paper, and the strontium assayed with the x-ray spectrometer. In Table VI1 results are given for sera, selected a t random from the clinical laboratory for determining the range expected in routine analysis. The calcium can be determined directly in serum with a high degree of precision ( *1%, 2 ~ ) . The strontium procedure exhibited a precision to *5.6y0 (2,) for 1 ml. of serum. This included the total error in sampling, ashing, transferring to a spot by the instrument of Figure 2, and reading. An additional error was introduced, in that the mean blank for serum ash (Tablr 11) was used as the blank for all these determinations. The mean Sr level (24.7) for this study may be compared with that of 25.6 found by a different technique using normal serum (Table 11). The mean Ca-Sr ratio of 393 in this study also compares ~ i t that h found in Table I1 (390). In Table VI1 sera 1 and 14 show 3 low strontium level, which is associated with a low calcium level. This is not true of serum 3. Averaging the values other than numbers 1. 3, 6, and 14 in this tablp, the mran is 26.27 with a standard deviation of ~t1.84. Kumbers 1. 3, 6, and 14 are more than 2 standard deviations from this mean.
Thus, in attempting to estimate the range most likely to be found, at the 5% level of probability, the mean would be 26.27 with a range of 13.68. This figure is not a normal range but probably approaches it. To establish a normal range i t would be necessary to study several hundred normal sera of various age groups of both sexes and on different diets. It mas to prepare for this study that the present investigation for a preferred procedure was undertaken. As far as we could determine, no previous study on the strontium content on human serum had been reported. However, in a recent study on bovine serum, three values obtained by emission spectroscopy (11) for three cows on the same feed are reported: Ca-Sr ratios of 170, 59, and 333. This wide variation does not agree with the much narrower range for Ca-Sr ratios we have observed in human serum (Tables I1 and VII). While one might expect variation in Ca-Sr ratio in serum depending upon the particular diet, such wide variation is improbable in healthy animals on the same diet. LITERATURE CITED
(1) Bedford,
J., Harrison, G. E., Raymond, W. H. A., Sutton, A., Brit. Med. J . 1960,589.
(2) Campbell, J. T., Shagolsky, H. I., h'ature 183, 1481 (1959). (3) Collin, R. L., J. .4na. Chena. Soc. 81,
5275 (1959). (4) Kulp, J. L., Schulert, 4. R., Hodges, E. J., Science 132, 448 (1960). (5) Likins, R. C., Posner, .4.S., Kunde, M. L., Craven, D. L., -4rch. Biochem. Bzophys. 83, 472 (1959). (6) MacDonald, S. S.,Spain, P. C., Ezmirlian, F., Rounds, D. E., J. Nutrition 57, 555 (1955). (7) Natelson, S., Richelson, 11.R., Sheid, B., Bender, S. L., Clin. Chem. 5 , 519 (1959'). \ - -
- I
(8) Eatelson, S., Sheid, B.. lbid., 6, 299 (1960). (9) Ibid., p. 314. 110) Roberts. W.11.B.. Suture 183. 887 . (1959). ' (11) Shalminoff, G. V., Conway, J. G., Pitzer, A., Appl. Spectroscopy 12, 120 (1959). (12) Sobel, A. E., Cohen, J., Kranier, B., Biochem. J . 29, 2640 (1935). (13) Sobel, A. E., Pearl, A., Gerchick, E., Kramer, B. A., J . Biol. Chem. 118, 47 (1937). (14) Wisserman, R. H., Proc. Soc. Exptl.
Biol. Med. 104, 92 (1960). (15) Wasserman, R. H., Comar, C. L., Papadoppoulou, D., Science 126, 1180 (1957). RECEIVED for review -4ugust 15, 1960. Accepted Eovember 30, 1960. Division of Biological Chemistry, 138th Meeting, ACS, New York, N. Y., September 1960. Work supported in part by a grant from the U. S. Public Health Service, S o . S-2829 (Cl). The fifth in a series on application of x-ray spectrometry in the clinical laboratory.
Spectrochemical Determination of Yttrium in Biological Materia Is CLARENCE L. GRANT Rufgers-The
Sfate University, New Brunswick, N. J.
b In a recent study of the transmission of dietary nonradioactive yttrium from hen to egg to chick, a need arose for an analytical method for the determination of this element in the mineral ash of bone and egg shells. A method has been developed which combines ion exchange enrichment with spectrochemical analysis. The coefficient of variation is h6.0 and the average recovery is 94% for yttrium concentrations of 2 to 35 p.p.m. in the ash. The main advantages of the method are its simplicity and versatility.
I
for marking the offspring of pheasants, an analytical method for the determination of yttrium in the ash of bone tissue and egg shells was needed. The expected concentration range of yttrium in the ash was 0.5 to 1000 p.p.m. N STUDYING TECHNIQUES
In most instances, sample size was limited to 2 grams or less of ash. In most studies of the metabolic behavior of yttrium, very small amounts of radioactive yttrium are used. Lack of sensitivity of existing analytical methods has seriously limited studies with nonradioactive forms of this element. MacDonald et al. (11) studied the skeletal deposition of yttrium in rats using a direct current arc spectrochemical procedure with a limit of detection of 80 p.p.m. Other direct current arc procedures (4-7, 10) for the determination of rare earths and yttrium in rare earth oxides are limited to the 50- to 100-p.p.m. range as a minimum. Butler (2) has also described a direct current arc method of rather low sensitivity for minerals containing appreciable quantities of these elements. The procedure of Waiing and Mela (15) for the analysis of
phosphate rocks for small amounts of rare earths involves multiple chemical manipulations. By combining solvent extraction and spectrochemical analysis, Feldman and Ellenburg (8) developed an excellent method for the determination of fractional part per million quantities of rare earths and yttrium in purified thorium and uranium prepar a t'ions. The procedure described in this paper employs a somewhat similar spark discharge. As yet their extraction procedure has not been tried by the author, but it should be adaptable to this problem. Hettel and Fassel (9), by combining ion exchange enrichment with direct current arc procedures (4-7, IO), determined certain rare earths in zirconium metal down to 0.003 p.p.m. To achieve this sensitivity, however, requires a 500-gram sample. Edge VOL. 33, NO. 3, MARCH 1961
401
g,ok
1
\\ LAI3245.12
Yr 3242.28
I-
z
LAI3245.12 1.0 0
60 90 120 TIME IN SECONDS
33
Figure 1 .
150
Volatilization curves
et al. (3)recently published a preliminary report on the combined use of ion exchange enrichment and spectrochemical determination of trace elements, including yttrium, in silicate rocks. Hydrochloric acid solutions are poured on Dowex 50-X8 resin in the hydrogen form and eluted with hydrochloric acid. These authors recommend using 5- to 10-gram samples, but do not give any limits of detection. APPARATUS AND REAGENTS
Standard commercially available spectrographic equipment was used, and is described in Table I with the excitation conditions. The ion exchange columns had a cross-sectional area of 1.54 sq. cm. and a height of 22 cm., giving a total bed volume of approximately 35 ml. Amberlite IR-120, 16- to 50-mesh, was
Table 1. Apparatus and Conditions for Obtaining Spectrograms
Spectrograph Electrodes Slit Optical system Analytical gap Excitation source
Exposure time Wave length region Emulsion Development
Densitometry
402
Jarrell-Ash 3.4-meter Wadsworth Mount High purity graphite rods 0.242 X 1.5 inches long 40 microns Source light focused on the slit, step filter directly in front of slit 2 mrn. National spectrographic poww source. High voltage spark ( C = 0.005 pf., L = 600 ph., R = 3 ohms, discharges per second = 840, current = 5 amp. r.f.)
Variable (see text) 2400 to 3600 A., first
order Spectrum analysis No. 1 4 minutes at 20' C. f 0.5, D-19, 15-second stop, 5 m'inutes in Kodaiix. A JarrellAsh Photoprocessor provided agitation Jarrell-Ash nonrecording microphotometer
ANALYTICAL CHEMISTRY
employed. The resin was purified with 6N hydrochloric acid a t 50' C. The total exchange capacity of the column, approximately 65 meq., was sufficient to handle an ash weight of 2 grams without breakthrough. Hydrochloric acid was redistilled in an all borosilicate glass distillation apparatus, and distilled water was further purified by passage through an Amberlite monobed resin column. Johnson, Matthey & Co., Ltd. high purity chemicals were employed. EXPERIMENTAL
Ion Exchange Enrichment. Schubert, Russell, and Farabee (14) isolated yttrium from urine acidified to 0.1N in hydrochloric acid, by passing the solution a t a flow rate of 8 ml. per minute through a column with a diameter of 2.5 cm. Mono- and divalent cations were eluted by passing 1500 ml. of 0.4.V hydrochloric acid a t a flow rate of 20 to 25 nil. per minute, and yttrium was removed by elution with 1000 ml. of 7 N hydrochloric acid. Exploratory studies used samples of a synthetic mixture approaching the composition of bone ash with 1000 p.p.m. of added yttrium. Since a smaller column than that employed by Schubert et al. (14) was used, a flow rate of 3 ml. per minute was employed for 1-gram samples in 100 ml. of 0.1N hydrochloric acid. For monitoring purposes, 25-ml. effluent fractions were evaporated to dryness and andyzed by a direct current arc spectrochemical procedure. Breakthrough was not observed in any case. It was found that 0.8N hydrochloric acid could be used a t a flow rate of 6 to 8 ml. per minute to elute mono- and divalent cations with no loss of yttrium. A volume of 1500 ml. is sufficient for this purpose. Since the rather large particle size of the resin and high flow rate cause considerable tailing in the removal of calcium, in future work the use of a resin of finer particle size and slower flow rate should be considered. When yttrium was eluted with 6N hydrochloric acid a t a flow rate of 6 to 8 ml. per minute, traces still appeared in the effluent after 1500 ml. Reduction of the flow rate to 2 ml. per minute did not eliminate this problem. By carrying out the elution at 50' C., all the yttrium could be removed with 1200 ml. of acid. The use of higher temperatures was not practical with hydrochloric acid because of gassing. After elution of the yttrium fraction with 6 N hydrochloric acid at 50" C., it is best to remove the resin and equilibrate it with 3 to 4 portions of water before replacement in the column. If the resin is washed in the column, considerable swelling occurs and the glass may shatter. Despite the large quantity of acid required for
YI
- I.Ot
324220'
LA13245 I2
!
I
I
0 500 1000 1500 moo MICROGRAMS C4 PER ELECTRODE PAIR
Figure 2. Effect of calcium on yttrium and lanthanum emission
complete elution of yttrium, the ease with which the separated yttrium is concentrated by evaporation makes it a very convenient eluent. Excitation Conditions. The advantages of a high inductance spark in combination with graphite electrodes have been discussed ( I d , IS) and will not be considered here. Internal Standard. The properties desired in the internal standard for yttrium were similarity of volatilization behavior, proximity of wave length of lines, and insensitivity of the intensity ratio to fluctuations in the excitation source. For a calcium matrix, lanthanum fulfilled these requirements when Y 3242.28 A was used as the analysis line and La 3245.12 A. was the internal standard line. Similarity of volatilization was demonstrated by a jumping plate study in which each series of exposures was made with 1, 10, and 200 pg. of yttrium, lanthanum, and calcium, respectively. The intensities and intensity ratios of yttrium to lanthanum as a function of time are shown in Figure 1. Each point is the average of six replicates. During the first 60 seconds, the relative intensity of lanthanum decreases somewhat faster than that of yttrium. This causes the yttrium to lanthanum intensity ratio to increase from 1.63 for the first 15-second interval to a value of 2.04 for the 60- to 75second interval, after which it remains constant. Therefore, a 30- to 60second prespark followed by a 60to 90-second exposure is recommended for maximum precision in those cases where the yttrium line intensity is adequate. For very low concentrations of yttrium, the prespark is omitted. Insensitivity of the intensity ratio to normal source fluctuations was studied by varying the source parameters. No significant shifts were found for the various conditions used. Matrix. Microgram quantities of calcium were always found in the yttrium fraction after separation. Since this would also hold true with
a solvent extraction procedure, unless repeated separations were employed, the effects of varying amounts of calcium were investigated. The results are presented in Figure 2 . Each exposure was made with 1 and 10 pg. of yttrium and lanthanum, respectively, and was replicated six times. Exposures were for 90 seconds with no prespark time. The relative intensities of both elements were greater in the presence of 50 fig. of calcium per electrode pair than when no calcium was present, but further increases in the amount of calcium caused the intensities to decrease. A similar phenomenon was recently reported by Owens (IS) in a study of the effects of gallium on the intensities of impurity elements. Unfortunately the yttrium to lanthanum intensity ratio also varied when less than 200 pg. of calcium was present. Consideration of the relative intensities suggests that the observed behavior of the intensity ratio was caused by greater enhancement of yttrium than lanthanum in the presence of 50 pg. of calcium, and less suppression of yttrium than lanthanum with 100 pg. of calcium. Thus, unless maximum sensitivity is necessary, it is best to use 200 pg. of calcium per electrode pair as a buffer. When maximum sensitivity is necessary, the amount of calcium must be controlled or the accuracy will suffer. Interelement Effects. Up to 500 pg. of iron was found in the yttrium fraction from 1 gram of bone ash. Much smaller amounts of other elements mere also present. Fortunately iron did not exhibit any detectable influence on the intensity ratio either with or without calcium present. PROCEDURE
Preparation of Electrodes. High purity graphite electrodes, 0.242 inch in diameter x 1.5 inches long, are treated with 1 drop of a 2% solution of Plicene in benzene (8). After drying, the process is repeated and the electrodes are placed in an oven a t 115' C. for 3 to 5 minutes, The solution to be analyzed is immediately delivered to this treated surface from a 50-p1. micropipet and dried. Thus, each pair of electrodes receives 0.1 ml. of solution. The electrodes must be kept dry until used. Preparation of Standards. Standards are prepared from stock solutions of high purity salts or oxides dissolved in 1N hydrochloric acid. When the standards contain 200 pg. per 0.10 ml. of calcium, a lanthanum concentration of 10 pg. per 0.10 ml. is appropriate. Under these circumstances, yttrium concentrations from 0.05 to 5.0 wg. per 0.10 ml. are readily handled. The author prefers to use six standards to cover this range. Other series of standards with less calcium may be prepared, but as the amount of calcium is reduced, the lanthanum concentration should be
decreased, or a weaker lanthanum line used. As expected, the useful concentration range of yttrium is extended to 0.01 pg. per 0.10 ml. or even less as the amount of calcium is decreased. Sample Preparation. Bone and egg shell samples are dry ashed a t 600' C. in platinum or other suitable vessels with the usual precautions. The white ash is ground to a fine powder in an agate mortar. For yttrium concentrations of 0.5 p.p.m. or greater, a 1.000-gram sample of ash is treated in a borosilicate glass beaker with an excess of concentrated hydrochloric acid. For lower levels of yttrium, a larger sample can be used, but a larger ion exchange column may be required. After evaporation of excess acid on a hot plate, the sample is dried in an oven a t 120' C. The dried salts are dissolved in 10 ml. of 1N hydrochloric acid with gentle heating, and made to 100 ml. with water. This solution is passed through the ion exchange column to separate the yttrium as previously described. The yttrium fraction is concentrated by evaporation to about 5 ml., quantitatively transferred to a small beaker with the aid of warm 6N hydrochloric acid, and evaporated to near dryness. If the resin is properly conditioned, only traces of soluble resin will be present and can be ignored. The residue is taken up in 1 ml. of a 2N hydrochloric acid solution containing 4 mg. of calcium and 200 kg. of lanthanum. The solution is quantitatively transferred with water to a Zml. volumetric flask and made to volume with water. This gives final concentrations of 200 pg. per 0.10 ml. of calcium, 10 pg. per 0.10 ml. of lanthanum, and IN hydrochloric acid, which are the same as for the standards. For 1.000-gram ash samples containing 1 to 100 p.p.m. of yttrium, the concentration in the final solution is 0.05 to 5.0 pg. per 0.10 m1.-the range covered by the standards. When the ash contains less than 1 p.p.m. of yttrium, increased sensitivity can be obtained by using larger samples, if available, reducing the volume of the final solution, reducing the calcium concentration of the final solution, or, preferably, by evaporating more of the final solution on the electrodes. As many as 20 successive evaporations of IN hydrochloric acid solutions can be made on a Plicene treated electrode without significant penetration into the graphite. Therefore, the whole final solution can be transferred to a pair of electrodes if desired. To retain constant quantities of 200 pg. of calcium and 10 pg. of lanthanum per electrode pair, the concentrations of these two elements in the final solution must be adjusted according to the amount of solution to be dried on the electrodes. With this procedure, the range of quantitative determinations can be extended to 0.05 p.p.m. for a 1.000gram sample. Still greater sensitivity can be obtained by reducing the calcium concentration, if necessary. For yttrium concentrations above 100 p.p.m. in the ash, the final solution volume can be increased t o dilute the
yttrium concentration, or a smaller initial sample can be employed. The latter suggestion has the merit of reducing the required size of the ion exchange column and the quantities of acids needed for elutions. RESULTS
Calibration data were obtained by making exposures of the six standards and a pair of iron pins on six different photographic plates, using a neutral filter in front of the slit. The plates were exposed on separate days over a 3-week period. An emulsion calibration curve was constructed by the two-step method (1) and an analytical curve was established by plotting intensity ratios of yttrium to lanthanum vs. concentration on log coordinates. The coefficients of variation of the intensity ratios ranged from 4.0 to 8.7 for the six standards individually. Both the precision and accuracy of the method were estimated by adding known amounts of yttrium to a large pooled sample of bone ash. Twenty individual 1.000-gram samples of ash were treated with four different levels of yttrium, giving five replicates a t each concentration. Each sample was re-ashed overnight a t 600' C. and then carried through the complete procedure. The concentration of yttrium in the original bone ash was not known but work to date proves that it was less than 0.1 p.p.m. Recoveries were calculated on the assumption that no yttrium was present except that added. Results are given in Table 11. These data show that recoveries are somewhat low on the average. Thus far, the source of this loss has not been investigated. One very possible reason would be adsorption of yttrium on the walls of the large beaker used in the evaporation step. Radioactive yttrium would be helpful in this connection. DISCUSSION
This procedure has been adequate for the problem for which it was developed. The manipulations required are simple,
Table II. Precision and Accuracy of Yttrium Determination CoeffiYttrium, pg. Found (av. of Taken 5 reps.) 2.18 8.70 17.40 34.80
2.08 8.85 15.0 32.5
cient of
Variation
6.93 4.32 5.24 7.49 Av. 6.00
VOL. 33, NO. 3, MARCH 1961
Recovery,
%
95 102 86 93 94
403
thereby making the method easily learned by a technician. One of the chief advantages of the method is the extreme versatility in handling samples of different sizes and yttrium concentration. Although the ion exchange procedure could be adapted to handle samples much larger than 1 gram, solvent extraction with thenoyltrifluoroacetone might be better for this purpose. It should also be possible to determine elements of the rare earth group by this method, but the author has done no work along these lines. ACKNOWLEDGMENT
The author is indebted to Hilbert
R. Siegler, Helenette Silver, and Laurance E. Webber for their cooperation in carrying out this study after the author had left the University of New Hampshire.
LITERATURE CITED
(1) Am. SOC. Testing Materials, Philadelphia, Pa., Photographic Photometry (E116-56T, 1956)in “Methods for Emission Suectrochemical Analysis.” Section 9, 1957. (2) Butler, J. R., Spectrochim. Acta 9, 332 (1957). (3)Edge, R. A., Brooks, R. R., Ahrens, L. H., Amdurer, S., Geochim. et Cosmochim. Acta 15, 337 (1959). (4) Fassel, V. A,, J . Opt. SOC.Am. 39, 187 (1949). (5) Fassel, V. A., Cook, H. D., Krotz, L. C., Kehres, P. W., Spectrochim. Acta 5,201 (1952). ( 6 ) Fassel, V. -4., Quinney, B. B., Krota, L., Lenta, C., ANAL. CHEM.27, 1010 (1955). ( 7 ) Fassel, V. A., Wilhelm, H. A., J . Opt. SOC.Am. 38.518 (1948). (8) Feldman, ‘C., Ellenburg, J. Y., ANAL. CHEM.30,418 (1958). (9) Hettel, H. J., Fassel, V. A., Ibid., 27, 1311 (1955). (10) Kinseley, R. N., Fassel, V. A., Tabeling, R. W., Hurd, B. G., Quinney, B. B., Spectrochim. Acta 13, 300 (1959). (11) MacDonald, N. S., Nusbaum, R. E., “
I
Alexander, G. V., Ezmirlian, F., Spain, P., Rounds, D. E., J . Biol. Chem. 195, 837 (1952). (12) Morris, J. M., Pink, F. X., “Symposium on Spectrochemical Analysis of Trace Elementa,” p. 39, Am. SOC. Testing Materials, Philadelphia, Pa., 1957. (13) Owens, E. B., Appl. Spectroscopy 13, 105 (1959). (14) Schubert, J., Russell, E. R., Farabee, L. B., Science 109,316 (1949). (15) Waring, C. L., Mela, H., ANAL. CHEY.25,432 (1953). RECEIVED for review February 15, 1960. Accepted December 2, 1960. Final report on one phase of Federal Aid to Wildlife Project FW-2-R, conducted cooperatively by the Research and Management Division of the New Hampshire Fish and Game Department, and the Engineering Experiment Station, University of Yew Hampshire. Twelfth annual Meeting-in-Miniature, Iiorth Jersey Section, ACS, February 1, 1960. Paper of the Journal Series, New Jersey Agricultural Experiment Station, Rutgers, the State University, Department of Soils, New Brunswick.
Spectrophotometric Determination of Technetium with Toluene-3,4-dithiol FRANCIS J. MILLER and PAUL F. THOMASON Analytical Chemistry Division, Oak Ridge National Laboratory, Oak Ridge, Tenn.
b A method has been developed for the spectrophotometric determination of technetium in the microgram range by reaction with toluene-3,4-dithiol in an aqueous medium that is 2.5N in acid. The colored complex that is formed is extracted into carbon tetrachloride. The intensity of the color of the carbon tetrachloride solution is measured at 4 5 0 mp. The molar absorptivity is of the order of 15,000. The interference of many metallic cations necessitates the preliminary separation of the technetium. The results of a study of the various factors that influence the formation and extraction of the complex are presented.
T
presented herein is the outgrowth of work done previously on the colorimetric determination of technetium with thioglycolic acid (4). Although the thioglycolic acid method suffices for the rapid determination of technetium and is especially suitable for a ranging procedure, it lacks sensitivity. Also, a colorimetric reagent for use in a solution of low pH R-ould be advantageous. d short search of the literature disclosed the long-known colorimetric reagent, toluene-3,4-dithiol, or simply dithiol. Sandell (6) lists this reagent HE STCDY
404
ANALYTICAL CHEMISTRY
and discusses its use as a colorimetric reagent for the determination of tin, tungsten, and molybdenum. Clark (6) has demonstrated the applicability of toluene-3,4-dithiol to the determination of many other metals. Morrison and Freiser (5) discuss the extraction of the bidentate complex of molybdenum with toluene-3,4-dithiol into an organic solvent. Gilbert and Sandell have characterized the Mo-dithiol complex as a tridentate complex in a thorough investigation (3). The investigation described herein has shown that toluene-3,4dithiol is also suitable as a colorimetric reagent for the determination of technetium when it is present as pertechnetate ion. Many elements interfere. Adequate means of separating and isolating technetium from other elements are known and have been discussed by Boyd ( 1 ) . A review article by Clark and Neville (6)list the conditions under which toluene-3,4-dithiol reacts with various ions to produce colored species. EXPERIMENTAL
Standard Solution of Potassium Pertechnetate. This solution contained 0.423 mg. of potassium pertechnetate, KTcO.,, per ml. Spectrographic analysis showed no impurities present.
Toluene-3,4-dithiol Solution. This solution was prepared by dissolving 5 grams of toluene-3,4-dithiol, CH8C6H3(SH)2,and 12.5 grams of thioglycolic acid, HSCH2COOH, in l liter of a 2.5 w./v. ?& , ,- solution of sodium hydroxide. This method of preparation is taken from a previous report (7‘). If prepared in this manner and stored in a polyethylene bottle, the reagent should be stable for a Deriod of at least 2 weeks. The dithiol solution does oxidize slowly on standing and should be checked against a standard calibration curve before use. Purified Carbon Tetrachloride. The analytical reagent grade carbon tetrachloride was further purified by shaking it first with a solution of ceric sulfate, then with a solution of sodium carbonate, and finally with distilled water. Apparatus. A Cary Model 14PJI recording spectrophotometer, with cells of 1-cm. light path, was used t o obtain and to record all spectral data. Procedure. Ten milliliters of purified carbon tetrachloride was placed in a 30-ml. separatory funnel; the funnel was equipped with a Teflon stopcock to avoid the presence of stopcock grease in the carbon tetrachloride. Two milliliters of water was added. Two milliliters of the toluene-3,4-dithiol reagent solution was placed in the funnel, and a suitable aliquot of the standard solution of
