218
T H E JOURiVriL O F I N D U S T R I A L A N D E N G I N E E R I N G C H E M I S T R Y
with dilute hydrochloric acid, washed again with water, transferred t o a weighed dish, evaporated, dried a t 1 1 0 ’ C. a n d weighed. This residue consists chiefly of unsaponifiable matter, b u t contains f a t t y acids as t h e ether has dissolved some soap which is afterwards decomposed by the hydrochloric acid. It is therefore taken up Kith neutral alcohol and titrated with standard alkali solution. T h e f a t t y acids thus found, calculated as oleic acid, are deducted from the weight before obtained. A third treatment with ether will extract so little more t h a t for ordinary analytical work i t may be omitted. If t h e f a t t y acids obtained from t h e grease, instead of t h e grease itself, have been used, t h e result must of course be calculated on t h e original grease. This method differs from t h a t of Allen and Thomson, given by Lemkowitschl as his preferred method, only in some details: chiefly t h e simple and accurate way of correcting for soap dissolved in t h e ether. Having this correction, i t is not necessary to be sparing in t h e use of alcohol for fear of causing t h e ether t o dissolve soap, b u t enough may be used t o cause a n almost instantaneous separation in the funnel of perfectly clear and sharply marked ethereal and aqueous layers. A determination of unsaponifiable matter can be easily completed in I I / ~hours. Ether has been found t o be t h e best general solvent fpr a variety of unsaponifiable matters found in fats and t h e objection t h a t i t dissolves some soap disappears when using this method. T h e uncertainty as t o t h e molecular weights of t h e f a t t y acids found by titration in t h e residue would lead t o error, but when t h e small percentage of these f a t t y acids calculated on t h e original sample of grease is considered i t will be seen t h a t this error is entirely negligible, and t h e f a t t y acids may be calculated as oleic acid, or, a n average molecular weight, as t h a t of tallow f a t t y acids, may be taken. The above method of determining unsaponifiable matter, when compared with other methods, has never given lower results. In some cases t h e results are higher, especially t h a n those obtained where petroleum ether is used as t h e solvent. T h a t the results are not too high has been repeatedly proven by a careful examination of t h e extract, particularly for neutral fat a n d for ethyl esters which might possibly have been formed from a little alcohol remaining in t h e ether when it is shaken with hydrochloric acid. The details of this method were worked out by Mr. C. Foerster, who has used i t in this laboratory, not only in t h e daily analysis of greases, but also with good results in extracting fats containing other t h a n t h e usual kinds of unsaponifiable matter. WYOMING, OHIO -_ _ _ ~ _ _ A NEW METHOD FOR THE DETERMINATION OF THE TOTAL FATTY ACIDS AND OTHER ETHER-SOLUBLE CONSTITUENTS OF FEEDSTUFFS By J B. RAT HER^ Received October 27, 1914
Determination of fats by direct saponification of 1 Lewkowitsch, “Chem. Technology and Anal of Oils, F a t s and Waxes.” 5 t h Ed. Vol I, p 458 Abstracted * U n d e r t h e general direction of G S. Fraps, Chemist by t h e author from Texas Exper. S t a , Bull 169.
VO\. 7 $ KO. 3
t h e sample has been proposed by Liebermann a n d Szekelyl and by Kumagawa a n d Suto.2 These methods are open t o several objections, chief among which are t h a t they make no adequate provision for t h e removal of non-fatty material from t h e extracts, a n d t h a t t h e methods, giving results comparable with ether extracts of t h e same samples, yield products not only impure b u t incompletely extracted. While i t has long been known t h a t ether does not extract all of t h e ether-soluble constituents of plant a n d animal products, t h e extraction is generally considered t o be complete enough for practical purposes. Dormeyer3 states t h a t ether extraction of meat i s far from complete a t t h e end of I O O hours. Browned states t h a t pepsin digestion of steer feces renders a larger amount soluble in ether t h a n can be obtained from untreated samples. Fraps and Rather5 show t h a t chloroform extracts considerable ether-soluble matter from hays which have previously been extracted with ether, and t h a t this extract contains f a t t y acids. We have been unable t o find any mention in t h e literature of t h e use of alcoholic soda or potash as a solvent for fats in plant or animal products. EXPERIME KTAL
A method was developed for t h e determination of total f a t t y acids and other ether-soluble constituents of feedstuffs. The experimental evidence on which t h e method is based may be found elsewhere.6 T h e following factors were considered in t h e development of t h e method (alcoholic soda method) : 1-Completeness of saponification; a-unsaponified material in t h e f a t t y acids; 3-fatty acids in t h e unsaponified material; 4-completeness of t h e extraction of t h e f a t t y acids b y petroleum ether; 5-completeness of extraction of the saponified residue; 6-saponified residue in f a t t y acids; ;-saponified residue in t h e unsaponified material; &--washing required t o remove hydrochloric acid after liberating acids in unsaponified material; 9-the checking of duplicate determinations run a t different times; 1-completeness of direct saponification; and 11-completeness of t h e extraction of t h e ether-soluble matter from t h e alcoholic soda extract. The following determinations were made on twelve samples of hays a n d excrements from them, and on twelve samples of concentrated feeding-stuffs: ether extract by t h e Official hlethod;’ constituents of t h e ether extract by t h e Precipitation a n d ethersoluble constituents by t h e Alcoholic Soda Method given below. The Precipitation Method is t h e same as t h e Alcoholic Soda Method with t h e principal exception of t h e method of extracting t h e ether-soluble matter from t h e sample. The 9lcoholic Soda Method, while very rapid, requires a knowledge of its technique for satisfactory results, so i t is given below in detail. 1
2
3 4 5
8 7
Pfiiigev’s Arch., 72 (1898), 360-366. Biochem. Zeit., 8, 212. Pfiiiger’s Arch., 61, 341-43. Proc. 20th Coss. A . 0. A . C. Texas Station, Bull. 162.
LOG.cit.
U.S. D. A,, Bureau Chem.. Bull. 107 (revised). Texas Experiment Station, Bull. 169.
Mar., 1915
T H E J O U R N A L OF I N D U S T R I A L A N D E N G I N E E R I N G C H E M I S T R Y
ETHER-SOLUBLE MATERIAL-Digest g. of t h e sample, or j g., if i t contains more t h a n 4 per cent ether extract, or is bulky, with 5 0 cc. of approximately 2 N alcoholic soda, for one hour under a reflux condenser. Filter with suction through asbestos in a carbon funnel of suitable size and wash I O times with boiling alcohol (redistilled). Transfer t o a dish with water and evaporate t o about I O cc. Transfer with hot water t o a pear-shaped separatory funnel. Acidify with I O cc. acetic acid, and extract the warm solution j times with jo cc. portions of ethyl ether (redistilled). D E T E R M I N A T I O N O F U N S A P O N I F I E D l\IATTER1-TranSfer the ether extract t o a 500 cc. separatory funnel, wash once with j o cc. water and remove t h e bulk of t h e suspended matter in t h e manner described under Determination of F a t t y Acids below. Add t o t h e etheral solution I O cc. of a warm I : 2 aqueous sodium hydroxide solution, t u r n t h e funnel on its side and shake gently. Allow t h e precipitate t o settle, a d d 2 5 cc. warm water, hold funnel in vertical position a n d give rotary motion. Allow t h e two layers t o separate, a n d draw off t h e clear aqueous solution into a joo cc. Erlenmeyer flask, leaving any emulsion in t h e funnel. Repeat and then shake gently as above with 5 successive 30 cc. portions of cold water, allowing a short time for t h e two solutions t o separate, and add t h e washings t o t h e soap solution in t h e flask. Transfer t h e ethereal solution t o a tared 2 0 0 cc. flask, evaporate or distil off t h e ether, a n d dry t o constant weight in a steam oven a t 100’ C. E X T R A C T I O N OF
IO
CORRECTION
FOR
FATTY
ACIDS
IN
UNSAPONIFIED
(For material high in f a t t y acids a n d low in unsaponified only)-Add 2 0 cc. of 0 . 2 N hydrochloric acid t o t h e ethereal solution of t h e unsaponified matter in t h e separatory funnel before evaporation, stopper a n d shake vigorously. Draw off the aqueous layer a n d discard. Evaporate t h e ethereal solution as above, dry a n d weigh. Heat t o boiling with 2 0 cc. alcohol, titrate with 0 . I N sqdium hydroxide a n d phenolphthaiein, running a blank on t h e alcohol. Multiply the corrected reading by 0 . 2 8 (or 0.56 if 5 g. sample is taken). The result is percentage f a t t y acids in t h e unsaponified matter. Subtract this figure from the percentage of unsaponified matter a n d a d d i t t o t h e f a t t y acids (below). The amount of f a t t y acids dissolved in t h e ethereal solution of t h e unsaponified matter is. on a n average, 16 mg. for substances very low in unsaponified matter, and this figure may be used if very accurate results are not desired. No correction for f a t t y acids in unsaponified matter is necessary for products containing 2 j or more per cent unsaponified matter. D E T E R M I N A T I O N O F THE F A T T Y ACIDS-Heat the soap solutlon on a steam bath t o remove dissolved ether, shaking gently from time t o time. This must be done carefully t o avoid frothing of t h e soap. Cool the soap solution nearly t o room temperature under the t a p , add 8 cc. of glacial acetic acid or its equivalent in weaker acetic acid, a n d extract in t h e separatory funnel with 4 0 cc. of redistilled petroleum ether, disMATTER
1
“Precipitation Method,’’ THISJOURNAL, 7, 34.
219
tilling below 7 5 ’ C., shaking violently. Draw off aqueous layer, hold funnel in a vertical position a n d give i t a rotary motion and let stand a minute. This will cause the suspended matter t o settle in a compact mass a t the bottom of t h e funnel. Draw off this portion into the flask containing t h e aqueous mixture. Extract the aqueous mixture 3 times more in a similar manner. By using a pear-shaped funnel and following* the above method, filtration t o remove suspended matter may generally be avoided. Shake well with two j o cc. portions of water t o remove suspended matter a n d traces of inorganic substances, allowing any emulsion t o go into aqueous mixture. Extract the latter a fifth time with petroleum ether. a n d after washing this extract twice with small portions of water, add to other extracts. Evaporate and dry t o constant weight. If t h e evaporation is carried t o completeness on a steam bath, t h e flask being turned on its side t o facilitate removal of gasoline fumes, t h e product m a y be dried t o constant weight in 3 or 4 hrs. This fraction is f a t t y acids. D E T E R M I N A T I O N O F S A P O N I F I E D RESIDUE-Acidify further t h e aqueous residue from t h e extraction of the f a t t y acids, with hydrochloric acid, warm a n d extract 5 times as above with 40 cc. portions of ethyl ether. Wash t h e combined extracts twice with 5 0 cc. portions of water, discard any suspended matter, evaporate and dry t o constant weight. The results are shown in t h e following table. PERCENTAGE TOTAL FATTY ACIDS AND OTHER ETHER-SOLUBLE CONSTITUENTS OF SOME FEEDSAND EXCREMENTS UNSAPON- TOTAL FATTY SAPONIRIED IBIED IN ACIDS IN RESIDUEIN Alc. Alc. Alc. Ether Ether soda Ether soda Ether soda No. extr. extr. extr. extr. extr. extr. extr. 1 Wheat shorts . . ... . . 3.79 0.23 0.24 3.08 4.43 0.39 0.71 2 Corn chops ..,... . . . . . 4.31 0.16 0.18 3.77 4.34 0.44 0.27 3 Cottonseed meal.. . . .. . 15.23 0.47 0.31 13.82 14.46 0.18 0.38 4 Rice bran.. . . ... . . . . . . 7.75 0.60 0.65 6.21 8.10 0.72 0.91 5 Milo maize choos...... , , 3.22 0.21 0.21 2.05 2.92 4. n 19 .- 0 .6. ... 6 Cold pressed coiton seed. 7.26 0.29 0.23 6.49 7.32 0.24 0 26 3.20 0.26 0.26 2.57 7 Kafir chops.. . , . . . . , . 3.18 0.60 0.24 8 Corn bran,. . . . . . . 8.59 0.29 0.34 5.50 8.13 1.70 0.59 9 Red rice.. . , . . . . . . 1.64 0.15 0.21 1.04 1.81 0.25 0.34 10 Wheat bran.. . . . . . . . . . . 4.10 0.25 0.25 3.50 4.41 0.14 1.01 11 Wheat shorts. . , . , . , , 2.65 0.17 0.23 2.22 2.84 0.12 0.31 10.29 0.55 0.50 12 Rice uolish.. . . . . . . . . . , 10.38 0.51 0.70 8.53 13 Toboia grass . . .. . . . . . . . 0.92 0 5 6 0.44 0.30 0.61 0.18 1.57 14 Excrement from No, 13... 1.06 0.70 0.75 0.25 0. 90 17 1 .R.5 .. 0 .. . . . 15 Prairie hav . . . . . , . . . . . . . 2.30 0.84 0.78 1.11 0.44 2.00
... .
... .
. . . .. . .. . ... .. . ... .
16
i7
18 19 20 21 22 23 24
. .. . . . .. No. 15... No. 16... Sorghum h a y . . . . . , , . . . . Excrement from No. 19.. . Moth bean h a y . . . . . . . . . Excrement from No. 20... Excrement from No. 22 ...
Average for concentrates .... Average hays and excrements
1.46 2.83 1.90 1.44 1.82 1.71 1.55 1.78 3.03 6.01 1.82
0.55 168 1.41 0.55 0.49 1.09 0.66 1.08 1.99 030 0 97
0.49 1.74 1.50 0.65 0.55 1.29 1.03 1.24 2.20 0.32 1.06
0.62 4.90 0.57
1.03 1.49 1.61 1.29 1.17 1.64 1.70 1.71 2.28 6.02 1.38
0.23 0.36 0.34 0.16 0.29 0.20 0.27 0.25 -. 0.46 0.50 0.28
1.71 3.02 2.62 1.75 1.60 2.44 1.25 2.87 2.53 0.48 2.10
T H E T O T A L F A T T Y ACIDS-This heading covers all f a t t y acids, whether free or combined, t h a t may be saponified, except volatile f a t t y acids, which are probably not present in appreciable amount. The f a t t y acids in t h e concentrates make up from 64 to 8 4 per cent of t h e ether extract. The f a t t y acids in t h e alcoholic soda extracts of these samples averaged 19 per cent higher t h a n those of t h e ether extract, or about t h e same as t h e total ether extract of t h e concentrates. The f a t t y acids in t h e ether extract of t h e hays a n d excrements make u p from 1 3 t o 49 per cent of t h e ether extract, with a n average of 31 per cent. The results by t h e Alcoholic Soda Method are higher in
a20
T H E J O U R N A L OF I N D U S T R I A L A N D ENGINEERING CHEMISTRY
every case, averaging 250 per cent as much as b y t h e Et her Extraction M et h o d . T H E U K S A P O N I F I E D MATTER-The unsaponified m a t t e r in t h e ether-soluble portion of t h e alcoholic soda extracts of t h e concentrates varied from 0 . 2 1 per cent in red rice t o 0.65 per cent in rice bran, and averaged 0 . 3 2 per cent. T h e unsaponified matter in the ether *>extract of t h e concentrates is essentially t h e same in amount as t h a t in the alcoholic soda extract, averaging 0.02 per cent lower t h a n t h e latter. The unsaponified matter in t h e alcoholic soda extracts of t h e hays and excrements from t h e m varied from 0.44 per cent in Tobosa grass t o 2 . 2 0 per cent in excrement from mothbean h a y and averaged 1.06 per cent. T h e unsaponified matter in t h e alcoholic soda extract averages 0.07 per cent (in per cent of sample) higher t h a n t h e ' corresponding fraction of t h e ether extract. THE S A P O N I F I E D RESIDUE-under this head we include all ether-soluble, saponifiable material difficultly soluble in petroleum ether. Practically all of t h e chlorophyll, when present, is found in this fraction. The saponified residue in t h e alcoholic soda extract of t h e concentrates varied from 0.19 per cent in milo maize chops t o 1.01 per cent in wheat bran, and average 0.48 per cent. The similar fraction in t h e ether extract varies considerably, b u t averages t h e same. T h e saponified residue in t h e alcoholic soda extract of hays and excrements varied from 1.25 per cent t o 3 . 0 2 per cent, and averaged 2 . 1 0 per cent. This is over twice as much as t h e average for the ether extracts of t h e same samples. The saponified residue of t h e ether extract of t h e hays and excrements was, in every case, much higher, averaging 0.28 per cent, less t h a n 15 per cent of t h e average for t h e saponified residue of t h e alcoholic soda extract. T h e nature of the f a t t y acids not extracted b y ether, and t h e reasons f o r considering t h a t t h e fraction, saponified residue, contains no f a t t y acids are discussed in another place.' It may be stated here, however, t h a t these f a t t y acids have mean molecular \?.eights of about t h e same magnitude as several of t h e most common f a t t y acids. The presented here shou' that On an average 14 per cent of t h e ether extract of these concentrates a n d 68 per cent of t h e ether extract of hays, and excrements from them, consist of non-fats. If anything more t h a n a n approximation of t h e f a t content of a h a y is desired, some method must be resorted t o which will remove unsaponifiable matter and saponifiable non-fats soluble in ether. We believe t h a t t h e Precipitation Method described elsewherel is rapid, accurate a n d simple enough t o be used as a routine as well as a research method for this purpose. The results show t h a t ether is a t best a very imperfect medium for t h e extraction of t h e total f a t t y acids of feedstuffs, and t h a t alcoholic soda gives much higher results in most cases. S C 3IMA R Y
A method for t h e determination and fractionation of t h e total f a t t y acids and other ether-soluble con8
Texas Experiment Station, Bull. 169.
Vol. 7 , S o . 3
stituents of plant products is proposed which gives much higher results t h a n does extraction with ether. Total f a t t y acids b y this process were one-fifth higher in t h e concentrates and more t h a n twice as high in the hays and excrements, t h a n b y extraction with ether. The ether extract of these concentrated feedingstuffs contained on an average 14 per cent non-fats, a n d t h a t of the hays and excrements 68 per cent non-fats. E t h e r extraction is a very imperfect method of determining fats or of total f a t t y acids in plant products. TEXAS EXPERIMENT STATION COLLEGESTATION,TEXAS
A CHEMICAL STUDY OF TWO DROUGHT-RESISTING
FORAGE PLANTS B y S LOMANITZ Received November 25, 1914
The two plants, t h e analyses of which are givcn below, have been introduced into Mexico for experimental purposes by Dr. Mario Calvino, now Chief of t h e Central Agricultural Experiment. Station of Mexico. The first, commonly known as "Rhodes Grass" (Chloris virgata or Ch. A b y s s i n i c a or Ch. G a y a n a ) , has been grown with great success in t h e dry arid regions of Australia, Costa Rica and Mexico. T h e second, Y h a l a r i s nodosa or Ph. bulbosa, called erroneously Ph. cornrnutata, has been grown in I'taly, Algeria and Mexico. Freezing has very slight effect on t h e former b u t none on the latter. Both are practically unaffected b y drought and are excellent forage plants since cattle eat them very readily and they exert no harmful effects upon t h e milk. The plants grow well under a wide range of conditions and give very good vields. Per cent Moisture (at 1000 c.), , . , . . . . . . Crude protein ( N X 6.25). , . . . ,
,...,....
RHODES GRASS Air- Dried a t dry 100OC. Green 8.30 0 . 0 68.83 6.87 7.49 2 . 3 3 1.64 1.79 0 . 5 6 11.39 12.42 3.87 28.42 30.99 9 . 6 6 43.38 47.31 14.75
PHALARIS NODOSA Air- Dried at dry l00'C. Green 9 . i s 0 . 0 77.84 1 0 . 5 4 11.60 2 . 5 7 2.66 2.93 0.65 12.77 1 4 . 0 5 3 . 1 1 25.82 28.42 6 . 3 0 39.06 4 3 . 0 0 9 . 5 3
1.70
7.81
8.59
pepsin method) . . . . . . . . . . . . . 3 . 2 5 3 . 5 4 Amide etc. n i t r o g e n . , . , , , , , , , 0 . 3 0 0 . 3 3 R ~ : ~ ~ , , $ j g a r s , , ( * l l t h n ' s ,, ,,
1.10 0.10 0,46
6.25 0.44
6.88 1.52 0 . 4 8 0,11 0,41
Sucrose (Clerget'smethod) . . . . . Soluble starch a n d d e x t r i n . , . . . .
0.44 0.26
2.02 1 19
2.22 0 . 4 9 1.31 0.29
1.31 0.76
1.43 0.83
1.90
T h e analyses given above were made of plants furnished b y Dr. Calvino, and grown in t h e experimental plots of the Division of Horticulture of this Experiment Station. T h e y were of about a meter length, the secds being already formed b u t not ripened. For t h e analyses the whole plant, stalk and ear, were taken. From the above analyses i t can be seen t h a t both these grasses have a comparatively high nutritive value, Especially is this t h e case with t h e Phalaris. The proportion of t h e digestible protein is especially high in this last-named plant, The proportion of ash is rather high b u t this seems t o be a characteristic of quite a number of grasses grown here. NATIONAL AGRICULTURAL EXPERIMENT STATION TACUBA, D. F., MEXICO
,
