ANALYTICAL CHEMISTRY
1460 ductometric titrations. The plots of Figures 3 and 4 are seen to be fairly linear over the entire ranges covered. The plot of Figure 2 is linear, except when the cell conductance drops well below about 200 X 10-6 mho, which corresponds to about 500 ohms. This is about ten times the Ra value, so a larger R3 value should be used for measurements in thiq range, as in Figures 3 and 4. I t is felt that these tests verify the validity of the circuit analysis described above.
-1
400
I
t
MHO X I os 300
L
too 2oo
I
”
ZOO
Figure 3.
I
/ ‘ I
J
/
required conductivity range is greater. I n an alternative procedure, the D setting was left constant and the off-balance vacuum tube voltmeter readings were obtained as a function of the cell conductivity. Although the V reading was found to be a linear function of cell conduciivity over a range adequate for considerable usefulness, i t is felt that the null-type readings are fundamentally more reliable. Tests in Titrations. A number of titrations were performed using the instrument of Figure 1, including strong acid us. strong base, strong acid us. salt of a weak acid, weak base us. weak acid, aniline in 50% ethanol us. strong acid, and others. The Helipot readings a t null were plotted directly against volume of added reagent, and conventional conductometric titration plots were obtained. Good results were obtained in all titrations. It was found possible, for instance, to titrate both hydrochloric and boric acid solutions with standard alkali using the same cell for both titrations.
400 600 HELIPOT OlAL
SUMiMARY AND CONCLUSIONS
I 800
1000
Data for Linearity Test with Ra of 250 Ohms
I
The Helipot dial readings a t null are linearly indicative of the conductivity of the titration cell over a wide range of values tested, and this dividing type of audio-conductometer may be used for satisfactory conductometric titrations. The Helipot dial provides 1000 useful divisions without interpolation and has great precision of reading because of the long scale length. The range of conductivity values covered can be adjusted by proper choice of RI. If extremely low conductivity values are to be measured, R1 and RI may be made of higher resistance values. The linearity of response is of interest for possible adaptation to automatic recording. The fact that the off-balance V reading changes sign on each side of null is of possible value in control work. The circuit is not dependent upon particular tube characteristics. A “magic-eye” tube could be substituted for the electron tube voltmeter, although the off-balance voltage reading feature would be sacrificed. The addition of a double-pole, double-throw switch in the circuit to interchange R, and RI would add the feature of optional conductance or resistance readings. LITERATURE CITED
Figure 4. Data for Linearity Test with Rs of 2500 Ohms
A similar test with a source voltage of 15 rather than 10, and with other parameters corresponding to those used in Figure 2, disclosed an even longer range of linearity for a given value of Ro. This suggests that the lower source voltage would be better where the range of conductivity change in a titration is small, but that the higher source voltage would be preferable when the
(1) Garman, R. L.,IND.ENQ.CHEM.,ANAL.,ED.,8, 146 (1936). (2) Seely, S.,“Electron Tube Circuits,” pp. 113-17, 146-62, New York, McGraw-Hill Book Co., 1950. (3) Shedlovsky, T., Chap. XXV in Weissberger’s “Technique of Organic Chemistry,” Vol. I, Part 11, 2nd ed., New York, Interscience Publishers, 1949. (4) Valley, G. E.,Jr., and Wallman, H., “Vacuum Tube Amplifiera,” Vol. 18 of M.I.T. Radiation Laboratory Series, pp. 44550,47980,New York, McGraw-Hill Book Co., 1948. RECEIVED for review January 10, 1952. Accepted June 20, 1952. Contribution No. 546 from the chemistry department of Indiana University. Abstracted from thesis submitted by Dale J. Fisher t o the graduate school in partial fulfillment of requirements for the degree of doctor of philosophy.
ldent ificat ion of Stabilizing Agents M. H. EWART A N D R. A. CHAPMAN Food and Drug Laboratories, Department of National Health and Welfare, Ottawa, Can.
E
XTENSIVE use of a variety of polysaccharides aa stabilizing or thickening agents in foods has created a need for
analytical methods for the identification of commercially available materials and for their detection and quantitative estimation in food products. A number of stabilizers, which are used or have been suggested for use in foods, are listed and their chemical nature is indicated in Table I. Throughout this report the t,erm “gums” is used in referring to all of these materials. Gelatin is included because it has many uses similar to those of the polysaccharides. As the initial step in a project concerned with the development
of the required analytical procedures, methods for the qualitative identification of a number of gums have been studied. Several procedures for the identification of gums are found in the literature. A method described by Jacobs and Jaffe ( 1 5 ) classifies the gums on the basis of physical characteristics or appearance of precipitates and thus requires considerable experience on the part of the analyst. Their outline does not include pectic substances, alginates, methylcellulose, carboxymethylcellulose, gum ghatti, starch, or gelatin. It also has the disadvantage of using an unstable reagent (llillon’s) which must be freshly prepared each day. A method developed by Cannon and adopted
V O L U M E 2 4 , N O 9, S E P T E M B E R 1 9 5 2
1461
The present work was undertaken to develop a method for the identification of stabilizing and thickening agents used in food products. The materials studied were pectin, de-esterified pectin, algin, Irish moss, gum tragacanth, gum karaya, locust bean gum, starch, agar, gum arabic, gum ghatti, carboxymethylcellulose, methylcellulose, and gelatin. A proposed identification scheme is based on precipitation reactions w-ith calcium chloride,
Iiy the Association of Official Agricultural Chemists ( 3 , 6) doe8 not provide for the identification of pectic substances, alginates, methylcellulose, carboxymeth) lcellulose, locust bean (carob) gum, or gum ghatti. Bryant ( 4 ) has described a procedure for distinguishing between pectin and certain gums, but it does not provide for positive identification of the gums. A number of other publications dealing with characteristic properties of these polysaccharides have been summarized by Mantel1 (16), but a systematic procedure is still needed for their identification. Such :I procedure would be useful for the identification of products used as thickeners or stabilizers in foods, drugs, and cosmetics and ultimately for the identification of polysaccharides isolated from these materials. In the present investigation, the manner in which the gums disperse in water after being %vetted with alcohol has been a valuahle index to the identity of unknown samples. Their solubility properties are summarized in Table 11. Use has also been made of the fact that many of the polysaccharides occur as salts of complex organic acids (Table I). The acidic properties may be due to the presence of uronic acid groups, as in gum arabic, or to the unesterified portion of sulturic acid molecules esterified \+ith the polysaccharide. When mineral acids are added to aqueous solutions or dispersions of these salts, the effective conrentrations of the polysaccharide anions are decreased. Thus, although the complex anions may yield insoluble salts with heavy
sodium hydroxide, barium hydroxide, and lead acetate. In addition, reactions of the stabilizing agents with a cationic soap, ammonium sulfate, mercuric nitrate, papain, and gelatin are listed. The proposed scheme should be useful for identification of unknown stabilizing agents. A number of the reactions reported might be employed for the identification of individual stabilizing agents in mixtures of these materials or isolated from foods.
metal cations, most are not precipitated from acid solutions. The amount the pH must be raised in order to precipitate the heavy metal salts-e.g., barium, mercury, or lead-is frequently characteristic of the individual polysaccharide. EXPERIMENTAL
During the present investigation 0.5 to l.Oyo aqueous dispersions of the polysaccharides were used for the tests. Aliquots of from 3 to 5 ml. were treated with varying concentrations of the reagents which it was hoped would give characteristic precipitation reactions. Initially the reagents used were those for which Jacobs and Jaffe (16) have described reactions with several polysaccharides. Subsequently a number of other reagents \-cere used.
-_
__
Table 11. Dispersion in Water of Gums, Wetted with Alcohol Gum Pectic acid Pectate ( N a , K , or salts) Pectate (Ca salts) Pectin Alginate
4”
Irish moss Agar
Table I. Source and Chemical Nature of Materials Commonly IJsed as Thickening Agents in Foods Material Pectic substances
Sourcp Fruits
Algin (sodium alginate) Irish moss
Seaweeds Seaweeds
haar
Seaweed5
Tragacanth
Plant gum
&Iethylcellulosr
Karaya
Modified cellulose Plants Modified cellulose Seed endosperm Seed endosperm Plant g u m
Arabic (acacia)
Plant gum
Ghatti
Plant gum
Gelatin
Modified protein
Starch Carboxymethylcellulose Locust bean gum (carob gum) Guar gum
Principal Components Galacturonic acid (occurs as methyl ester) Xannuronic acid ( N a salt) Galactose, galactose 4sulfate ( K and C a salts) Galactose (D- and L-) galactose 6-sulfate ( C i and M g salts) ].-Fucose, D-xylose, galacturonic acid, L-aiabinose, D-galactose
References
Tragacanth Methylcellulose Starch Carboxymethylcellulose
(18) (16)
Locust (carob) Karaya Arabic (acacia)
(18)
.-
Ghatti
(18)
Gelatin
‘Manner of Dispersal in Water Insoluble Forms either clear or turbid solution on heating Insoluble Swells in cold water and dissolves on heating Dissolves slowly in cold water or quickly on heating t o form viscous solution Dissolves slowly in cold water, rapidly on heating t o form viscous solution Swells in cold water, dissolves on heating, gels on cooling Swells t o form viscous dispersion in cold or hot water, b u t does not form true solution Dissolves slowly in cold water b u t becomes cloudy or gels on heating Disperses o n h e a t i n g Dissolves slowly in cold water, rapidly on heating giving clear viscous solution with some fin: fibrous suspended material Forms viscous suspension b u t not a true solution Forms viscous suspension. Insoluble particles settle on standing Dissolves in cold water t o form a clear only slightly viscous solution Dissolves t o form almost clear solution but some insoluble material may remain a s fine suspension Swells in cold water and dissolves on heating
(18)
Jfethyl-D-glucose
(18)
D-Glucose Carboxymethyl-D-glucose
(18)
Jlannose and galactose
(15)
Mannose and galactose
($0)
Galactose, acetic acid, galacturonic acid, rhamnose. tagatose D-Glucuronic arid, D-galactose, L-arabinose, rhamnose (mixed C a , Mg. and K salts) L-Arabinose. galactose, galacturonic acid (Ca salt) Amino acids
(14)
(18)
(18)
(16)
Reactions which were found useful for characterizing the gums are summarized in Tables I11 and IV. Only those materials having anionic components, such as alginates, or potential anionic components, such aspectin, give pronounced reactionswith cationic soap (Table 111). As in the case of precipitates withheavymetals, the precipitates with the cationic soap quickly disperse on acidification of the medium. Ammonium sulfate is of interest, in that it gives pronounced precipitation tests with several of the gums but not with alginates, pectin, tragscanth, karaya, arabic, or ghatti, each of which probably contains uronic acidcomponents. The reactions with Stokes’s acid mercuric nitrate illustrate the effects of low pH on precipitation of heavy metal salts of the polysaccharide acids. An excess of the reagent makes the solutions strongly acidic and thus the weakly dissociated acids redisperse. Alginic acid and pectic acid are insoluble and thus are not dissolved by excess Stokes reagent. Papain and gelatin give pronounced precipitation reactions only with those gums having anionic components. These precipitates are found only if the
Table 111. Precipitation Reactions of Polysaccharide Gums and Gelatin 1 Val. 1% Solution of
0.5 Vol. Saturated Ammonium Sulfate Gelatinous translucent precipitate
Gum De-esterified pee tin
Cationic Soapa Fine opaque precipitate
Alginate
Fine opaque precipitate
Nil
Pectin
Flocculent precipitate
Nil
Irish moas
Stringy or Bocculent precipitate
Gelatinous precipit a t e or gel
Agar
Gelatinous precipitate . Fine opaque precipitate
Flocculent precipitate Nil
Methylcellulose Starch
Si1 Xi1
Precipitate Precipitate
Carboxymethytcellulose 1,ocust
Gelatinous clotted precipitate Nil
Gelatinous precipitate Precipitate (voluminous)
Karaya
Flocculent precipitate
Nil
Arabic (aeacia)
precipitate fine)
Nil
Ghatti
Fine prccipitate
Tragacanth
(very
Nil
Dilutedb Stokes's Acid Mercuric Nitrate Added Dropwise Gels (almost opaque). Insoluble in excess reagent Gels (almost opaque). Insoluble in excess reagent
b c
4 Vol. 95% CiHaOH
+ 2urated - 3 Drops SatNaCl
Gelatine Precipitate
Precipitate
Precipitate
Cloudy
No definite effect
Precipitate
Precipitate
Cloudy
Precipitate
Precipitate
Precipitate
Nil Nil
Nil Nil
Precipitate dissolves in excess reagent No pronounced effect
Precipitate
Precipitate
No pronounced effect
Nil
Flocculent precipitate dissolves in excess reagent Flocculent precipitate dissolves in excess reagent Si1
Precipitate
Precipitate
Precipitate
Precipitate
Fine opaque nonsettling precipitate
Nil
Precipitate
Fine precipitate, nonsettling (2-3 vol.) Finely flocculent precipitate, coagulates
Forma almost opaque gel which dissolves in excess reagent Transparent gel. Redispersed b y excess reagent Turbid or cloudy Flocculent precipitate. Dissolves in excess reagent Si1 Si1
Gelatinous precipitate, gels (1 vol.) Gelatinous precipitate (1 vol.) becomes stringy with 4 vol. alcohol Transparent gelatinous precipitate. Gels ( 1 vol.) Stringy precipitate Fine flocculent preoipitate Voluminoua precipitate, jellylike Nil Opaque flocculent precipitate Voluminous olotted precipitate Voluminous opaque stringy precipitate, forms clot Flocculent precipitate, discrete particles
Nil Precipitate Nil Nil Precipitate in alkaline medium (88) RodJon (alkyl dimethyl benzyl ammonium chloride), Fairfield Laboratories, Plainfield, S . J. Mercury dissolved in twice its weight of concentrated nitric acid and diluted,to 100 times its volume with distilled water. Precipitates with papain and gelatin are observed only in weakly acidic medium and most exhibit properties of coacervates rather than true preolpitates
Gelatin 0
1 Vol. J"/.
1 VOL 2%c Papain ( 6 ) Precipitate
pH of the mixture is belorr the isoelectric point of the protein and it is possible that they would be more correctly called coacervates. They are usually dispersed by a few drops of mineral acid or of dilute ammonium hydroxide. The characteristic manner in which some of the gums are precipitated by alcohol may also be of value in their identification. The remtions described in Table IV form the basis of a proposed procedure for the systematic identification of the gums. REAGENTS
Calcium chloride (CaCh), 3% solution (weight/volume). Ammonium hydroxide, 3.0 iV solution. Sodium hydroxide, 3.0 N solution. Barium hydroxide, saturated solution stored in a bottle equipped with a siphon and a soda-lime tube. Basic lead acetate, 20% suspension (weight/volume). Heat to boiling, cool, and use supernatant solution. Hydrochloric acid (or other mineral acid), 3.0 N solution. Methylene blue, 0.1 % aqueous solution. Tincture of iodine (U.S.P., 14). Iodine-potamium iodide stock solution, containing 0.5% iodine and 1.0% potassium iodide. Iodine-potassium iodide test solution, consisting of stock solution diluted 1 to 5 . Cupric sulfate (CuSOc. 5H,O), 15% solution (weight/volume). Borax (N&2B~O7.10H20), 4% solution. Ruthenium red test solution ( 3 ) . Picric acid, saturated aqueous solution. IDENTIFICATION PROCEDURE
\Vet a 0.25- to 0.5-gram sample of the material to be identified with 1 to 2 ml. of 95% alcohol and add 50 ml. of distilled water. Suspend the solid material in the water by shaking or stirring. Heat the suspension, with frequent shaking, on a hot plate or over a burner. If the sample dissolves, discontinue heating immediately; otherwise hold a t 85' to 95" C. for 15 minutes. Group A. I. Treat a 3- to 5-ml. aliquot of the solution with 0.2 volume of 0.25 M calcium chloride. A gelatinous precipitate or gel indicates alginates or de-esterified pectin. If no reaction is apparent with calcium chloride alone, add 1 vol-
ume of 3 N ammonium hydroxide to the calcium chloride treated sample. Slow formation of a gel or gelatinous precipitate indicates pectin. 11. If either test in A I was positive, mix a fresh 3- to 5-ml. aliquot of sample with 1 volume of 3.0 N sodium hydroxide. Observe the reaction and then heat the mixture in a boiling water bath for 10 minutes. Immediate formation, in the cold, of a gelatinous or flocculent precipitate indicates either pectin or de-esterified pectin. No precipitate indicates aliginates. All three mixtures become yellow on heating, but the precipitates with pectic subqtanres do not dissolve.
Group B. If the material is not an alginate or a pectic suhstance, carry out the following tests.
I. Mix a 3- to 5- ml. aliquot of the sample with 0.1 volume of saturated barium hydroxide. Observe in the cold and heat in boiling water bath for 10 minutes. Formation in the cold of a nonsettling, almost opaque, gelatinous precipitate indicates Irish moss. Carry out confirmatory test for Irish moss. A small amount of flocculent precipitate or cloudiness in the cold and a definite lemon yellow color on heating identify gum tragacanth. Color changing during heating to yellow, then to green, and finally to gray indicates agar. Carry out confirmatory test for agar. If the mixture becomes cloudy or forms a gel on heating, but becomes clear on cooling, methylcellulose is indicated. Carry out confirmatory test for methylcellulose. An opaque flocculent precipitate which may tend to redisperse on heating and reprecipitate on cooling indicates starch. Carry out confirmatory test for starch. Precipitates which disappear when the barium hydroxide is thoroughly mixed with the sample may be disregarded a t this point. 11. If the material has not been identified, mix a fresh 3- to 5ml. aliquot of sample with 1 volume of saturated barium hydroxide. Observe whether there is an immediate precipitation and examine again after standing 5 minutes a t room temperature.
V O L U M E 24, N O . 9, S E P T E M B E R 1 9 5 2
b
0
a
1463
ANALYTICAL CHEMISTRY
1464
Table V.
Samples Examined by Proposed Identification Procedure
Gum Sodium pectate Pectic acid Pectin Sodium alginate Irish moss Tragacanth Agar
T>pe Powdered Granular Powdered Powdered Powdered Powdered Sliiedded Granular Fibrous Whole wheat florir Tapioca flour Soluble starch Cornstarch Amioca (amylopectin)" Clear jel" Clear-1'10-H (sodium salt of starch acid esterla Dry-Flo (starch ester)" Vulca (starch ether)u &\Ielojel" Nu-film (starch acid ester)-
Methylcellulose Starch
GELATIN. Add 2 to 3 drops of gum solution to 2 ml. of saturated picric acid. .4 fine yellox precipitate confirms gelatin.
No. of Samples 1
1 I 3 1 1
1
1 1
I 1 1
Sodium carboxymethylcellulose Locust bean (carob) Guar Karaya Arabic (acacia)
Powdered Powdered Powdered Powdered Powdered Lump Powdered Gbatti Lump Gelatin Granular u Products of Piational Starch Prodiicts, X e w York, N. Y.
A voluminous opaque, stringy precipitate which tends to form a clot indicat,es locust bean gum. This precipitate may appear flocculent if the mixture is shaken vigorously. Carry out' confirmatory test for locust bean gum. .Z voluminous opaque flocculent prc8cipitate which forms immediately indicates carboxyInethylcell~ilose. Carry out confirmatory test for carboxymethylcellulose. An opaque flocculent. precipitate xhich forms slowly and is not voluminous indicates guni karaya. Carry out confirmatory test for karaya. Group C. If the sample hits not been identified it may be guni arabic, gum ghatti, or gelatin. I. Mix a fresh 3- to 5-ml. aliquot of sample with 1 ml. of basic lead acetate solution. Immediate formation of a voluminous opaque precipitate indicates gum arabic. If there was only a small amount of flocculent precipitate, or no precipitate, with the basic lead acetate add 1 ml. of 3.0 N ammonium hydroxide to the lead-containing sample. -4 voluminous opaque flocculent Precipitate indicates gum ghatti. If there is no precipitate, the sample probably is gelatin. Carry out confirmatory test for gelatin. CONFIRMATORY TESTS
ALGINATES A N D DE-ESTERIFIED PECTIXS.Add 0.2 volume of 3 N hydrochloric acid (or other mineral acid) to 3 t o 5 ml. of the sample, A gelatinous precipitate confirms alginates or de-esterified pectin. IRISH Moss. Add 2 t o 3 drops of 0.5% methylene blue in water to 1 ml. of the sample solution. Precipitation of purple-stained fibers confirms Irish moss. Mix 5 ml. of sample with 25 ml. of 95% METHYLCELLULOSE. alcohol and 2 to 3 drops of saturated sodium chloride. S o wecipitate confirms meth$lcellulose. AGAR. Precipitate gum from 5 nil. of sample with alcohol and stain with tincture of iodine (3). Starch is also stained blue by this reagent. STARCH. Add 1 to 2 drops of the iodine potassium iodide test solution to 1 ml. of sample. A blue or purple color confirms starch. Some samples of gum tragacanth may give a faint blue test here. CARBOXYMETHYLCELLULOSE. Add 2 ml. Of 1.0 h! Cupric SUIfate to 3 to 5 ml. of sample solution. An opaque, slightly bluish, clotted precipitate confirms carboxymethylcellulose. LOCCSTBEANGUM. Add 1 ml. of 4% borax t o 3 t o 5 ml. of gum solution. If mixture gelatinizes, locust bean gum is confirmed. Guar gum also forms a gel here. KARAYA.Precipitate gum from 5 ml. of solution with alcohol and stain with ruthenium red (3). If sample swells considerably and is stained pink, karaya is confirmed.
DISCUSSION
The proposed procedure has been tested with the gums listed in Table V. It was possible to identify the modified starches as starch products, because the solubility of all these material8 is decremed by barium hydroxide and all give positive tests with the iodine-potassium iodide reagent. It is possible that not all the thickening agents or gums employed in food product,s a t the present time have been included in this study. Cherry gum and quince seed gum, for example, have been suggested in the liternture for use in foods. HowevPr, the proposed procedure invludes a11 the gums that were available during t,he investigation. The proposed scheme for identification of stabilizing and thickening agents is applivable only when they have not been mixed with other materials. To identify thickening agents in ioods by this method it would first he necessary to separate them from the foods, but separatisn techniques might alter the reaction characteristics of the gums. Much work has been done on methods for the separation and detection of gums in particular foods, such as mayonnaise and Frrnch dressing (8, 7 , 10, l l ) , soft curd cheese (1, 8, 10, 11, 13, l.Y), tomato products (10, 13), starchy foods ( Z l ) , cacao products (13, 17, E?),ice cream and frozen desserts ( g - f f ) , canned chicken ( l o ) , and meat products (12 ) . Additional references to methods for separating gums from foods may be found in reviewe by Jacobs and Jaffe (15) and Mantel1 (16). The emphasis in most procedures has been on detection of the gums without identification. However, Wyler ( 2 4 ) has outlined methods for the detection and identification of locust (carob) bean gum, methylcellulose, carhoxymethylcellulose, starch, pectin, and alginate in meat products. Thus identification tests proposed in the present paper may be useful for the identification of gums separated from foods hy methods already described in the literature. However, it is probable that many special techniques will be required for the analyses of particular combinations of foods and thickeners. A great deal more work must be done before it will be possible to identify all of the gums in the various foods in which they may be used. LITERATlIRE CITED
(1)
Agr. Chemists, "Official Methods of Analysis," 7th ed., pp. 266-7, 1950. (2) Zbid., p. 486. (3) Zbid., pp. 631-3. (4) Bryant, E. F., IND.ENG.CHEM.. .\NAL. ED.,13, 103 (1941). (5) Cannon, J. H., J . Assoc. Ofic.A g r . Chrmists, 22, 726-8 (1939). (6) Ene, G. E., J . Am. P h a r m . Assoc., 30, 19-20 (1941). (7) Fine, S.D., J . Assoc. Ofic.A g i . Chemists. 28, 249-51 (1945) (8) Gnagy, M. J., Ibid., 34, 361-8 (1951). (9) Hart, F. L., Am. J . Pub. Health, 33, 599-601 (1943). (10) Hart, F. L., J . ilssoc. Ofic.Agr. Che?nzsts, 20, 527-34 (1937). (11) Zhid., 23, 597-603 (1940). (12) Zbid., 25, 718-22 (1942). (13) Zhid., 33, 741-2 (1950). (14) Hirst, E. L., Hough, L.. and Jones, $1. K. N., J . Cheni. SOC., 1949, 3145-51. (15) Jacobs, M. B.. and Jaffe, L., IND.ENG.CHEM.,AXAI..ED., 3, 210-12 (1931). (16) Mantell, C. L., "Water-Soluble Gums," New York, Reinhold Publishing Corp., 1947. (17) Mendelsohn, F. Y., J . Assoc. Ofic.Agr. Chemists, 34,361 (1951). (18) Pigman, W.IT., and Goepp, hi. R., "Chemistry of the Carbohydrates," New York, Academic Press, 1948. (19) Racicot, P. A., and Ferguson, C. S., J . Assoc. Ofic.Agr. Chemists, 21, 110-12 (1938). (20) Rafique, C. hi., and Smith, F., J . Bm. Chem. Soc., 72, 4634-7 (1950). (21) Redfern, S., J . Assoc. Ofic. Agr. Chemists, 29, 250-5 (1946). (22) Steigmann, A., J . Soc. Chem. Znd., 44, 88 (1945). (23) Winkler, W.O., J . Assoc. Ofic.Agr. Chemists, 22, 600-5 (1939). (24) Wyler, O., Mitt. Gehiete Lebensm. Hyg., 41, 46-55 (1950). dssoc. Offio.
RECEIYED foi review February 4. 1952. .Iccepted July 16, 1952.
