Potentiometric Quantitative Determination of Sodium Aminoethylthiosulfates and A m inoethylth iosulf uric Acids SIR: The protective utility of 8-aminoethylthiosulfuric acid against ionizing radiation has been established (3). The physiological investigation of this radio-protective effect of the acid and its derivatives requires analytical methods for assaying these compounds. Heretofore, no simple analytical methods have been applied to aminoethylthiosulfuric acid or sodium aminoethylthiosulfate determinations. Saville (6) devised an analytical method which involves oxidation with bromine water to the corresponding sulfonyl bromide, followed by reaction with cyanide, giving cyanogen bromide which is estimated colorimetrically. Thiols, disulfides, thiocyanates, and related compounds interfere. Displacement of sulfite from thiosulfuric acid by cyanide or thiol has been studied as a possible method for quantitative estimation of the thiosulfate group, but yields of sulfur dioxide greater than 8OOj, could not be obtained (4). No references to aminoethylthiosulfate analyses were found.
The feasibility of titrating these compounds in nonaqueous solvents (2) has been investigated and new, accurate analytical methods for the quantitative determination of these compounds have been perfected. The thiosulfuric acid group is too weakly acidic for aqueous titration but the proton can be titrated effectively in pyridine solvent. The anion of sodium aminoethylthiosulfates is too weakly basic for aqueous titration, but the anion can be titrated effectively in ethylene glycol solvent. The titrations are standard procedures for weak acids ( 1 ) and acid salts (6) but have not been heretofore applied to aminoethylthiosulfuric acid derivatives. EXPERIMENTAL
Modified Reference Electrode. A Beckman sleeve-type calomel electrode containing methanol saturated with KCl was used. Preparation and Standardization of 0.1 N Tetrabutylammonium Hydroxide. Prepare the titrant pre-
cisely as directed in reference ( I ) . Standardize the titrant by potentiometric titration of benzoic acid. Preparation and Standardization of 0.1N HC104. Dissolve 7.2 ml. of perchloric acid (72%) in ethylene glycol and dilute to 1 liter. Standardize the titrant by potentiometric titration of a 10-ml. aliquot of standard 0.1N N a O H . Analytical Procedure for Aminoethylthiosulfuric Acids. Accurately weigh a sample which will require a titration of 2 to 10 ml. into a 250-ml. beaker. Add a few drops of water t o dissolve the sample. Add pyridine solvent (50 ml.) and titrate potentiometrically with standard 0.1N tetrabutylammonium hydroxide. Pyridine contains acidic impurities and a blank titration is necessary. Analytical Procedure for Aminoethylthiosulfates. Accurately weigh a sample which will require a titration of 2 to 10 ml. into a 250-ml. beaker. Add a few drops of water to dissolve t h e sample. Add ethylene glycol (50 ml.) and titrate potentiometrically with O.1N HC10, in ethylene glycol, as in the above procedure. Blank corrections are unnecessary. RESULTS A N D DISCUSSION
Table I.
1.
2. 3. 4. 5. 6.
7. 8.
9.
Analysis for Arninothiosulfuric Acid Derivatives Microanalysis -Potentiometric titration calcd., found, Grams Grams Purity, 70 70 added found 70 Compound , . 0,2652 0.2660 100.3 NHZCHzCHzSzOaH 0.2712 0.2709 9 9 . 9 HOCHzCHzOCH( OH )CHzNHCHzCHzSzOsH 23.3 23.5 0.1648 0.1650 100.1 0.0793 0.0783 98.7 HOCHzCH(OH)CH2NHCH2CH~S2OSH 27.7 27.9 0.0934 0.0924 9 8 , 9 0.1090 0.1087 99.7 CHaCH( 0H)CHzNHCHzCHzSzOaH 2 9 . 8 30.0 0,0916 0.0922 100.6 0.1018 0.1016 99.8 (HOCHz)zC(0H)CHzNHCHzCHzSzOaH 24.5 2 4 , 8 0.0639 0,0645 100.9 0.0554 0.0555 100.2 HOCHzCHzCH~NHCHzCH~SzO~H 29.8 2 9 . 9 0,0670 0.0679 101.3 0.0696 0.0701 100.7 HOCHzCHzNHCHzCHzSz03H 31.9 32.0 0,0408 0.0416 102.0 0.0535 0.0549 102.6 HOCHzCH(OH)CH(0H)CHzNHCHzCHzSzOsH 24.5 23.7 0.0141 0.0156 110.6 0.0172 0.0189 109.9 HOCHzCHzOCHzCHzNHCHzCHzSzOZH 26.1 26.1 0.0337 0.0335 9 9 . 4 0.0529 0.0531 100.0
10. HOCHzCHzOCHzCHzNHCHzCHzNHCHzCHzSzOaH C&On 15.9 14.9 0,0237 0.0258 108.9 0.0210 0.0231 110.0 11.
HOCHzCHzCHzNHCHzCHzNHCHzCHr Sz03H ' C4H606
12.
l 170
ANALYTICAL CHEMISTRY
15.7 15.8 0.0770 0.0546 0.1031 0.1573 0.1235
0.0753 9 7 . 8 0.0529 96.7 0.1030 9 9 . 9 0.1580 100.4 0.1241 100.5
These procedures were applied to several compounds and the data are given in Table I. Compounds 1 through 10 were titrated with the standard base. Compounds 11 and 12 were titrated with the standard acid. The purity factors indicate that these two potentiometric titration techniques are adequate for assay of aminoethylthiosulfuric acid derivatives. The poorer results with compounds 8 and 10 are expected from the sulfur microanalyses; although the carbon, hydrogen, and nitrogen microanalyses of compounds 8 and 10 checked, the sulfur analyses indicated that contamination was present. Acidic impurities, neutralized at potentials similar to thiosulfuric acid, interfere and must be removed. The most likely contaminants in this procedure are mercaptans from thiosulfate reduction, and these do not interfere. The difference in equivalence potential between mercaptans and thiosulfuric acids was 200 mv., the mercaplan being a weaker acid. The simultaneous determination of mercaptans and thiosulfuric acids is readily possible by this titrimetric procedure. Thymol blue has the same equivalence point as thiosulfuric acid, and the titrimetric procedure is simplified by using two drops of o.3yOthymol blue in isopropyl alcohol as a visual end point indicator. The visual end point, how-
ever, is less precise than the potcntiometric end point (*0.05 ml. ml.). There is one class of aminoethylthiosulfates which cannot be acid-titrated in ethylene glycol. The N-acetyl and N-formyl derivatives of aminoethylthiosulfates were neutral salts in ethylene glycol, and hence could not be assayed by titration. These titrations are now performed on a routine basis in laboratory to assay research quantities Of these pounds.
(3) Holmberg, B., Sorbo, B., Nature 183, 832 (1959). (4) Mulligan, B., Swan, J. M., Rev. Pure A p d . f%m. 12,82 (1962). (5ilE;if: S. R., ANAL. CHEM. 18, 246
ACKNOWLEDGMENT
Derivatives of 0-aminoethylthiosulfuric acid used in this investigation ere synthesized by ~ 1H.. ~ ~ l lJ.i E. ~ , ~ ~and J. ~C. J ~ ~ i ~ ~ ~ , ~ (6) Saville, . B., Chem. Znd. 1956, p. 660. JOHN C. MACDONALD ( I ) Cundiff, R. H., Markunas, P. C.,
Boston Laboratory Monsanto Research Corp. Everett, Mass. 02149
ANAL. 28,792 (1956). (2) Fritz, J. S.,“Acid-Base Titrations in Nonaqueous Solvents,” G. Frederick Smith Chemical Co., Columbus, Ohio, 1952.
WORK supported by the United States Army Medical Research and Development Command, under Contract No. DA49-193-MD-2 109.
LITERATURE CITED
Amperometric Titration of Primary Amides SIR: I n a n earlier paper (d) a method was described for the spectrophotometric titration of primary aliphatic amides with hypobromite in basic solution. Although this procedure is satisfactory for aliphatic amides, it cannot be used for the analysis of aromatic amides because of the instability of the primary reaction product, the corresponding aromatic .Y-bromamide, in basic solution. The decomposition products of the N-bromamide are rapidly oxidized by hypobromite, and no suitable end point can be found. This communication deals with a new titrimetric procedure for amides which can be utilized for the analysis of both aliphatic and aromatic amides. The sample of amide is dissolved in a dioxane-water mixture which is 1M in hydrochloric acid, and titrated with a standard solution of calcium hypochlorite using a n amperometric end point. EXPERIMENTAL
Reagents and Apparatus. An approximately 0.5‘V solution of calcium hypochlorite was prepared by dissolving reagent grade calcium hypochlorite in water, followed by filtration t o remove solid calcium carbonate. T h e solution was standardized iodometrically. KO change in titer was evident over a 1-month period, in contrast to t h a t evident with t h e more dilute solution used previously ( 2 ) . The amides used were of the highest purity obtainable from commercial sources and were used without further purification. The amperometric titration apparatus consisted of a dry cell potential source, a divider, an RCA Model WV-84B microammeter, a Leeds and Yorthrup pH meter, a rotating platinum electrode, and a saturated calomel electrode. Procedure. -4 25-ml. aliquot containing 0.5 mmole of amide in dioxane was diluted to exactly 150 ml. with
aqueous HCl so t h a t the final solution was -1M in HCI and 20’3, dioxane. The applied potential was set to f0.4 volt us. S.C.E., and the calcium hypochlorite titrant was added in 0.5-ml. increments. T h e resulting current readings were taken after t h e addition of each increment of titrant, corrected for dilution, and plotted against volume of titrant. T h e best straight lines were drawn through the experimental points, a n d the intersection of these two straight lines was taken as the end point.
hypochlorous acid may be involved The sequence of reactions suggested is:
+ H+ HOCl + H + HOCl + HC1OC1-
+
Table I. Amperometric Titration of Primary Amides
No. of Amide trials Benzamide 8 o-Toluamide 9 p-Toluamide 5 Acetamide 6 Propionamide 3 n-Butyramide 3
Av . apparent Std. purity, dev., % % 99.6 0.6 100.6 0.9 97.0 0.6 99.6 100.0 100.4
1.3
0.7 0.5
HOCl
H20Cl+
C12
+ H2O
0
I1
R-C-NH2
+ Cl2
4
O
H
I1 / R-C--N \ c1
RESULTS A N D DISCUSSION
The results of the titration of a number of primary amides are given in Table I. Although these results were obtained with a 0.5-mmole sample of amide, as small a sample as 0.05 mmole was titrated with 0.05N calcium hypochlorite with only a slight decrease in accuracy and precision. The time required to complete a titration was about 10 minutes. The reaction utilized in this procedure is the N-chlorination of the primary amide in 1M hydrochloric acid with a chlorinating agent produced by the addition of calcium hypochlorite. The exact nature of the chlorinating agent, however, is not known, and it is probable that both chlorine and protonated
+
+HC1
0
It
R-C-NHa
+ H20C1’ O
+
H
Undoubtedly chlorine is the predominant chlorinating species when hydrochloric acid is used, because the reaction between hypochlorous acid and hydrochloric acid is rapid ( 1 ) . However, similar titrations were performed using I N sulfuric and 1N perchloric acids instead of hydrochloric, and identical titration results were obtained, even though the rate of chlorination was markedly slower than when hydrochloric acid was used. Because the calcium hypochlorite titrant contained a n appreciable amount of free chloride, chlorine is probably still the major chlorinating agent, but the protonated hypochlorous acid may be involved also. The excess chlorinating agent slowly decomposes after the end point of the titration has been passed. The rate of decomposition of 1JI hydrochloric acid is approsimately 1% per minute. A number of methods were attempted to stabilize the chlorinating agent, but none was successful. For example, if VOL. 37,
NO. 9, AUGUST 1965
1171
