ANALYTICAL CHEMISTRY.
112 ride values were 1.637’i lower while the E;:; X 2000 were 17.11% higher (except in two cases which gave atypical colors). On the unsaponifiable fractions the per cent deviations of the antimony trichloride values were 4.11% lower and the E;?&, X 2000 values 26.25Y0 higher. Since there is good agreement between the values obtained by the glycerol dichlorohydrin and antimony trichloride methods, and since activated glycerol dichlorohydrin possesses many advantages over the Carr-Price reagent, the use of activated glycerol dichlorohydrin in the determination of vitamin il in fish liver oils is recommended. ACKNOWLEDGMENT
The authors are indebted to Daniel Melnick of the Food Research Laboratories, R. H. Kreider of Mead Johnson and Company, and S. M. Gordon of Endo Products, Incorporated, who supplied the samples used i n this study.
LITERATURE CITED
(1) Gridgeman, N. T., “Estimation of Vitamin A”, pp. 11-19. Lever Bros. and Unilever Limited, Port Sunlight, Cheshire England, 1944. (2) Oser, B. L., Oil,Paint Drug R e p t r . , 139,4 (1941). (3) Oser, B. L., Melnick, D., and Pader, M., IKD.ENG. CHEM.. ANAL.ED.,15,717 (1943). (4) Ibid., 15, 724 (1943). (5) Oser, B. L., Melnick, D., Pader, M.,Roth, R., and Oser, M., Ibid., 17, 559 (1945). (6) Rawlings, H. W., and Wait, G. H., Oil and Soup, 33,83 (1946). (7) Sobel, A . E., and Werbin, H., IND.ENG.CHEM.,ASAL. ED.,18, 570 (1946). (8) Sobel, A. E., and Werbin, H . , J . Biol. Chela., 159, 681 (1945). (9) Twyman, F., and Allsopp, C. B., “Practice of Absorption Spectrophotometry”, 2nd ed., p. 57, London, Adam Hilger, 1934. (10) Vandenbelt, J. M., Forsyth, J., and Garrett, A., INP. ENQ. CHEM.,ANAL.ED. 17,235 (1945). (11) Wilkie, J. B:, J . Assoc. Oficial A g r . Chem., 28, 547 (1945). PRESENTED before the American Society of Biological Chemists, Atlantio City, X. J. This study was aided by a fund granted by the American Home Products Corporation, and by a grant from the Cnited Hospital Fund of the City of Greater New York.
Analysis of Mixtures of Mercurous and Mercuric Mercury and Sulfuric Acid BENJ. WARSHOWSKY AND PHILIP J; ELVING, Publicker Industries, Znc., Philadelphia, Pa.
A procedure is described for the determination of mercurous and mercuric mercury and of sulfuric acid in mixtures such as are found in used hydration catalysts of the sulfuric acid-mercuric sulfate type. Afercurous mercury is determined iodometrically, total mercury after cerate oxidation by thiocyanate titration, and sulfuric acid by neutralization in the presence of large amounts of chloride.
A
SOLUTION of mercuric sulfate in sulfuric acid is often used as a catalyst’ for the hydration of organic compounds, as in the addition of water to unsaturated carbon to carbon linkages. After some use, part of the mercuric salt is reduced to mercurous sulfate which is catalytically inactive. It is desirable, therefore, to be able to determine rapidly and simply the concentrations of mercurous and mercuric mercury in the catalyst solution, so as to know when the catalyst needs regeneration and the extent of regeneration necessary. Since the sulfuric acid concentration must be carefully adjusted for the particular hydration reaction used to obtain optimum yields, a rapid method for its determination in the presence of the mercury salts is necessary in order to keep the concentration of acid at the proper level. The solution of the problem was found by adapting known methods for the determinat,ion of mercurous and mercuric mercury, and devising a simple procedure for the determination of the sulfuric acid, I n a solution containing mercurous and mercuric mercury and sulfuric acid, the total mercury content’ is first determined by oxidizing the mercurous mercury to mercuric mercury with a sulfato-ceric acid solution and titrating the resulting mercuric ions with standard thiocyanate solution (2, 4 ) . The mercurous mercury is determined in another sample by oxidizing it to the mercuric iodide complex with iodine liberated from an excess of standard iodate solution and determining the excess iodine by titration with standard thiosulfate solution (1, 3 ) . Sulfuric acid is determined by titration with standard sodium hydroxide solution in the presence of excess sodium chloride. DISCUSSION
Willard and Young ( 5 ) describe a method for the determination of mercurous mercury in.the presence of mercuric mercury based
on the oxidation of the former in hot sulfuric acid solution by excess standard sulfato-ceric acid solution and titration of the excess electrometrically with standard ferrous sulfate solution. T h e authors of the present paper obtained poor and erratic results when the method was applied to synthetic mixtures. Since t h e values obtained depend on the reduction of ceric to cerous ions, any substance which is oxidized under the conditions used interferes with the determination; apparently, in the experiments made, some substance may have been present which erratically reduced ceric ions; Willard and Young indicated that large amounts of mercuric ion caused a slight decomposition of ceric ion in hot solution. Blank corrections were not determined. T h e possible presence of organic matter in actual catalyst samples was another reason for avoiding the use of a method for measurement which involved boiling with a n acid ceric solution for 30 minutes, HoTvever, the procedure employing oxidation with sulfato-ceric acid was used as a preliminary oxidation step in the determination of the total mercury content, sirace in this case the results Tvere not based on the consumption of the oxidant, Pure samples of mercurous sulfate were analyzed by the iodometric method (3) and a precision of 5 parts and an accuracy of 2 to 3 parts per thousand were obtained. The presence of mercuric sulfate did not affect the results. Since the consumpt’ionof iodine is employed as a measure of the mercurous mercury content, any substance which reacts with the iodine or the thiosulfate under the conditions used will give erroneous results. Accordingly, i t is necessary t o check the procedure in the presence of any organic sludge that may be formed in the hydration reaction and be present in the catalyst sample. I n many cases extraction with organic solvents or simple distillation can be used to remove interfering organic materials.
V O L U M E 19, NO. 2, F E B R U A R Y 1 9 4 7 Table I. Sample Weight Mg.
145.0 164.9 196.9 174.7 170.2 141,5 159.9 177.2 148.5 144,8
113
Determination of Total Mercury by Thiocyanate Titration after Ceric Oxidation Mercury Present Mercurous Mercuric Mg
.
Mg.
0 98.0 0 111.4 47.6 93.2 26.3 96.0 60.0 64.8 55.9 48.8 91.2 31.6 115.3 23.2 119.8 0 116.8 0 Av. Av. deviation h v . deviation of mean Standard deviation, u
Total M g.
98.0 111.4 140.8 122.3 124.8 104.7 122.8 138.5 119.8 116.8
Found Mg
.
RIercury Recovered
97.5 111.3 140.6 122.5 124.3 105.0 122.1 138.5 120.1 116.2 99.9 0.3 0.1 0.11
% 99.5 99.9 99.9 100.2 99.6 100.3 99.4 100.0 100.3 99.5
The difference between the total mercury content and the mercurous mercury found equals the mercuric mercury present. The difficulty in determining the acidity of a solution containing mercury salts is due to formation of hydroxy compounds which not only remove hydroxyl ions from slightly acid solutions but also act as buffers. Therefore, before the sulfuric acid content of a solution containing mercury salts can be determined by titration with a standard solution of a base, it is necessary either t o remove the mercury salts or to convert them to forms which will not appreciably affect the p H of the solution. An efficient method for accomplishing this purpose was the addition of an excess of chloride ions as sodium chloride. The mercurous ions were precipitated as insoluble mercurous chloride, while the mercuric ions were removed from action as t'he soluble mercuric chloride complex. The acid concentration could then be determined by titration with standard sodium hydroxide solution. REAGENTS
.
Standard thiocyanate solution, 0.1 N . Dissolve 9.7 grams of analytical reagent grade potassium thiocyanante in water and dilute t o 1 liter. Standardize against pure silver nitrate or standard silver nitrate solution, using the Volhard method. Standard iodate solution, 0.1 N . Dissolve 1.784 grams of carefully dried analytical reagent grade potassium iodate in water and dilute t o 500 ml., resulting in a 0.1000 N solution. Standard thiosulfate solution, 0.1 LV, Dissolve 24 grams of C.P. grade sodium thiosulfate pentahydrate and dilute to 1 liter with preboiled distilled water. standardize against the standard iodate solution. Standard base solution, 0.1 A7, Dissolve 4.0 grams of C.P. grade sodium hydroxide in water and dilute to 1liter. Standardize against analytical reagent grade potassium acid phthalate. For most accurate results, a carbonate-free base solution should be used. Ferrous solution, approximately 0.2 N . Weigh out 39 grams of C.P. grade ferrous ammonium sulfate hexahydrate and dilute t o 500 ml. with 0.1 N sulfuric acid. Ceric solution, approximately 0.1 AN. Dissolve 33 grams of anhydrous ceric sulfate in 30 ml. of concentrated sulfuric acid and dilute to 1 liter with water. Sulfuric acid solutions. Prepare two sulfuric acid solutions, (1) diluted 1t o 1with water and (2) 0.1 N . Chloride solution. Dissolve 10 grams of C.P. grade sodium chloride in 90 ml. of water. Indicator solutions. Customary indicator solutions of ferric sulfate (40%), methyl red, o-phenanthroline ferrous indicator, and starch. Potassium iodide, solid, C.P. grade. Mercury salts. Baker's C.P. analyzed grade mercurous and mercuric sulfates were used; analysis showed these salts to have the theoretical composition. Water. All solutions are prepared with distilled water, which is also used whenever water is mentioned in the procedures. PROCEDURE
Total Mercury. T o a sample of catalyst mixture containing approximately 0.1 gram of mercury salts, add 100 ml. of water, 20 ml. of 1 to 1 sulfuric acid, and 50 ml. of the ceric reagent.
Bring the resulting solution t o a boil and keep it a t the boiling
point for 20 to 30 minutes. This treatment dissolves any insoluble mercury salts and ensures presence of d l mercury in the mercuric state. Cool the solution and add 3 drops of o-phenanthroline ferrous indicator. Remove the excess ceric ions by adding from a buret ferrous sulfate solution almost to the equivalence point, so t h a t the solution retains a slightly greenish color and not the brown color of the reduced form of the indicator. Determine the mercuric ions present by titrating the solution with standard thiocyanate solution. Although the ferric ions present in the solution can serve as a n indicator, add 2 ml. of 40% ferric sulfate solution t o ensure a satisfactory end point. Th: temperature of the solution during titration must be between 15 and 20 O C. The end point of the solution occurs when the color of the solution changes t o a brown hue; the indicator blank is negligible and may be neglected. One milliliter of 0.1000 N thiocyanate solution is equivalent to 10.03 mg. of total mercury content of the sample. Near the equivalence point a precipitate of mercuric thiocyanate may appear but this does not affect the end point. The precipitate is due to the fact t h a t while mercuric thiocyanate is only slightly soluble a t room temperature (0.07 gram per 100 ml. of water a t 25' C.), it forms a soluble complex ion with excess mercuric ions; depletion of mercuric ions due t o conversion t o the complex may result in precipitation with a high enough mercury concentration. Mercurous Mercury. Transfer a sample containing approximately 0.25 gram of mercurous mercury to a 125-ml. glassstoppered Erlenmeyer flask. 4 d d 25 ml. of standard 0.1 AV iodate solution, 1 gram of potassium iodide dissolved in a few milliliters of water, and 10 ml. of 1 S sulfuric acid, stoppering the flask as soon as the acid is added. Shake the flask and its contents vigorously until the precipitate which first forms is completely dissolved. Remove and rinse the stopper with water, and immediately titrate the excess iodine with standard 0.1 N thiosulfate, adding starch indicator solution near the end of the t'itration. One milliliter of 0.1000 N iodate solution consumed is equivalent to 20.06 mg. of mercurous mercury present in the sample. Sulfuric Acid. Add 10 ml. of the 10% sodium chloride solution to a sample containing approximately 0.25 gram of sulfuric acid. Dilute the sample solution to a volume of 150 ml., and titrate with standard 0.1 N sodium hydroxide solution, using methyl red as indicator. DATA
Synthetic mixtures of mercurous and mercuric sulfate containing varying proportions of each were prepared and the total
Table 11. Determination of Mercurous Mercury by Iodometric Titration Sample Weight
Mercury Present .\I ercurous 11ercuric
lug.
lug.
M g
747.8 728.0 1064.8 995.4 533.9 585.7 347.0 424.7 270.3 191.9
505.4 492.0 511.9 503.0 178.0 186.0 65.3 61.5 0 0
0
.
Mercurous Mercury Found Recovered Mg.
1.4 2.0 249.0 203.2 218.1 250.0 201.9 275.0 216.9 155.5
0 247.9 202.5 218.1 251.1 202.0 269.2 218.1 154.8
70
..
:
100 4
100.3 100.0 99.6 . ~ 100.0 102.2 99.4 100.5
.
Av. 100.3 h v . deviation 0.5 Av. deviation of mean 0.2 0.47 Standard deviation, c (omitting 102.2 value)
Table 111. Determination of Sulfuric Acid in Presence of Mercurous and Mercuric Mercury sample Weight Mo. 435.5 429.5 444.5 445.5 428.5 437.5
Mercury Present Mercurous Mercuric
Present
iug.
1wg.
lug.
0
60.8 56.8 36.3 44.4 0 0
345.5 345.5 345.5 345.5 345.5 345.5
0 36.5 30.4 67.0 74.2
Sulfuric Acid Found Recovered Mg.
344.9 345.8 345.5 345.5 345.1 345.1 100.0 0.1 0.0 0.04
% 99.8 100.1 100.0 100.0 99.9 99.9
~
ANALYTICAL CHEMISTRY
114 mercury content was determined by the thiocyanate titration after the ceric oxidation as described. The results are shown in Table I. Table 11shows the results obtained by the iodometric procedure for the determination of mercurous sulfate in the presence of mercuric sulfate. Titration of the sulfuric acid in samples containing sulfuric acid and varying amounts of mercurous mercury, mercuric mercury, and mixtures of both showed that in the presence of excess chloride ion the volume of standard base solution required for neutralization of the acid was identical with t h a t used in the absence of mercury salts (Table 111). If sodium chloride was not added prior to the titration with sodium hydroxide solution, a definite color change or end point could not be detected, because of the hydrolysis of the mercury salts and the resulting buffering action. The presence of the chloride, by removing the mercurous and mercuric ions, permits the acid content to be measured with
an accuracy and precision of 2 parts per thousand or better. In preparing the synthetic samples the mercury salts were added to the solution cpntaining the measured amount of sulfuric acid which prevented hydrolysis. ACKNOWLEDGMENT
The authors would like to express their appreciation to Joyce E. Mandel for her aid with the experimental work. LITERATURE CITED
(1) Hempel, W., Ann. Chem., 110, 176H (1869). (2) Kolthoff, I. M., and Furman, N. H., “Volumetric Analysis”, Vol. 11,p. 442, New York, John Wiley & Sons, 1929. (3) Ibid., pp. 263-6.
(4) Kolthoff, I. M.,and Sandell, E. B., “Textbook of Quantitative Chemical .4nalysis”, rev. ed., pp. 480, 575, New York, Macmillan Co., 1943. (5) Willard, H. H., and Young, P., J . Am. Chem. SOC.,52, 567-9 (1930).
Hemoglobin in Meat Scraps and Tankage RAYMOND REISER Texas Agricultural Experiment Station, College Station, Texas The optimum concentrations of alkali, alcohol, and pyridine required for the solution of packing-house dried blood were studied and described. The extinction coefficients of the alkaline pyridine hemochromogen of hemoglobin of dried bloods were calculated from the hemoglobin content as determined by iron analyses and the density of the hemochromogen color in alcoholic solution. These ratios w-ere found to fall in two groups, depending on the time and temperature used to dry the blood. They are, therefore, indirect measures of the relative value of a blood meal as a feed. They may also be used as standards for determination of hemoglobin in meat and bone scraps, which, by definition, should contain no added blood.
A
CCORDIKG to the definitions of the Association of American Feed Control Officials ( I ) , meat scrap differs from tankage in t h a t blood is excluded from the former but not the latter. It follow t h a t the presence of blood in meat scraps “except in such traces as might occur unavoidably in good factory practice” constitutes adulteration. There is evidence, furthermore, that t,he biological value of blood meal protein for growth is low and its biological value, palatability, and digestibility decrease with increase in temperature of preparation ( 2 , 6 ) . There is need, therefore, of a method for the determination of blood meal in meat scraps and tankage. Since palatability and digestibility of blood meal decreases with increase in temperature of drying ( 2 , 6 ) , i t is desirable also t o have a measure of the heat treatment received by a sample of blood meal. A method for the determination of blood in packing-house byproducts has been reported from this laboratory ( 3 ) . The sample w?s extracted with ether and boiled in 1% sodium hydroxide solution. The mixture was added t o pyridine in a volumetric flask, made to volume with water, and filtered. A portion of the filtrate was reduced with sodium hydrosulfite and the increase in density a t 550 mp determined by comparing the reduced solution with the unreduced solution in a spectrophotometer. The average extinction coefficient of ten samples of blood meal was used as the standard, the results being given in terms of blood meal. Since publication of that work, it has been found t h a t a n alcoholic solut,ion has a number of advantages over a n aqueous solution. The dried blood dissolves more readily, a greater amount of color per unit amount of hemoglobin is produced, no loss of color occurs during boiling, and preliminary extraction of fat is unnecessary, since the alcohol dissolves the soaps which, in a n aqueous solution, froth during boiling and give a cloudy solution.
The absence of frothing permits refluxing a fixed volume of solvent containing pyridine and eliminates the necessity of making the solution to a final volume after boiling. The standard has also been reinvestigated. Variations in densities of hemochromogens of blood meal are so great that the validity of using the average figure described above is questionable. S o n e of the customary standards can be used. The oxygen capacity (5) obviously cannot be used on dried material and the iron method of Wong ( 7 ) gives low results with blood meal. Although total iron may be used for blood meal, i t is inadequate in the case of meat and bone scraps because a large part of the iron may be of nonhcmoglobin origin. Colorimetric methods based on acid or alkaline hematin or hemochromogens are also inadequate. Acid hematin is insoluble and cannot be precipitated in the proper colloidal dispersion from a solution of the dried blood. Attempts to use published alkaline hemochromogen methods were unsuccessful because i t was found that the extinction coefficient of blood meal is only about one third to one fifth that of fresh blood. It thus became necessary to develop a new method of determining the extinction coefficient of the hemoglobin in blood meal. This method is described below. EXPERIMENTAL
Conditions Affecting the Solubility of Blood Meal. The effects of alcoholic concentrations, alkalinity, pyridine, particle size, and boiling period on solubility of blood meal and on the intensity and stability of the resulting hemochromogen were studied. The criterion of solubility was the relative amount of hernochromogen color produced under conditions inadequate to dissolve the sample completely, all factors except the one being studied being
I
