Vol. 6, No. 1
ANALYTICAL EDITION
46
free from carbonate. To each aliquot in a flask add methyl red and a drop of hydrochloric acid just sufficient to turn it red. Then from the titration buret add hydroxide until the methyl red just turns a full lemon-yellow. Do not stop a t the so-called neutral tint, The solution will then be alkaline to hydrochloric acid, a t a pH of about 10, and will be practically neutral to phenolphthalein and boric acid a t about the same pH. The differences are well within the color indicator errors of titration. (Allen and Zies ( 1 ) working on borosilicate glass found a difference of 0.3 mg. Bz03 between the end point with p-nitrophenol and the beginning with phenolphthalein. The author has not been able to confirm this error using methyl red, although theoretically some alkali should be consumed.) To this neutralized solution add an excess of mannite and titrate to a permanent red with phenolphthalein, a color which is not bleached by further addition of mannite. If sufficient mannite is added a t the beginning, the solution will be nearly colorless until the final red. I n absence of excess mannite a pink color will maintain during much of
the titration and a true end point will be reached only when a single drop of the alkali gives a red that does not fade. Using 0.2 N alkali, titrations will habitually check to 0.1 cc. equivalent to 0.7 mg. of BzOs or less than 0.1 per cent on 1 gram titrated. Check analyses in this laboratory recently ran as follows: 38.80, 33.07, 29.67, 29.55, 44.61, 44.10, 41.01, 42.19, 42.07, 38.81, 33.22, 29.77, 29.62, 44.69, 44.25, 41.15, 42.19, 42.14, routine work. Two drops of 0.2 N alkali will always indicate an end point and the quantitative reaction between sodium hydroxide and boric acid in the presence of a polyhydric alcohol has never been brought into dispute, LITERATURECITED (1) Allen, E. T., and Zies, E. G., J . Am. Ceram. Xoc., 1,739-86 (1918). (2) Dodd, Analyst, 54,715 (1929). (3) Scott, W.W., IND.EXG.CHEM.,Anal. Ed., 4,306 (1932). (4) Sullivan and Taylor, IND.ENQ.CEIEM.,6, 897 (1914). (5) Wherry, J . Am. Chem. Soc., 30, 1684 (1908). RECEIVED July 24, 1933.
Determination of Iron Adaptation of the Mercaptoacetic Acid Colorimetric Method to Milk and Blood GLADYSLEAVELL AND N. R. ELLIS Nutrition Laboratory, Animal Husbandry Division, Bureau of Animal Industry, U, S. Department of Agriculture, Washington, D. C. HE mercaptoacetic acid colorimetric method of Lyons
T
(9) for the determination of small quantities of iron offers advantages over the thiocyanate colorimetric method of Elvehjem (3) as adapted from Kennedy ( 6 ) , in that smaller samples may be used, thus making wet-ashing practicable and avoiding the losses and contaminat,ions encountered in the dry-ashing procedure, and in that the color produced is more stable. The authors’ experiments confirmed observations of Andreasch ( I ) , Ginsburg and Bondzynski (4), and Claesson (2) on the formation of an intensely blue or purple compound by the reaction of mercaptoacetic acid (thioglycollic acid) with ferric chloride in ammoniacal solution, the tendency of the color to fade, and the ready restoration of the color when the solution is shaken with air. Preliminary experiments with a standard iron solution (0.005 mg. of iron per cc.) showed that for quantities of iron between 0.0025 and 0.025 mg., the colored solutions could be conveniently compared in tubes of uniform diameter, such as Nessler tubes, and that it was not necessary to dilute to volume, as the addition of distilled water to the solutions in the tubes caused no noticeable change in color intensities of the columns when observed through their entire depths. For quantities of iron from 0.015 to 0.1 mg., dilution to volume and the use of the colorimeter are recommended. Larger quantities of iron necessitate dilution and the production of color in convenient aliquots. Comparison of color-development by the use of 2 drops of undiluted mercaptoacetic acid, 1 cc. of a 1 to 15 aqueous solution of the acid, 1 cc. of a 1 t o 15 alcoholic solution, and 1 cc. of a 1 to 15 aqueous solution rendered alkaline with ammonium hydroxide (the reagent described in the next section) favored the last as it combines the advantages of nearly
eliminating the unpleasant odor of the mercaptoacetic acid with good control of the amount of reagent added. Experiments on wet-ashing of milk with sulfuric acid, sulfuric and nitric acids (8, IO), and sulfuric and perchloric acids (5, 7) were performed. The ashing with sulfuric acid alone was too slow, and with nitric acid resulted in muddiness and other irregularities in the colors developed, whereas the colors with perchloric acid were like those developed when standard iron solutions were used. METHOD REAGENTS.Iron-free concentrated sulfuric acid, 60 per cent perchloric acid, concentrated ammonium hydroxide, standard iron solutions, and the mercaptoacetic acid reagent are necessary. The standard iron solutions are prepared by dissolving 1 gram of pure iron wire in dilute sulfuric acid and oxidizing with concentrated nitric acid. The oxides of nitrogen and excess nitric acid are expelled by boiling. The solution is diluted to 1 liter, with further dilution as needed. I n all dilutions it is advisable to add extra sulfuric acid to prevent hydrolysis of the dissolved iron salts to insoluble basic compounds. The mercaptoacetic acid reagent is prepared by the addition of 4 cc. of mercaptoacetic acid to a solution of 8 cc. of concentrated ammonium hydroxide in 50 cc. of water. DETERMINATION OF IRON IN MILK. Samples* of 5 cc. of milk (or 0.5 gram dried milk) are digested with 3 cc. of con1 With samples of this size, there wa8 rarely any turbidity from precipitation of alkaline earth phosphates in the final ammoniaoal solution. Filtration of occasional turbid samples through small iron-free filter paper gave clear solutions with colom matohing those of the duplicates vhioh were not turbid. Filtration of very turbid solutions from larger samples pave entirely satisfactory results.
January 15, 1934
INDUSTRIAL AND ENGINEERING CHEMISTRY
centrated sulfuric acid and 2 cc. of perchloric acid in microKjeldahl flasks on an electric heater until colorless and the excess of perchloric acid has been driven off. The perchloric acid is added in 0.5-cc. portions, one after the sulfuric acid, the second to the cooled flask after the charring is quite decided, and the remaining portions if, and as, needed. The digestion requires from 30 minutes to 2 hours, When the digestion is complete, the contents of the flask are transferred t o a 150-cc. beaker, the flask is rinsed two or three times with small volumes of distilled water, the liquid cooled, and the acidity adjusted by the addition of concentrated ammonium hydroxide from a dropping bottle until just alkaline to litmus (a very small piece of litmus paper may be kept in the solution), then made just acid with concentrated sulfuric acid and an excess of from 5 to 10 drops of acid added. One cubic centimeter of the mercaptoacetic acid reagent is added and the solution made strongly alkaline by the addition of 1 to 2 cc. of concentrated ammonium hydroxide. The colored solution thus formed is compared with a series of standards in narrow tubes (12 X 20 mm.); therefore the volume should be kept below 20 cc. If the volume is too large for these tubes, other tubes of uniform diameter and appropriate capacity, such as Nessler tubes, may be used. TABLEI. RESULTSOF IRON DETERMINATIONS BY MERCAPTOACETIC ACID METHOD SAMPLE
VOLUME IRON ADDED IRON FOUND
cc Goat’s milk A Goat’s milk A Goat’s milk B Goat’s milk B Goat’s milk C Goat’s milk C Goat’s milk D Jersey COW’B milk Jersey cow’s milk Milk from local market
.
5 5 5 5 5 5 5 5 5 5
QTam
Dried miIk Dried milk Dried milk Dried milk
0.563 0.507 0.584 0.577
Hog’s bloodb Hog’s bloodb
0.020 0.01c
Hog’s bloodb
0.02d
cc .
Mg.
Mg. 0.0035,0.0035
0 665 0.008+
0.004--,0.0040 003 0.006540.003,0.0035 0 003 0.006 0.0035,0.003,0.003 0.0035,O.004 0:003 0.0065 0.0035 0.003.0.003 -
: :...
... ..... ....
... ... ... ... ...
0.0058 0.0056 0.0058 0.0059 0.01C0,0.0104 0.0050, 0.0053 0.0053,0.0052 0.0054,0.0052 0.0052 0.0100,0.0103 0.0104,0.0104
IRON^
Mg./-
100 cc.
0.07
0.06+ 0.08-
0.07+ 0.065 0.06
0.0063 0.075 0.07 0.06 1.03 1.10 0.995 1.02 51.0 52.3
... ... ...
52:s
Guinea pig’s bloodb 41.0 0.28 0.080,0.086,0.0S0 Guinea pig’s bloodb 0. If 0.045,0.045,0.045 45.0 Guinea -Die’s O.lf 41.5 0.02 0.06. 0.063 - bloodb a The added iron was subtracted before the calculation of mg. per 100 and the results are for 100 cc. excepting in the case of dried milk where they are for 100 grams. b See also section “Comparison with Other Methods.” Measured with blood.pipet. d 2 cc. of 1 to 100 dilution. e 2 00. of 1 to 10 dilution. f 1 CC. of 1 to 10 dilution.
The standards for color comparison are prepared by the substitution of 1 to 5 cc. of appropriate standard iron solutions for the sample to be analyzed. The entire procedure in the preparation of the colored standards must be identical with that for the sample. The standards occasionally fade within an hour; however, the color is satisfactorily restored if the solution is then shaken with air. Standards are often satisfactorily revived even after they have been allowed to stand for 24 hours or more. It is, however, advisable to replace some of the members of the aeries of standards every day to verify the values of the older aolutions. Table I gives results obtained by the application of this procedure to goat’s milk, cow’s milk, and milk powder. DETERMINATION OF IRON IN BLOOD. Samples of 0.01 or 0.02 cc. of blood are taken with the blood pipet, transferred with dilution to the micro-Kjeldahl flasks, and digested with 1 cc. each of concentrated sulfuric and perchloric acids. The remainder of the procedure is the same as described above.
47
Table I includes data for determinations on hog’s blood by this procedure, and by using 1-and 2-cc. portions of a 1 to 100 dilution, and also data from some earlier experiments with guinea pig’s blood in which amounts equivalent t o 0.1 and 0.2 cc. were taken from a 1 to 10 dilution. In the latter case, the final volume of the colored solutions was 25 cc. and the colors were compared in the colorimeter. COMPARISON WITH OTHER METHODS. Kennedy’s thiocyanate method applied to the sample of guinea pig’s blood, ranging from 0.1 to 0.4 cc., with and without added iron, gave results which when calculated for 1-cc. samples are 0.42, 0.39, 0.40, 0.395, 0.435, and 0.414 mg., respectively. The average is 0.412 mg., compared with an average of 0.423 mg. for nine determinations by the mercaptoacetic acid method, which are given in the table. Hog’s blood was used for a macrodetermination of iron by the digestion of 25-cc. samples with 60 cc. of concentrated sulfuric acid, dilution, reduction of the ferric iron to ferrous by pure zinc, and titration with standard permanganate solution. The iron found was 13.375 and 13.350 mg., respectively. Calculated for 100-cc. samples, the average is 53.75 mg., compared with 52.0 mg. for the mercaptoacetic acid method (Table I). OTHERAPPLICATIONS.This method was applied to eggs and to feces. Because of the larger quantities of iron the procedure for milk was modified as follows: two-gram samples were used, the product of digestion was diluted to 50 cc., and 10-cc. aliquots were used for color development without the adjustment of the acidity prior to the addition of the mercaptoacetic acid reagent. Either tubes or the colorimeter may be used. SUMMARY The mercaptoacetic acid method for the determination of small amounts of iron has been adapted to the analysis of milk and blood. ’ Wet-ashing with sulfuric acid and perchloric acid has been found satisfactory and avoids the contaminations and losses encountered in the dry-ashing procedures. ACKNOWLEDGMENT The authors desire to express their appreciation to Paul E. Howe for advice during the progress of the work and to A. K. Besley for the samples of milk. LITERATUBE CITED Andreasch, Rudolph, Ber., 12, 1391 (1879). Claesson, Peter,lbid., 14,412 (1881). Elvehjem, C. A., J. Bid. Chem., 86, 463 (1930). Ginsburg, J., and Bondzynski, S., Bet-., 19,116 (1886). Henriques, Valdemar, and Roche, Andree, Bull. SOC. chim. biol., 11, 679-92 (1929). (6) Kennedy, R. P., J. Biol. Chem., 74, 385-91 (1927). (7) Lematte, L., Boinot, G., and Kahane, E., J. pharm. chim., 5, (1) (2) (3) (4) (5)
325-31, 361-73 (1927).
( 8 ) Lorber, Leo, Biochem. Z., 181,391-4 (1927). (9) Lyons, Edward, J. Am. Chem. SOC., 49,1916-20 (1927). (10) Neumann, A., Z. phyaiol. Chem., 37, 115 (1902); 43, 32 (1904). RECEIVED November 18, 1932.
JAPAN TO MANUFACTTJR~ SILICONCARBIDE.Large-scale production of silicon carbide is proposed by Japanese interests, according to the Department of Commerce. Last ear Japan’s imports of “mineral substance for grinding or polistin amounted to 3120 metric tons, valued at 939,000 yen, of whicfihe United States supplied approximately 50 per cent. Annual consumption of silicon carbide in Japan is approximately 1000 metric tons. Two Japanese companies have started production on a commercial basis, and it is expected that their combined output will materially reduce imports of artificial abrasives.
