By optical coincidence, two Keydex serial numbers are selected, namely 0080 and 0086, which correspond respectively with diethyltryptamine and dimethyltryptamine. Both substances can easily be differentiated by TLC and GLC. Example 2 is shown in Figure 7. Next cards are taken from the Keydex system: 235-240 nm, pH 7; 235-240 nm, pH 12; 240-245 nm, pH 12; 245-250 nm, n-heptane; 250-260 nm, pH 2; 250-260 nm, pH 7; 250-260 nm, ethanol; 250-260 nm, n-heptane; 260-270 nm, pH 2; 260-270 nm, pH 7; 260-270 nm, ethanol; and 260-270 nm, n- heptane. The number of peaks in the zone 240-300 nm are 240300 nm, pH 2 , 3 peaks; 240-300 nm, p H 7 , 2 peaks; 240-300 nm, pH 12, 1 peak; 240-300 nm, ethanol, 3 peaks; 240-300 nm, n- heptane, >5 peaks. By optical coincidence, 1 Keydex serial number becomes visible, namely 0015; pentobarbital-amphetamine, sold on the Belgian market as Pentoadiparthrol. This can further be affirmed by TLC and GLC.
CONCLUSIONS The examples prove that this kind of Keydex system means progress in the rapid and safe identification as far as narcotic and psychoanaleptic drugs are concerned. Examples are shown where complete recording of a spectrum was possible. Sometimes drugs do not dissolve in a given medium or do not go over to the n-heptane phase; these “negative” spectra are programmed as well since solubility and partition are determining for the drug itself and therefore offer supplementary information. On the other hand, the question of mixed powders may arise; of course the spectra of the present compounds overlap and cannot be identified by the presented system, unless some often-encountered
mixtures are programmed, like the amphetamine-barbiturate complexes for instance. The relative extinctions of the different peaks of one spectrum are not considered in the Keydex system; of course this would complete the total of UV characteristics. The one-year experience we now have, shows that this kind of UV characteristics need not be programmed. When one Keydex analysis points out four serial numbers, we can still compare the overall shape of the spectra of the selected numbers with the spectrum of the unknown. To reduce the influence of concentration on the shape of U.V.-curves, a log absorbance plot us. wavelength may be introduced ( I ) . At our department, parallel to this method, a similar system has been worked out for the morphological identification of pharmaceutical preparations with special attention to tablets. The morphological section combined with the UV spectra programming offers reliable and rapid determination possibilities.
ACKNOWLEDGMENT We acknowledge Oscar Van den Broecke for his excellent cooperation in building up the system. We thank the Firm Van Ermenghem, Leuven, Belgium, for turning the idea of the microseparator into a practical form. LITERATURE CITED (1) L. W. Bradford and Brackett J. W., Mikrochim. Acta, 1958/3, 353-381. (2) C. McArdle, The General Hospital, Birmingham, U.K., personal communication, 1973. (3) J. F. Lorentz, Centre Hospitalier de Nancy, Nancy, France, personal communication, Symposium on tablet identification, July 1973, Nancy, France. (4)J. V. Jackson, Forensic Toxicology Department, Metropolitan Police Laboratories, London, U.K., personal communication, 1972.
RECEIVEDfor review April 29, 1974. Accepted September 12, 1974.
Rapid, Sensitive Spectrophotometric Method for the Determination of Ascorbic Acid Mohamed A. Eldawy,’ A. S. Tawfik, and S. R. Elshabouri Department of Pharmaceutical Chemistry, Faculty of Pharmacy, University of Assiut, Assiut, A. R. Egypt
A spectrophotometric procedure based on the interaction of dimethoxydiquinone with ascorbic acid was developed. A buffered ascorbic acid solution was reacted with the reagent and the resulting color was measured at 510 nm. Absorbance vs. concentration was linear up to 80 Fg/ml. Replicate analyses showed good agreement, and an average recovery of 99 f 0.6% was obtained for analysis of synthetic mixtures. Other reducing substances, preservatives, stabilizers, minerals, vitamins, and hormones, likely to be present along with ascorbic acid do not interfere with precision of the method or the development of the color. The method is applicable to single as well as multicomponent formulations. Assay results on various commercial samples were reported.
To whom all correspondence should be addressed.
Numerous methods for, and excellent reviews on, the analysis of ascorbic acid are available in the literature ( I 3 ) . The official methods adopted for the analysis of this vitamin depend upon its reducing properties ( 4 , 5 ) . These methods whether iodometric, 2,6-dichlorophenol-indophenol, or the ceric sulfate, in spite of many modifications, still reflect analytical procedures which suffer from lack of specificity. Other reducing substances are oxidized by iodine which is the reagent adopted by U.S.P. XVIII ( 4 ) ,for the analysis of this vitamin. The standard dichlorophenolindophenol procedure used for the official assay of ascorbic acid injection is not a specific assay for the title vitamin (2). Among other substances, sulfohydryl compounds, thiosulfate, riboflavin, and ferrous compounds do interfere with the determination by this method (2). The oxidation of ascorbic acid by ceric sulfate does not correspond to any definite oxidation stage and is largely influenced by time of contact of the reactants, the relative ratio of acidity of the medium and the temperature (6).
ANALYTICAL CHEMISTRY, VOL. 47, NO. 3, MARCH 1975
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These shortcomings initiated many investigations to develop simple colorimetric methods for the determination of this vitamin (7-10). In spite of their simplicity, some of these methods are rather elaborate, of low sensitivity, or subject to interferences by other substances likely to be present along with ascorbic acid. The method developed by Aragones-Apodaca ( 7 ) ,necessitates prior chromatographic separation. The rather low sensitivity of the pyrrole color reaction ( 8 ) , with 100 pg/ml as lower limit for detection, render this method less suitable for the analysis of ascorbic acid in biological fluids. Vitamins B2 and B12 interfere with the photometric N-bromosuccinimide method, which is also applicable to vitamins B1 and B6 (9).The color formed by the silver complex of sodium-p- sulfonamidobenzoate which is employed by Rusu ( 1 0 )for the analysis of ascorbic acid in spite of its high sensitivity, is subject to many interferences. I t has been observed in these laboratories ( 1 1 ) that DMDQ (I) interacts with certain amines to form chromogenic products, namely carbazoloquinones (11).
DMDQ
II
1
This observation was investigated to find out its potential applicability as a means for the analysis of pharmaceutical amines. These investigations revealed that the reaction can be quantified to be used as a simple analytical tool for the determination of many pharmaceutical amines (12,13). Among the vast series of amines and related compounds that were investigated was vitamin B1. This investigation was undertaken as part of a program directed toward the development of simple procedures for the analysis of the active ingredients in geriatrics without prior separation of individual components. It was found that vitamin B1 reacts quantitatively with this reagent and a satisfactory method for its analysis was developed ( 1 3 ) .However, when interference studies were carried out, it was found that vitamin C does interfere with the development of the color for thiamine hydrochloride. Vitamin C, when present, produces an instantaneous reddish violet color. This interference was circumvented by a simple oxidation process of vitamin C, and we became interested in investigating this interference so as to develop it into an analytical tool for the determination of vitamin C. As a result of these investigations, a rapid, sensitive spectrophotometric method for the determination of ascorbic acid has been developed. The developed method, unlike other methods, offers a good degree of specificity in that other reducing substances likely to be present along with vitamin C in pharmaceutical formulations do not interfere.
EXPERIMENTAL Apparatus. A Carl Zeis Jenna 223346 spectrophotometer was used in this study, but any other suitable spectrophotometer may be used. Dimethoxydiquinone ( I ) (DMDQ). This was prepared according to a reported procedure ( 1 4 ) . Several crystallizations from dioxane yielded an analytical sample. Reagents and Solutions. p H 6.6 phosphate buffer ( 1 5 ) . (DMDQ), 0.1% solution in peroxide-free dioxane. Ascorbic acid was purchased from E. Merck and used as supplied. Dehydroascorbic acid was prepared i n situ according to a reported procedure (16). All chemicals used were reagent grade. Distilled water was used throughout. Standard Ascorbic Acid. Weigh accurately 50 mg of ascorbic acid in a 25-m1 volumetric flask, dissolve, and complete to volume 462
with p H 6.6 phosphate buffer. From this stock solution, different dilutions were made. This stock solution must be freshly prepared. Preparation of Assay Solutions. For Pure Ascorbic Acid. Appropriate volumes of standard ascorbic acid are used as the assay solution. For Ascorbic Acid Tablets. Weigh and powder 20 tablets. Transfer an accurately weighed quantity of the powder equivalent to about 50 mg of ascorbic acid to a 25-1111 volumetric flask. Dissolve as completely as possible in pH 6.6 phosphate buffer and complete to volume with the same buffer; either filter and discard the first portion of the filterate, or transfer the contents of the flask to a centrifuge tube and centrifuge for 10 min. The clear solution obtained is the assay solution. For Synthetic Mixtures. Transfer an accurately weighed amount of the powdered synthetic mixture equivalent to about 50 mg of ascorbic acid to a 25-m1 volumetric flask and proceed as under ascorbic acid tablets. For Ascorbic Acid Injections and for Syrups Containing Ascorbic Acid. Transfer to a 25-m1 volumetric flask an accurately measured volume of injection or syrup equivalent to about 50 mg of ascorbic acid. Complete to volume with pH 6.6 phosphate buffer and mix. This is the assay solution. For Injections and Syrups by Addition Method. Add an accurately weighed amount of ascorbic acid to an accurately measured volume of injection or syrup equivalent to the known weight of ascorbic acid contained in a 25-ml volumetric flask, complete to volume with p H 6.6 phosphate buffer, and mix. Development of Color. Into separate 10-ml volumetric flasks, each containing 2 ml DMDQ solution, pipet 1 ml of the standard solution, 1 ml of the assay solution of appropriate dilution, and 1 ml of p H 6.6 phosphate buffer. Dilute to volume with dioxane. Determine the absorbance of the standard and sample solution at 510 nm in 1-cm cells us. the blank using a suitable spectrophotometer.
RESULTS AND DISCUSSION The absorption spectrum of the interaction product of DMDQ with ascorbic acid is shown in Figure 1. I t exhibits an absorption maximum a t 510 nm. The molar absorptivity a t 510 nm is 1738. The relationship between absorbance at 510 nm and concentration was quite linear up to 80 pg ascorbic acid per ml solution as shown in Figure 2 . The limit for detection is 10 pg per ml. The color is immediately produced in the cold and is stable over 24 hours when kept in the dark. Heating results in lower absorption intensities for the same standard solution and gradual fading of the color is observed. This is by itself an advantage which would allow determination of ascorbic acid in the cold without interference from compounds containing amine functional group which need heating to produce the chromogenic carbazoloquinone (11). The specificity of the method for ascorbic acid in the presence of several frequently encountered excipients, preservatives, common reducing agents, and stabilizers is shown in Table I. A placebo was prepared to contain some common tablet excipients or additives. Several different weights of this excipient or additive, each approximating sample weights of commercial tablets, were mixed with a known amount of ascorbic acid and the assay was performed. The average recovery ranged from 98-101.2 f 0.8%. Further, certain minerals, hormones, and other vitamins that are likely to be present along with vitamin C in multivitamin and geriatric formulations were added to a known amount of ascorbic acid and, upon carrying out the analysis, the percentage recovery (Table 11) was found to range from 99 to 101%. Riboflavin, if present in more than threefold of the average amount found in commercial pharmaceutical formulations, will give slightly higher absorbance values. The relatively low percentage recovery obtained when copper sulfate was the additive might be due to the promoting effect of copper upon oxidation of ascorbic acid ( 1 7 ) . Ascorbic acid samples containing ferrous sulfate as
ANALYTICAL CHEMISTRY, VOL. 47, NO. 3, MARCH 1975
I
Table I . Determination of Ascorbic Acid in the Presence of Common Reducing Agents, Excipients, Antioxidants, a n d Preservatives
07t
4scorbic acid, mg
__-Founda
4dded
Substance added, m g
Recovery, 96
101.0 * 1 Aspartic acid 50 50.5 100 100.0 * 0.2 Cyst e in 50 50.2 50 101.2 i 0.6 50.6 Glutamic acid 50 50 99.8 * 0.2 50 49.9 I3enzoic acid 200 100.0 * 0.3 50 50 Citric acid 100 99.9 * 0.1 49.95 Tartaric acid 50 50 100.1 * 0.1 50 Fructose 50.1 100 99.8 i 0.2 50 Glucose 49.9 200 100.2 * 0.2 Mannose 50.1 50 200 100.0 0.0 50 50.0 Sucrose 100 100.2 * 0.1 50 Starch 50.1 100 100.0 f 0.1 50 Lactose 50.0 100 101.0 1.0 50 Urea 50.5 200 100.2 i 0.2 50.1 50 Thiourea 100 Sodium hy 98.0 * 0.8 49 drosulfite 50 50 Sodium meta99.0 * 0.2 bisulfite 50 49.5 50 Sodium thio 50 100.0 * 0.1 su If at e 50 50 100.0 i 0.01 50 50.0 G lv ce r o 1 250 100.0 * 0.01 Alcohol 50 50.0 150 The found values are average of 3 determinations.
410
430
450
470
490 510 1L17,
552
5x1
5s0
610
630
Figure 1. Absorption spectrum of ascorbic acid color product
,,P’
Table 11. Determination of Ascorbic Acid in the Presence of Minerals, Vitamins, a n d Hormones Which Usually Occur i n Combination with Ascorbic Acid in Pharmaceutical PreparaLions Ascorbic acid, rng Added
Substance added
Thiamine HC1 Ribof lavine Pyridoxine
25 nig 5.5 mg
50 50
Founda
50 50.5
Recovev, %
100.0 101.0
f
0.1
+ 0.2
50 HC 1 49.95 99.9 * 0.2 2 mg 49.5 99.0 * 1.0 50 Folic acid 2 nig C yanoco balaniin 50.1 100.2 * 0.2 50 1 iLg Nicotinamide 50 50.0 100.0 i 0.0 10 mg Ferrous s u l 50 50.0 101.0 i 0.1 fate 100 nig Cobalt sulfate 50.1 100.2 * 0.2 50 1.55 mg 50 49.9 99.8 rt 0.2 Zinc sulfate 3 mg Copper s u l 50 fate 48.0 96.0 * 1.0 4.5 mg Mag ne sium 50.1 100.2 * 0.2 50 sulfate 5 mg Ethinyl estra50.0 100.0 + 0.1 50 diol 100 pg Methyl tes tosterone 50 2.5 mg 49.9 99.8 i 0.1 Calcium gluconate 50.0 100.0 * 0.1 50 100 mg Dehydr o ascorbic acid 50 49.95 99.9 r O . l 18 mg The found values are average of 3 determinations.
the additive produced a precipitate when the sample was dissolved in pH 6.6 phosphate buffer. The precipitated ferrous oxysalt was removed by centrifugation. The resulting
Figure 2. Standard calibration curve for ascorbic acid color product, A, 510 nm
clear solution gave a negative test for ferrous ion, and no interference was observed upon subsequent assay. Dehydroascorbic acid which is the main oxidative degradation product of ascorbic acid does not interfere. The method presented here is quite applicable to the analysis of ascorbic acid content in commercial pharmaceutical formulations. Results for measurements of commercial vitamin C tablets were quite good. Results obtained (Table 111) are comparable to the official B.P. 1968 method (5 ). Different commercially available vitamin C injections were satisfactorily analyzed by this method (Table IV). While product B, which contains analgin along with ascorbic acid, and Product C gave percentage assay close to that of the label claim, product A which contains calcium gluconate in conjunction with ascorbic acid, analyzed for 91.5% of the declared amount. Though Table I1 shows that calcium gluconate does not interfere with the developed method, nevertheless different amounts of ascorbic acid were added to each of the injections, and the analysis was performed. Percentage recoveries of the added ascorbic acid were 99.6% or better in all injections indicating that product A actually contains less than the claimed amount. ANALYTICAL C H E M I S T R Y , VOL. 47, NO. 3 , M A R C H 1975
463
:I
:.
3 06
'
//4
p5
/$,,!
\
0:
L5cCCC-3,C CC,'
0
1 9
2UC210
Table V shows the results obtained for the analysis of ascorbic acid content in three commercial multivitamin syrups, which have different label declaration amounts of vitamin C, along with vitamin B,, B6, calcium pantothenate, and nicotinamide. Formulation C contains folic acid in addition. To investigate the mechanism of this coior formation, a mole ratio study of the interaction of ascorbic acid with DMDQ was carried out. Figure 3 shows that maximum absorption occurs when the ratio of reagent to vitamin is 1:l. Though the chemistry of this interaction is not clear, it might be safely assumed that a redox reaction is involved,
2
8
,
,
,
3 7
4 6
5 5
ae!ai,ve
Mole
6
3
4
2
~~
'
0
Percent
Figure 3. Continuous variation plot for ascorbic acid-DMDQ color product.
Table 111. Application of the Developed Method to the AssaT of Tablets Found in the Market and Comparison of the Results with the B.P. Methoda
a
Ascorbic acld found per tablet mg (DMDQ ðod)
Manufacmer
Ascorbic acld claimed per tablet, mg
A
500
502
B
500
459.5
C
500
477.45
D
500
500
hlean of found amount ( D l I D Q method)
Ascorbic a i - d found per tablet m (B.P. method
100.4 = 0.96 S . D . 1.048 91.9 f 1.1 S.D.1.204 95.49 i 0.46 S . D . 0.651 100.0 i 0.26 S . D . 0.547
496.5
hlen, of found amount (B.P. method)
99.3 i 0.32 S . D . 0.45 92.1 z 0.23 5 . D . 0.315 9 5 . 3 = 0.9 S . D . 1 014 99.56 T 0.33 S.D.0.905
460.5 467.5 497.8
The found values are ateraee of 5 determinations
Table I\'. Determination of Ascorbic Acid in Commercial Ampoule Formulations and Recovery of Ascorbic Acid Added to These Formulations Ascorbic acid, mg Ascorbic Acid, niii Manufacturer
Claimed per ampoule
A
1000
B
C
a
1000
500
Found per ampoule0
915
1016
506.3
Found,
.j
91.5 i 0.66 S.D. 0 . 7 7
101.6
i 0.49 S . D . 0.519
*
101.26 0.99 S.D. 1.09
Average of 6 experiments. Average of 3 experiments.
.\ddid
Found h
25
24.96
15
14.99
25
24.99
15
15.01
25
25 .O
15
15.01
____
R L L e?, ~
99.85 = 0.15 S . D . 0.219 100.14 = 0.102 S.D.0.302 99.96 * 0.4 S . D . 0.519 100.34 f 0.31 S.D.0 . 3 9 100.03 = 0.017 S . D . 0.043 100.14 z 0.27 S.D. 0.37 _______.
Table V. Determination of Ascorbic Acid in Commercial Syrup Formulations and Recovery of Ascorbic Acid Added to These Formulations ii'orhli
Ascorbic acid, mg hlanufacturer
A
B
C
a
464
Claimed
200mg / 1 5 nil 5 0 mg / 5 1111
100 mg / 1 5 ml
Fowid"
199.8
49.92
102.5
bound,
99.9 * 0.1 S . D . 0.1
99.84 I 0.06 S . D . 0.1
*
102.5 0.55 S . D . 1.02
Average of 5 experiments. Average of 3 experiments.
ANALYTICAL CHEMISTRY, VOL. 47, NO. 3, MARCH 1975
Lid,
1
Lil
___
1ddr.d
I amid
25
24.92
15
14.98
25
24.995
15
14.99
25
24.99
15
14.99
QICC\
'39.7
, 0.6
dn
-L
5.n. 0.26 99.7 i 0.33 S . D . 0.44 99.98 I 0.02 S . D . 0.014 98.4 z 0.06 S . I). 0.066 99.96 I 0.046 S.I.,. ) 104 99.98 x 0.3 S . D . 0.467 __
.-
LITERATURE CITED
resulting in oxidation of ascorbic acid to dehydroascorbic acid and partial reduction of DMDQ to the "Indigoid" quinhydrone (III),as shown here.
Ascorbic acid
(1) H. Elsourdy, Ph.D. Thesis, University of Assiut, 1968. (2) Takeru Higuchi and Einar Brochmann-Hanssen. Pharmaceutical Analysis," lnterscience Publishers, New York. London, 1961, p 689. (3) W. H. Sebrell. Jr., and Robert Harris, "The Vitamins," 2nd ed., Vol. I, Academic Press, New York and London, 1967, p 305. (4) "The United States Pharmacopeia" 18th rev., Mack Publishing Co., Easton, Pa., 1970, pp 51-53. (5) British Pharmacopeia, 1968, pp 64-65. (6) G. G. Rao and G. S. Sastry, Anal. Chim. Acta, 56, 325 (1971). (7) R. Aragones-Apodaca, lnform. Quim. Anal. 21, 224 (1967); Chem. Abstr., 68, 722792 (1968). 18) Y. Kochi and Y. Kaneda, Bitamin. 41 (3), 240-4 (1970); Chem. Abstr., 73, 289582 (1970). (9) Basch Serrat, Ars. Pharm., 11, 267 (1970); Chem. Abstr., 75, 25466t (1971). (10) Rusu et a/., Univ. Timisoara, 9 (2), 109 (1971); Chem. Abstr. 78, 1435569 (1973). (1 1) A. S. Hammam M.Sc. Thesis, Univ. of Assiut, 1961. (12) M. A. Eldawy, Abstracts of papers, 12th Congress of Pharmaceutical Sciences. Cairo. Nov. 1971. D 31. _.. M. A. Eldawy, A. S. TawfiK,'and S. R . Elshabouri, Abstracts of papers 33rd International Congress of Pharmaceutical Sciences, Stockholm, September 3-7, 1973, p 153. Erdtmann, Roc. Roy. Soc., Sec. A, 143: 211 (1934). "The United States Pharmacopeia" 18th rev., Mack Publishing Co.. Easton, Pa., p 939. J. Davidek et ab, Scientific papers of the institute of Chemical Technology, Prague ESO, 1971, p 17. M. P. Lamden, And. Chem., 22, 1139 (1950). A. Robertson et a/., J. Chem. SOC., 11 (1955).
I11
~I
Structure I11 is assigned to the violet intermediate reduction product of DMDQ (18). However, this assumption is far from conclusive, and a thorough investigation of the chemistry of this reaction, which is beyond the scope of this manuscript, is currently being undertaken and will be the subject matter of a future report.
SUMMARY A fast, facile, spectrophotometric method for determining ascorbic acid is described. The procedure is based on interaction between DMDQ and vitamin C to give a stable reddish violet color. The method is sensitive and offers a good degree of specificity to allow determination of ascorbic acid in the presence of other substances likely to be present along with vitamin C in pharmaceutical dosage forms. Single component and multivitamin formulations are satisfactorily analyzed by this method. Ordinary tablet excipients, antioxidants, preservatives, and stabilizers do not interfere. Dehydroascorbic acid also does not interfere.
RECEIVEDfor review June 13, 1974. Accepted October 8, 1974. Abstracted from a thesis presented by S.R. Elshabouri in partial fulfillment of the Ph.D. Degree, University of Assiut. Presented, in part, a t the 33rd International Congress of Pharmaceutical Sciences, Stockholm, Sept. 3-7, 1973. Abstracts p 153.
Improved Rhodanine Method for the Spectrophotometric Determination of Gold 1. E. Lichtenstein Corning Glass Works, Corning, N. Y. 14830
Drawbacks of the rhodanine specfrophotometric method for gold currently in use are limited solubility of the reagent and instability of the reaction product. These disadvantages have been overcome by dissolution of the reagent in pyridine and by use of a mixed aqueous-pyridine reaction system. At 515 nm, a plot of absorbance vs Au concentration Is linear In the range 0.2-0.8 X 10-5M Au. The molar absorptivity based on Au concentration is 3.8 X l o 4 in the waterpyridine system, or about twice that In the 0.12M HCI medium commonly used. The effects on color development of significant reaction variables have been studied, as well as interference by associated noble metals. An analytical procedure utilizing the improved rhodanlne method, applicable to the determination of gold In various matrices, has been developed.
A popular spectrophotometric method for gold ( 1 ) utilizes the reagent p - dialkylaminobenzylidenerhodanine (the alkyl can be methyl or ethyl). whose name shall be shortened to "rhodanine" from now on in this article.
NH-C=O
s=c,
I
I LH3 , C = C H ~ N'CH3
S RHODANINE (DIMETHYL DERIVATIVE)
In acidic medium, tetrachloroaurate ion reacts with rhodanine to form an intensely red product whose absorbance, as measured a t 515 nm, is proportional to the concentration of gold present. The reaction product is obtained as a colloidal suspension, as are most colored gold-organic compounds that have been reported for the spectrophotometric determination of gold in aqueous medium (2, 3 ). As is generally the case with colloidal systems, the intensity of color developed in the Au-rhodanine reaction is very sensitive to reaction conditions, notably electrolyte concentration and pH. Color maximizes within 1-2 minutes of initiation of reaction, but fades rapidly after that; hence, color stability is a problem. Another serious drawback of the recommended method is limited solubility of the reagent in aqueous medium. Rhodanine is very soluble in pyridine and in mixed aqueous-pyridine media. Further, an aqueous solution of
ANALYTICAL CHEMISTRY, VOL. 47, NO. 3, MARCH 1975
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