error in the presence of permanganate was due to the fact that MnOa- was precipitated via the reaction: Mn04-
+ AsC#14+= AsC#14Mn04(s)
(2)
The reason for the interference by chlorate (while bromate had no adverse effect) has not been ascertained; possibly, tetraphenylarsonium chlorate (which is soluble per se) might have coprecipitated with tetraphenylarsonium perchlorate under our experimental conditions. Fluoroborate interfered drastically yielding sigmoid titration curves without any welldefined end point. While the effect of fluoroborate was not studied further, it appears consistent with the reasonable assumption that a kinetically slow process of the type BF4-
+ AS+^+ = BF~AsC#I~ (s)
(3)
might have prevailed.
The method described in this paper is remarkably catholic; it represents ZL preferred novel instrumental approach to the rapid and convenient determination of perchlorate in the presence of most other common anions and cations. In the present work, titrant addition was automated and titration curves were-likewise-automatically recorded. Even more complete automation, i.e., digital readout of the end point, is readily feasible using appropriate derivative electronic circuits (18).
RECEIVED for review September 17, 1971. Accepted January 6,1972. Based on a Ph.D. thesis by Peter W. Carr. Supported by Research Grant GP-11386 from the National Science Foundation. (18) P. T. Priestley, Analyst, 88, 194 (1963).
Determination of Sulfinic Acids in the Presence of Thiols by Titration with Aqueous Sodium Nitrite James P. Danehy and Victor J. Elia Department of Chemistry, Unicersity of Notre Dame, Notre Dame, Ind. 46556 SPECIFIC METHODS for the quantitative determination of sulfinic acids based on the reaction between the latter and nitrous acid have been previously published (1-3). The reaction is free from interference by the presence of other species which do not react with nitrous acid (e.g., sulfonic acids, thiolsulhates, thiolsulfonates). 2RS02H
+ HONO
+
(RS02)zNOH
+ H20
(1)
It has been proposed that, in the case of organic disulfides possessing certain structural features, alkaline decomposition occurs by a direct nucleophilic attack of hydroxide ion on one of the sulfur atoms to give thiol and sulfinic acid as the ultimate products (4). While the relatively stable sulfinic acids have actually been isolated subsequent to the alkaline decomposition of aromatic disulfides (5), 2RSSR
+ 40H-
+
3RS-
+ RS02- + 2Hz0
(2)
in the corresponding cases of aliphatic disulfides only sulfonic acids and thiols have been determined as ultimate products (4). Therefore, it was considered worthwhile to attempt to establish whether or not sulfinic acids are formed in these latter cases by using the method of titration with nitrous acid. For this method to be feasible, it had first to be established that titration with nitrous acid can be used in the presence of thiol and disulfide, both of which would be present in significant amounts. Kresze and Winkler (6) have reported that tert-butyl mercaptan reacts quantitatively with nitrous acid to form stable thionitrites. Ashworth and Keller (7) have extended this (1) C . S. Marvel and R. S. Johnson, J . Org. Chem., 13, 822 (1948). (2) J. L. Kice and K. W. Bowers, J . Amer. Chem. Soc., 84, 605 (1962). (3) B. Lindberg, Acta Chem. Scnnd., 17,383 (1963). (4) J. P. Danehy and W. E. Hunter, J. Org. Chem., 32,2047 (1967). (5) J. P. Danehy and K. N. Parameswaran, ibid., 33, 568 (1968). (6) G. Kresze and J. Winkler, Chem. Ber., 96,1203 (1963). 39, 373 (1967). (7) G. W. Ashworth and R. E. Keller, ANAL CHEM.,
observation to other thiols and have developed a spectrophotometric method based thereon for the quantitative determination of thiols. RSH
+ HONO
--*
RSNO
+ HzO
(3)
Since the concentration of thiol in a mixture of thiol and sulfinic acid can be determined without interference from the latter by the use of Folin’s reagent (8), it should be possible to determine the concentration of sulfinic acid in such a mixture by titration with nitrous acid provided that: the thiol under investigation is titrated quantitatively by nitrous acid; the disulfide does not interfere; and sulfinic acid reacts quantitatively in the presence of thiol. EXPERIMENTAL
Materials and Reagents. The thiols and disulfides were purchased, received as gifts, or synthesized by us, as related in earlier reports ( 4 , 5). Folin’s reagent (a phosphotungstic acid) was prepared and used as previously described (8). The sodium nitrite and other chemicals were all of ACS reagent grade. Titration of Aliphatic Thiols and of p-Chlorobenzenesulfinic Acid by Nitrous Acid. An accurately weighed sample of thiol or of p-chlorobenzenesulfinic acid was dissolved in 10 ml of water, stirred magnetically, and 5 ml of 5N H2S04 was added. The solution was then titrated with continuous stirring by addition of standardized sodium nitrite solution until an external end point was obtained: when one drop of the solution gave an instantaneous blue color when added to a starch-KI solution. Titration of Aromatic Thiols by Nitrous Acid. The titrations of p-chloruthiophenol, thiophenol, and m-mercaptobenzoic acid were carried out in the following manner. A sample of the corresponding crystalline disulfide was dissolved in 10-15 ml of dimethylformamide (DMF), and 3 ml of (8) J. P. Danehy and J. A. Kreuz, J . Amer. Chem. SOC.,83, 1109 (1961). ANALYTICAL CHEMISTRY, VOL. 44, NO. 7, JUNE 1972
1281
Titration of Thiols, Alone and in Mixtures with p-Chlorobenzenesulfinic Acid, in 1.67N Aqueous Sulfuric Acid with Aqueous Sodium Nitrite Sodium nitrite Thiol Sulfinic acid Name Wt, mg mmol Wt,mg mmol ml mmol 0.244 29.8 ... ... 4 , 72b 0.240 L-Cysteine 61.4 ... ... 0.507 10.00 0.509 60.6 0.496 ... ... 9.82 0.500 99.3 0.814 ... 15.90 0.810 1.. 0: 465 ... 81.9 4.44 0.226 83.1 0.490 0.472 59.9 14.16 0.720 0.106 8.58 ... ... 2.04~ 0.102 2-Mercaptoethanol 0.207 ... 16.85 3.92 0.197 ... 11.7 0 067 0.64 0.032 ... ... 23.4 0.133 1.34 0.067 ... 31.2 0.177 ... 1.72 0.086 23.4 0:063 0.133 5.15 2.50 0.126 11.7 0.063 0.067 5.15 1.88 0.094 0.106 9.4 0.053 2.52 0.127 8.58 0.085 Mercaptosuccinic 12.7 ... ... 1.69d 0.084 0.170 ... acid 25.4 3.38 0.168 ... 9.15 0: 052 0.52 0.026 ... 9.15 0.085 12.7 0.052 2.18 0.108 0.104 18.30 0.085 2.74 0.136 12.7 0.246 28.0 2-Mercaptoethyl... 4.66" 0.243 0.258 ammonium chloride ... ... 4.80 0.250 29.3 0.412 46.8 ... ... 7.80 0.407 0.588 ... ... 10.91 0.570 66.8 0.51 59.2 2,5-Dimercapto... ... 7.94d 0.394 0.64 67.7 adipic acid ... ... 8.92 0.452 0.234 3.46 0.172 24.6 ... ... 0.245f p-Chlorothio... ... ... 4.729 0.245 0.737 ... ... phenol 14.30 0.744 ... 0.257f ... ... ... Thiophenol 4.70 0.244 0.771 ... 14.61 0.760 ... ... 0,4361 m-Mercaptoben... ... ... 8.45 0.439 zoic acid mmol RSOlH a On assumption that mmol HONO = mmol RSH 0.0509M NaN02. 0 0.0502M NaN02. d0.0496MNaN02. e 0.0522111NaNG. f Spectrophotometric determination with Folin's reagent. 0 0.0520111NaN02. Table I.
:
.
.
I
Sulfur acctd for by HONO" 98 100 100 100
98 99 96 95 97 100 98 98 98 96 99 99 100 97 100 99 97 99 97 78 71 73 100 101 95 99 101
+
1N HC1 was added. The disulfide solution was reduced by zinc amalgam and HCI and the resulting solution was made up to 15 ml with sufficient D M F and water to give an 80% (v/v) DMF-water solution. A 1-ml aliquot of the reduced solution was diluted to 10 ml and analyzed with Folin's reagent for the concentration of thiol. Aliquots of the stock thiol solution were removed, and added to a solution consisting of 5 ml of 5N HzS04and 10 ml of DMF. The solution was then titrated with a standard sodium nitrite solution. Alkaline Decomposition of Aromatic Disulfides. The alkaline decompositions of p-chlorophenyl disulfide, phenyl disulfide, and 3,3 '-dithiodibenzoic acid were carried out as follows. An accurately weighed sample of disulfide was placed in a 50-ml volumetric flask, dissolved in DMF, and the flask was made up to near volume with sufficient D M F and water to give an 80 (v/v) D M F aqueous solution. The solution was thoroughly aspirated with nitrogen and kept under nitrogen for the duration of the reaction. T o the mixture was added 3 ml of 2.50N sodium hydroxide and the flask was made up to volume with D M F to give a final reaction mixture consisting of 0.150N sodium hydroxide in 80% (v/v) D M F . The alkaline decompositions of p-chlorophenyl disulfide, phenyl disulfide, and 3,3 '-dithiodibenzoic acid were allowed to proceed for 45 minutes, 24 hours, and 3-20 hours, respectively. A 1-ml aliquot was then removed, quenched in 1 N HCl, made up to volume in a 10-ml volumetric flask, and 1282
ANALYTICAL CHEMISTRY, VOL. 44, NO. 7, JUNE 1972
subsequently analyzed for thiol using Folin's reagent. The remainder of the decomposition solution was quenched in 5 ml of 5N Hi301 and immediately titrated with standard sodium nitrite solution. Alkaline Decomposition of Aliphatic Disulfides. The alkaline decompositions of the aliphatic disulfides were carried out using the general procedure previously reported ( 4 ) . At the same time that aliquots were removed for determination of thiol and disulfide with Folin's reagent, an aliquot was removed, quenched in 5N HzS04, and immediately titrated with standard sodium nitrite solution. RESULTS AND DISCUSSION
No one of several disulfides tested consumed any nitrous acid under the titration conditions employed. The results of the titration of known concentrations of several thiols and of p-chlorobenzenesulfinic acid, alone and in pairs, are given in Table I. Of the eight thiols examined only one, 2,5-dimercaptoadipic acid (a dithiol), failed to react quantitatively with nitrous acid. In those cases in which mixtures of thiol and pchlorobenzenesulfinic acid were titrated with sodium nitrite solution, the results show clearly that Reactions 1 and 3 are generally additive. It appears feasible, then, t o determine sulfinic acids in
Table 11. Alkaline Decomposition of Aromatic Disulfides in 0.150N Sodium Hydroxide in 80 (v/v) Aqueous Dimethylformamide at 35.2 "C, Followed by Titration of Sulfinic Acid and Thiol with Nitrous Acid mmol of mmol of mmol RSH/ mmol of RSH ml of 0.0520N NaN02 = mmol NaNOz mmol RS02H RSOIH Compound (colorimetric) NaNOz HONO -mmol RSH 0.224 3.01 p-Chloro0.673" 15.10 0.785 0.112 14.90 0.775 0.114 0.228 2.90 phenyl 0.661° 0.214 3.14 disulfidec 0 .673b 15.00 0.780 0.107 0.216 2.93 0. 633b 14.25 0.741 0.108 av 3.00 2.74 0.302 Phenyl 0.827 18.80 0.978 0.151 3.12 0.266 disullided 0.830 18.50 0.963 0.133 2.97 0.284 18.85 0.984 0.142 0.842 3.47 0.242 18.50 0.963 0.121 0.843 19.40 1.009 0.142 3.05 0.284 0.867 2.90 0.294 0.854 19.25 1.001 0.147 av 3.04 3.26 0.379 8.40 0.437 0.058 0.116 3,3'-Dithio8.30 0.432 0.058 3.22 0.116 dibenzoic 0.374 8.38 0.436 0.060 3.14 0.120 acide 0.376 3.24 0.118 0.382 8.48 0.441 0.059 av 3.21 4 Initial concentration of disulfide, 10.66 X 10-3M. bInitial concentration of disulfide, 10.54 X lW3M. The disulfide was 4 W 2 % decomposed in each of the runs. d Initial concentration of disulfide, 16.84 X 10-8M; disulfide was 32-34Z decomposed, 61nitial concentration of disulfide was 9.16 X 10-3M; extent of disulfide decomposition was 27%. Table 111. Alkaline Decomposition of 2,2 '-Dithiodiethanol. Titration of SulBnic Acid by Nitrous Acid moles RSH/ [RSOZH]~ Time, hr [RSSR] M X lo4 [RSH] M X lo4 M x 104 moles RSOzH decompn S acctd for 454 ... ... 0.00 ... 0.0 100.0 401 62 28 3.0 2.21 11.6 98.4 6.0 392 76 37 2.04 13.7 98.8 385 98 41 10.0 2.37 15.2 100.0 23.0 352 132 57 2.31 22.4 98.4 478 ... ... 0.Ob ... 0.0 100.0 3.0 409 92 40 2.30 14.4 99.4 380 121 56 6.0 2.17 20.4 98.0 10.0 352 149 64 2.33 26.3 96.0 24.0 308 208 76 2.73 35.6 94.2 Medium, 1 ,050N sodium hydroxide. Medium, 1.20N sodium hydroxide.
the presence of thiol and disulfide in dilute sulfuric acid by titration RSH
+ 2RSOzH + 2HONO RSNO
+
+ (RS0z)zNOH + 2H20
(4)
with aqueous sodium nitrite. Of course, if the amount of thiol present is large compared to the s u l h i c acid, the difference between the millimoles of nitrite used in the titration and the millimoles of thiol determined independently would be subject to poor accuracy and precision. From the quantitative determination of alkali consumed, of thiol formed (Folin's reagent), and of the relatively stable sulfinic acid formed (isolation in high yield in preparative experiments), it has been unequivocally established (5) that the aqueous alkaline decomposition of aromatic disulfides occurs by direct nucleophilic displacement of sulfur from sulfur by hydroxide ion so that two moles of disulfide give three moles of thiol and one mole of sulfinic acid (Reaction 2). Therefore, it seemed that a brief restudy of the alkaline decomposition of several aromatic disulfides would be a good test of the validity and usefulness of the titrimetric method described in this report.
It has already been shown in Table I that the titrations of three aromatic thiols are in agreement with their spectrophotometric determinations with Folin's reagent, and that pchlorobenzenesulhic acid is titrated quantitatively. Accordingly the alkaline decompositions of the corresponding disulfides (pchlorophenyl disulfide, phenyl disulfide, and 3,3'dithiodibenzoic acid) were carried out in 80% (v/v) aqueous dimethylformamide solutions at 35.2 "Cand followed both by spectrophotometric determination of thiol and residual disulfide, and by titration of acidified samples with aqueous sodium nitrite. The results (Table 11) show average values of 3.00, 3.04, and 3.21 for the ratio of thiol to sulfinic for the three disulfides, respectively. In earlier studies on the alkaline decomposition of aliphatic disulfides, sulfonic acids, rather than sulfinic acids, have been isolated. The former may have arisen fortuitously as the result of aerial oxidation of the latter or may be more closely linked mechanistically to the alkaline decomposition itself. Therefore, the present titrimetric method has been used to investigate whether or not sulfinic acids are present at any stage of the alkaline decomposition of aliphatic disulfides. Cystine is of particular interest because the disulfide linkages in proteins are integral parts of cystinyl residues. PreANALYTICAL CHEMISTRY, VOL. 44, NO. 7, JUNE 1972
1283
viously published information (4) on the alkaline decomposition of cystine itself appeared to be consistent with the direct nucleophilic displacement mechanism, but the chromatographic data were interpreted in favor of cysteic acid (RS03H), though it now appears that P-sulfinoalanine (RS02H) would probably have given the same signal. While cysteine is titrated quantitatively with sodium nitrite (Table I), unfortunately, an authentic specimen of 0-sulfinoalanine gave very erratic results when titrated with the same reagent. Thus the method is not available for settling this question. The alkaline decompositions of 2,2’-dithiodiethanol, 2,2’dithiodiethylamine, and 4,4’-dithiodibutyric acid have previously been rationalized in terms of direct nucleophilic displacement (4). Reinvestigation of the alkaline decomposition of these disulfides, using the present titrimetric method for
determining sulfinic acid, revealed that only in the case of 2,2’-dithiodiethanol did the amount of nitrous acid consumed exceed the amount of thiol determined colorimetrically. In the other two cases, the amount of nitrous acid consumed was equivalent to the amount of thiol present as determined by Folin’s reagent. From the quantitative data for 2,2’-dithiodiethanol (Table 111), thiol and sulfinic acid appear to be formed in a molar ratio ranging from 2.0 to 2.7, and from 95 to 100 of the total sulfur is accounted for.
RECEIVED for review October 15,1971. Accepted January 11, 1972. We acknowledge gratefully the support provided by the National Institutes of Health (AM-13109). V.J.E. was a postdoctoral research associate, 1969-71.
Thin-Layer and Paper Chromatography Analysis of the Reaction Products of Urea and Formaldehyde W. Y. Lee Research Centre, Monsanto Australia Limited, West Footscray, Victoria 3012, Australia THE REACTION of urea (U) and formaldehyde (F) is complicated; the precise nature of the U F reaction is still not fully understood ( I ) . It has been generally accepted, however, that the primary products of the U F reaction are methylol and methylene ureas. As these compounds are nonvolatile and comparatively unstable, a need for their identification existed. This is especially of interest in the field of U F chemistry and U F moulding compounds production. In this communication, eight model compounds, prepared in various stages of the U F reaction have been identified by using thin-layer and paper chromatography technique.
EXPERIMENTAL Thin-Layer Chromatography (TLC). Glass plates, 200 X 200 mm, were coated with silica gel (Merck Silica Gel G ) or cellulose (Merck Micro Crystalline) with the spreader set at a thickness of 0.3 mm. After air-drying for 30 minutes, the silica gel plates were activated at 100 “C for 30 minutes and the cellulose plates at 105 “C for 10 minutes. All plates were stored over silica gel (desiccant). The chromatographic tanks were lined with filter paper and equilibrated 2 to 3 hours before the plates were chromatographed. The aqueous solutions of sample were applied to the chromatoplates by means of capillary pipets (Microcaps, Drummond Scientific Co., U.S.A.) of 1-p1 capacity. The solvent was allowed to travel t o a height of 100 mm for silica gel plates and 150 mm for cellulose plates. Paper Chromatography (PC). Whatman Chromapaper Nos. 1, 4, and 54 were employed. Solutions of 1 p1 were applied as in the TLC technique mentioned above. Descending solvent was allowed to travel to a distance of 370 mm. Solvent Systems. The solvent systems used for silica gel TLC were (A) pyridine-water-chloroform (17: 3 :8, VjV), (B) n-propanol-water-chloroform-acetic acid (80 :4 :16 :4, VjV), and (C) n-propanol-toluene-water-acetic acid (70 :20: 3 :2, VjV). The solvent system used for cellulose TLC and PC was pyridine-water-chloroform (20:2.5 :8, VjV). (1) C. P. Vale and W. G. K. Taylor, “Aminoplastics”, Iliffe, London, 1964, pp 20-43. 1284
ANALYTICAL CHEMISTRY, VOL. 44, NO. 7, JUNE 1972
Chromogenic Reagents. The chromogenic reagents used were: (A) 1 gram of p-dimethylamino benzaldehyde (B.D.H. Analar) dissolved in a mixture of 30 ml of ethanol and 3 ml of concd HC1 diluted with 180 ml of 1-butanol; (B) a solution of p-dimethylamino benzaldehyde in HzS04prepared according to Rink and Gehl(2), and (C) one volume of 10% (WjV) aqueous solution of chromotropic acid (Ajax Chemicals, Australia) mixed with 5 vol. of concd HzSOa-HzO(5:3, VjV) solution. Reagents A and B were used for TLC and PC giving bright yellow spots. Reagent C was used for silica gel TLC and heating at 100 OC for 5-10 min after spraying was required to develop violet spots. Standard Compounds. Urea (May & Baker reagent) was used as received. Monomethylolurea and dimethylolurea were prepared by the method described by deJong and deJonge (3) and Scheibler et al. (4). remectively. Bis(monomethylo1obtained by reacting 2 moles of urea and 1 mole of formaldehyde at p H = 7-8 at room temperature for 2 days (5). This compound may also be prepared by the method of Zigeuner and Pitter (6). Methylene diurea, trimethylene tetraurea, methylolmethylenediurea, and methylene bismethylolurea were prepared essentially according to Kadowaki (7). Dimethylene triurea was obtained in the preparation of methylene diurea by careful fractional precipitation. All the standard compounds obtained were identified by elemental analysis, IR, and NMR.
RESULTS AND DISCUSSION Table I gives the mean R f values (5-10 runs) of urea and its derivatives. In the silica gel TLC, solvent A gives better resolution for U, MMU, and DMU but gives poor resolution (2) M. Rink and A. Gehl, J. Chromatogr., 20, 415 (1965). (3) J. I. deJong and J. deJonge, Rec. Trav. Chim., 71, 643 (1952). (4) H. Scheibler, F. Trostler, and E. Scholz, Angew. Chem., 41, 1305 (1928). ( 5 ) W. Y.Lee, unpublished results, 1970. (6) G. Ziegeuner and R. Pitter, Monatsh., 86, 57 (1955). (7) H. Kadowaki, Bull. Chem. SOC.,Jap., 11,248 (1936).
