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 C H E M I S T R Y
43 6
with no signs of deterioration, either in flavor, odor, or appearance, and have been able to cultivate both aerobes and anaerobes from them after t h a t time. Under-processing in favor of sealing as a preserving agent should not be advocated, but we feel sure t h a t many products have been placed on the market which were under-processed and have not spoiled. We hope t o determine this point later. Table XI1 shows a comparison between numbers of bacteria destroyed b y sterilization and the keeping quality. This table is a compilation from Tables I X and XI. The two tables were not made on the same date or from the same lots of corn. We have, however, made several tests of this sort with similar results. Unfortunately no counts were obtained from sealed tubes, but they probably would differ b u t very little from those sealed with cotton. TABLE XI1 1.0 Per cent Salt 38 8 2 44 19 6 2
Heated Water 60 min. 1 d a y . . 34 120 min. 1 day 8 180 min. 1 d a y . , , , 4 15 min. twice 1 day 50 30 min. twice 1 day 20 60 min. twice 1 dav.. 8 90 min. twice 1 da$ 4
...... ........ . . ... .. ....
... 60 min. 1 d a y . , ...... + 120 min. 1 d a y . . ...... +
+ + Sealed with Rubber + + + -
-
180 min. 1 d a y . , . . . . . . 15 min. twice 1 day.. . 30 min. twice 1 day.. . 60 min. twice 1 day ... 90 min. twice 1 day ... 60 min. 120 min. 180 min. 15 min. 30 min. 60 min. 90 min.
Number of Bacteria 0.05 2.0 Per Per cent cent Salt Acid 27 32 4 6 1 3 32 32 10 14 2 2 1 0 Sealed with Cotton
........ + ......... ... .-
1 day 1 day.. ...... 1 day., twice 1 day twice 1 day twice 1 day.. . twice 1 day..
++ + --
per Cc. 0.1 Per cent Acid 24 3 1 28 10 1 0
--
-
-
0.3 Per cent Acid 18 1 0 21 8 0 0
-
-
These results show t h a t in several cases spoilage did not occur even when there were, no doubt, a few organisms present. This is shown particularly in the case in which acid is used. The influence of sealing is also very marked. Even in cases in which considerable spoilage occurred in cotton-sealed tubes no spoilage appeared in tubes properly sealed with rubber. EXPERIMENT 13-This experiment was devised t o show influence of rubber alone upon the keeping quality. i
TABLE XI11
0.2 0 . 0 5 0'.1 2.0 0.5 1.0 Per Per Per Per Per Per cent cent cent cent cent cent Water Salt Salt Salt Acid Acid Acid Heated Plugged with one-hole rubber stoppers, the hole being plugged with cotton 15 min. 1 day . . . . . . . . . . . . . . 4. 30min.lday.............. 460 min. 1 d a y . . . . . . . . . . . . . . Plugged with one-hole rubber stopper and glass rod 15 min. 1 d a y . . . . . . . . . . . . . . + 30 min. 1 d a y . . . . . . . . . . . . . . 60 min. 1 day.. . . . . . . . . . . . . Plugged with cotton only 15min.lday.............. 430 min. 1 d a y . . . . . . . . . . . . . . 60 min. 1 day . . . . . . . . . . . . . . f
+ _+ +
+ + + - _+ - - - -- -
-
-
_ - _ - _-- - ++ + + + + - -
Since we had obtained such marked results by sealing tubes with rubber as compared t o sealing with cotton it was considered possible t h a t the rubber stoppers might contain some substance toxic t o the bacterial cell and thus prevent growth. Beets were used in this case and were packed in large mouth bottles and treated as indicated in Table X I I I . The
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plus sign means spoilage. The minus sign means good after I O days. We may conclude from Experiment 13 t h a t i t was not the influence of the rubber alone t h a t could account for the preserving action in rubber-sealed containers. Table XI11 serves again t o emphasize the great value of sealing and the use of acid in the canning industry. CONCLUSIONS
I-Blanching is of no value in reducing the time necessary t o properly process canned foods. 2-Small amounts of salt are of little value in preventing the growth of bacteria in canned foods. 3-Small amounts of organic acid (acetic acid) have a distinctly retarding action upon the growth of bacteria in canned vegetables. The use of small amounts should be advocated in all cases in which it will not injure the texture, flavor, or appearance of the product. 4-In many cases an unsterile product will keep indefinitely if properly sealed. This, however, is not true in all cases and sealing should not be expected t o take the place of proper processirig because of the danger of loss due t o spore-forming anaerobes. DEPARTMENT OB BACTERIOLOGY KANSAS STATEAGRICULTURAL COLLEGE MANHATTAN, KANSAS
DETECTlON O F ADDED COLOR IN BUTTER OR OLEOMARGARINE By HERBERTA. LUBS Received December 12, 1917
I-OBSERVATIONS
ON
SOME
QUALITATIVE
TESTS
FOR
THE D E T E C T I O N O F A D D E D C O L O R S I X F A T S
A study of the various methods described for the detection of added colors in butter and butter substitutes reveals a number of misleading statements which might lead a more or less inexperienced analyst t o false conclusions. For example, certain tests described in the literature lead t o the false conclusion t h a t some aniline colors are vegetable colors, and vice versa. Furthermore, certain azo colors cannot be detected by methods which are supposed t o reveal their presence. Some of the tests described in the literature for the detection of added color are perfectly satisfactory when certain compounds are present, but fail t o reveal the presence of added color when other dyes are used, and for this reason some changes must be made in the procedure, These modifications will be discussed under the tests in question. The analyst should make a combination of tests with the modifications subsequently recommended t o obtain reliable results. L O W ' S TEST-According t o Low,' if a fat containing an azo color is shaken with a mixture of four parts of glacial acetic acid and one part of concentrated sulfuric acid the acid layer will settle o u t with a winered color. I t is quite true t h a t in some cases a winered color is obtained, but in other cases a yellow, brown, or even a blue color is imparted t o the acid layer in the presence of various azo colors. For 1
J. A m . Chem. Soc.,
20 (1898), 889.
June, 1 9 1 8
T H E JOURNAL OF INDUSTRIAL A N D ENGINEERING CHEMISTRY
instance, Yellow 0 B, Yellow A B,’Sudan I, Sudan 11, Butter Yellow, Aniline Yellow, and some other dyes will give reddish colorations to the acid layer, while with Sudan G and Aniline-azo-phenol a yellowish brown color is obtained. Sudan I11 imparts a bluish tinge to the acid layer. Although turmeric is not often used for t h e coloration of butter, its presence has been occasionally detected, and in this test will impart a brilliant violet-red coloration t o t h e acid layer. However, if turmeric is present i t will be detected upon extracting t h e fat with aqueous alkali and identifying by the usual methods.2 When the test is made as originally described by Low, t h e separation of t h e two layers is very slow and indistinct and, particularly in the case of oleomargarine, t h e color due t o the reaction of t h e fat practically obscures t h e color due t o t h e presence of a n azo dye. A decided improvement on the method of Low consists in first diluting the fat, about 2 0 g., with a n equal volume of petroleum ether, and shaking in a separatory funnel with I O cc. of t h e acid mixture, consisting of one volume of concentrated sulfuric acid and I O volumes of glacial acetic acid (99.5 per cent). I n this way a more rapid and clean-cut separation of t h e tm-o layers is effected and there is less decomposition of t h e constituents of t h e fat. Pure butter fat will impart no color t o the acid layer, except upon very long standing. Some specimens of oleomargarine after standing for a short time will impart a brownish coloration t o the acid layer, t h e intensity of which increases upon standing. If a n azo color be present t h e coloration of the acid layer is very distinct and appears immediately. DOOIJTTLE’S TEST3-A small portion of the f a t is dissolved in ether, t h e solution divided into two equal parts and one portion is shaken with dilute alkali and t h e other with dilute hydrochloric acid. If t h e aqueous alkaline layer is colored yellow, i t is stated t h a t a vegetable color is present; and if the acid layer is colored pink, the presence of a coal-tar color is assumed. Since this test was developed t h e list of coal-tar, oil-soluble colors has been considerably augmented a n d some of these colors will give reactions by t h e above method which might lead t o false conclusions. For instance, Aniline-azo-phenol and Sudan G will impart a yellow color t o the alkaline layer and furthermore, many oil-soluble azo colors do not give a pink coloration with dilute hydrochloric acid. I n order t o determine the limitations of this method solutions of t e n different oil-soluble azo colors in butter were prepared. Two imparted a pink color t o the acid layer when dilute acid was used; with the remainder the acid layer was colorless. When concentrated hydrochloric acid was used, eight imparted color t o the acid layer. Six of t h e eight gave a red coloration, and two, a yellow coloration. I n making Doolittle’s test i t is advisable t o use I O t o 20 g. of fat and in t h e acid extraction t o substitute concentrated for dilute hydrochloric acid. 1 These are t h e trade names for o-toluene-azo-&naphthylamine and For butter coloring a mixture of t h e two benzene azo-&naphthylamine. is usually used. 2 Allen’s “Commercial Organic Analysis,” 6 (1911), 415. 3 U. S. Dept of Agr., Bureau of Chemistry, Bull. 66 (1902), 152.
43 7
GEISSLER’S TEsT1-Geissler states t h a t if a few drops of clarified fat are mixed with a small amount of fuller’s earth, a pink t o red coloration will be produced in t h e Several azo colors will presence of various azo dyes. give this test quite satisfactorily, but the, majority will not and hence this test cannot be relied upon as a general method for the detection of azo colors. PROCEDURE RECOMMENDED FOR PRELIMINARY EXAMINAT I O N O B F A T .FOR T H E D E T E C T I O N O F ADDED COLOR
Dissolve about 20 g. of t h e fat in 5 0 cc. of petroleum ether, and 2 0 g. in jo cc. of ethyl ether. Shake out t h e ethyl ether solution in a small separatory funnel with dilute sodium hydroxide solution. If the aqueous solution is colored yellow a vegetable color is indicated and the alkaline extract must be tested for such colors. Add I O cc. of a mixture of one volume of sulfuric acid and I O volumes of glacial acetic acid t o t h e petroleum ether solution and shake vigorously. The acid layer will settle out in a few minutes with a decided coloration if a n azo dye is present. I n this test annatto, if present in sufficient concentration, will impart a momentary green color which changes over t o brown. Turmeric imparts a fairly permanent violet-red coloration similar to t h a t given by certain azo dyes, but its presence will be detected in the alkaline extract. The azo colors impart a yellow, brown, red or blue color t o the acid layer. A N IMPROVED METHOD F O R T H E DETECTION
4F ANNATrO
Perhaps the most extensively used vegetable color for butter a t the present time is annatto. I t s presence can be readily determined by a slight modification of described tests. If t h e aqueous alkaline extract from about 2 0 g. of fat dissolved in jo cc. of ether is passed through a filter paper several times, the excess of alkali washed off, the red coloration produced by a solution of stannous chloride is very readily obtained. It is advisable t o add a small amount of hydrochloric acid to the stannous chloride solution. I n applying t h e Massachusetts State Board of Health method2 t o fats which contain very small amounts of annatto, about 30 g. of fat should be warmed with 60 cc. of z per cent sodium hydroxide and filtered through a funnel surrounded by warm water. The aqueous filtrate is returned through the filter paper repeatedly for 3 or 4 hours. After washing t h e fat and alkali from the paper the test for annatto is made in the usual way. The paper need not be dried. 11-THE
S E P A R A T I O N O F T H E AZO COLORS F R O M F A T S AND T H E IDENTIFICATION O F YELLOW 0 B AND YELLOW A B
I n a bulletin3 recently issued from this Bureau, Mathewson has summarized the various methods previously used for the separation of azo dyes from oil solutions and described several new methods which he developed. For t h e Sudan dyes particularly, he suggests extraction with a mixture of phosphoric and sulfuric acids and states t h a t this method is not 1
J. A m . Chem. S O L ,20 (1898), 110.
2
Bureau of Chemistry, Bull 107 (1912), rev., 126 I b i d , 448 (1917).
3
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 C H E M I S T R Y
438
applicable t o such colors as o-toluene-azo-b-naphthylamine, since they are destroyed by treatment with strong acids. This statement is not quite correct, as will be shown below. A solution of Yellow 0 B, one part in 12,500, was made as follows: z cc. of 0.4 per cent alcohol solution of 0 B were diluted t o I O O cc. with a mixture of 90 cc. glacial acetic acid, I O cc. concentrated sulfuric acid and I O cc. of water. The spectrophotometric curve of this solution was read immediately in a Hufner type spectrophotometer.’ Since the extinction coefficients are proportional t o the weight of light-absorbing material per unit of volume, by determining these extinction coefficients after certain definite times had elapsed, it was possible t o calculate the percentage decomposition of the dye. The loss expressed as per cent is plotted against time and it will be seen from the curve that even after 5 hrs. have elapsed, a time surely sufficient for any analyst t o have completed this step of the procedure, there is still 8 j per cent of the original dye undecomposed, while after half an hour, the time usually required, the loss is only 2 per cent. The points on the curve are each the average of seven observations a t different wave lengths on the absorption spectrum of the dye solutions. 70
50
4 40 t30 20 10
0
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No. 6
venient t o use a different method. For example, if a dye is easily removed by dilute acid or alkali from t h e f a t such a procedure would be more convenient. T h e proper procedure t o be used should be decided by t h e judgment of the analyst from a few preliminary tests on small amounts of the fat. It might be suggested a t this point t h a t if it is found preferable t o extract with dilute acid or alkali, dilution of t h e fat with ethyl ether is more preferable than dilution with gasoline having a low boiling point, since a more satisfactory extraction can be secured when the former solvent is used. OF THE ACID AND ALKALINE EXTRACTS FROM SOLUTIONS O F VARIOUS OIL-SOLUBLE DYES
TABLE I-COLORS
BUTTER
Butter solutions containing 1 part of dye in SO 000 of fat were prepared. Before extraction with hydrochloric acid or )alkali these solutions were diluted with an equal volume of ethyl ether: before extraction with t h e sulfuric-acetic acid mixture the solutions were diluted with petroleum ether, The colors of the acid or alkaline extract are listed in the following table. I n the case of butter the shades are permanent for a fairly long period, but with oleomargarine the change of shade is comparatively more rapid. 1VOl. 1 Vol. Conc. HCI HIIS04 4 Vols. 1 Per cent 10 Vols. Dye Hz0 Conc. HCl NaOH Acetic Acid Aniline-azo-phenol,. . No color Yellow Yellow Yellow-brown Sudan G. No color Yellow-brown Yellow-brown Yellow-brown Aniline Yellow.. . . . . Red color ...... Brownish red o Toluene - azo B napht h y 1a m i n e (Yellow 0 B). . . . . No color Red ...... Red Benzene-azo-8-naohthylamine IYeliow A B). . . . . . . . . . . . . No color Red ...... Red Sudan I . . . . . . . . . . . No color Very faint red ...... Cherry-red Sudan 11... . . . . . . . . No color Very faint red ...... Violet-red No color Blue Sudan 111... . . . . . . . No color N o color Butter Yellow. . . . . . Red ...... Red Amino-azo- a-naphthalene.. . . . . . . . . . No color Violet ...... , Violet
-
.......... -
......
......
h60
6
Vol.
J
/o
2s
7.5 f//C
/N hwP3
FIG. I-CURVE SHOWING THE PERCENTAGE DECOMPOSITION OF YELLOW 0 B IN ACID SOLUTION WITH TIME
A concentration of one part in 50,000 of Sudan I, under the same conditions, showed absolzttely n o decomposition during the same time interval, namely 7 5 hrs. The specific action on Yellow 0 B is possibly a direct result of the presence of the free amino group in the dye molecule. I n the same bulletin2 it is stated t h a t o-tolueneazo-P-naphthylamine (Yellow 0 B) and its benzene analog can be slowly extracted from the gasoline solution of the fat by 4- t o 6-normal hydrochloric acid. This is true if the dye is present in relatively large amount, but if the quantity of dye in the fat is about one part or less in 50,000,practically no extraction of Yellow 0 B can be effected. Very rarely would an amount of dye greater than this be used. The phenol method of extraction3 is, as stated, “somewhat inconvenient” and particularly so if one attempts to work with a pound or so of the fat. Though the method suggested in this paper isof general application in the separation of azo dyes from fats in a fairly pure state, yet very often i t is more con1 The spectrophotometric determinations were made by A. B. Clark of this laboratory, t o whom the writer wishes to acknowledge his indebtedness. 2 Bureau of Chemistry, Bull. 448 (1917), 7 . a I b i d , 107 (1917).
It is not the purpose of this paper t o present a systematic scheme for the separation and identification of all of the possible oil-soluble colors, but to present a method for the separation from fats of some oil-soluble azo colors not easily removed by dilute acids or alkalies, and principally t o enable t h e analyst t o determine whether the dye present is Yellow A B or Yellow 0 B, either singly or in combination, or some other azo dye. TABLE11-A LIST OF COMMON NAME Yellow A B . . ............... Yellow 0 B . . ............... Aniline Yellow. Butter Yellow. Spirit Yellow R . . ........... Benzene-azo-phenol .......... Sudan I .................... Sudan 11... . . . . . . . . . . . . . . . . Sudan 111.. Sudan G . . . . . . . . . . . . . . . . . . . Benzene-azo- a-naphthylamine Amino-azo- .-naphthalene.
............. .............
................
...
SOMEOIL-SOLUBLECOLORS(a) COMPONENTS Schultz Aniline &naphthylamine o-Toluidine @-naphthylamine Aniline aniline dimethylaniline Aniline o-Toluidine 4- o-toluidine Aniline phenol Aniline &naphthol Xylidine &naphthol Amido-azo-benzene &naphthol Aniline resorcin
No. (b)
+ + .. + ii + 32 68 + .. ++ 36 76 + 23 + 35 Aniline a-Naphthylamine + a-naphthylamine + a-naphthyl- .. amine @-Naphthylamine + a-naphthol .... Aniline + a-naphthol a-Naphthylamine + a-naphthol + &naphthol 165 106 a-NaDhthvlamine - -
6-Naphthalene-azo- a-naphthol Benzene-azo- a-naphthol.. Sudan Brown.. Carminaph Garnet.. (a) Probably the most used :are: Butter Yellow, Aniline Yellow, Yellow A B and 0 B and the Sudans. (b) Farbenstofftabellen, Schultz, 1914.
.... ............. ........
The writer has effected quite satisfactory separations and identifications of Yellow A B and Yellow 0 B from butter and oleomargarine containing the dye in the proportion of one part in I O O , O O O by means of the procedure described. PROCEDURE F O R T H E SEPARATION FROM FATS AND T H E IDENTIFICATION
O F YELLOW A B AND YELLOW 0 B
If the preliminary tests have shown the presence of azo colors the analyst must first examine the fat for the presence or absence of certain azo colors. For this purpose about 4 0 g. of the melted fat are dissolved in
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 C H E M I S T R Y
June, 1918
about I O O cc. of ethyl ether and the solution divided into two portions. One portion is extracted with 1 2 per cent hydrochloric acid and the other with 5 per cent sodium hydroxide. If the acid extract is colored, the presence of any of the following dyes is indicated: Aniline Yellow, Amino-azo-a-naphthalene, Benzene - azo - a - naphthylamine, and Butter Yellow. The coloration of the alkaline extract would indic a t e Sudan G, Aniline-azo-phenol or /3-Naphthaleneazo-a-naphtho1.l If the absence of dyes which can be extracted by dilute acid or alkali has been shown, proceed in the following manner: Melt one or two pounds of f a t 2 (it is usually desirable t o use a n amount of fat containing about I O mg. of dye) on a water b a t h ; allow the water and solids t o settle; pour off the liquid fat and dilute with an equal volume of petroleum ether. Filter through a Buchner funnel or large folded filters. Place the filtrate in a separatory funnel and extract with a mixture composed of I O cc. of concentrated sulfuric acid, 90 cc. of glacial acetic acid (99.5 per cent) and I O cc. of water. Use about I O O cc. of the acid mixture for zoo cc. of petroleum ether solution of the fat. If Yellow A B or Yellow 0 B a r e present the acid layer will separate with a wine-red coloration.3 Separate the acid layer, and for each I O O cc. of acid mixture originally used add I O cc. of water and I O cc. of concentrated hydrochloric acid; add about IOO cc. of ether and shake; if the ether layer does not separate out add more ether and shake again; separate the aqueous layer and repeat extraction with one-half the amount of ether originally used. Beside the f a t t y material, Sudan I, I1 and I11 and Carminaph Garnet are removed by t h e ether. Yellow 0 B and Yellow A B remain in the acid solution. The acid layer is then diluted with water, about two volumes, and carefully neutralized with strong alkali, cooling during the process. T h e solution is then extracted with ethyl ether, the ether solution washed first with water and then with an excess of dilute alkali, drawn off, a n d evaporated t o dryness. Care must be taken t h a t all of t h e acid is removed from the ether solution before evaporation t o dryness. The residue consists of Yellow 0 B or Yellow A B, or a mixture of both. Further confirmation of the identity of these two dyes can be obtained in several ways. CONFIRMATORY
TESTS
FOR
YELLOW 0 B Ah’D
THE
IDENTIFICATION
OF
YELLOW A B
The residue obtained from the sulfuric-acetic acid extraction is then dissolved in several cc. of alcohol, transferred t o a test tube and reduced with hydrochloric acid and zinc dust. I t is t o be emphasized t h a t the volume of alcohol and hydrochloric acid used should be as small as possible. This reduction mixture 1 For a systematic scheme for separating and identifying many of the oil-soluble colors see Mathewson, Bureau of Chemistry, Bull. 137 (1910), 54, or Allen’s “Commercial Organic Analysis,” 5 (1911), 666; see also Bureau of Chemistry, Bull. 448 (1917). 2 One experienced in t h e method can satisfactorily separate and identify 2 mg. of dye in 200 g. of f a t , As a rule it is more satisfactory t o use larger amounts of fat. Of course, the amount of dye used is variable. Only occasionally will 2 lbs. of butter contain more than 10 mg. of dye. 3 By noting t h e color of the acid layer, often a very good idea of the nature of t h e dye present can be obtained. See Table I
43 9
is then diluted with a few cc. of water, m:tde alkaline with sodium hydroxide solution, cooled and quickly extracted with ether. The ether solution after filtration through a small, dry filter paper into a small separatory funnel is then treated with a few drops of a 0 . ; per cent ferric chloride solution and shaken vigorously. If Yellow A B or Yellow 0 B is present a brilliant green coloration of the ether will be evident. Upon adding several cc. of water and again shaking, the green layer settles out beneath the ether. This color reaction depends upon the effect of ferric chloride upon I-2-diaminonaphthalene, but is riot specific for this compound, since i t will be given by a number of diaminobenzenes and diaminonaphthalenes. Though the ethereal solution of the reduction products of Sudan I11 will give a reaction with ferric chloride identical with t h a t obtained from Yellow A B or Yellow 0 B, as previously pointed out, the presence of Sudan I11 is indicated by the very characteristic blue color of the acetic-sulfuric acid extract, and furthermore this dye would have been previously eliminated. I t is because of the lack of specificity of the ferric chloride reaction that a preliminary exclusion of a large number of dyes must be effected as described. In order t o obtain a further check on the identity of the isolated dye a portion should be used in making a dyeing test. The colors of many oil-soluble dyes on silk and the reactions of the dyed fiber are tabulated by Mathewson.’ Yellow 0 B and Yellow A B dye silk a yellowish brown. The dried dyed fiber gives a violet coloration with concentrated sulfuric acid and a red coloration with concentrated hydrochloric acid. COGORINVESTIQATION LABORATORY BUREAUOF CHEMISTRY WASHINGTON, D. C.
AN ACCURATE LOSS-ON-IGNITION METHOD FOR THE DETERMINATION OF ORGANIC MATTER IN SOILS2 BY J. B. RATHER Received December 10, 1917
The loss-on-ignition method for t h e determination
of organic matter in soils gives highly erronleous results which are due, as is well known, t o hydraied mineral constituents of the soil, carbonates and unoxidized minerals. The organic carbon method for t h e determination of organic matter requires the determination of total and inorganic carbon and ari arbitrary factor for the calculation of the carbon t o organic matter. Since the carbon content of t h a t portion of the organic matter which has been separated from the soil may vary from 44 t o 6 4 per cent3 many workers content themselves with reporting organic carbon. I t is evident t h a t if the hydrated, unoxidized, and carbonaceous minerals were removed from the field of action the loss-on-ignition method would be superior Bureau of Chemistry, Bull. 448 (1917), 4 5 . Abstracted by the author from Bull 140, Arkansas Experiment Station, to which publication the reader is referred for additional details and further data. The material under the heading “Application of t h e Method t o Abnormal Soils” does not appear in the publication referred to. This article was read in part before the Association of O 8 c i a l Agricultural Chemists, Washington, D . C., November, 1917. 3 See Fraps and Hamner, Texas Experiment Station, Bull. 129. 1 2
