Colorimetric Determination of Iron .with Kojic Acid M. L. MOSS WITH M. G . MELLON Purdue University, Lafayette, Ind.
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I n preparing solutions for transmittancy meamrements the iron solution was measured and buffered with 10 ml. of the acetate solution. Ten milliliters of the rea ent were then added and the solution w m diluted to 50 ml. jddition of buffer preceded extraneous ions in determining the extent of interference from these sources. It is recognized that this sequence differs from the situation prevailing in an actual analysis, but it eliminates from the color measurements several undesirable effects, such as precipitation of the iron on addition of basic solutions.
N A recent paper (1) Barham mentioned that Corbellini
and Gregorini (2) and Tamiya (6) had studied the course of certain fermentations producing kojic acid by means of colorimetric determinations of the acid with ferric chloride. It seemed probable, therefore, that this process might be reversed by using the organic compound as a reagent for iron. As tests confirmed this prediction, a spectrophotometric study of the reaction was undertaken in the hope that this new colorimetric method for determining iron would have advantages over existing procedures. Kojic acid (2-hydroxymethyl-5-hydroxy-y-pyrone)is unique as a reagent for iron, in that no organic compound previously so used is a pyrone derivative.
Apparatus and Solutions A standard solution containing 0.05 mg. of iron per ml. was pre ared by dissolving iron wire of reagent quality in nitric acid anxdiluting with redistilled water. One milliliter of this solution is equivalent to 1 p. p. m. of iron when diluted to 50 ml. An aqueous solution containing 0.1 per cent of kojic acid was used as the reagent. More concentrated solutions are susceptible to mold formation on continued standing, although this may be inhibited by using 20 per cent ethanol as solvent or by adding 1 p, p. m. of phenyl mercuric nitrate. Standard solutions for the study of the effect of diverse ions on the color reaction consisted of alkali metal salts for the anions and of nitrates, chlorides, and sulfates for the cations. These contained 10 mg. of the ion in question per ml. of solution. A solution 1 M in ammonium acetate and 0.35 M in acetic acid waa used for buffering the iron solutions.
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Transmittancy measurements were made in 1.000-cm. cells with a General Electric recording spectrophotometer adjusted for a spectral band width of 10 mp. A glass electrode assembly (4) was used for pH measurements.
Color Reaction KATUREOF REACTION.Data obtained according to the method of Vosburgh and Cooper ( 7 ) indicate that the iron and kojic acid react in a molecular ratio of 1 to 3 a t a p H of 5 to 6. It has been shown that the hydroxymethyl group is not concerned in the color reaction, but that the enolic hydroxy is essential (9). This suggests formation of a salt with possible implication of the carbonyl group in a five-membered ring. An inner complex salt in accordance with the above ratio would be consistent with the coordination tendencies
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of iron, although such nonelectrolytes, as a rule, are insoluble (3). Other colored constituents may be present, especially in solutions of lower pH. With kojic acid, EFFECTOF REAGENTCONCENTRATION, as with ferron, the intensity of the color developed with iron depends upon the concentration of the color-forming reagent. This effect, shown in Figure 1, necessitates a relatively careful measurement of the quantity of reagent. Ten milliliters of 0.1 per cent solution are sufficient to produce a deep color with 10 to 20 p. p. m. of iron in 50 ml. and little is gained by using excessive amounts. Transmittancy curves EFFECT OF IRON CONCEKTRATION. for solutions containing from 0.5 to 50 p. p. m. of iron and 10 ml. of 0.5 per cent reagent in 50 ml. of solution are shown in Figure 2. A linear relationship between iron concentration and log transmittancy prevails, in accordance with Beer’s law, for concentrations up to about 40 p. p. m. A permanent Calibration curve may thus be established by determining the
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transmittancy for a given iron concentration and drawing a straight line through the determined point to 100 per cent a t zero concentration, on semilogarithmic paper. Conformity to Beer’s law should not be a critical consideration in evaluating a method, but it is an advantage in fulfilling the requirements for use of a variable-depth comparator of the Duboscq type. This law is not valid for solutions containing equal amounts of iron but different quantities of reagent. The concentration range over which the method is applicable depends upon the cell thickness and the means of comparison, One can distinguish 0.05 p. p. m. of iron from a blank in 30-cm. Kessler tubes. Comparison of amounts greater than 5 p. p. m. is difficult. Photoelectric instruments can detect 0.1 p. p. m. in a 1-cm. cell and the upper limit is about 20 p. p. m. The sensitivity is improved by acetone, but the effect is much less than in the thiocyanate method (8). The change of transmittancy with concentration is more pronounced than that of the trichromatic or monochromatic
values, as is usually the case in such systems. The red and green values are practically equal. EFFECT OF ACID CONCENTRATION. As acid concentration exerts considerable influence on the intensity and hue, it should be controlled within rather narrow limits. Strong acids or bases destroy the color, the change in basic solution being irreversible. Figure 3 represents solutions containing 10 p. p. m. of iron and 10 ml. of reagent in 50 ml. of solution at p H values from 1to 9. The orange hue a t p H 5 was stable for more than 5 weeks. -4low p H is desirable in order to avoid precipitation of iron, although the tendency to fade increases with the acid concentration. It is desirable to maintain the p H between 5.5 and 7 during the color measurements, since this range is best for achieving optimum color stability, small variation of transmittancy with pH change, and high absorption in the blue region. Ammonia may be added for adjustment of pH only after development of the color. Otherwise, precipitation of some iron is likely to occur. Ammonium acetate is a convenient buffering agent. EFFECT OF DIVERSEIONS (Figure 4). An outstanding disadvantage of colorimetric methods for ferric iron is the fact that certain ions react with the iron to form stable complexes which decrease the ferric-ion concentration. This is encountered especially with phosphates, fluoride, and several organic ions. Other types of interference include formation of colorless complexes by the reagent with various metals, precipitation of insoluble products, presence of colored constituents, and development of a color by the reagent with some ion other than iron. Reduction of part of the iron by certain ions may lead to low results for total iron, although this effect has no relation to the color reaction itself. No reagent free from all these kinds of interference has been reported for ferric iron. Ordinarily, the treatment of the sample is designed to circumvent interference by other substances as
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far as possible, but it is desirable to have a reagent which reacts specifically and quantitatively with the desired constituent. I n this study the effect of 500 p. p. m. of the ion in question was determined and, if interference was observed, smaller quantities were used until the change in transmittancy a t 440 mp was reduced to about 5 per cent. The extent of interference can thus be determined accurately in terms of iron concentration. An error of 2 per cent is not prohibitive for colorimetric methods.
TABLEI. EFFECTOF DIVERSE IONS Ion
Added as
Permissible Concentration P . p . m. 3 30 170 250 25 130 50
500 500 10 5 19 15 100 25 330 250 20 100 10 5 25 200 5 30 140
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S o effect on the color was observed in solutions containing 500 p. p. m. of each of the following ions: acetate, silver, barium, borate, benzoate, bromide, calcium, cadmium, chloride, chlorate, perchlorate, carbonate, formate, mercuric, potassium, lithium, magnesium, manganese, sodium, ammonium, nitrate, thiocyanate, sulfate, and strontium. Table I shows the maximum permissible quantities of various ions not already mentioned which may be present without causing a n error greater than 2 per cent. Reducing ions, such as cyanide, iodide, nitrite, and sulfite, did not interfere if added to the iron solution after addition of the acetate buffering solution. If the ion was added before buffering the solution a t pH 5 , however, low results mere obtained. Such ions would be oxidized in the normal treatment of the sample and thus present no difficulty. LOF results are caused by certain ions, including phosphates, fluoride, and a fen- organic acids. Citrate, oxalate, and pyrophosphate must be entirely absent. The extent of fluoride influence may be applied for determining this element, although the sensitivity is probably too low for the method to be practical in visual work. The relation between fluoride concentration and transmittancy of a solution containing 1 p. p. m. of iron and kojic acid is close to linear up to 100 p. p. m. of fluoride. A change in transmittancy of only 1 per cent corresponds to 6 p. p. m. of fluoride, however, IT hich would necessitate very accurate measurement of the color. Aluminum, zinc, and a few other metals form colorless complexes n ith the reagent, thus lowering its effective concentration and leading to low results. This source of interference cannot be eliminated satisfactorily merely by adding more reagent, since the color reaction is not stoichiometric. All highly colored compounds should be absent unless their
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absorption bands can be eliminated by proper setting of the monochromator.
Recommended Procedure Procure a representative portion of the material to be analyzed and subject it to the necessary preparative treatment, Weigh or measure by volume a quantity of sample containing 1 mg. of iron or less. TREATMEm OF SAMPLE. Ores may be dissolved in hot hydrochloric acid. Chlorostannous acid should not be added to hasten dissolution unless the solution is to be diluted to 1 liter or more. Organic matter combined with the iron necessitates the use of ashing procedures. After dissolution of the sample, oxidize the iron by boiling with a little concentrated nitric acid. Other oxidants, such as persulfate or hydrogen peroxide, may be used. Dilute the oxidized solution with iron-free Tvater and add 1 gram of ammonium acetate. Filter if the solution is not clear. Transfer the solution to a 100-ml. volumetric flask, add 10 ml. of 0.1 per cent kojic acid solution, dilute to the mark, and mix well. MEASCREMEKT OF DESIRED CONSTITWEST.The color, which is developed immediately, may be measured by the usual methods. Standards for visual comparison, prepared with the same acid concentration as the sample, may be kept for a week. If a filter photometer is used, the curves of Figure 1 indicate that the filter should be a blue or blue-green, such as Corning glasses Sos. 533, 554, and 429.
Summary A spectrophotometric study of the reaction between iron and kojic acid indicates that this compound is a suitable reagent for the colorimetric determination of ferric iron, Measurements were made on low-iron waters and on ores containing as high as 50 per cent of iron. Values for the ores agreed within a few tenths of a per cent with the titrimetric values and required somewhat less time. Although this method is not ideal, it is free of some of t h e interferences of other methods, and the sensitivity is adequate, especially with blue filters in a photometer. The color is relatively stable and conforms to Beer's law over a wide range. The chief limitation of the method is the necessity of controlling the acidity. d variation between pH 5.5 and 7 is permissible, and this range yields an orange hue suitable for visual comparisons. These limits are much wider than those for reagents such as ferron ( 6 ) . hlaximum sensitivity is obtained if the iron concentration is between 1 and 20 p. p. m. for a cell thickness of 1 cm. The color is somewhat less intense than that formed with thiocyanate or o-phenanthroline, thus permitting determinations on samples higher in iron than these other methods can accommodate n-ithout dilution. The method is not applicable to samples containing aluminum, citrate, oxalate, or pyrophosphate.
Acknowledgment
H. X, Barham of Kansas State College very kindly furnished the kojic acid. Literature Cited (1) Barham, IND.EXG.CHEM.,A s s L . E D . , 11, 31 (1939). (2) Corbellini and Gregorini, Gazz. chim. ital., 60, 244 (1930). (3) Diehl, Chem. R e t s . , 21, 39 (1937). (4) Mellon, "Methods of Quantitative Chemical Analysis", p. 413,
New York, Macmillan Co., 1937. (5) Swank and Mellon, I X D .ENG.CHEM.,SAL. ED.,9 , 406 (1937). (6) Taniiya, Acta Pnytochim. (Japan), 3, 51 (1927). (7) Yosburgh and Cooper, J . A m . Chem. SOC.,63,437 (1941). (8) Woods with hleliun, ISD. E N G .CHEhf., A N . ~ LE . D . , 13,551 (1941). (9) Yabuta, J . Chem. M C . , 125,575 (1924). ABSTRACTED from a thesis presented by Jf, L. Moss t o t h e Graduate School of Purdue University in partial fulnllment of the requirements for t h e degree of master of science, June, 1940.
