\
ANALYTICAL EDITION
January 15, 1944
3’1
Recovery experiments were run by adding known amounts of xanthone to apples that had been sprayed with lead arsenate, and analyzing by the described procedure (Table I). The average recovery from seven :rtmples was 99.0 per cent, which is not statistically different from complete recovery. The completeness of removal of xanthone from Winesap and Rome apples (collected 6 weeks before harvest) was also studied by submitting samples to a second stripping with toluene. The amount of xanthone removed by the second treatment was calculated after allowing for the amount of toluene left on the apples from the first stripping. Six samples, having from 6 to 13 mg. of xanthone per sample, treated in this manner, showed a removal of 99 per cent or better with the first 5-minute stripping treatment.
toluene is in contact with the acid layer overnight and is probably due to temperature changes. Ground-glass joints are preferred for the reflux reduction. Rubber stoppers may be used if they are boiled in normal alkali for 15 minutes, then in normal sulfuric acid for 15 minutes longer, and finally rinsed well with distilled water. One standard graph obtained in ACCURACY AND PRECISION. this investigation is given in Figure 2. The absorption cells used were standard test tubes (2-cm. inside diameter). The line was fitted by the method of least squares. The standard error of estimate for the points on this line is *6.7 micrograms. Aliquots can be so chosen as to permit readings on 200 to 500 micrograms of xanthone, thus allowing reduction of the percentage standard error to 3.3 or less. More accurate and precise results can be obtained if the colored solutions are read in cells with flat optical windows, since the test tubes used in this work vary about 0.5 per cent in light transmission.
LITERATURE CITED (1) F a h e y , J. E., Cassil, C. C., and Rusk, H. W., Agr. C h m . , 26, 150-5 (1943).
J. Aasoc. Oficicll
PHENOL STUDIES Qualitative Tests for Phenol and
0-,
m-, and p-Cresol
WM. B. DEICHMANN Kettering Laboratory of A p p l i e d Physiology, College
of Medicine, University of Cincinnati, Cincinnati, O h i o
Qualitative tests for phenol employing ferric chloride, hypochlorite, or the reagents of Melzer, Millon, Liebermrnn, Guareschi, and Cotton have been modified to permit differentiation between phenol and 0 - , m - , and p-cresol.
T
H E manufacture and use of phenol and cresol on a large scale have furnished an incentive for investigating the toxicity and metabolism of these compounds, and these investigations, in turn, have called for a review of analytical methods useful in their detection and estimation. A review of quantitative methods for the estimation of phenol in biological material, including a spectrophotometric procedure for the quantitative estimation of free, conjugated, and total phenol in tissues and fluids, has been published (2, 3). I n this paper qualitative tests for 0-, m-,and p-cresol which are modifications of well known qualitative tests for phenol are described. The hypochlorite test for o-cresol offered here has apparently not been recorded before. A single test or a combinatidn of several of these color tests can be used very effectively for the identification of phenol or e,m-, or p-cresol, if the unknown solution contains only one of these compounds. If the unknown contains two or more of these substances, positive identification of each compound is not always possible; m-cresol, when present in mixtures in a low concentration, is particularly apt to escape recognition. I n analyzing for these compounds, even though each substance gives very similar color reactions in high and in low concentrations, it is best to prepare and test dilutions that approach the ranges of sensitivity. These concentrations will furnish in addition some rough idea of the quantities present. The phenol used was Merck’s reagent quality, and the 0-,m-, and p-cresol, obtained from the Eastman Kodak Company, was believed to be from 96 to 98 per cent pure. The melting points of these cresols are 30-31”, 10-1lo, and 32-34” C.,respectively. QUALITATIVE TESTS
DETECTIONOF PHENOLAND DERIVATIVESCONTAINING PHENOL-HYDROXY GROUPING.The sensitivity of Millon’s test ( 7 ) depends to some extent upon the quantity of mercury
used and the manner of preparing the reagent. For these studies the latter was prepared by dissolving 497 grams of mercury in 700 ml. of nitric acid (sp. gr. 1.42) and diluting this solution with 2 volumes of water. One milliliter of the test solution is added to 2 ml. of Milton’s reagent. Phenol and a-cresol when present to about 1.0 mg. per ml., and m- and p-cresol when present to about 0.5 mg. per ml., produce a red color almost immediately a t room temperature. When reduced to about 0.05 mg. per ml., ea h of these compounds produces in the cold or on careful heating a straw-yellow color which is destroyed by further heating. MODIFICATION OF MELZER’SBENZALDEHYDE TEST (6) FOR DETECTION OF PHENOL A N D a-, m, AND p-CRESOL. Mix 1 ml. of the aqueous solution to be tested with 2 ml. of concentrated sulfuric acid (sp. gr. 1.84), and after cooling under the tap add 2 drops of benzaldehyde. Heat over a flame, cool, and add 10 ml. of water and 20 ml. of 40 per cent potassium hydroxide. The sensitivity of this test is about 1 mg. per ml. of solution. MODIFICATION OF GUARESCEI’S TEST( 4 ) FOR DISTINGUISHING PHENOL AND P - ~ R E S O L FROM 0- AND m-CREsoL. Add about 0.5 gram of solid potassium hydroxide and 3 drops of water to 3 ml. of a chloroform extract containing phenol or cresol, then warm and observe. Straw-colored (yellow) globules will rise and the potassium hydroxide and water layer will assume a yellowish tinge on warming if phenol or p-cresol is present, In the presence of oor m-cresol, the potassium hydroxide and the water layer will assume a pinkish or rose-red color. The sensitivity for each of these compounds is about 4 mg. in 3 ml. of extract. FERRIC CHLORIDETEST(8) FOR DEITECTION OF 0- AND p-
Table
I. Color Changes
PHENOL O-CRESOL CRESOL P-CREBOL Changes observed in 5 minutes after addition of aulfuric acid and benzaldehyde Cloudy olive
Cloudy orange
Cloudy yellow
Milky white
After heating Cloudy reddish-brown
Cloudy brownish-red
Cloudy Cloudy yellowish-brown brownish-green
After cooling and addition of water and potassium hydroxide Blue or violet solution and precipitate
Colorless or faintly tan colored solution and precipitate
38
Vol. 16, No. 1
INDUSTRIAL AND ENGINEERING CHEMISTRY
CRESOL. Add 2 drops of a freshly prepared 10 per cent aqueous solution of ferric chloride to 5 ml. of the test solution. Phenol and m-cresol produce clear bluish-purple colors, ocresol produces in about 10 minutes a slightly cloudy urine yellow or brown solution, while p-cresol produces a cloudy blue solution. The sensitivity for each compound is about 10 mg. in 5 ml. MODIFIC.4TION O F LIEBERMAKN'S TEST( 5 ) FOR DETECTION OF CRESOL. To 3 ml. of the unknown aqueous solution add slowly and with shaking 1 ml. of the reagent (6 per cent solution of sodium nitrite in concentrated sulfuric acid). A cloudy orange solution develops in about 5 minutes if p-cresol is present. Phenol and m-cresol yield clear, and o-cresol very slightly cloudy brown or yellowish-brown solutions. The sensitivity of this test is about 5 mg. in 3 ml. MODIFICATION OF COTTON'S TEST(1) FOR DETECTION OF' pCRESOL. To 3 ml. of an aqueous solution, add 1 ml. of concentrated ammorliam hydroxide (sp. gr. 0.901) and 4 drops of the freshly prepared reagent (10 ml. of concentrated hydrochloric acid and 0.5 gram of potassium chlorate added to 40 ml. of water). Phenol and 0- and m-cresol produce in 5 to 10 minutes clear light blue colors; p-cresol produces a clear light straw-yellow color. The sensitivity is about 10 mg. in 3 ml. of solution. HYPOCHLORITE TESTFOR DETECTIOK OF O-CRESOL.To 5 ml. of the test solution, add one drop of sodium hypochlorite solution. In the presence of o-cresol the solution will immediately turn a cloudy yellowish-n-hite; in the presence of phenol, and wa- and p cresol, it will remain clear and colorless. The sensitivity k about 4 mg. in 5 ml. of solution. (Excess of hypochlorite must be avoided because it may produce faint cloudiness with p cresol.)
Stability
OF
DISCUSSION
It is reasonable to assume that all reagents discussed in this paper will also react with some compounds other than phenol, or 0-,m-,or p-cresol. Therefore one must make certain that the test solution is comparatively free from compounds related to phenol or cresol. This may require preliminary precipitation, extraction, or distillation procedures. Millon's test, even though it makes specific differentiation between phenol and the three cresols difficult, is of value because of its simplicity, as a preliminary test. This should be followed by the modified tests of Melzer and Guareschi, which will identify the compound. The conclusions drawn from these latter two tests may be checked by the hypochlorite and ferric chloride tests or by the modified procedures of Liebermann and of Cotton. LITERATURE CITED
(1) Cotton, S., BUZZ.soc. chim., 21,8(1874). ( 2 ) Deichmann, Wm., and Schafer, L. J., Am. J. Clin. Path., 12,129 (1942). (3) Deickmann, Wm., and Scott, E. W., IND.ENG. CHEM.,ANAL. ED.,11, 423 (1030). (4) Guaresclii, cited by H. Schiff in Correspondensen, Ber., 5, 1055 (1872). (5) Liebermann,C.,Ber., 7,248,1098 (1874). (6) Meher, H., 2.anal. Chem., 37,345 (1898). (7) Millon, M.E., Compt. rand., 28, 40 (1849). (8) Schiff, H.,Ann., 159,158(1871).
Standard Solutions OF Copper Perchlorate and Potassium Iodate JOSEPH J. KOLB Lankenau Hospital Research Institute, Philadelphia, Penna.
W
HENEVER sodium thiosulfate is used in titration work
of high accuracy frequent restandardisation is necessary. I n order to avoid the troublesome and wasteful necessity of preparing fur each standardization a fresh solution of a primary standard [iodine, potassium iodate (S),copper perchlorate (8)1, a standard in the form of a solution of a primary substance of dependable stability is desirable. The work reported here deals with the possibility of using potassium iodate or copper perchlorate (8) for such a purpose. In considering the stability of such solutions it is necessary to distinguish between changes due to chemical instability, presumably resulting in decrease of active concentration, and changes due t o evaporation from the container, which will cause increases of concentration. In the study of Berman ( I ) , for instance, on the stability of potassium iodate, it is impossible to distinguibh the role of these two factors. However, his data suggest thrct evaporation has been an appreciable factor in his results, and that, in some cases, an apparent stability has resulted from the opposing effects of evaporation and decomposition. In a recent paper (4) on the stability cf sodium thiosulfate solutions no account was taken of the possible effect ol' exgoration. A graphical study of the data on stability shows that in the first 60 days there is approximately a 0.3 per cent increase in normality. After that the values drop. Again two antagonistic tendencies, evaporation and decomposition, tend to produce a false picture of the stability of thiosulfate solutions. The evaporation factor can be eliminated if, a t the beginning of the experiment, samples of the solution to be examined are pipetted into separate flasks, and some of these are titrated a t once, while others are titrated after a suitable lapse of time. The extent of evaporation, on the other hand, can be measured by
suitable weighings of the vessels containing stock solutions. If the solution is chemically stable, it is then possible to calculate the theoretical normality a t any time from the initial normality and the loss of weight that has occurred. A P P A R A T U S AND REAGENTS
A 50-cc. buret and one 10-cc. pipet were carefully calibrated and were used throughout the experiments. Details of the preparation and use of the copper perchlorate and potassium iodate are given in (2) and ( 3 ) ,respectively. EXPERIMENTAL
All titrations were carried out in duplicate. The amount of active substance present in solutions when they were fresh, and after they had stood for various lengths of time, was always determined by titration with thiosulfate (0.025N), newly standardized against two freshly prepared cupric perchlorate solutions (8). ~~
Table
~
1. Stability of O.1N Copper Perchlorate and Potassium Iodate Solutions
Solution Cu(C1OSr
KIOI
5
b
Days 6r~1,d.n: 565 565 289 454 565 565 565 289 454
Normality Initial Final" 0.1007 0.1007 0.0993 0,0993 0.1027 0.1027 0,1027 0.1007 0.1007
0.1002 0.1004 0.0992 0.0993 0.1021 0.1013 0.0983 0.1002 0.1004
Conditions Thymoi, glass atoppers Glass stoppers Cork stoppers, spore6 on corks Glass stoppers Thymolb, glass stoppers Cork stoppers, spores on corks
Final normality calculated on basis of its initial volumo. Only one sample; others are average value found for two samples.
