Determination of Iron
A
Study of the o-Phenanthroline M e t h o d SELMA L. BANDEMER AND P. J. SCHAIBLE
Chemistry Section, Michigan Agricultural Experiment Station, East Lansing, Mich.
A critical study was made o l the o-phenanthroline method. The rate of color development was influenced by the.order of, and time interval between, additions of reagents, temperature of solutions, type and amount of phosphate, and the length of time the solutions stood before being read in the photometer. If the reaction was adjusted with sodium citrate instead of acetate and the citrate was added aker the hydroquinone and o-phenanthroline a t temperatures above POo C., iron was completely recovered.
NG some nutrition experiments, the iron content of the D""1. rations and of individual yolks and whites of eggs was determined by the o-phenanthroline method ( 6 , 9 , 4 ) , In this method, the ashed materials are dissolved in dilute hydrochloric acid. Sodium acetate is added to aliquots to adjust the p H to 3.5, hydroquinone to reduce the iron, and o-phenanthroline to develop an orange-pink color. Iron in the form of ephenanthroline complex is evaluated by means of a photometer using a Corning No. 430 blue-green filter. Certain difficulties developed and a study of the method was therefore made. EXPERIMENTAL
The relative order in which hydroquinone and o-phenanthroline were added was unimportant. If solutions stood for 120minutes, the order of addition of reagents was not critical. The time that elapsed between the addition of the acetate and other reagents was of no consequence when iron salts by themselves were used. With solutions of ashed egg yolk and white, however, this time interval was important; the longer the interval, the less iron was determined. T o investigate the possibility that the phosphorus, which was present in the materials to an appreciable extent in proportion to the iron, might have caused these low results, ferrous sulfate and sodium pyrophosphate solutions were mixed, dried on the steam bath, and ashed overnight in the muffle. Only 34% of the iron was recovered. Other salts of phosphoric acid were then investigated in a similar manner. In Table I1 are given the data on the recovery of iron from solutions when these salts were present in an iron-phosphorus ratio similar to that of egg white. I n general, if the adjusting solution, whether acetate or citrate, was added first, poor recover. ies were obtained; these were even worse with ashed samples, When the acetate or citrate was added last, recoveries were com. plete in the case of unashed but not ashed materials. A possible explanation for the failure to determine the iron in the unashed solutions when the pH-adjusting solution was added first is that an iron-phosphate complex is formed which does not react with the o-phenanthroline. In the ashed samples, it is probable that an insoluble iron meta- or pyrophosphate is formed which does not dissolve in the dilute hydrochloric acid. Cowling and Benne ( 1 ) reported that if solutions of ferrous sulfate and sodium pyrophosphate were heated in a water bath
The addition of acetate to solutions of the ash of the materials used frequently produced turbid solutions which could not be read directly in the photometer (a Cenco Sheard-Sanford Photelometer was used). Other acetates besides sodium were tried with the same result. Cowling and Benne (I), working with plant ash, overcame this by adding ammonium citrate before the p H was adjusted to 3.5 by acetate. It occurred to the authors that citrate might replace the acetate in adjusting the p H and thus eliminate the turbidity. T o check this hypothesis, aliquots Table 1. Effect of Order of Addition of Rea ents upon Iron of an acid solution of the ash of a poultry ration were adjusted to Determination in Solutions of.Ashed Egg Yolk, Egg White,. pH 3.5 with sodium acetate, sodium citrate, or potassium citrate and Poultry Ration Egg Poultry solution. The acetate gave a cloudy solution which required White, Rations, Egg Yolk centrifugation, whereas the citrates were clear. All three, howAcetate Citrate Citrate Citrate Order of Addition of Reagents 30 60 30 120 30 120 30 120 ever, gave correct iron values. The rate of color development 1st 2nd 3rd min. min. min. mm. rnin. min. min. rnin, with sodium citrate was the same as with the acetate but it was c P e r cent recoaerv of i r o n somewhat slower with potassium citrate. 100 95 92 100 0-P 77 84 75 81 HQ 100 74 91 96 89 100 0-P 83 89 HQ Since sodium citrate was more satisfactory than the acetate 94 100 99 78 91 100 100 0-P 99 A 95 100 100 99 82 83 A 100 100 for adjusting the pH, the range in p H over which it could be used 100 100 100 100 0-P 100 100 100 100 and still retain maximum color development was ascertained. 100 100 100 100 A 100 100 100 100 HQ To aliquots of ashed egg white, yolk, or poultry ration, 0.5 to A solution used to adjust to pH 3.5, HQ = hydroquinone. 22 ml. of a 25% sodium citrate solution were 0-P o-phenanthroline, added and the p H and iron determined. Maximum color development occurred above p H 2.5 Table II. Effect of Phosphates upon Recovery of Iron from Ferrous Sulfate Solution and it remained maximum to p H 5, which was (Fe:P 1:100, read after 30 minutes) as high as \\-as obtained with the amounts of Y S o d i u m Citrate Added-Sodium Acetate Added-citrate used. First Last First Last First Lust First Laat If the final solutions stood either 30 or 60 Phosphate UnushLd Unashkd Asheh Ashed Unashld UnushLd Ashed Aahed % % % % % % % % minutes before reading in the photometer, the 100 98 100 100 100 100 100 100 order of addition of reagents was found to be of 91 100 92 90 80 100 88 100 96 100 98 88 48 100 92 96 significance. Whenever the solution used t o 83 85 86 100 48 76 80 100 adjust the reaction to p H 3.5 was added first, as 76 82 100 40 52 88 72 100 96 97 100 88 88 60 100 100 in the Hummel and Willard procedure (S),or was 67 88 86 100 84 12 72 100 79 80 100 83 24 80 100 80 added second, results were inconsistent (Table I). 52 74 100 86 20 100 72 100 Both the acetate and citrate behaved in the same 71 95 100 90 100 72 48 100 52 80 88 100 ... ... ... ... way. If, however, both the hydroquinone and 55 69 100 90 ... ... ... ... 52 67 100 88 ... ... ... o-phenanthroline were added before the pH... adjusting solution, determinations checked well. 2 317
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CITRATE FIRST
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CITRATE LAST I
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Inasmuch as individual titrations to determine the amount of citrate required to adjust the reaction to p H 3.5 slow up the procedure considerably, it was decided to find out if an average volume of citrate could be used when samples of similar materials of about the same size are analyzed. A number of samples of egg yolk of approximately the same weight were ashed, dissolved in acid, and made up to the same volume. Two aliquot. of each solution were analyzed for iron. T o the first was added the amount of citrate required to give pH 3.5 as determined by titration; to the second, the approximate
can be found by titrating about a dozen samples and using the whole number of milliliters just greater than the average of these values. This can be done because the maximum color develops over a fairly wide pH range. Much labor, therefore, can be saved if an average volume of citrate is used instead of the varying volumes found by titrations when approximately equal samples of like materials are being analyzed. The procedure ~ t 9outlined below is simple and has been found satisfactory by the authors.
Cibitr d d i i either lint or Ins1
for one hour after the addition of the reagents, color developed conipletely in the presence of considerable phosphorus as pyrophosphate. But they added the acetate before the other reagents to unashed substances. The authors show in Table I1 that, if either acetate or citrate is added last to unashed materials, heating is unnecessary. In an additional experiment, it was determined that, with solutions of ashed materials, heating as long as 60 minutes on a steam bath did not produce maximum color REAGENTS when the pH-adjusting solution was added first; furthermore, the longer the interval between the addition of this solution and Iron Standard Solution (1 mg. of iron per ml.). Dissolve 1 the other reagents, the less the color developed. gram of electrolytic iron in 50 ml. of 10% sulfuric acid and dilute to 1 liter with distilled water. The effect of temperature of the solutions before mixing waa also investigated (Figure 1). At 14" C., less than half of the iron waa determined in egg white ash when the citrate was added Table 111. Comparison of Amounts of Citrate Obtained by first and over 80% when the citrate was added last. At 21' and Individual Titrations with Average of These Titrations 25' C.,about 75 and 85%, respectively, were found if the citrate (Based upon determination of iron in a series of egg yolk 8amDles IronFound was added first, whereas complete recoveries were obtained if Citrate Individual Average of the citrate was added last. At 31" C., practically complete reYolk Reaured titrationu titration Grams MI. Y Y coveries were obtained no matter whether the citrate waa added 3.39 5.7 40.5 41 . O first or last. These data were obtained from readings after the 6.0 3.41 39.5 39.2 33.5 5.4 3.45 33.7 solutions stood 30 minutes. If the solutions stood 120 minutes, 5.2 33.0 3.50 32.5 the order of addition of reagents was immaterial except a t 14' C. 5.5 35.5 3.51 34.7 43.7 4.7 3.51 44.0 Similar results were obtalned with egg yolk end a poultry ration. 6.0 30.7 3.53 37.2 4.6 34.0 3.66 33.5 From this it is apparent that the temperature of the room or of 5.4 3.58 48.0 47.5 5.6 the solutions is an important factor in the conduct of the pro3.59 51.2 51.0 4.9 40.7 3.67 41.0 cedure, particularly if the adjusting solution is added first. 5.1 45.2 45.0 3.81 T o find the relative effect of temperature and the amount of Av. 5.34 40.1 40.1 citrate necessary for proper pH upon the rate of color develop ment.. vanring - auantities of 1 to 4 hvdrochloric acid were added to aliquots of a socution of an ash from a poultry ration. These aliquots were adjusted to p H 3.5 with sodium citrate and the color was developed with hydroquinone and o-phenanthroline. Four different temperatures were used. If readings were made after 30 minutes, at any one temperature, the greater the amounts of citrate, the less the percentage of iron determined (Figure 2). For any given concentration of citrate, the lower the temperature, 4the less the percentage of iron determined. In 3 other words, those solutions that required large amounts of citrate t o produce a p H of 3.5 also z3required a higher temperature to produce maximum color. When the solutions stood 120 minutes, except at 14' C., neither the temperature nor volume of citrate affected the recovery of 7. I RON DETERMINED iron. If the citrate is added last, these complications do not arise. Figure 2. Effect of Temperature and Amount of Citrate upon Iron Determination I
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ANALYTICAL EDITION
May, 1944
Sodium Citrate Solution. Dissolve 250 grams of sodium citrate in distilled water and make up to 1 liter. Hydroquinone Solution. Dissolve 1 gram of hydroquinone in 100 ml. of distilled water. Store in refrigerator and discard if any color develo s. o-Phenanthroine Solution. Add 150 ml. of almost boiling distilled water to 0.5 gram of o- henanthroline in a 200-ml. up to volume. Store in volumetric flask. When‘cool, ma,! refrigerator and discard if any color develops. PROCEDURE
Pipet an aliquot of the unknown solution containing an iron concentration suitable for the range of the photometer into a 25-ml. volumetric flask, add 1 ml. of the hydroquinone, 2 ml. of the o-phenanthroline, and the proper amount of the citrate solution, and make up to volume. Let stand 30 minutes at a temperature above 20’ C. and read in the photometer, using a blank made fom the reagents in the same way for the 100 setting (this eliminates correcting for iron in the reagents). Use 1-cm. absorption cells and a 12.5-mm. No. 430 dark-shade blue-green Cornin molded glass filter. Convert readings into concentrations ofiron by referring to a curve made from the iron standard anlution in exactly the same manner.
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If the pH was adjusted before the introduction of o-phenanthroline, the rate of color development was influenced by such factors as the time interval between the addition of reagents, temperature of the solutions, type and amount of phosphate present, amount of citrate, and length of time the solutions stood before being read in the photometer. If the sodium citrate was added after the hydroquinone and o-phenanthroline at temperatures above 20’ C., these factors did not adversely affect the recovery of iron. Under these conditions, maximum color developed when the solutions stood only 30 minutes. For samples of similar materials of approximately the same size, i t was found expedient to use an average volume of citrate rather than to titrate each sample individually. The procedure for the c-phenanthroline determination of iron, modified as a result of the study is presented. LITERATURE CITED
(1) Cowling, Hale, and Benne, E. J., J . Assoc. Oficial Ag?, Chem.,
25,555(1942). Fortune, W. B., with Mellon, M. G., IND. ENG.CHEM.,ANAL. ED.,10,60 (1938). (3) Hummel, F. C.,and Willard, H. H., Ibid., 10,13 (1934). (4) SayweU, L.G.,and Cunningham, B. B.. Ibid., 9, 67 (1937). (2)
SUMMARY
Sodium citrate was found more satisfactory than the acetate in adjusting the reaction for the development of the color of the iron-o-phenanthroline complex.
JOURNAL Article No. 666 (n.6.) Michigan Agricultural
Experiment Station.
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Furf ural Determination Iodine Method for Hydrolyzed W o o d Liquors HUGH R. ROGERS, Department of Research and Development, Masonite Corporation, Laurel, Miss, A rapid method has been developed for the determination of furfural. It is based on the oxidation of furfural to furoic acid b y iodine in alkaline solution, and with aqueous solutions of pure furfural shows excellent precision and is highly accurate. With samples that contain other iodine-consuming constituents in addition to furfural slightly high results are obtained, but the precision is satisfactory. The method is well suited for control work in which accuracy is second in importance to the rapidity with which determinations may be made.
rompensate for their iodine consumption. Furfural in solution with the heads is determined by carrying out a blank reaction on each sample in slightly alkaline solution in which the iodine preferentially reacts with essentially all the heads or lower alcohols, aldehydes, etc., but with only 12.5% of the furfural present, the furfural being oxidized to furoic acid. Then by the regular sample reaction in a 1 N sodium hydroxide solution the iodine required by all the furfural and heads is found and thus the total furfural in the sample is calculated. DEVELOPMENT OF M E T H O D
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ECAUSE of the large number of furfural analyses required for control work in the author’s laboratory, a simple and rapid method with a reasonable degree of accuracy was desired. The rapid bisulfite method of Jolles (3) and the bromine method of Hughes-Acree (Z),both volumetric, were not practical, owing to the nature of the samples and the specific control conditions required by the methods. The gravimetric phloroglucinol (1) and 2,4dinitrophenylhydrazine ( 7 ) methods gave sufficiently accurate results, but were much too slow. The phloroglucinol precipitation method was found to be the most practical and was used until the development of the present procedure. A slight excess of iodine in strongly alkaline solution reacts quantitatively with furfural. In an approximately 1 S sodium hydroxide solution the hypoiodite, which is formed from the iodine, oxidizes the furfural quantitatively t o furoic acid. By a method bared on this reaction most of the difficulties found with the other methods have been eliminated. Pure furfurdl in aqueous solution can be determined accurately by this reaction. However, furfural used by the author is obtained from hydrolyzed wood liquors and is present in aqueous solution with lower boiling constituents which are termed “heads”. Owing to the presence of these heads in the furfural samples, it has been necesssry t o devise a method that would
REGULAR SAMPLE REACTION.Pervier and Gortner (6) first tried the use of iodine in alkaline solution as a method for determining furfural. Although they gave very few details of their work, they reported that the results could not be duplicated, Later Kline and Acree ( 4 ) also tried iodine in alkaline solution for determining furfural according to their method for determining aldose sugars (6). They gave no details of their work and reported only that negative results were obtained. Although very little information on the work of the previous investigators is given in the literature, it seems that their lack of succew was due mainly to use of too large furfural samples and too low an alkalinity. In the present work it has been found that the oxidation of furfural to furoic acid by iodine in alkaline solution is a function of the alkalinity. Approximately 100 mg. of pure furfural is as large a sample as can be oxidized quantitatively, regardless of the alkali concentration. With samples of furfural much larger than this, the oxidation is not quantitstive, apparently because of the rapid formation of iodate and iodide before the hypoiodite originally formed has a chance to react with all the furfural present. Figure 1 shows the effect of alkalinity on the oxidation of furfural to furoic acid by iodine when the reaction is carried out for 20 minutes a t room temperature in a reacting volume of
