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vertase inversion method proposed by Hudson1 should be used conjointly with the acid inversion. Under such circumstances, should the quantity of total sugars agree b y both methods, one may safely say, with our present knowledge, that the increase in reducing sugars is due t o sucrose, raffinose, or both. As the latter sugar is present in notable quantities in cottonseed-meal, and may be in other materials, it is not altogether safe t o calculate this increase as sucrose with the idea that only sucrose is present. By the invertase method many other substances are eliminated. It seems better, therefore, to use the expressions ( I ) “reducing sugars calculated as dextrose,” or “ reducing sugars calculated as invert sugar;” ( 2 ) “total sugars b y acid hydrolysis (or by invertase) calculated a s dextrose,” or “total sugars by acid hydrolysis (or by invertase) calculated as invert sugar,” and (3) “increase in reducing sugars b y acid hydrolysis (or b y invertase) calculated as sucrose.” SUMMARY.
A method of preparing an extract for sugar determinations in grains, cattle foods, and vegetable material in general is given on page 487, depending on boiling the product with 5 0 per cent. alcohol. A comparison of the results obtained b y this method using various classes of materials is given with that of a method depending on the extraction of the sugars with water a t loom tempsrature t o which a percentage of sodium carbonate is added to inhibit enzymic action. A study of the limitations of the two methods :s made, in which it is shown that the alcohol method will give satisfactory and comparable results on material, uhether it is analyzed immediately or after standing for some time, while t o obtain true and concordant results with the sodium carbonate method the most careful attention must be :iven to certain details of manipulation. I n the sodium carbonate method, after neutralizing the sodium carbonate by clarifying with alum or lead, the work must be carried on with dispatch i s the enzyms again become active; ( 2 ) the acidity of the sample must be determined and the quantity of sodium carbonate thereby regulated, or the accuracy of the results will probably be affected; (3) normal lead acetate should be used instead of alum for clarification under most circumstanc. s, otherwise an error is introdmed. Taking these points into consideration, it is the opinion of the authors t h a t the lcohol method is to be preferred in general work, but when a long, comparable series of results is to be obtained on samples of the same kind of material the sodium carbonate method might vantageously, provided it is run with dispatch and the greatest care is exercised in its operation; and, most important of all, the results obtained by its use should compare with those obtained b y alcohol digestion. Some materials, however, can not be analyzed by the sodium carbonate method because clear filtrations are not obtainable even with lead clarification. 1 U. S. Dept. Am.. Bureau of Chemistry, Circ. 60; THIS JOURNAL, 2, 43-5 (1910).
[CONTRIBUTIONFROM
THE
July, 1 9 r i
HAYEMEYERLABORATORIES OF COLUMBIA UNIVERSITY. NO. 196.1
THE DETECTION OF SALICYLIC ACID. B y H. C. SHERMAN AND A. GROSS. Received May 8 . 1911.
For the detection of salicylic acid, especially when present in small amounts, it is customary in many laboratories to rely upon the violet reaction with ferric chloride, almost t o the exclusion‘of other tests, The popularity of the ferric chloride test is readily explained b y its simplicity and delicacy, Using fresh one per cent. ferric chloride as reagent the test is delicate in our hands to a dilution of about I : 400,000 when applied t o I O cc. of solution, about I : 750,000 to I : I,OOO,OOO if 25 cc. of solution be tested. The violet color obtained with such small amounts of salicylic acid must be observed quickly as i t fades rapidly, passing through a rose-red color. A faint rose color may also be obtained on addition of ferric chloride t o solutions containing salicylic acid in amounts too small t o show violet reaction. Unfortunately, however, the formation of a violet color with ferric chloride is a reaction b y no means confined t o salicylic acid. Mulliken’s tables1 include many colorless compounds which give more or less distinctly violet reactions with ferric chloride and some of these also resemble salicylic acid in solubilities, That this may lead t o error in the testing of foods for salicylic acid has been pointed out b y several writers.a Among the tests for salicylic acid, other than the ferric chloride reaction, are the formation of the methyl ester or the nitro-compound, the reactions with bromine water and with Millon’s reagent, and the J orissen test. The adoption by Mulliken of the methyl-ester and nitration tests for the identification of salicylic acid is sufficient evidence of their value for cases in which enough salicylic acid is involved to make them available; but these tests a n d also the test with bromine water seem not t o be sufficiently delicate for the detection of very small amounts. I n tests with Millon’s reagent it was found that heating for some time increases considerably the delicacy of the test. When two drops of Millon’s reagent were added t o the solution t o be tested, shaken in a test-tube, and immersed in a boiling-water bath with a blank test for comparison there was developed in the course of 45 minutes’ heating a delicate reddish or pink color even in the presence of only minute amounts of salicylic acid. With practice and with blank tests for comparison no difficulty was found in detecting the presence of I part salicylic acid in 2,000,000 of water when 2 0 cc. were tested; when only I O cc. were tested, the pinkish tint was barely perceptible a t this dilution. Longer heating and variations in the amount of reagent added were tried without appreciably altering the result. The limit of delicacy 1 “The Identification of Pure Organic Compounds.” 2 Brand, Ztschr. f . d . ges. Brauw., 16, 3 0 3 ; Ber.. 27, 8 0 6 ; Erich, DET Bierbrauer. 24, 465; Munsche, Woch. f. Brauerei, 10, 739; Abraham, J O Z L T ~ L . de Pharmacie de L i l ~ e 6, . 1 7 3 ; Backe, Annales des Falsificaiions, Nov.. JOURNAL, 2, 2 4 ; Backe. Comfit. r d . , 110, 540; 1909; Sherman, THIS 78.
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of the test with Millon’s reagent as here used seems, therefore, to be reached by heating in boiling water for 45 minutes and to lie a t a dilution of about I
:
2,000,000.
The Millon reaction also has the advantage over the ferric chloride test that the color produced even with very small amounts of salicylic acid shows no evidence of fading on standing over night; but on account of the large number of substances which respond t o the Millon reagent’ it seems unlikely that this reaction will prove ‘as useful as that of Jorissen. The Jorissen rea.ction2 has been used by a number of European investigators3 for the identification of small amounts of salicylic acid and was found useful by one of us4 as a means of distinguishing between salicylic acid and maltol or isomaltol when present in small quantity in foods. In further experiments with this reaction we have found that by diminishing the amount of copper used and increasing the time of heating, the test can be made much more delicate than appeared in our earlier work in which the test was used in its original form, and considerably more delicate than the ferric chloride reaction. The longer heating is necessary to fully develop the characteristic color, a t least when only very small amounts of salicylic acid are present, and the reduction in the amount of copper diminishes the slight green color due to the reagent which otherwise may interfere with the more delicate tests. The test as n o w used for very small amounts of salicylic acid is as follows: Bring the solution t o be tested into a test tube, add 4-5 drops of I O per cent. sodium or potassium nitrite, 4-5 drops of j o per cent. acetic acid, and I drop of one per cent. copper sulphate. Shake after addition of each reagent and finally place in a boiling-water bath in such position that the test liquid is completely immersed in the boiling water and allow t o stand for 45 minutes, then remove, allow t o cool and examine against a white background, viewing the tube both vertically and horizontally and comparing with a blank test in which the same amounts of reagents have been added t o pure water. I n this way, the presence of as little as 0.005 t o 0.01 milligram of salicylic acid in pure water solution can be detected. Faint but perceptible reactions were obtained with 5 to 8 cc. of a solution of I : I,OOO,OOO and with 18 t o 2 5 cc. of solutions of I : 3 , 0 0 0 , 0 0 0 to I : 3,500,000. No advantage has been found in a brine-bath over a water-bath, in longer heating than 45 minutes, nor in varying the amounts of nitrite and acetic acid used. When larger amounts of salicylic acid are present a drop of stronger copper sulphate solution may be used,
’
Vaubel. Zt.whr. angew. Chem., 1900, 1 1 2 5 ; Nasse, PRiiger’s Archiv. j . d . ges. Physsiol., 83. 361 (1901) ; Mann, ‘‘Physiological Histology,” pp. 321-23, and ‘‘Chemistry of the Proteids,” p , 7 , Jorissen, Bulletins de I’Academie Royal des Sciences, des Lettres, et des Beaux-wts de Beleique, 3rd series, 3, 259. da Silva, Combt. rend , 131, 423; Klett, Pharm. C e n t r . , 41, 4 5 2 ; Ztschr. Unlers. h’uhr. Genussin., 4, 4 6 9 ; Portes and Desmouli&res,Annul. chin$ anal., 6 , 4 0 1 ; Ztschr. L‘nters. .Vahr. Genussm., 5, 4 6 8 ; Windisch, Ibid.,
I,447. Sherman, T H x s JOURNAL, 2, 24.
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up t o a ten per cent. solution as originally recommended. Except with very small amounts of salicylic acid the red color of the Jorissen reaction develops quickly on heating and the long immersion in the water-bath then becomes unnecessary if only qualitative results are required. A feature which will be of great importance in colorimetric estimations of small amounts of salicylic acid is that while the violet color of the ferric chloride test fades rapidly, the red color of the Jorissen test is quite stable. Even the faint colors obtained by long heating, where only very minute amounts of salicylic acid are involved, have shown no deterioration when allowed to stand over night. I t has also been found that the ferric chloride and Jorissen tests may be applied to the same solution of salicylic acid. After making the ferric chloride test the liquid may be diluted until the violet color disappears and then submitted to the Jorissen reaction, when, if salicylic acid is present, pink color will appear. N o extended study seems to have been made to determine what other substances will give red or pink reactions in the Jorissen test. Jorissen, in describing his reaction, stated that phenol behaves in the same way as salicylic acid but benzoic acid does not. Allen states that neither benzoic, cinnamic nor tartaric acid responds to the Jorissen test, which statement we have confirmed. Special importance attaches t o the behavior in the Jorissen test of those substances which give violet reaction with ferric chloride. Maltol and isomaltol have already been considered; a few others have been tested with the following results: Phenol, in our hands, gives the same color as salicylic acid in both the Millon and the Jorissen tests, but the limits of delicacy are quite different. Phenol can be detected by the Millon reaction to about I : 2,000,000. In the Jorissen test, phenol I : I O O , O O O gives practically the same color as salicylic acid I
:
1,000,000.
Saligenin gives, in the Jorissen reaction, a red color a t I : 10,000;a yellowish tint a t I : I O O , O O O ; no reaction a t I : I,OOO,OOO. The limit of delicacy for the ferric chloride reaction with saligenin lies between I : 10,000and I : 2 0 , 0 0 0 . 2-oxy-isophthalic acid gives the Jorissen reaction up to a dilution of I : I O O , O O O but is easily distinguished from salicylic acid in the color which it gives with ferric chloride. Methyl-ethyl-aceto-acetate, which gives, with ferric chloride, a violet-red color in concentrated solutions, gives neither the hlillon nor the Jorissen reaction when tested a t a dilution of I : 1,000. Orcin, arbutin, resorcin and phlorizin, which give blue, violet, or red-violet reactions with ferric chloride do not respond to the Jorissen test. THE P R E S E R V A T I O N O F EGGS. By R. BERCER Received March 2 7 . 1911.
While the preservation of eggs on a commercial scale by the cold storage system is universally adopted,
