ANALYTICAL CHEMISTRY
1010 fied in this fashion, the improvement was not sufficient t o warrant the additional time required. If the Cellosolve is received in metal cans, it should be transferred t o glass containers to prevent contamination by the container. .4n electrolyte 1.5AVwith respect to hydrogen chloride was chosen in preference t o the I S solution employed by Hansen and coworkers. This choice was based on the fact that slightly low results occasionally obtained with the weaker acid solution seemed to be eliminated by increasing the acidity of the solution. Where a large number of samples are t o be processed in a relatively short period of time, it has been found desirable to prepare the Cellosolve-hydrogen chloride solution 4 N with respect t o the hydrogen chloride and store at 0’ C. As the supporting electrolyte is required, a portion is withdrawn and diluted with cool Cellosolve t o 1.5N. A 4AVstock solution prepared in this fashion is stable for about 3 months: beyond this time a slight decrease in the normality of the solution may be observed, but otherryise there should be no apparent change. Hansen, Parks, and Lykken ( 3 ) pointed out that their procedure gave erroneous results with some samples of gasolinee which had been blended with cracked material that had been stored for a period of time, and suggested that interference from peroxide compounds might be responsible. This hypothesis was confirmed, a t least in part, by substituting iso-octane containing known amounts of organic hydroperoxides or peroxides. During the present investigation it was found that such reduction waves could he eliminated entirely if water was added t o the Cellosolve-hydrogen chloride mixture following heating, and the aqueous layer employed for analytical purposes. Employing the procedure described, tests on samples of gasoline containing known amounts of tetraethyllead t o which various peroxide compounds had been added substantiated the desirability of adding water, and demonstrated that quantitative recovery of the tetraethyllead as lead could be assured. Subsequent evperiments proved t h a t this
modification did not introduce undesirable side effects from unsaturated hydrocarbons or other substances which are considered normal components of commercial gasolines. Moreover, the addition of water seemed to improve the consistency with which diffusion and residual currents could be measured. While this increased precision may be reflected in several ways, it is believed that by elimination of the hydrocarbon phase from the test solution the capillary tip is maintained in a more reproducible state, which assures a more consistent drop time. The failure of the half-wave potential to remain independent of the tetraethyllead concentration in the original Cellosolve procPdure has likewise been eliminated by the proposed modification. .4 large number of leaded gasoline samples have been analyzed and compared with the results obtained by the referee method of test (1, 2 ) . A portion of these data, covering a period of over 2 years (Table I), illustrate. the applicability of the modified Cellod v e procedure to petroleum testing. ITith the possible exception of gasoline samples I’ and TTI,the agreement of results by the two methods is extremely good. At the time this work was done, it was thought that the polarographic results for samples V and VI were in error; however, now nearly 3 years later, there is some ieason to doubt whether or not this conclusion is warranted. Recently it was found desirable to revise ASTM method D 526 ( I ) , because it gave known low iesults in some samples. The two Gamples in question may have been of a similar type. LITERATURE CITED
(1) din. Soc. Testing Materials, “Standards on Petroleum Products and Lubricants.” I S T l I Designation D 526-48T, S o r e m b e r
1950. ( 2 ) Ibid., Designation D 52B -5XT. November 1953. ( 3 ) Hansen, K. A . , Parks. T. D.. and Lykken. L., ANAL.CHEJI.. 22, 1232-3 (1950). RECEIVEDfor review .4ugust 30. 19.54,
Accepted December 30, 1954
Quantitative Spectrographic Analysis of Rare Earth Elements Determination of Holmium, Erbium, Yttrium, and Terbium in Dysprosium, Determination of Yttrium, Dysprosium, and Erbium in Holmium, and Determination of Yttrium, Dysprosium, Holmium, Thulium, and Ytterbium in Erbium VELMER A. FASSEL, BEVERLY QUINNEY, LAIRD C. KROTZ, and CARL F. LENTZ institute for
Atomic Research and Department o f Chemistry, lowa State College, Ames, lowa
Emission spectrometric methods are described for quantitative determination of rare earths commonly associated with purified dysprosium, holmium, and erbium. The concentration range from the detection limit up to 1% is covered by these methods. The procedures are based on direct current carbon arc excitation of rare earth oxide-graphite mixtures. The unique similarity in excitation behavior among many of the rare earths provides a high degree of internal standardization of variables inherent in direct current carbon arc excitation. An unusual example of self reversal in the Ho 3456.00 A . line is noted.
I
N PREVIOUS papers of the series (3, 4 ) , emission spectro-
metric methods for the quantitative determination of the rare earths commonly found as impurities in purified lanthanum, cerium, praseodymium, neodymium, and samarium were described. The present article is on the extension of the same basic method to the determination of the other rare earths commonly associated with purified dysprosium, holmium, and erbium. -4s in the pro-
cedures discussed previously, the methods described cover the concentration ranges from the detection limits up to about 1%. Impurity concentrations above 1% can usually be determined by spectrophotometric measurements (8, 10, 11). 4PP4R4TUS
The spectrograph, e\ternal optical system, excitation source, electrode assembly, photographic processing, and microphotometer employed in this investigation have been described ( 4 ) . EXCITATIOU CONDITIONS
The considerations involved in t h e selection of direct current carbon arc excitation of the samples in the form of rare earth oyide-graphite mixtures have been given in detail (2-4). Briefly stated, these factors were as follon s: the desirability of exciting the samples under conditions requiring a minimum of preliminary sample preparation, hence the choice t o employ the oxide form as obtained from niost flactionation procedures; the convenience and sensitivitj- of the direct current carbon arc for exciting the spectra of refractory oxides; the enhanced stability
V O L U M E 27, NO. 6, J U N E 1 9 5 5 ot the arc discharge on the addition of powdered graphite to the electrode charge; and the high degree of internal standardization obtainable from rare earth systems because of the unique similarity in excitation behavior among man\- of the rare earths. SELECTION O F AiXALYSIS LINES
Table I.
1011
Wave Lengths of Analytical Line Pairs, Concentration Ranges Covered, Estimated Limits of Detection and Precision
% Rare E a r t h Oxide Ai.. Dev. from Mean. Concn. range Limit of Line Pair, Background covered detection Impurity Corrections A. 52 Th T b 4318.85 Yes 0 2 -2.0 0.1 5.7 Dy 4319.21a 3.2 Tes or nob 0 . 0 2 -1.0 Y Y 3601.92 0.007 D y 3611.90 Yes H O Ho 345fi.00 0 . 0 2 -1.0 0.02c 1 2 D y 3443.46 Yes Er E r 4007,97 0 02 - 1 . 0 0.02 4.9 D y 4005.48a Ho Y Y 4374 94 NO 0 . 0 1 -1.0 0 00.5 3.1 Ho 4379.14d 1Tes or nob 0 . 0 1 -1.0 0.008 Y 3982.69 4.5 Ho 3966.86d Tes 0.0.5 - 1 . 0 0.01 D y 4221.10 5.8 D? Ho 4221.6Za El E r 3906.32~- Yes or no 0 . 0 1 -1.0 0.01c 4.8 Ho 3910.30d Er Y T 4371 9 4 NO 0.01 -1.0 0.0007 5.8 E r 4382.17 Yes 0 . 0 1 -1.0 0,005 Dy 3898.546.3 D? -~ E r 8891.65" Ho Ho 3456 00 5-0 0 01 - 1 . 0 0.005c 2.7 E r 3436.95d Tm T m 3362.61 NO 0 01 -1.0 0,001 4.5 E r 3328.30d Yb Yb 3987.99 K O 0.005-0.2 0 0001 5.9 Er77157d Tb T b 2470 .5iH so 0.01 -1.0 .,.. 2.9 E r 2986 (i9* " W a r e length measured by authors. b Background corrected curve used for determination at lower concentrations. and curves uncorrwted for baokground used in upper concentration range. C Quantitative measurement of intensity ratio necessary because of interference. d Wave lengths reported by Gatterer and Junkes (6). All other wa\.e lengths from ( 7 ) . Rare Earth Matrix D?
Since the spectra of the rare rarths under consideration in this paper are among the most complex of the rare earth group, the selection of analysis lines ip beset with the same basic problems discuvsed in previous papers. These are the location of interference-free analysis lines possessing adequate sensitivity for deterniining the impurity rare earths helow O . l % , and t,he differentiation of persistent lines of the impurity rare earths and weak unidentified lines of the matrix rare earth. Unless one is positive that the sample of rare earth is entirely free of the SUFpected rare earth impurity, this differentiation cannot be made direct,ly. .is outlined earlier ( 3 ) ,visual observations of line intensities and quantitative measurements of intensity ratios of selected line pairs in successive samples from ion-exchange column fractionations of highly purified rare earths provide t h e most definitive information for making thtxse differentiations. These observations indicate many erroneous spectral line identifications in t,he "1I.I.T. Wavelength Tables" ( 7 ) , especially in the spectra of erbium anti holmium. Some of these misidentifications have been recognized and tabulated by Gatterer and Junkes ( 5 ) ,and a few have been listed by Smith and Wiggins ( 1 2 ) . The number of lines found to he erroneously identified now total about 160 (a tahulation of these n-ill be presented in a subsequent communication.) Contrary to the listings in t h e 1I.I.T. tables, the erbium 4007.967 .i,analysis liiie is completely free of dysprosium interand ference, the dysprosium 3898.544 .4.,yttrium 4374.935 -4., ytterbium 3987.994 -4.analysis lines are completely free of orbium interferences, and dysprosium 4221.104 -4.analysis line lins no holinium interference, T h e analytical intensitj- ratios for all of these lines extrapolate t o zero intensity ratio for zero conccxntration of the respective impurities. lines selrcted (see Figure 5 and Table I ) present what is believed t o be the optimum compromise between maximum sensitivitj- of detection of the impurity rare earth and minimum spectral interference from lines of the matrix or other impurities whirh may he encountered in the samples. A few of the selected analj-sis lines are not completely free of interference from verj- weak lines of the matrix rare earth. The magnitude of the interference is so small, however, t h a t the chosen lines still represent maximum sensitivity of detection. The analysis lines falling in this category are holmium 3456.00 A . for the determination of holmium in dysprosium and erbium matrices, and erbium 8906.32 h.for the determination of erbium in holmium. Since constant analytical intensity ratios are obtained on repeated ion exchange fractionations of highly purified
materials, these interferences ai ise fi om very w a k lines of the matrix. Although erbium 3906.32 A. u a s ilelecttd for the determination of this element in pure holmium, erbium 4007.97 ;i.can be visually deterted to a slightl!, lower rrhiuni concentration. T h e erbium 4007.97 A. line, however, cannot be used for quantitative measurements becauqe of it. proximity to the holmium 4008.28 A. line.
Table 11. Operating Conditions for Analysis of Rare Earths Sijectrograpli Upper electrode (cathode) 1.11werelectrode (anode)
Jariell-.lsh 3.4-meter stigmatic grating spectrograph Graphite rod, l/s-inch diameter and 1 inch long, pointed a t one end Sliallow thin-walled grapliite electrode ('/,-inch diameter graphite witli 2-mm. deep cavity and wall thickness of I/? nini.) containing 15 nig. of 1 t o 1 mixture of ignited rare earth oxides and 200-mesh powdered grarjhite. .inode is dupported on '/pinch graphite pedestal .4nalytical gap 4 mni. D.c. arc, 2.50 volts, 17 t o 18 ampereExcitation source Length of expo.sure Sample arced t o complete consumption. See Figure 3 for time required for coinr>lete samlile consumption Emulsion Spectruni analysis S o . 1 13000-4800 .4.) Kodak 1\Ia (1000-5000 .4.) Slit width 0.04 nim. Wave-length region 2400-4600 A , , second order 4 minutes at 21° C. in Eastman Kodak D-19 with Development continuous agitation Den-itonietry .\*plied Research Laboratories Coinpalator-Densitonieter Eiiiulsion calibration Two-step sector, preliminary curve method Kodak A1 plates should not be confused with class RI spectral sensitivity. The Class B panchromatic sensitivity, high resolving power. and high contracrt of Kodak M plates make them very useful plates for analytical speotroscopy in \%a\e-length region between 4300 and 6700 4. -
~~~
~
~~
-
-
~
ANALYTICAL CHEMISTRY
1012 I n a few cases selection of several different malytiosl lines wm found to he desirable. For the determination of ytterbium in erbium, the most sensitive ytterbium line (3987.99 A,) showed excessive self reversal above 0.2% ytterbium; therefore, a weaker line (yb 2970.56 A,) showing less self-reversal was selected for the determination of ytterbium up t o 1%. For t h e determination of yttrium in holmium, the most sensitive holmium-free yttrium line (4374.94A.) is subject to interference from a dysprosium line when the dysprosium content is greater than 2%. When the dysprosium content is in this range, the alternative analysis line a t 3982.59 A. is used. UNUSUAL EXAMPLE OF SELF-REVERSAL
T h e holmium 3456.00 A. line used for t h e determination of holmium in dysprosium and erbium undergoes strong self-reversal in a most unusual manner. When the spectrum of holmium is
I
A
excited under conditions not conducive t o self-reversal, this line appears &s a broad line (Figure 1, A ) which even under high resolution conditions (Figure 2, A ) shows no detectable structure. (Echellogrms were obtained in the 625th order of a 200-lineper-inch Bausch & Lomb Optical Co. echelle, externally crossed (6) with a Hilger E492 Littrow quartz spectrograph.) When holmium is excited under conditions producing moderate selfreversal, the intensity of the line is decreased and hyperfine structure becomes apparent (Figure 2, B). Under excitation conditions conducive to strong self-reversal, fine structure is still detectable in the reversed portion of t h e line (Figure 2, C ) . Under t h e same excitation but moderate dispersion the line assumes the appearance of a pair of weak, sharp lines, separated approximately 0.25 A. (Figure 1, B). The unusually steep intensity contour of these lines is unlike the broadening generally observed in reversed lines emitted in arc and spark discharges. Thus, in spectrograms obtained under strong reversal conditions, the high intensity and reversed character of this line may pass unnoticed. The nature of this line-reversal is being investigated in an effort to provide an explanation for this phenomenon. SELECTION OF INTERNAL STANDARD LINES
The great similaity in physical properties of many of the rare earths offers unique opportunity to compenmte for uncontrolled excitation variables in a direct current carbon arc by in-
B F i g u r e 1.
S p e c t r o g r a m s showing H o 3456.00 A. line
A.
Mixture of 1 part of pure HoaOl to 20 parte of powdered graphite excited in 3- to 4 - a m w e direct cment carbon arc 8. Pure HolOl excited i n s conventional 10 ampere direot onrbon arc
Y 3601.92 Dy 361 I.90
0.5
Y 3982.59 H03966.85 Y 4374.94
Ho4379.14 E1390632 ~0391~30 Dy4221.10 Ho4221.62
Y 4374.94 Er4382.17
20
- ---
Dy3898.54 Er3891.65
1.0
H O 3455.70
Ho3456.00
F i g u r e 2. M i c r o p h o t o m e t e r recordings of eohellograms of Ho 3456.00 A. line A. Mixture of 1 part of pure Hoax to 20 parts of powdered
B.
graphite uoited in conventional 3- to 4-ampere direot current carbon are Mixture of 1 part of pure HosOa to 10 parts of powdered plaphife excited in conventional IO-ampore direct carbon
neir-reversau
C. Pure €lnlO, excited in oonventionJ IO-ampere direct carbon arc (strons self-rerersal)
0.I I
I
I
t
35
45
55
ti5
‘\
5
16
25
Yb3987.99 Er3977.57
TIME (SEC.) F i g u r e 3.
V a r i a t i o n of a n a l y t i c a l i n t e n s i t y ratios d u r i n g vaporization period Arrow denotes complete sample consumption
V O L U M E 27, N O . 6, J U N E 1 9 5 5
1013
I Ha3456.00 Dy3443 46 Er 4007 97 Dy4005.48 Y 3601.92
Dy3611 9 0 Tb4318.85 Dy43 19.21
s
-
I
0
0 7 t
n
"
"
Y 437494 Ho4379 14
0
Er 3906 32 Ho391030
D 4221.10 HZ4221 6 2
z
Id 0 4 t
t-
0
"
0
"
4
Y 3982.59 Ha 3966.85
~
k= -c -c -.-
Ho 3456 00 Er 34 36 9 5 Y 437494 Er 4382 I 7 Dy 3898 54 Er 3891 65
w \c 2 p -~ 2 -
> \
20
\
lo[
n
,
I
~
Yb3987 99 Er3977.57 Yb 2970.56 Er 2986.69
Tm 3362 6 I 0.5
Er 3328 30 6
8
IO
12
14
16
18
20
ARC CURRENT (AMPS) Figure 4.
Variation of analytical intensity ratios with arc current
ternal standardization (9-4). The relative constancy of analytical intensity ratios during the excitation period is a direct measure of the degree of similarity in volatilization behavior of the pair of elements. Figure 3 shows that in a few cases (thulium us. erbium, ytterbium us. erbium) significant differences in volatilization rates are evident, but that these differences are no greater than those usually encountered when direct current
carbon arc methods are applied to nonmetallic mixtures (1). For many of t h e line pairs (erbium us. dysprosium, holmium vs. erbium, holmium 21s. dysprosium, terbium us. dysprosium), virtually ideal similarity in vaporization behavior is indicated, and for the remainder, only minor selective volatilization is detected. Under these conditions, the selection of line pairs which respond similarly to excitation temperature fluctuations has been shoyn to lead to a high degree of internal standardization ( 1 , 2 ) . I n the absence of excitation potential data to guide in the selection of internal standard lines of similar excitation requirements, recourse y a s taken to observations of the effect of a major excitation variable, the arc current, on the intensity ratios of line pairs chosen on the basis of intensity and wave-length proximity. The line pairs showing minimum intensity ratio variations with arc current (Figure 4)were selected for analytical purposes. CALIBRATION EXPERIMEhTS
The synthetic standard samples were prepared by chemical procedures deecribed in previous communications (3, 4). Pertinent experimental details used in obtaining the calibration data are summarized in Table 11. The procedures used in photo-
. _ _
~
lable 111.
Results f r o m RecoFerj Experiincnts liiiriuiIt>
Concn 7% initial Detn. analysis €Io?OainDytOa 0.018 0.130 >-?osin Dy,Oj 0 . 0 2 2 0.057 1.~03in HozOa
DyrOsinHo208 Ergot in H0203 T203 in Erg03
0.170 0.410 0.480 0 071
D3.20~in
0.043
0 ,O i l E1203
0 047
€To?Oain Er?Oa
0 020 o.0~0 0.020
Ti11203 in
Er203
Yb?Oa i n Erg03
Added,
%
X
Found 0.124
0.330
0.320
0.200
0.220
0.230
0.100
0.157
0,150
0.106 0.106 0.106 0.230 0,500 0,250 0 500 0 010
0 278
0.26;
0.516 0.586 0 821
0.535 0.560 0 310 0.510 0.300 0.543
0.100 0.200
o 0
n20
nx
0.041) 0,040
0.020 0.050 0.100
0 027 0.027 0.027
0.010 0 020 0 100
0.049
Impurity,
Calcd. 0.118
@.si1
0 29'3 0 543 0 OXO 0.040 0.0i0
0 029
0 onn 0.0!1!1
0 068 0 OP8 0 154
O 149
0.017 0.047
0,127
o nw o
071
0.036 0.087 0 117
E
~
70 5.1 3.0 4.5 4.5 4.0 3.9 4.4 3.4 10.7 2.4 0.6 1.6 2.6 1.4 1.4 1.O 3.3 2.7 0.0 4.7
O O T
003c
I
PER CENT RARE E A R T H OXIDE (LOGARITHMIC SCALE)
Figure 5.
Analytical curves for determination of other rare earth impurities in dysprosium, holmium, and erbium Curves labeled with (BC) are corrected for background
~
~
~
1014
ANALYTICAL CHEMISTRY
graphic photometry, intensity ratio evaluations, and background correction followed standard practices (9). The 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 data express the average per cent deviation from the mean of quadruplicate exposures of the standards on individual plates. ACCUR&C\
The 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
The 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) (2) (3) (4)
Ahrens, L. H., “Spectrochemical Analysis,” Chap. 6 and 7, Addison-Wesley, Cambridge, Mass., 1950. Fassel, V. A., J . Opt. SOC.Amer., 39, 187 (1949). Fassel, V. A., Cook, H. D., Krotz. L. C., and Kehres, P. W., Spectrochim. Acta, 5 , 201 (1952). Fassel, V. h..and Wilhelm, H. A , , J . O p t . SOC.Amer., 38, 518 (1948).
(IO)
Gatterer, d.,and Junkes, J., “Spektren der Seltenen Erden.” Specola Vaticana, Citta del Vaticano. 1945. Harrison, G. R., J . Opt. SOC.Anrer., 39,522 (1949). Harrison, G. R., ed., “M.I.T. Wavelength Tables,” Wiley, New York, 1939. XIoeller, T., and Brantley, J. C., ANAL.CHEM.,22, 433 (1950). Nachtrieb, N. H., “Principles and Practices of Spectrochemical Analysis,” Chap. 6, McGraw-Hill, New York, 1950. Prandtl, W., and Scheiner, K., 2. anorg. allgem. Chem., 220, 107
(11)
Rodden, C. d., J . Research Satl. Bur. Standards, 26,557
(5)
(6) (7) (8) (9)
(1952). (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 1N 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. To 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 nater 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-
