642
ANALYTICAL CHEMISTRY
These data shovi that a minimal temperature of 45” C. is necessary for the development of maximum color. and that the analysis of pure tryptophan requires an amino acid environment such as casein hydrolyzate, to prevent its destruction during alkaline treatment. Making use of these precautions, the results obtained with various materials are given in Table 111. Results would indicate that the modification of the method of Sullivan and Hess for the determination of tryptophan is adaptable to use with microquantities of protein. LITERATURE C l T E D
(1) Alderton, G., Ward, IT. H., and Fevold, H. L., J . B i d . Chem., 157, 43 (1944).
(2) Bates, R. W., Proc. Am. SOC.B i d . Chem., J . Bid. Chem., 119, vii (1937). (3) Block, R. J., and Bolling D.. “Amino Acid Composition of Proteins and Foods,” Springfield,Ill., Charles C Thomas, 1945. (4) Gunness, M., Dwyer, I. M., and Stokes, J. L., J . B i d . Chem., 163, 159 (1945). (5) Kunita, M., J . Gen. Physiol., 24, 15 (1940). (6) Swag, M .G., and Steers, E., Federation Proc., 7, 310 (1948). (7) Straus, E., Dingle, J. H., and Finland, &I., J . ImmirnoZ., 42, 331 (1941). (8) Sullivan, M. X., and Hess, IT. C., J . B i d . Chem., 155, 444 (1944)
RECEIVED April 13, 1948. Supported by a research grant from the Dilision of Research Grants and Fellowships, Sational Institute of Health, U. S. Public Health Service.
Spectrophotometric Determination of Iron in Ores with Kojic Acid J. P. MEHLIG AND M. J. SHEPHERD,
JR.
Ordcon State College, Corivdlis, Ore.
has pointed out that, in view of the very general MELLOX belief ’ that(8)colorimetric determinations are limited to maximuni concentrations of a few parts per million of the desired constituent, there is need for further study regarding the upper limit for reliable work viith modern instruments. This point of view deserves more attention than it usually receives. Some spectrophotometric work of this nature has been reported by the senior author and his co-workers (5-7). The purpose of the work described in this paper was to apply to the speckojic acid, 5-hydroxy-2-(hydroxymethyl)-l,4-pyrone, trophotometric determination of iron in ores in an effort to furnish further proof that such determinations are not limited to a micro scale, but are practical for macroquantities as well. The reaction between kojic acid and ferric iron to produce a yellom-ish-orange complex found its first analytical application in the colorimetric determination of the acid with ferric chloride (1, IO). Moss and Mellon (9) reversed this process, using kojic acid as the reagent for the colorimetric determination of iron, and made a critical spectrophotometric study of the effect of diverse ions and other factors upon the color system. Kojic acid is the first pyrone derivative t o be used as a reagent for iron. APPARATUS AND SOLUTIONS
A11 spectrophotometric measurements were made with a CencoSheard spectrophotelometer. All p H measurements were made with a Beckman p H meter. An aqueous solution containing 1 grain of kojic acid per 1000 ml., a commercial 3y0 solution of hydrogen peroxide, and an aqueous solution containing 250 grams of ammonium acetate per 1000 ml. were used. Standard iron solutions, containing 1, 2, 3, 4, and 5 mg. of iron per 1000 ml., were prepared by dissolving the calculated weights of 99.95% ferrous ammonium su1fat)ehexahydrate in water, and adding 25 ml. of 12 M hydrochloric acid and enough hydrogen peroxide to ensure complete oxidation of the iron to the ferric condition. The excess of peroxide was decomposed by boiling and each solution after cooling LYas accurately diluted to 1000 ml. R E F E R E Y C E CURVE
T o produce the color system 1 mI. of the appropriate standard solution of iron was carefully measured with a microburet into a 100ml. volumetric flask and 10 ml. of kojic acid solution were added, followed by 10 ml. of ammonium acetate solution to provide a pH value of 6.3 to 6.5. The reagents must be added in the order given; if the ammonium acetate solution is added before the kojic acid the reddish-brown color of ferric acetate interferes. The volume was then made up to 100 ml. .4 yellowish-orange color developed immediately. The transmittancy a t 440 mp (9)
was determined for each solution in a 1-em. cell. The entrance slit was set at a width of 2.5 mm. and the exit slit at 20 mp. T h e transmittancy was calculated by dividing the transmittancy of the standard solution by the transmittancy of the blank solvent. A reference curve was plotted correlating the logarithm of the extinction with the concentration of iron in milligrams per liter. No portion of this curve was a straight line. The deviation from a straight line was undoubtedly caused by the limitations of t h e instrument, as Moss and Mellon (9) have shown by using a General Electric spectrophotometer that the system does obey Beer’s. law for concentrations up to at least 20 mg. of iron per liter.
Table I. Sample To. 1 2 3 4
5 B 7 8 9 10 11
12 13
Results Obtained w - i t h Kqjic Acid Iron by Dichromate Xethod
Iron by I
