The results of the titration of a range of microgram amounts of hydrogen peroxide a t p H 3.0 are summarized in Table 11. Down to submicrogram levels, the mean error was about 0.1% and the average deviation was about 1%. With samples smaller than 1 pg. (0.05 peq.) the error increased to about 4y0 with an average deviation of 5 to 6%. I n titrations a t low current, the end point break was rounded and results became less precise. The titration of 0.05 peq. was also performed using platinum wire indicating electrodes. The noise level was reduced but the current response was much smaller. The accuracy using the platinum wires was slightly better than xhen platinum foils were used.
The author thanks William C. Purdy for suggesting this problem. He is indebted to Richard E. Wolf for technical assistance. LITERATURE CITED
(1) Bard, A. J., Lingane, J. J., Anal. Chim. Acta 20,463 (1959). (2) Bradburv, J. H., Hamblv. A. N.. Australian ’ J . Sci. ’ Research“’A5. 541 (1952). ( 3 ) Christian, G. D., Microchem. J . 9, 16 f1965).
(4)Christian, G. D., Purdy, W. C.,
J . Electroanal. Chem. 3, 363 (1962). Kolthoff, I. M., Sandel, E. B., “Text-
book,, of Quantitative Inorganic Analysis, 3rd ed., p. 600, MacMillan, New York, 1952. (6) Malmstadt, H. V., Pardue, H. L., ANAL.CHEM.32,1034 (1960).
(7) Mattenheimer, H., “Mikromethoden Fur Das Klinish-Chemisch Und Biochemische Laboratorium,” p. 9, Walter De Gruyter & Co., Berlin, 1961. (8) Ramsey, W. J., Farrington, P. S., Swift, E. H., ANAL.CHEU.22,332 (1950). (9) Rowley, K., Swift, E. H., Ibid., 26, 373 (1954). (10) Sakurai, H., Kagyo Kagaku Zasshi 64,2119 (1961). ( 1 1 ) Takahashi, T., Sakurai, H., Talanta 9.189 (1962). (12j Tutundzic, P. S., Paunovic, M. M., Anal. Chim. Acta 22,201 (1960).
GARYD. CHRISTIAN^ Division of Biochemistry Walter Reed Army Institute of Research Walter Reed Army Medical Center Washington, D. C. 20012 Present address, Department of Chemistry, University of Maryland, College Park, Md.
S pe ct ro phot o met ric Dete r min a ti o n of LevuIinic Aci d with Hy d razi ne SIR: Phenylhydrazine and its 2,4dinitro analog are not suitable for the gravimetric determination of levulinic acid because of the appreciable solubility of the hydrazones and because the reagents tend to precipitate gummy materials from impure levulinic acid solutions. From a spectrometric standpoint, the absorbance of the reagent would be expected to interfere with that of the derivative. Hydrazine itself has a low molar absorptivity so that if it formed a strongly absorbing derivative quantitatively, it would be most suitable. Hydrazine was reacted with levulinic acid, and the result exhibits a strong maximum at 242 mw. On the basis of this finding, it was possible to determine extremely small amounts of levulinic acid with good precision and without preliminary isolation. EXPERIMENTAL
Apparatus. T h e absorbances were measured with a Beckman DK-2 spectrophotometer using 1-cm. cells. Reagents. HYDRAZINE MONOHYDROCHLORIDE, 50% SOLUTION. This was prepared from t h e commercially available salt or by neutralizing hydrazine hydrate with the appropriate amount of hydrochloric acid, with cooling. LEVULINICACID. Molten levulinic acid was cooled and allowed to crystallize slowly until about one half of it was solid. The melt was separated and the process repeated on the crystals. The recrystallizations were then carried out in carbon tetrachloride containing 7% chloroform, using Korite at first. The resultant colorless hygroscopic crystals were considered pure and used as a standard. 1420
4,5 - DIHYDRO - 6 - METHYL - 3(2H) This was prepared according to Poppenberg (4) and recrystallized from benzene. The long colorless needles melted at 105’ C. and became opaque on exposure to air because of the formation of the monohydrate. Procedure. A sample containing 10-200 mg. of levulinic acid in a 20 x 150 mm. test tube is treated with 1.0 ml. of 5070 aqueous hydrazine monohydrochloride and enough water t o make about 10 ml. T h e mixture is heated in a boiling water bath for 30 minutes and then diluted to at least 1 liter. Further dilution may be necessary t o bring t h e absorbance within t h e optimum range for the spectrophotometer. T h e p H of the final solution is adjusted to 1-2 with concentrated hydrochloric acid. A blank is prepared containing an equal amount of sample and hydrochloric acid but no hydrazine. It is diluted the same as the sample, and the absorbance, A , of the sample, when run against the blank, is determined at 242 mp. Then: PYRIDAZINONE.
0.00156 X A = levulinic acid, grams/100 ml. of final solution RESULTS A N D DISCUSSION
The outstanding feature of this method is that the levulinic acid does not need to be isolated or partly purified before being determined. The use of the increase in absorbance over that of the blank, and the high dilution employed, allows the determination to be carried out on extremely dark, impure solutions. Reagent Absorption. T h e molar absorptivity of levulinic acid at 242 mp is only 20, so its presence in the blank does not interfere. The molar
absorptivity of hydrazine a t this wavelength is 43, but in strongly acid solution (pH 1-2), it falls to about unity. Because of this dependency on pH, and to minimize the absorption due to the large excess of hydrazine used, the absorption measurements are made on strongly acid solutions. Even so, with the amount of hydrazine used, it is necessary to dilute the reaction mixture to at least 1 liter to reduce the absorption due to the hydrazine salt to less than 1-270, which is about the limit of accuracy of our instrument. These precautions are particularly important when working in the range of 1-20 mg. of levulinic acid, where the hydrazine excess is about 100-fold. Probably the method could be extended to lower levels of levulinic acid by using double cells to keep hydrazine and levulinic acid separate in the blank, but this approach was not pursued. The absorption curve of the pure reaction product of levulinic acid and hydrazine was scarcely affected a t all by strongly acidifying its aqueous solution. Mineral Acid Concentration. With hydrazine alone, the reaction did not proceed to completion. I n t h e presence of mineral acid it did, but low values were also obtained with too much acid. A satisfactory amount of hydrochloric acid corresponded to t h a t in hydrazine monohydrochloride, which was used. The p H a t the beginning of the reaction could vary from 3 to 7 without significantly affecting the results. Time and Temperature. T h e reaction proceeded to completion in 30 minutes in a boiling water bath. Longer time (up to 1 hour) and lower temperature (70’ C.) did not seem to be harmful, b u t the boiling water bath was most convenient. Very little reaction occurred at room temperature in 30 minutes.
Determination of Levulinic Acid
Found - -
0.0111 0.0444 0.1110 0,2220 5 ml. cirude NO. 5 -t 0.0444
1:1000 1:4000 1:10,000 1:20,000 1: 5000 1:5000
2 3 4
Absorbance at 242 mp 0.706 0.711
70 of theory
Gram 0.0110 0.0444 0.1119 0,2212 5.5" 14.5"
0.717 0.709 0.35 0.93
99.0 100.0 100.8 99.6 100; 7
Mg./ml. of original sample.
Concentration. T h e final diluted solutions followed Beer's law. T h e y were stable for several hours, b u t their absorbance decreased appreciably overnight. T h e use of much more t h a n 200 mg. of levulinic acid in the sample is inconvenient because of t h e extensive dilution necessary t o keep t h e measured absorbance less t h a n one. An increased amount of hydrazine monohydrochloride reagent might also be required. Calibration. T h e analysis was r u n on pure levulinic acid t o determine t h e factor for converting absorbances t o concentrations in grams/100 ml. After this factor had been determined, five replicates of a solut'ion made u p t o contain 23.2 mg. of levulinic per milliliter were run. T h e average of these five results was 23.3 mg. of levulinic acid per ml. with a standard deviation of only 0.13 mg./ml. After synthesizing a pure sample of the hydrazine derivative of levulinic acid, its absorbance was measured and found to correspond exact1.y to that found for an equivalent amount of levulinic acid. Experiments 1 to 4 (Table I) show that the method is accurate to within 1% over the recommended range, when known solutions of pure levulinic acid are used. Application. Tlhe method was shown t o be applicable to impure solutions as follow;: h normal levulinic acid preparation from sawdust mas carried out (S), and the filtrate and washings were concentrated. The resulting solution was turbid and nearly black. Five milliliters of this solution were carried t,hrough the present analytical procedure and diluted 1: 1. Because the resulting solution was turbid, it was filtered, and a further dilution of 1 : 5 was carried out on an aliquot. X blank was prepared by diluting and filtering similarly. The result of the analysis was a value of 5.5 mg./ml. (Table I:, ?io. 5). Another 5 ml. of the original sample was then taken and treated with 2 ml. of a pure levulinic acid solution containing 2.22 grams/100 ml. This ivas an increase of 8.9 mg. of levulinic acid per ml. of the 5-ml. sample. Analysis of this mixture then showed 14.5 mg./ml. (Table I, No. 6). The calculated value is 14.4 mg./ml. Thus, while the absolute amount of levulinic acid in the impure sample is not known, the addition of a known amount of levu-
linic acid was determined within 1%. The fact that impure and dilute samples may be analyzed directly is one of the main advantages of this method. Interfering Substances. T h e reaction of levulinic acid and hydrazine has been studied before and proceeds according to t h e equation:
N Hz I
This cyclic hydrazide hydrazone (4,sdihydro-6- methyl - 3(2H)pyridazinone) melts at 104-105' C.; in air it forms a monohydrate, m.p. 83' C. The latter has the same composition as levulinic acid hydrazide, and Curtius ( 2 ) , who was the first to prepare the material, thought that was what he had. This misconception was corrected by Wolff (5) and again confused by Bennett (1). A pure sample of the material was readily prepared from levulinic acid and hydrazine, according to Poppenberg (I),and its spectrum measured. It was found to correspond exactly with the curves from the analytical procedure. The molar absorptivity at 242 mp was calculated to be 7460. Since the formation of cyclic derivatives with hydrazine is not limited solely to levulinic acid, it would be expected that other substances would interfere with the analysis. This was the case, but, fortunately, none of the interfering substances are normally associated with levulinic acid in appreciable amounts. On the other hand, because of the acidic nature of the reaction conditions, it would be expected that the analysis would be equally suitable for levulinic acid salts, esters, amides, etc., as for levulinic acid itself. This was verified for the case of n-butyl levulinate. However, a-angelica lactone, the internal anhydride of levulinic acid, gave only 67y0 of the value expected when it all converted to levulinic acid. ,&Angelica lactone, the conjugated isomer, gave only about 5y0of the theoretical value.
I n this type of differential analysis, i t is clear that a substance may interfere in two ways. It may be converted into a material absorbing strongly a t 242 mp or i t may itself absorb a t that wavelength, but be converted by the analysis into a less strongly absorbing substance. I n this case the blank will have more absorbance than it should have to compensate for impurity, The following substances were carried through the standard analytical procedure to test their interference, FORMIC, ~ E T I C and , SUCCINICACIDS. No interference. FURFURAL. A brown resin separated during the reaction and was removed b y filtration after the first dilution, There was no absorption a t 242 mp, and no interference. ETHYLACETOACETATE.This material gave an absorption curve almost identical with that from levulinic acid, The maximum was a t 237 mp with a molar absorptivity, e = 7400. It would be expected that any P-ketoacid would similarly interfere, since the formation of pyrazolones by reaction with substituted hydrazines is well known. ACETONYLACETONE. This material gave rising absorption from 260 to 220 mp with c = 1900 a t 242 mp, which is 25% of the value obtained when running levulinic acid. Hence, 1,4diketones would also be expected to interfere strongly, presumably forming dihydropyridazines. MESITYL OXIDE. This compound also interferes by having a more strongly absorbing blank than sample. 7-VALEROLACTONE. Rising absorption to beyond 200 mp would cause this compound to interfere only if its concentration equaled or exceeded that of levulinic acid. GLUCOSE.A peak a t 280 mp (e . = 255) and one a t 226 ( E = 1470) with the minimum a t 264 were shown by glucose. The e at 242 mp was 900 or 12y0 of that of levulinic acid in the test. Hence, if there is 10% as much glucose as levulinic acid, some interference will occur. However, the peaks at 226 and 280 mp serve as warnings and might even be used to compensate for the presence of glucose. LITERATURE CITED
(1) Bennett, C. W., J. Am. Chem. SOC. 50, 1748 (1928). (2) Curtius, Th., J . Prakt. Chem. 50,  522, 524 (1894). (3) Frost, J. R., Kurth, E. F., T a p p i 34, 80 (1951). (4) Poppenberg, O., Ber. 34, 3263 (1901). (5) Wolff, L., Ann. 394, 98 (1912).
WILBURL. SHILLING BRUCET. HUNTER Chemical Products Division Crown Zellerbach Corp. Camas, Wash. DIVISIONof Analytical Chemistry, 133rd Meeting, ACS, San Francisco, Calif., April 1958. VOL. 37, NO. 1 1, OCTOBER 1965