracts following ether extraction to ascertain whether ether, followed by chloroform, followed by alcohol, would extract a total of fatty acids equal to that extracted by alcoholic soda alone. The process was as follows: . Extract the dried residue from the ether extraction with chloro- form for 16 hours. Remove solvent from extract and residue, and ex- tract further in the same flask with alcohol for 16 hours. Determine the total fatty acids by the Precipitation Method, bringing the soap into solution as described under Alcoholic, Soda Method. In the case of the samples of hays and excrements, the fatty acids were determined in the chloroform extract alone, and the Digestion Method was used. 16 TEXAS AGRICULTURAL EXPERIMENT STATION. The ether, chloroform, and alcohol used in this Work were redistilled in this laboratory. THE TOTAL FATTY ACID-S. In Table 1 are shown the results of the determination of fatty acids by the various methods. The total fatty acids represent the free fatty acids in the original sample, the fatty acids from the glycerides, from saponifiablewaxes, and lecithin. All feeding stuffs contain free fatty acids in appreciable amounts, and the free acids often make up the bulk of the fat. Like- wise the glycerides may not be all triglycerides. For these reasons, and since fats are valuable as a food only because of their fatty acid content, we believe that it is better to express fat in terms of fatty acids. TABLE 1. Percentage 0f Fatty A01 ds m Feedstufis and Ecvc-remeozts by Venous Methods. 7U I C! d Fatty acids in 5 a i5 /\ '5'? .’* ether extract. % E e 3 :38 Labora- ~ Ether ————-——_—_ ESP-g . ._ “'7'; fig Differ- tvfy extract. Diges- Precipl- i,’ §.g B .23 3.8 g *5’ ence No. tion tation 3 5,9 3 9. 13g P‘ (B—A) m l-l ' Method. Method. p“ ° °"‘° h‘ "3 12996 Wheat Shorts . . . . . . . . . . . . . . . . . . . . .. 3 .79 3.08 0.37 3.45 4.43 0.98 12999 Corn chops . . . . . . . ..' . . . . . . . . . . . . . .. 4.31 3 .77 0.16 3.93 4.34 0.41 13021 Cottonseed meal . . . . . . . . . . . . . . . . . . . 15.23 13.82 0.69 14.51 14.46 -0.05 13023 Rice bran . . . . . . . . . . . . . . . . . . . . . . . .. 7.75 6.21 0.39 6.60 8.10 1.50 13030 Milo maize chops . . . . . . . . . . . . . . . . . . . 3.22 2.05 0.37 2.42 2.92 0.50 13045 Cold pressed cottonseed . . . . . . . . . . . . . 7.26 6.49 0.46 6.95 7.32 0.37 13091 Kaflir chops . . . . . . . . . . . . . . . . . . . . . .. 3.20 2.57 0.34 2.91 3.18 0.27 13140 Corn bran . . . . . . . . . . . . . . . . . . . . . . . .. 8.59 5.50 0.36 5.86 8.13 2.27 13150 Red rice . . . . . . . . . . . . . . . . . . . . . . . . . .. 1.64 1.04 0.44 1.48 1.81 0.33 13166 Wheat bran . . . . . . . . . . . . . . . . . . . . . . .. 4.10 3 .50 0.55 4.05 4.41 0.36 13172 Wheat shorts . . . . . . . . . . . . . . . . . . . . . . 2.65 2.22 0.27 2 .49 2.84 0.35 13190 Rice polish . . . . . . . . . . . . . . . . . . . . . . .. 10.38 8.53 0.45 8.98 10.29 1.31 6288 Tabosa grass . . . . . . . . . . . . . . . . . . . . . .. 0.92 0.15 0.30 0.08 0.38 0.61 0.23 6290 Excrement from tabosa grass . . . . . . . . 1.06 0.23 0.25 0.11 0.36 0.90 0.54 7724 Prairie hay . . . . . . . . . . . . . . . . . . . . . . .. 2.30 0.54 0.65 0.09 0.75 1.11 0.36 7763 Sudan grass . . . . . . . . . . . . . . . . . . . . . . .. 1.46 0.71 0.63 0.06 0.69 1.03 0.34 7799 Excrement from prairie hay . . . . . . . . . . 2.88 0.43 0.90 0.17 1.07 1.49 0.42 7970 Excrement from Sudan grass . . . . . . . . . 1.90 0.29 0.25 0.27 0.52 1.61 1.09 7980 Sudan straw . . . . . . . . . . . . . . . . . . . . . . . 1.44 0.45 0.67 0.06 0.73 1.29 0.56 7991 Sorghum hay . . . . . . . . . . . . . . . . . . . . .. 1.82 0.63 0.89 0.07 0.96 _1.17 0.21 7999 Excrement from Sudan straw . . . . . . .. 1.71 0.35 0.39 0.14 0.53 - 1.64 1.11 8002 Moth bean hay . . . . . . . . . . . . . . . . . . .. 1.55 0.35 0.62 0.15 0.77 1.70 0.93 8011 Excrement from sorghum hay . . . . . . . . 1.78 0.40 0.62 0.05 0.67 1.71 1.04 8013 Excrement from moth bean hay . . . . . . 3.03 0.26 0.62 0.06 0.68 2.28 1.60 Average for concentrates . . . . . . . . 6.01 4.90 0.40 5.30 6.02 0.72 Average for hays and excrements 1.82 0.57 0.11 0. 69 1.38 0.70 The fatty acids in the concentrates varied from 13.82 per cent. in cottonseed meal to 1.04 per cent. in red rice. They average 4.90 per cent, about 19 per cent. less than the average for the corresponding total ether extracts. The difference, however, in individual cases varies from 36 per cent. less with red rice to 16 per cent. less with wheat shorts, N0. 13172. The fatty acids in the hays and excrements varied from 0.25 per cent. in the excrement from tabosa. grass and the experiment from Sudan grass to 0.96 per cent. in excrement from prairie hay. They FATTY Aorns AND ETHER-SoLUBLE CoNsrrrUENrs or FEEDSTUFFS. 1'7 average 0.54 per cent., which is about 56 per cent. of the average of the corresponding total ether extracts. Chlorofonn and alcohol extracted from the concentrates, which had previously been extracted with ether, from 0.16 to 0.69 per cent. of * fatty acids, averaging 0.40 per cent. The fatty acids in the acoholic soda extract of the concentrates varied from 14.46 per cent. in cottonseed meal, to 1.81 per cent. in red rice, with an average of 6.02 per cent. The difference between the sum of the fatty acids obtained with ether, chloroform and alcohol, and the fatty acids by the Alcoholic Soda Method. of extraction varied from 0.05 per cent. in the cottonseed meal to 2.27 per cent.. in corn bran. The average is 0.72 per cent. The sum of the fatty acids obtained by successive extractions with ether, chloroform, and alcohol is less than the acids extracted from the concentrates by alcoholic soda alone. It will be noted that the fatty acids by the Digestion Method are lower than by the Precipitation Method. This may be explained as fol- lotvsf When the Precipitation Method was being developed for the concentrates, it was found that 5 extractions with violent shaking were required to remove the fatty acids. For the sake of uniformity this was applied to the hays and excrements. The only difference between the two processes consists in one additional extraction and more vig- orous shaking by the Precipitation Method. It appears that the sepa- ration of the two saponifiable fractions is not entirely satisfactory. The fatty acids extracted by chloroform only, after ether extraction, were determined in the hays and excrements. The fatty acids ex- tracted ‘by this solvent, after previous extraction of the sample with ether varied from 0.05 per cent. in excrement from sorghum hay to 0.27 per cent. in excrement from Sudan grass, and averaged 0.11 per cent. The percentage of fatty acids as determined by the Alcoholic Soda Method in the hays and excrements was in every case higher than the sum of the fatty acids extracted by ether followed by chloroform. The fatty acids varied from 0.61 per cent. in excrement from sorghum hay to 2.28 per cent. in excrement from moth-bean hay, and averaged 1.38 per cent. This is about 250 per cent. of the fatty acids in the ether extract and about 210 per cent. of _the fatty acids extracted by both ether and chloroform. The fatty acids extracted by the Alcoholic Soda Method from the 24 feeds and excrements were higher in every case ‘than the fatty acids in the ether extract of these samples, averaging 123 per cent. as much for the concentrates and 250 per cent. as much for the hays and excre- ments from them. _ The Alcoholic Soda Method proposed in this Bulletin gives higher results for the total fatty acids in feeding stuffs than does the method involving extraction of the sample with ether. The higher results may be due in part to ether insoluble, alcohol solu- ble, incrustations on the fats and in 1iart to fattyacids combined as lime or other ether-insoluble salts. This may be especially true of the excrements. _ An Alcoholic Soda Method has been devised for the determination 18 TEXAS AGRICULTURAL EXPERIMENT STATION. of total fatty acids and other ether-soluble constituents of plants, by which process are extracted more fatty acids from plant products than ether and, in hays and excrement, twice as much as fatty acids as can be obtained by successive extraction of the sample xvith ether, chloroform, and alcohol. _ An improved method of determining the total fatty ELCIdSELIlCl other constituents of ether extracts has been developed which is believed t0 be verv accurate and rapid. Six determinations each of unsapo-nified matter, total fatty acids, and saponified ‘residue, in-ether extracts may be determined in about 24 hours, and require only about 3 or 41 hours of urork. This method is believed to meet a need for a. convenient analytical process for the determining‘ fats as fattv acids in ether ex- tracts when more than approximations or comparative results are re- quired. ‘ THE UNSAPONIFIED BIATTER. In Table 2 are collected the determinations of ether extract, un- saponified matter in the ether extract by the Precipitation and Di- gestion Methods, and unsaponified matter in the alcoholic soda ex- _ tract. _ The imsaponified matter in the ether extract varied from 0.15 per cent. in red rice, to 0.60 per cent. in rice bran, and averaged‘ 0.30 per cent. The ether extract in the concentrates "varied from 1.64 per cent in red rice, to 15.23 per cent. in cottonseed meal, and averaged 6.01 per cent. Expressed in terms oi’ percentage of ether extract, cot- tonseed meal is lowest in unsaponified matter, with 3.1 per cent; and red rice is highest with 9.2 per cent. The average is 5.8 per cent. i Stellwaag finds the unsaponified matter in the ether extract of corn grain to be 3.7 per cent; in cottonseed cake, 1.1 per cent.; and in wheat bran 7.5 per cent. Our results on corn chops, cottonseed meal, and wheat bran are 3.7, 3.1, and 6.1 per cent., respectively’. The unsaponified matter in the alcoholic soda extract of the con- centrates is essentially the same in amount as that in the ether extract. It averages 0.32 per cent. of the sample. It appears that, while the unsapoiiifiecl matter in the ether extracts of the concentrates does not make up a very large percentage of the extract, it is large enough, wfhen expressed in percentage of sample, to deserve attention in the case of rice bran, cottonseed meal, and rice polish. ' _ In co-nsidering- the results with the hays and the excrements there- from, we find the unsaponified matter present in relatively large amounts. The ether extract of these products varies from 0.92 per cent. to 3.03 per cent., and averages 1.82 per cent. The unsaponified matter in the ether extract svarieswfrom 0.49 per cent. in sorghum hay- to 1.99 per cent. in excrement from moth-bean hay, and averages 0.97 per cent. Expressed in percentage‘ of extract, ‘the unsaponified matter varies from 26.9 per cent. in sorghum hay to 74.2 per cent. in excre- ment from Sudan grass, and. averages 52.7 per cent. On an average, more than half of the ether extract of these hays and excrements con- sists of non-fats. These results are similar ‘to those already reported from this Station (Fraps and Rather, Z00. c1125.). They emphasize the conclusion made in the publicationmeferred to, that “it is not correct FATTY Aorns AND ErHER-SoLUBLE OONSTITUENTS on Fnnnsrurrs. 19 to use the term ‘fats’ or ‘oils’ to designate the ether extract of hays and fodders.” The Linsaponified matter in the alcoholic soda extract is about. the same as that in the ether extract in some cases, but averages 0.09 per cent. higher. The results by the former method of extraction, how- ever, are considerably higher in the case of moth-bean hay and excre- ment from Sudan straw and excrement from moth-bean hay. The sum of the averages of determinations of llllSilpOll1fi€Cl matter in the ether extracts of these samples, and- ‘that in the chloroform extracts following ‘ether is 1.10 per cent, and the average for the alcoholic soda extracts is ]..06 per cent. 'l‘hese results show that in order to determine the fats of hays and excrernents by extractions with ether, with anything more than the merest approximation of the truth, there must be some method incor- porated in the analytical p-rocess to eliminate the unsaponified matter. Likewise, the determination of fats in concentrates by extraction With ‘ether would have a more positive value it the unsaponifialale were re- moved in the process of determination. TABLE 2. lfnsaponified Matter in Some Feed Stuyts and Exe1'e112ents b]; VrJirioizs Methods. +5 >1; é; +5.” =-- °°@ 3o’? Unsa onified matter “g +L o . E 565E in Either extract 5% §§ ‘Labora- 4 {g5 V? (Precipitation) I5 1'3. 3 tory ' Ether m E _ g m =.. i5 No. extract. g9. g In per cent- In per cent g5 c6 5. Q . g t‘; 8.9. of sample. of ether g 5'8 q E ,':>*’*’*° extract. ;3*“m"‘ m 12996 Wheat shorts (bran) No. 1 . . . . . . . . . . . . . . . . . . . 3.79 0.23 0.23 6.1 ' 0.24 12999 Corn chops . . . . . . . . . . . . . . . . . . . . . . . . . .. 4.31 0.25 0.16 3.7 0.18 13021 Cottonseed meal . . . . . . . . . . . . . . . . . . . . . . . . . . . 15.23 0.28 0.47 3.1 0.31 13023 Rice bran‘ . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 7.75 0.18 0.60 7.7 0.65 13030 Milo maize chops . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.22 0.13 0.21 6.5 0.21 13045 Cold pressed cottonseed . . . . . . . . . . . . . . . . . . . . . 7.26 0.16 0.29 4.0 0,23 13091 Kafir chops . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 3.20 0.56 0.26 8.1 0.26 13140 Corn bran . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 8.59 . . . . . . . . .. 0.29 3.4 0.34 13150 Red rice . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 1.64 0.14 0.15 9.2 0.21 13166 Wheat bran . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 4.10 0.18 0.25 6.1 0.25 13172 Wheat shorts . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 2.65 0.07 0.17 6.4 0.23 13190 Rice polish . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 10.38 0.19 0.51 4.9 0.70 ' 6288 Tabosa grass . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.92 0.33 0.56 60.9 0.44 6290 Excrement from tabosa grass. . . . .- . . . . . . . . . . . 1.06 0.50 0.70 66.0 0.75 $24 graiirie hay . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 63 u an grass . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6 . . . . 7799 Excrement from prairie hay . . . . . . . . . . . . . . . . . . 2.83 1.78 1.68 59.4 1.74 7970 Excrement from Sudan grass . . . . . . . . . . . . . . . . . 1.90 1.32 1.41 74.2 1 .50 7980 Sudan straw . . . . . . . . . . . . . . . . . . . . . . . . . . ..’... 1.44 0.43 0.55 38.2 0.65 7991 Sorghum hay . . . . . . . . . . . . . . . . . . . . . . . . . . . . .._ 1.82 0.58 0.49 26.9 0.55 7999 Excrement from Sudan straw . . . . . . . . . . . . . . . . 1.71 1.18 1.09 63 .7 1.29 F8002 Moth bean hay . . . . . . . . . . . . . .. . . . . . . . . . . . . . . 1.55 0.73 0.66 42.6 1.03 ‘8011 Excrement from sorghum hay . . . . . . . . . . . . . . . . 1.78 0.80 1.08 60.7 1.24 8013 Excrement from moth bean hay . . . . . . . . . . . . . . 3.03 1.76 1.99 65.7 2.20 Average for concentrates . . . . . . . . . . . . . . . . 6 .01 0.22 0 .30 5.8 0 .32 Average for hays and excrements . . . . . . . . . 1.82 0.92 0.97 52.7 1.06 Comparison of the Digestion and Precipitation Processes for the Separation 0t the D"1i»sapon=ified itlatter: The determination of the ‘unsaponified matter in the ether extract of the concentrates by the Precipitation Method and by the Digestion Method gave results which 2O TEXAS AGRICULTURAL EXPERIMENT STATION. were, in most cases, in fair agreement. The averages of the determin- ations on the concentrates were 0.30 per cent by the Precipitation Method and 0.22 per cent by the Digestion Method. The results tend to be lower by the Digestion Method than by the Precipitation Method. In a few cases the differences were very great. We believe that the low results are due to incomplete extraction of ‘the unsaponified. The Digestion Method, which was devised for roughages, proved very tedi- ous on the concentrates on account of the formation of emulsions. In one case it was necessary to include the emulsion in the unsaponifiable and to extract the latter again the same number of times, after evap- oration of the ether and dilution with water, in order to separate it with reasonable completeness from the soap solution. In the case ‘of No. 13140, Corn Bran, the determination was not completed because of the formation of a very persistent emulsion. No difficulty was experienced with emulsion in determinations by the Precipitation Method, and We believe it is a much more convenient, rapid and accurate process for the determination of unsaponifiable mat- ter in fats and oils than has heretofore been proposed. With the hays and excrements, the Precipitation Method gave, in some cases, higher results than the Digestion Method. The averages are, however, 0.97 per cent. for ‘the former and 0.92 per cent. forthe latter. We have shown that these higher results are not due to incom- plete saponification, or to soap in the unsaponified, and it appears that even the vigorous treatment of the soap solution to remove unsaponified matter as directed in the Digestion Method was not entirely success- ful. So far as We know, the Digestion Method is themost vigorous treatment ‘to remove unsaponifiable matter from the soap solution ever reported. It seems much more desirable, both from a standpoint of speed and of accuracy, to remove the fatty acids from theunsap-oni- fiable, by the precipitation process, than vice versa. We believe that ‘the Precipitation Method described in this Bulletin is sufficiently accurate, speedy, and simple to allow it to be used as an t analytical as well as a research method. THE SAPONIFIED RESIDUE. The results of the determinations of saponified residue in the ether extract and by the alcoholic soda. method are sho-wn in Table 3, together with the percentage of ether extract in the samples for comparison. FATTY AcIns AND ETHER-SoLUBLE CONSTIITUENTS or FEEDSTUFFS. 21 TABLE 3. Percentage 0f Saponified Residue in Feed Stu/ts and Excre-ment by Var/ious Jllethods. Saponified residue in Saponified ether extract. residue by Labora- Ether alcoholic tory extract. Precipitation Digestion soda N0. method. method. method. 12996 Wheat shorts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12999 Corn chops . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13021 Cottonseed meal . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13023 Rice bran . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13030 Milo maize chops . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13045 Cold pressed cottonseed . . . . . . . . . . . . . . . . . . . . . . . 13091 Kafir chops . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13140 Corn bran . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13150 Red rice . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13166 Wheat bran . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13172 Wheat shorts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13190 Rice polish . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ' ' ' ' ' ' ' ' into-ob ooo: oacnrowwkiiobokw 8$$§omg$g$o>oo>w ooowsre-cooovnoowooi-zo . . . . . . . . . i“ maiq $00 mwwwml-oooawinww CIII-ll-J K105 Id oaoi-qococam-ovwiawoo U1l.\')|-P~Ul@©ri>r-Pl\DO0|-P.-Q a >= a extract. E’ ‘Kg g +3 f2’ “Qg L; r E *3 e as Q’ 3 *3 s es S 8 =3 S 8 e *3 0+5 '5 T“ i; ‘£6 >. '5 F f; 2% >. °° (D ‘gem: ~ °’ q» gap E. .5 m .5 E m Period N0. 24, feed tobosa grass. Sheep No. 4, feed N0. 6288. Fed 4000 gm . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 36.8 11.0 24.4 . . . . . . .. 7.2 62.8 . . . . . . .. Residue 20 gin, N0. 6288 . . . . . . . . . . . . . . . . . . . .. 0.2 0.1 1.0 . . . . . . .. 0.0 0.3 . . . . . . .. Eaten . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 36.6 11.9 24.3 12.4 7.2 62.5 55.3 Excreted 2049 gm, No. 6290 . . . . . . . . . . . . . . . . .. 21.7 5.1 18.4 13.3 5.5 37.9 34.4 Digested . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 14.9 6.8 5.9 —0.9 3.7 24.6 20.9 Percentage digested . . . . . . . . . . . . . . . . . . . . . . . . . 40.7 57 .1 24.3 0.0 51.4 39.4 37.8 Period N0. 38, feed prairie hay. Sheep No. 3, food No. 7724. . . Fed 4000 gm . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 92.0 26.0 44.4 . . . . . . .. 17.6 80.0 . . . . . . .. Residue 97 gm, No. 7724 . . . . . . . . . . . . . . . . . . . .. 2.3 0.6 1.1 . . . . . . .. 0.2 1.9 . . . . . . ._ Eaten . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 89.7 25.4 43.3 17.9 17.4 78.1 60.7 Excreted 2127 gm, N0. 7799 . . . . . . . . . . . . . . . . .. 60.2 19.2 31.7 12.5 7.7 64.2 56.5 Digested . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 29.5 6.2 11.6 5.4 9.7 13.9 14.2 Percentage digested . . . . . . . . . . . . . . . . . . . . . . . . . 32.9 24.4 26.8 30.2 55.7 .17.8 6.9 Period No. 39, feed Sudan grass. Sheep No. 1, food No. 7763. Fed 4000 gm . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 58.4 26.2 41.2 . . . . . . .. 9.2 68.4 . . . . . . .. Residue25 gm, N0. 7763 . . . . . . . . . . . . . . . . . . . .. 0.4 0.2 0.3 . . . . . . .. 0.1 3.8 . . . . . . .. Eaten . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 58.0 26.0 40.9 14.9 9.1 64.6 55.5 _ Excreted 1662 gm, N0. 7970 . . . . . . . . . . . . . . . . 31.6 4.2 26.8 22.6 5.7 43.6 37.9 Digested . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 26.4 21.8 14.1 —7.7 3.4 21.0 ‘ 17.6 Percentage Digested . . . . . . . . . . . . . . . . . . . . . . . . . 45.5 83.8 34.5 0.0 37.4 31.1 31.7 Period No. 42, feed Sudan grass. Sheep No. 1. feed N0. 7980. . Fed 4000 gm . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 57.6 26.8 - 51.6 . . . . . . .. 6.4 70.0 . . . . . . .. Residue 122 gm, No. 7980 . . . . . . . . . . . . . . . . . . .. 1.8 0.8 0.2 0.2 2.1 . . . . . . .. Eaten . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 55.8 26.0 51.4 20.4 6.2 67.9 61.7 Excreted 2119 gm, N0. 7999 . . . . . . . . . . . . . . . . .. 36.2 8.3 34.8 26.5 4.2 51.7 47.5 Digested . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 19.6 17.7 16.6 —1.1 2.0 16.2 14.2 Percentage digested . . . . . . . . . . . . . . . . . . . . . . . . . 35.1 68.1 32.3 0.0 32.3 23 .9 23.0 Period No. 43, feed sorghum hay. Sheep No. , food N0. 7991. Fed4000 gm . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 72.8 35.6 46.8 . . . . . . .. 11.2 64.0 . . . . . . .. Residue 133 gm, N0. 7991 . . . . . . . . . . . . . . . . . . .. 2.4 1.2 1.6 - . . . . . .. 0.4 2.1 . . . . . . .. Eaten . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 70.4 34.4 45.2 10.8 10.8 61.9 51.1 Excreted 1600 gm, N0. 8011 . . . . . . . . . . . . . . . . .. 28.5 9.9 27.4 17.5 4.0 _ 45.9 41.9 Digested . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 41.9 24.5 17.8 -6.7 6.8 16.0 9.2 Percentage digested . . . . . . . . . . . . . . . . . . . . . . . . . 59.5 71.2 39.4 0,0 63.0 25.8 18.0 Period No. 44, feed moth bean hay. Sheep No. 1, food No. 8002. Fed 3000 gm . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 46.5 18.6 51.0 - - . . . . .. 8.2 37.5 . . . . . . .. Residue 154 gm, No. 8002.2. . . . . . . . . . . . . . . . .. 2.4 1.0 2.6 - . . . . . .. 0.4 1.9 . . . . . . .. Eaten . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 44.1 17.6 48.4 30.8 7.8 35.6 27.8 Excreted 1174 gm, N0. 8013 . . . . . . . . . . . . . . . . .. 35.6 - 7.3 26.8 19.5 5.4 29.7 24.3 igested . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 8.5 10.3 21.6 11.3 2.4 5.9 3.5 Percentage digested . . . . . . . . . . . . . . . . . . . . . . . _. . 19.3 58.5 44.8 . . . . . . . . 30.8 16.6 12.6 30 TEXAS Aenrourrtnar EXPERIMENT STATION. SUMMARY AND CONCLUSIONS. 1. An improved method for the determination of the total fatty acids and other constituents of ether extracts has been developed. 2. A new method has been developed for extracting the total fatty acids and other ether-soluble constituents of feeding-stuffs and excre- ments, which, by means of alcoholic soda, removed more fatty acids from feeding-stuffs than did successive extractions with ether, chloro- form and alcohol. 3. Ether extracts of the concentrates contained saponifiable mate- rial which does not appear to be fatty acids, averaging about 8 per cent. and unsaponifiable matter averaging about 6 per cent., a total of approximately 14 per cent. of non-fats in the ether extract of concen- trates. Ether extracts of hays and excrements from them contain saponified material which does not appear to be fatty acids, averaging about 15 per cent. of the ether extract. Together with the unsaponi- fiable matter, they made a total of approximately 68 per cent. of non- fats in the ether extract of roughages. 4. Molecular weight determinations and other evidence indicate that the ether soluble, petroleum-ether soluble acids in the alcoholic soda extracts of feed stuffs are probably fatty acids. 5. The digestibility of the various ether-soluble fractions was deter- mined in six hays with sheep. The fatty acids are digested on an average of 60.5 per cent. in the ether extract; the fatty acids in the alcoholic soda extract were digested 33.7 per cent. 6. The digestibility of the fatty acids extracted by alcoholic soda but not by ether had an average digestibility of 11.2 per cent. The diges- tibility in four cases was zero. '7. The saponified residue of the ether extract were digested, on an average, 45.1 per cent., and in the alcoholic soda. extract 25.8 per cent. 8. The nitrogen-free extract of feed stuffs contains considerable material soluble in ether, which can be extracted by alcoholic soda. This ether-soluble matter consists of unsaponfiable matter, fatty acids, and, principally, of non-fat organic acids, in the case of hays and excre- ments from them. It made up from 2.72 to 12.39 per cent. of the _ nitrogen-free extract of those samples, and averaged 5.97‘ per cent. In the concentrates it make up from zero to 3.84 per cent. of the nitrogen~ free extract, and averaged 1.49 per cent. of the nitrogen-free extract.