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Spectrophotometric Determination of bis(O,O-dialkyldithiophosphoryl) disulfides
MICROCHEMICAL
JOURNAL
27, 544-548 (1982)
Spectrophotometric Determination of Bis(O,O-dialkyldithiophosphoryl) disulfides
INTRODUCTION Bis(O,O-dialkyldithiophosphoryl)disulfides [(RO),PSS], are used as vulcanization accelerators and ultraviolet light stabilizers and are formed in the antioxidation reactions of zinc salts of dialkyldithiophosphates (1). Zinc dialkyldithiophosphates [(RO), PSSJ2Zn have been widely used as lubricating oil additives yet the detailed mechanism of their antioxidant activity is still uncertain. No reports in the literature were found describing the determination of bis(O,O-dialkyldithiophosphoryl)disulfides. I have found that dithiofluorescein, which contains thiols and chromophore groups, can be used for spectrophotometric determination of these disulfides. The interaction between dithiofluorescein excess and disulfides involves the formation of mixed disulfide as shown below,
SRI.!-
s s /I II + (RO),PS-SP(OR)2
S II (RO), PS-SR,S-
+
S /I + (RO),PS-,
where R, denotes fluorescein. In this article the presented method determination of disultide is indirect and based on the decrease of absorbance of blue color of dithiofluorescein by disulfide action. MATERIALS
AND METHODS
Apparatus
The absorbance of solutions was measured by means of a Spekol spectrophotometer (Carl Zeiss, Jena, Germany) with cells of 10. 0 mm optical length. Reugent
und Solutions
All the chemicals of analytical-grade cation. 544 0026-265x/82/040544-05$01.00/0 Copyright All rights
@ 1982 by Academic Press, Inc. of reproduction in any form reserved.
were used without
further purifi-
DETERMINATION
METHOD
FOR
DISULFIDES
545
Sodium and zinc salts of diakyldithiophosphates were prepared from the corresponding alcohols in accordance with the literature (2). Bis(O,O-dialkyldithiophosphoryl) disulfides were obtained by oxidizing sodium salts of dithiophosphates with iodine in potassium iodine solution: [(Iso-C,H,O),PSS],, after recrystallization from ethanol, melted at 90-91”C, in the literature at 90-91°C (3); [(C,H,O),PSS], oil, Z- 1.5580: [(iso-C,H,,O),PSS], oil, n, 20- 1.5146, in the literature corref;onding to 1.56 and 1.5148 (3). Dithiofluorescein (POCh Gliwice, Poland) was prepared according to Wroriski (4) as follows: Approximately 4 mg of dithiofluorescein was dissolved in 2 ml water solution; 20 g of EDTA (disodium salt), and 20 g triethylamine in 1 1; then the solution was diluted with water to 100 ml and filtered. The solution should be stored in a dark bottle and protected from light in the best way. The concentration of reagent decreases at a rate of about 5%/24 hr as a result of air oxidation. Amherlite IRA-400 (Serlvcr Feinhiochemie, Heidelberg, Germany). A stock solution (5 x lo-” M/liter) was made by dissolving the proper disulfide in 100 ml of ethanol. The stock solutions of disulfides were diluted to give the desired concentration level. The buffer solution (borax/NaOH) oj’pH 10. A solution of zinc salt of dithiophosphate was prepared by dissolving the accordant amount of this compound in ethanol. Recommendedprocedurefor the determination. Disulfide (0.5-70 nM) in 2 ml, or less, was added to the 0.5 ml buffer, afterwards mixed with the solution of dithiofluorescein, and next diluted to 5 ml by ethanol and the solution was allowed to stand for 30 min in a dark place at room temperature. Then the absorbance was measured at 588 nm vs ethanol. Sepamtion ofzinc srrlts qf dithiophosphate. About 10 g of Amberlite in OH form was washed with three 25-ml portions of anhydrous ethanol to remove water. About 20 ml of solution of zinc dialkyldithiophosphate and disulfide together with about 1 g Amberlite IRA-400 were added to a loo-ml Erlenmeyer flask. The mixture was stirred (magnetic stirrer) for 5 min, the resin allowed to settle, and supernatant liquid discarded. The residue was then washed by stirring with three successive 15-ml portions of ethanol. The connected solutions were diluted to 100 ml in a volumetric flask by ethanol, and then the concentration of disulfide was determined. RESULTS
AND DISCUSSION
EjTect of Time
To study the rate of dithiofluorescein color decrease, the concentration of disulfides used was 6 nMlm1 for bis(O,O-diisopropyldithiophosphoryl)disulfide (Fig. 1, curve 1) and 14.5 n/V/ml for bis(O,O-
546
STANISJLAW
Time,
PCAZA
men
FIG. 1. Color reduction rate of dithiofluorescein using bis(O,O-diisopropyldithiophosphoryl)disulfide (curve 1) and bis(O,O-diisoamyldithiophosphoryl)disulfide (curve 2).
diisoamyldithiophosphoryl)disulfide (Fig. 1, curve 2) with the absorbance measured at different reaction times. Reaction times of 30 min were found to be adequate for the disulfides tested. Longer delays did not effect the absorbance.
Effect of PH Since dithiofluorescein is most stable at pH 10, the effect of pH on the rate of reaction was not studied. Determination of Disulfide A plot of the absorbance against the concentration of disulfide was prepared (Fig. 2), the linear relationship in range up 16 M/ml obeying Beer’s law. The molar extinction coefficient was determined from the slope of the line; the average value obtained was 4.1 x lo4 k 2500 liters M-’ cm-‘. On the molar basis disultides gave practically identical absorbance readings
FIG. 2. Calibration with dithiofluorescein.
curve for the assay of bis(O,O-diisoamyldithiophosphoryl)disulfide
DETERMINATION
METHOD
FOR
DISULFIDES
547
(within experimental errors). The unknown concentration of disulfides is deduced by comparison with standard curves for known concentration of disuhides. A molar extinction coefficient may also be used to calculate the concentration of disulfide but dithiofluorescein must then be freshly prepared. This reagent is oxidized during storage to dithiofluorescein disulfide; generated in thiol-disulfide interchange reaction the dithiophosphate ion is capable of cleaving the dithiofluoresceine-disulfide bond yielding a new sulfhydryl color anion as shown below, S S II
Ii
SR,S-SR,S-
+ (RO),PS- + (RO),PS-SR,
+ -SR,S:
hence measured absorbance is not adequate for the concentration of disulfide. It was observed that the dithiofluorescein, after storing for a long time in reaction with very small amounts of disulfides, gave absorbances higher than the blank solution of reagent. As an extinction of 0.01 can still be measured, the sensitivity of the determination of disulfides will amount to approximately 0.2 M/ml; this can be taken as the sensitivity of the method suggested in this work. The relative standard deviation is in the range 0.5 l.S!%. All oxidizing compounds bring about the disappearance of blue color in dithiofluorescein. It turned out that dithiophosphate anion appeared to be a compound introducing serious interference and therefore the results calculated for disulfides decrease with increasing amounts of this anion. Study of the influence of (RO),PSS was carried out by preparing various samples, all
0
250
500
x15nv
3lthlophosphnte FIG. 3. Effect of varying concentration of zinc diisoamyldithiophosphate on the determined concentration of bis(O,O-diisoamyldithiophosphoryl)disulfde (concentration IS nM/ml): 1. after Amberlite IRA-400 treatment: 2. without separation of diverse anion.
548
STANISCAW
PCAZA
with 15 nMlm1 of disulfide and varying concentrations of zinc diisoamylodithiophosphate. The results obtained were presented in Fig. 3, curve 2. To eliminate interference of this compound, anion-exchange resin has been used with the results shown in Fig. 3, curve 1. Nearly quantitative separation of the interfering anion was acheived; 500-fold molar excess of zinc diisoamylodithiophosphate decreases the amount of the determined disulfide about 8%. As it can be gathered from the above presented studies the method can be recommended for simple, sensitive, and rapid analysis of bis(O,Odialkyldithiophosphoryl)disulfides. This method also seems to constitute a useful tool for assays of disulfides in the study of antioxidant activity of zinc dialkyldithiophosphates in lubricating oils. SUMMARY A new simple method has been developed for determining the bis(O,Odialkyldithiophosphoryl)disulfide content up to 16 n&I/ml with good reproducibility. The procedure is based on the dithiofluorescein-disulfide interchange reaction. The average value of the molar extinction coefficient obtained was 4.1 x lo4 f 2500 liters M Icm -I. The use of ion-exchange separation seems to be quite satisfactory for determination of this disulfide in the presence of dithiophosphate salts interference.
REFERENCES I. Burn, A. J., The mechanism of the antioxidant action of zinc dialkyldithiophosphates. Tcrruhedron 22, 2153-2161 (1966). 2. Brazier, A. D., and Elliott, J. S., The thermal stability of zinc dithioph0sphates.J. In.tr. Pet. 53, 63-76 (1967). Hsueh Pao 22, 215-224 (1956); Cheer. 3. Ping-Fang, Hu, and Wan-Yi, C, Herr Hsurh Ahstr. 52, 7816~. 4. Wronski, M., Submicrodeterminations of thiols, disulphides and thiol esters in serum by using o-hydroxymercuribenzoic acid and dithiofluorescein. Bioclrcm. J. 104,978-986 (1967).
JOURNAL
27, 544-548 (1982)
Spectrophotometric Determination of Bis(O,O-dialkyldithiophosphoryl) disulfides
INTRODUCTION Bis(O,O-dialkyldithiophosphoryl)disulfides [(RO),PSS], are used as vulcanization accelerators and ultraviolet light stabilizers and are formed in the antioxidation reactions of zinc salts of dialkyldithiophosphates (1). Zinc dialkyldithiophosphates [(RO), PSSJ2Zn have been widely used as lubricating oil additives yet the detailed mechanism of their antioxidant activity is still uncertain. No reports in the literature were found describing the determination of bis(O,O-dialkyldithiophosphoryl)disulfides. I have found that dithiofluorescein, which contains thiols and chromophore groups, can be used for spectrophotometric determination of these disulfides. The interaction between dithiofluorescein excess and disulfides involves the formation of mixed disulfide as shown below,
SRI.!-
s s /I II + (RO),PS-SP(OR)2
S II (RO), PS-SR,S-
+
S /I + (RO),PS-,
where R, denotes fluorescein. In this article the presented method determination of disultide is indirect and based on the decrease of absorbance of blue color of dithiofluorescein by disulfide action. MATERIALS
AND METHODS
Apparatus
The absorbance of solutions was measured by means of a Spekol spectrophotometer (Carl Zeiss, Jena, Germany) with cells of 10. 0 mm optical length. Reugent
und Solutions
All the chemicals of analytical-grade cation. 544 0026-265x/82/040544-05$01.00/0 Copyright All rights
@ 1982 by Academic Press, Inc. of reproduction in any form reserved.
were used without
further purifi-
DETERMINATION
METHOD
FOR
DISULFIDES
545
Sodium and zinc salts of diakyldithiophosphates were prepared from the corresponding alcohols in accordance with the literature (2). Bis(O,O-dialkyldithiophosphoryl) disulfides were obtained by oxidizing sodium salts of dithiophosphates with iodine in potassium iodine solution: [(Iso-C,H,O),PSS],, after recrystallization from ethanol, melted at 90-91”C, in the literature at 90-91°C (3); [(C,H,O),PSS], oil, Z- 1.5580: [(iso-C,H,,O),PSS], oil, n, 20- 1.5146, in the literature corref;onding to 1.56 and 1.5148 (3). Dithiofluorescein (POCh Gliwice, Poland) was prepared according to Wroriski (4) as follows: Approximately 4 mg of dithiofluorescein was dissolved in 2 ml water solution; 20 g of EDTA (disodium salt), and 20 g triethylamine in 1 1; then the solution was diluted with water to 100 ml and filtered. The solution should be stored in a dark bottle and protected from light in the best way. The concentration of reagent decreases at a rate of about 5%/24 hr as a result of air oxidation. Amherlite IRA-400 (Serlvcr Feinhiochemie, Heidelberg, Germany). A stock solution (5 x lo-” M/liter) was made by dissolving the proper disulfide in 100 ml of ethanol. The stock solutions of disulfides were diluted to give the desired concentration level. The buffer solution (borax/NaOH) oj’pH 10. A solution of zinc salt of dithiophosphate was prepared by dissolving the accordant amount of this compound in ethanol. Recommendedprocedurefor the determination. Disulfide (0.5-70 nM) in 2 ml, or less, was added to the 0.5 ml buffer, afterwards mixed with the solution of dithiofluorescein, and next diluted to 5 ml by ethanol and the solution was allowed to stand for 30 min in a dark place at room temperature. Then the absorbance was measured at 588 nm vs ethanol. Sepamtion ofzinc srrlts qf dithiophosphate. About 10 g of Amberlite in OH form was washed with three 25-ml portions of anhydrous ethanol to remove water. About 20 ml of solution of zinc dialkyldithiophosphate and disulfide together with about 1 g Amberlite IRA-400 were added to a loo-ml Erlenmeyer flask. The mixture was stirred (magnetic stirrer) for 5 min, the resin allowed to settle, and supernatant liquid discarded. The residue was then washed by stirring with three successive 15-ml portions of ethanol. The connected solutions were diluted to 100 ml in a volumetric flask by ethanol, and then the concentration of disulfide was determined. RESULTS
AND DISCUSSION
EjTect of Time
To study the rate of dithiofluorescein color decrease, the concentration of disulfides used was 6 nMlm1 for bis(O,O-diisopropyldithiophosphoryl)disulfide (Fig. 1, curve 1) and 14.5 n/V/ml for bis(O,O-
546
STANISJLAW
Time,
PCAZA
men
FIG. 1. Color reduction rate of dithiofluorescein using bis(O,O-diisopropyldithiophosphoryl)disulfide (curve 1) and bis(O,O-diisoamyldithiophosphoryl)disulfide (curve 2).
diisoamyldithiophosphoryl)disulfide (Fig. 1, curve 2) with the absorbance measured at different reaction times. Reaction times of 30 min were found to be adequate for the disulfides tested. Longer delays did not effect the absorbance.
Effect of PH Since dithiofluorescein is most stable at pH 10, the effect of pH on the rate of reaction was not studied. Determination of Disulfide A plot of the absorbance against the concentration of disulfide was prepared (Fig. 2), the linear relationship in range up 16 M/ml obeying Beer’s law. The molar extinction coefficient was determined from the slope of the line; the average value obtained was 4.1 x lo4 k 2500 liters M-’ cm-‘. On the molar basis disultides gave practically identical absorbance readings
FIG. 2. Calibration with dithiofluorescein.
curve for the assay of bis(O,O-diisoamyldithiophosphoryl)disulfide
DETERMINATION
METHOD
FOR
DISULFIDES
547
(within experimental errors). The unknown concentration of disulfides is deduced by comparison with standard curves for known concentration of disuhides. A molar extinction coefficient may also be used to calculate the concentration of disulfide but dithiofluorescein must then be freshly prepared. This reagent is oxidized during storage to dithiofluorescein disulfide; generated in thiol-disulfide interchange reaction the dithiophosphate ion is capable of cleaving the dithiofluoresceine-disulfide bond yielding a new sulfhydryl color anion as shown below, S S II
Ii
SR,S-SR,S-
+ (RO),PS- + (RO),PS-SR,
+ -SR,S:
hence measured absorbance is not adequate for the concentration of disulfide. It was observed that the dithiofluorescein, after storing for a long time in reaction with very small amounts of disulfides, gave absorbances higher than the blank solution of reagent. As an extinction of 0.01 can still be measured, the sensitivity of the determination of disulfides will amount to approximately 0.2 M/ml; this can be taken as the sensitivity of the method suggested in this work. The relative standard deviation is in the range 0.5 l.S!%. All oxidizing compounds bring about the disappearance of blue color in dithiofluorescein. It turned out that dithiophosphate anion appeared to be a compound introducing serious interference and therefore the results calculated for disulfides decrease with increasing amounts of this anion. Study of the influence of (RO),PSS was carried out by preparing various samples, all
0
250
500
x15nv
3lthlophosphnte FIG. 3. Effect of varying concentration of zinc diisoamyldithiophosphate on the determined concentration of bis(O,O-diisoamyldithiophosphoryl)disulfde (concentration IS nM/ml): 1. after Amberlite IRA-400 treatment: 2. without separation of diverse anion.
548
STANISCAW
PCAZA
with 15 nMlm1 of disulfide and varying concentrations of zinc diisoamylodithiophosphate. The results obtained were presented in Fig. 3, curve 2. To eliminate interference of this compound, anion-exchange resin has been used with the results shown in Fig. 3, curve 1. Nearly quantitative separation of the interfering anion was acheived; 500-fold molar excess of zinc diisoamylodithiophosphate decreases the amount of the determined disulfide about 8%. As it can be gathered from the above presented studies the method can be recommended for simple, sensitive, and rapid analysis of bis(O,Odialkyldithiophosphoryl)disulfides. This method also seems to constitute a useful tool for assays of disulfides in the study of antioxidant activity of zinc dialkyldithiophosphates in lubricating oils. SUMMARY A new simple method has been developed for determining the bis(O,Odialkyldithiophosphoryl)disulfide content up to 16 n&I/ml with good reproducibility. The procedure is based on the dithiofluorescein-disulfide interchange reaction. The average value of the molar extinction coefficient obtained was 4.1 x lo4 f 2500 liters M Icm -I. The use of ion-exchange separation seems to be quite satisfactory for determination of this disulfide in the presence of dithiophosphate salts interference.
REFERENCES I. Burn, A. J., The mechanism of the antioxidant action of zinc dialkyldithiophosphates. Tcrruhedron 22, 2153-2161 (1966). 2. Brazier, A. D., and Elliott, J. S., The thermal stability of zinc dithioph0sphates.J. In.tr. Pet. 53, 63-76 (1967). Hsueh Pao 22, 215-224 (1956); Cheer. 3. Ping-Fang, Hu, and Wan-Yi, C, Herr Hsurh Ahstr. 52, 7816~. 4. Wronski, M., Submicrodeterminations of thiols, disulphides and thiol esters in serum by using o-hydroxymercuribenzoic acid and dithiofluorescein. Bioclrcm. J. 104,978-986 (1967).