1014
ANALYTICAL CHEMISTRY
graphic photometry, intensity ratio evaluations, and background correction followed standard practices (9). T h e steps of the sectored spectrograms showing optimum exposure intensities were measured and the results averaged to reduce random photographic errors. Background corrections were made if the background per cent transmittance was less than 95. The analytical curves obtained are shown in Figure 5 as composite log-log plots. I n view of the general stability of t h e intensity ratios under changing excitations, these curves probably could be employed directly for semiquantitative purity determinations in other laboratories if samples are excited under comparable conditions. A summary of wave lengths of the analytical line pairs, background correction information, concentration ranges covered, estimated limits of detection, and precision data are summarized in Table I. T h e precision d a t a express the average per cent deviation from the mean of quadruplicate exposures of the standards on individual plates. ACCUR&C\
T h e nonexistence of other analvtical methods for performing these determinations precluded the comparison of analytical results as verification of t h e accuracy of the spectrographic results. As a r ~alternative, the accurarj- of the results was demonstrated by a series of recovery experiments M hich are sunimarized in Table 111.
ACKNOWLEDG\IENT
T h e authors wish to express their appreciation to F. H. Spedding and associates for providing the pure rare earths used in this investigation. LITERATURE CITED
(1) Ahrens, L. H., “Spectrochemical Analysis,” Chap. 6 and 7, Addison-Wesley, Cambridge, Mass., 1950. (2) Fassel, V. A., J . Opt. SOC.Amer., 39, 187 (1949). (3) Fassel, V. A., Cook, H. D., Krotz. L. C., and Kehres, P. W., Spectrochim. Acta, 5 , 201 (1952). (4) Fassel, V. h..and Wilhelm, H. A , , J . O p t . SOC.Amer., 38, 518 (1948). (5) Gatterer, d.,and Junkes, J., “Spektren der Seltenen Erden.” Specola Vaticana, Citta del Vaticano. 1945. (6) Harrison, G. R., J . Opt. SOC.Anrer., 39,522 (1949).
(7) Harrison, G. R., ed., “M.I.T. Wavelength Tables,” Wiley, New York, 1939. (8) XIoeller, T., and Brantley, J. C., ANAL.CHEM.,22, 433 (1950). (9) Nachtrieb, N. H., “Principles and Practices of Spectrochemical Analysis,” Chap. 6, McGraw-Hill, New York, 1950. (IO) Prandtl, W., and Scheiner, K., 2. anorg. allgem. Chem., 220, 107 (1952). (11) Rodden, C. d., J . Research Satl. Bur. Standards, 26, 557 (1941); 28, 265 (1943). (12) Smith, D. hl., and Kiggins, G. AI., Analust, 74, 95 (1949).
RECEIVED for review October 1, 1954. Accepted November 2 6 , 1954. Contribution No. 361 from the Institute f o r Atomic Research and Department of Chemistry, Iowa State College. Work was performed in Ames Laboratory of the Atomic Energy Commission. Paper VI11 in a series of papers on quantitative spectrographic analysis of rare earth elements.
Method for Direct Colorimetric Determination of Oxalic Acid JULIO BERGERMAN and JAMES S. ELLIOT Solano Laboratory, Berkeley, Calif., and Department of Surgery, Division of Urology, University of California M e d i c a l School, San Francisco, Calif.
Under suitable conditions, oxalic acid and indole react to form a red- or pink-colored compound which conforms to Beer’s law-. Photometric comparison with standards permits the determination of oxalic acid in concentrations as low as 0.050 mg. per ml. The method is simple and possesses a high degree of sensitivity, the average being within zt2qc.
REAGENTS
Indole Reagent. Dissolve 100 mg. of indole in 100 ml. of concentrated sulfuric acid. For the best results, this reagent should be prepared fresh daily. Solutions which have been prepared for 24 hours or longer before use will yield high blanks. Standard Solutions. Dissolve oxalic acid or sodium oxalate in 1N sulfuric acid ranging in concentration from 0 100 to 1.00 mg. of oxalic acid (H2C20a)per ml. PROCEDURE
I
N T H E course of a laboratory investigation pertaining to
urolithiasis, a search of the literature revealed no available method for the direct colorimetric determination of oxalic acid or oxalate ion. Known methods are indirect, and involve the conversion of oxalic acid to another substance which is subsequently determined. I n 1938, Paget and Berger ( 3 ) described a procedure in which oxalic acid is first reduced by metallic zinc in acid solution, and the resulting product determined colorimetricall\with phenylhydrazine. They noted that uric acid and allantoin were also reduced by pondered zinc to substances producing a color with phenllhydrazine. Calkins (I), in 1943, described ai1 indirect method of determination in which oxalic acid is first reduced to glycolic acid by powdered magnesium and sulfuric acid. Using 2.7-naphthalenediol. the glycolic acid is then determined colorimetrically. In the authors’ experience, accurate and reproducible results have been difficult to obtain with these indirect methods. I n the present paper a method is reported for the direct colorimetric determination of oxalic acid based on its reaction with indole. A similar reaction was described in 1899 by Gnezda ( d ) , who observed the formation of a pink-colored compound by a reaction between indole and oxalic acid.
Dissolve the unknown consisting of oxalic acid in a measured amount of 1 N sulfuric acid. Place a 2.0-ml. aliquot of t h e unknown containing from 0.100 to I .OO mg. per ml. of oxalic acid in a test tube. Place 2.0 ml. of each of the pure oxalic acid standard solutions in test tubes. T o prepare a reagent blank, place 2.0 ml. of LV sulfuric acid in a test tube. T o each tube add 2.0 ml. of indole reagent, allowing t h e reagent to run down t h e side of the tube to minimize heat development. Wait 60 seconds. Mix each tube thoroughly. Place in a water bath a t 80’ to 90” C. for 45 minutes. Cool and measure absorbance of each tube in a photometer with wave length a t 525 mp, setting t h e blank, consisting of indole reagent and 1N sulfuric arid, a t zero. T h e amounts given are suitable for m e in a NIodel DU Beckman spectrophotometer using 1.00-em. cells, and requiring a total volume of about 4.0 ml. for determination. Total volumes may be varied for use in other types of photometer so long as the 1 t o 1 ratio of indole reagent and standard solution is maintained. For example, if less accuracy is desired, total volumes of 5.0 ml. may be used and measured in a Klett-Summerson colorimeter using a green filter a t 540 mp. After complete color development, the colored solutions may be quantitatively diluted with distilled n a t e r or I N sulfuric acid for photometric romparison without loss of accuracy. EXPERIMEh-TAL
T h e reaction of the indole reagent and oxalic acid as described produces n pink- or red-colored compound depending on the con-
V O L U M E 27, N O . 6, J U N E 1 9 5 5
1015
centration of osalic acid. The reagent blank consisting of L\sulfuric acid and indole is yellow. T h e fully developed coloi. ij stable for 24 hours. -2fter this period there is c,onsiderahle increase in value of t'he blsnk. A spectral absorption curve of the colored compound obtaincd by the reaction hetlveen the indole reagent and oxalic acid in lA\snlfuric acid was determined on the Beckman Model DI. spectrophotometer in a cell of 1.00-cm. depth. Ilaximuni nbsorptiori w:i< found to occur a t 525 nip ( P W Figwe 1 ) .
400
500
600
r\ number of organic acids were tested for color development with indole. Formic acid x a s found to produce a pink- or redcolored compound. However, acetic, propionic, tartaric, citric. henzoic and uric acids in amounts of 10.0 mg. per ml. did not produce any color Xvhen treated with indole. Approsimately 10% of henzoic acid had not dissolved a t the close of the re:tction. Color interference was also investigated. Large amounts of chloride ion were found to produce a black turbidity of the clear pink solution. Standard solutions were prepared containing 1.00 mg. of osalic acid per nil. and amounts of sodium chloride ranging from 0.04 to 4.0 mg. per mi. S o interference was noted in this range. However, definite interference was noted with a ratio of 1.0 mg. of oxalic acid to 6.6 nig. of sodium chloride per nil. S o interference was noted n-ith relatively large amounts of phosphate. Monobasic potassium phosphate \vas added i n amounts of 0.40 to 8.0 mg. per nil. t o st,andards corit:titiing 1.00 m g . of oxalic. arid per nil. without interference.
700
WAVE L E N G T H ( m p )
Figure 1. Spectral absorption of colored conipound produced by reaction between indole and oxalic acid Concentration of oxalic acid. 0.25 mg. per nil.
()salic acid standards containing 0.100 to 1.00 mg. of oxalic acid per ml. dissolved iri 1 S sulfuric acid were prepared and treated with indole reagent. A curve conforming to Beer's lau- was obtained. T h e slope of the curve is indicative of the seiisitivity of the method (see Figure 2). AJ a n index of precision, standard solutions of osalic acid were prepared in triplicate and treated with indole reagent (sei' Table I). Per cent deviation from the mean absorbance reading varied from +4.3 to -4.0 with a n average of npprosimatel!. Lkt2.0. Concentrations of os:ilic w i d ranging from 0.050 to 1.00 nip. per ml., corresponding to the limits of accuracv of the spectrophntorneter. have been determined n-ith the pame precision
Table I .
~
0.2
0.8 mg. H2C1O4/ml. 0.4
46
1.0
Figure 2. Reaction of indole reagent and standard solutions of oxalic acid
Sulfuric acid has been employed in the method described because osalic acid is commonly separated as calcium osalate which is then dissolved in acid. T h e authors have employed l.\sulfuric acid because the use of higher concentrations of sulfuric acid-e.g.. 3 . i S sulfuric acid-results in less color development and reduced sensitivity. T h e concentration of indole in the indole reagent \vas chosen hecause it gave masinium color development with lovest blank value.
Ahsorbance Readings from Reaction between Indole and Oxalic AcidU D I S C U S S I O S A S D SU.1IhIARY
0 37:
0 82 1.8 1 "
The method descrihed has so far been applied only to osalic. acid standards and to aliquots of calcium oxalate dissolved in known amounts of 1N sulfuric acid. It permits the estimation of osalic acid in the presence of acetic, propionic, tartaric, citric. henzoic, and uric acids. Chloride ion, escept iri relatively high c,oncentrations, and phosphate do not interfere. T h e method allows the determination of osalic acid in coiicentrations ranging from 0.050 to 1.00 mg. per nil. Larger amounts niu\- be determined by quantitative dilation. LITER4TURE CITED
1
on
1 XI
1 . 1; 2.3
0.0 Three nieawrements were n':tde a t each conc,entration of oxulic acid. -
(I) Calkins, V. P., ISD. ENG.CHEM..~ A L ED., . 15, 76% (1943: (2) Gneeda, Julius, Compt. rend., 128, I584 (1899). ( 3 ) Paget, AIaicel. and Berger. Raoul, Ibid., 207,800 (1938).
