illg indicator blank corrections in dilute solutions. LITERATURE CITED
i l 'i A a s t i m . R..
Weherline. R.. Pallila. F.. i).
(3) Bruckenstein, S., Xelson, D. C., ANAL.CHEW33, 439 (1961). 14) Clark. B. L.. Tooten. L. A.. J . Phus. ?'hein. 33. 1468 11929).' ( 5 ) Connor;, K.- Ak,Higuchi, T., ANAL. CHEM.32, 93 (1960). (6) Fort,une, W. B., Mellon, 11. G., J . Am. Chem. Soc. 60, 2607 (1938). ( 7 ) Goddu. R. F., Runie. D. Y.. ANAL. CHEW26, 16T9 (1954). 18) Himichi. T . Rehm. C.. Barnstein. --a C., Zbid., 28, 1506(195k). (9) Hiskey, C. F., Ibid., 21, 1440 (1949). (10) Hiskey, C. F., Rabinowitz, J., Young, I. G., Ih&, 22, 1464 (1950). (11) Hiskey, C. F., Young, I. G., Ibid., 23, 1196 (1951). \ - I
~~
~ - >
~J
~~
(12) ,Humnielstedt, L. E. I., Hunie, D. S., Ibzd., 32, 576 (1960). (13) Kolthoff, I. M., Bruckenstein, S., J . Am. Chem. Sac. 78. 1 119561. (14)Kolthoff. I. iI.. 'Ro&nbl&n. G..
(24) Reilleg, C. X., Schweizer, B., Zbid., 26, 1124 (1954). (25) Ringborn, 8., Sundman, F., 2. anal. Chem. 116. 104 11939). 126) Rinehoh. A'. 1-anninrn. E.. .?no?.
(15) Kortum, G., Anqew. Chem. 50, 193 (1937). (16) Loomeijer, F. J., .-Lna/. Chim. d c t a 10. 148 (1954). 1171 'hlacInnes.'D. A.. Jones. P. T.. J . ' A m . Chem. Aoc. 48, 5831 (lb26). (18) ?\Ialmst,adt, H. V., Rec. Chew. Piogress 17, l(1956): (19) Malmstadt, H. T. ., Roberts, C. B., ANAL.CHEZI.28, 1408 (1956). (20) Mrllon, l f . G., Ferner, G. IT-., J . Phus. Chem. 35. 1025 11931). (21) kellon, 31.G,, Mehlig, 'J. P., Zbid., 35, 3397 (1931). ( 2 2 ) Sichols, 11.L., Kindt, B. H., .%SAL. CHEM.22, 785 (1950). (23) Rehm, C., Higuchi, T., Ibid., 29, 369 (1957).
Schooley, M . R., J . ilk. C', 70, 732 (1948). (28) SchJLarzenbach, G., *'Coniplexonietric Titrations." 1). 5 5 . Intrrscience. Sew Tork. 1957. (29) Ibid., p.' 57. (30) Ibid.,p. 58. (31) JToodland, W. C., Carlin, R. B., Warner, J. C., J . d m Chem. Soc. 75, 5835 (1953). RECEI\EDfor review Februarv 9. 1962. Accepted April 19, 1962. Takkn in part from a thesis submitted by 11. 11. T. K. Gracias in partial fulfillment of the requirements for the degree of master of science. Work sponsored by the Office of Ordnance Research, U. S.Army.
Spectrophotometric Determination of Microgram Quantities of DivinyI Sulfone in Aqueous Media C.
R. STAHL
Cenfral Research laboratory, General Aniline 8, Film Corp., Easton, Pa.
b An alkaline aqueous solution of benzenethiol exhibits an absorption maximum at 262 mp in the ultrais violet region. This method based on the reaction of divinyl sulfone with benzenethiol to form bis (phenylthioethyl) sulfone which precipitates causing a decrease in the ultraviolet absorption of the benzenethiol solution. The decrease in absorbance i s a measure of the amount of divinyl sulfone present. Although benzenethiol reacts with many compoundsi this method i s specific for divinyl sulfone since this i s the only compound which i s known to precipitate under the conditions of this procedure.
D
studies of cross-linking agents for cotton, a method was needed for the determination of trace quantities of divinyl sulfone in n-ater. .1 literature survey produced no niethods for the determination of divinyl sulfone in the parts per million range, and only one method for determining higher concentrations. Ford-Xoore. Peters, and Kakelin ( 4 ) outline a method for assaying divinyl sulfone gravimetrically as the bicysteine derivative. This method cannot be adapted to the determination of small concentrations of divinyl sulfone because of the solubility of the bicysteine derii-atil-e and the gravimetric nature of the method. Corrections for solu-
980
URING
ANALYTICAL CHEMISTRY
bility must be made even when high concentrations of the sulfone are being determined. Stahmann and covorkers ( 5 ) in studying reactions of divinyl sulfone follon-ed the disappearance of the vinyl groups by determining the decrease in thiosulfate titer or by measuring the drop in benzenethiol by titration with iodine. S o details of their procedures are given. Divinyl sulfone is a very reactive compound, and its reactions have been 1%idely discuwed ( I , 3-5). The reaction with thiols appeared to be the most promising for our purpose since it is rapid and complete. Stahmann et al.( 5 ) found that the reaction of divinyl sulfone n i t h benzenethiol, in the presence of base, is 99% complete in i minutes. The method of Beesing et ul. ( 2 ) for acrylonitrile by reaction n ith dodecanethiol \\as tried for dkinyl sulfone and gave good results for relatively high concentrations of the sulfone with slight modification to prevent the precipitation of the partially reacted sulfone. 13y using 0.001S iodine solution as titrant for the e w e v thiol, i t is possible to determine dil inyl sulfone in the parts per million range; honever, the accuracy and precision are poor because of the small titrations. 13is(phenylthioethyl)sulfone is very insoluble in n ater and precipitates when microgram quantities of divinyl sulfone react with benzenethiol in aqueous sodiuni hydroxide solution. The re-
moval of phenyl groups from solution by this process causes a decrease in absorption in the ultraviolet region. A rapid, precise method for determining microgram quantities of divinyl sulfone was developed on the basis of this decrease in ultraviolet absorption. PROCEDURE
A sample, containing 50 to 200 fig. of divinyl sulfone, is diluted to 5 nil. with distilled nater in a 50-nil. voluiiietric flask. One milliliter of a solution of benzenethiol in I S sodium hydroxide containing approximately 60 mg. of thiol per 100 nil. of SaOH is added. The flask is stoppeied and allowed to stand for 15 minutes a t rooni temperature. The contents of the flask are then diluted to 50 nil. with distilled nater. The absorbance of the solution is determined at 340 and 262 nip in a 1.0-em. cell. X blank of 5 nil. of distilled n ater is treated in t h r same manner. The difference in the absorbance a t 340 and 262 mp is subtracted from the blank absorbance to obtain the decrease in absorbance due to the precipitation of the bia(pheny1thioethyl)sulfone. Llicrograms of diviiiyl sulfone are read from a standart1 curve of decrease in absorbance 2's. micrograms of divinyl sulfone prepared from knomn quantities of the sulfone, and parts per niillion divinyl sulfone are found by dividing micrograms of sulfone by grams of sample. .1lternately, parts per million divinyl sulfone ran be calculated using the expression
r
Figure 1.
Plots of decrease in absorbance vs. micrograms b. Divinyl sulfone * Vinyl sulfonyl ethanol Sample volume = 5 ml. Sample volume = 10 mi.
o and
c.
0
Decrease in absorbance (:rams of sample X 0.0031 p.p.m. divinyl sulfone The value 0.0031 is the decrease in absorbance due to 1 pg. of divinyl sulfone. DISCUSSION
The precipitation of bis(pheny1thioethy1)sulfone is dependent on t'he volume of solution present during the reaction. Dilution after reaction does not dissolve the precipitate. Increasing the volume of the sample before reaction results in the formation of less precipitate and smaller changes in absorbance for the loner concentrations of sulfone. For a particular sample volume, as the concentration of sulfolie is reduced, a point is reached belon- which the change in absorbance per microgram of sulfone decreases, aiid the, plot of decrease in absorbance us. sulfone cwicentration deviates from a st'raight line. This can be seen in F'igurcl 1 in d i i c h decrease in absorbance 2's. iiiicmgrams of sulfone plots are given for sample volumes of 5 ml. and 10 ml. The change in absorbance is linenr a t a concentration of approximately 10 p g . per nil. in both cases. This permits determination of dil-inyl sulfone in c*oncent'rationsof 10 p.p.m. or higher. Thiols in basic solution are oxidized to disulfides by air. However, the :imomit of oxidation of the benzenethiol which occwrs in determining divinyl sulfone by this procedurr, is so small t h a t it does not affect the results. During the dc.velopment of the method, at't'enipts were made t'o reniove the bis(pheny1thioetliy1)sulfone precipitate by filtration to eliminate the correction for the rise in base line due to the scattering
effect of the precipitate. During this filtration, hoa ever, the oxidation of the thiol was sufficient to cause erratic results. Reliable results can be obtained without removing the precipitate by measuring the absorbance a t 340 nip as a correction for scattering due to the precipitate. The rise in base line due to the precipitate is illustrated in Figure 2, b. Curve a is the spectrum of the benaenethiol blank, and curve b is the spectrum of an equal quantity of benzenethiol after reaction with 159 pg. of divinyl sulfone. The absorbances at 340 and 262 m p are 0.01 and 1.81 in curve a and 0.08 and 1.38 in curve b. The differences obtained by subtraction :ire 1.80 and 1.30, respectively, and the decrease in absorbance due to the divinyl sulfone is 0.50. Values for thrinyl sulfone obtained by calculation of the decrease in absorbance in this manner exhibit good accuracy and precision. Results of repeat determinations on aqueous solutions of kno\qn concentration are given in Table I. Small caoncentrations of other unsaturated compounds, such as acrylonitrile and vinyl sulfonyl ethanol, which react with benzenethiol in basic iolution but do not form precipitates, cause qome decrease in absorbance. This decrease in abqorbance is much less than due to the reaction of divinyl d f o n e . Curve e, Figure 1, gives the decrease in absorbance due to vinyl sulfonyl ethanol uhich is the only reactive impurity which would be expected in dii ins1 sulfone solutions. If vinyl sulfonyl ethanol or other reactive compounds are present, high values are obtained for divinyl sulfone Table I1 s h o w the effect of vinyl sulfonyl ethanol and acrylonitrile on results obtained for divinyl sulfone. The increases in the divinyl sulfone
Figure 2. a
b
Ultraviolet spectra
Benzenethiol Benzenethiol plus divinyl sulfone
values are less than would be mpected. From the decreases in absorbance caused by vinyl sulfonyl cthanol and acrylonitrile in the absence of divinyl sulfone, values of 26.8 p.11.m. aiid 30.8 p.p.ni. would be predicted for the samples containing 22.5 p.p.ni. vinyl sulfonyl ethanol and 24 p.p.m. acrylonitrile, respectively. The presence of these reactive compounds does not cause serious errors if their concentrations are no greater than the divinyl sulfone concentration; however, relative high concentrations of these compounds produce very high divinyl sulfone results.
Table 1. minations
Results of Repeat Deteron Aqueous Solutions of Divinyl Sulfone
1)ivinyl Sulfone, P.P.hI. Added Found 11.2 19.1 22.5 :3:3 . 7
11.2 18.8 22.6 33.6
KO.
of
IjeterminaPlv. tions Dev., C,; 1.8 2.0 1.B 1.2
4 4 8 4
Table II. Effect of Vinyl Sulfonyl Ethanol and Acrylonitrile on Divinyl Sulfone Analysis
Solution Composition, P.P.M. Vinyl Uivinyl sulfonyl Acrylosulfone ethanol nitrile 22 3 22 5 22 5 22 .5 22 5 22 5 22.5 22.5 22.5
0
7 5 15 22 5 6000 0 0 0 0
1)ivinyl Sulfone Ipound, P.P.Xl,
0 0
22 .t
0 0 0 8 16 24 5000
22 8 22 8 14 0 22 8 24.2 24.8 53.2
VOL. 34, NO. 8, JULY 1962
22
8
e
981
Values for divinyl sulfone will not be obtained if none is present since the absence of a precipitate a t the end of the 15-minute reaction period demonstrates the absence of divinyl sulfone in the sample being analyzed. . h y decrease in absorbance n-ithout the formation of a precipitate is due to the reaction of the benzenethiol with some compound other than divinyl sulfone. I n the absence of a precipitate i t can be assumed that no divinyl sulfone is present without any absorbance measurement. It is possible to determine divinyl sulfone in the presence of small amounts of other reactive compounds turbidimetrically, since the other compounds
do not form precipitates. The relative amounts of divinyl sulfone present can easily be estimated visually from a series of samples containing varying amounts of the sulfone. By visual comparison v i t h a series of standards the divinyl sulfone contents of aqueous solutions can be determined within 2 p.p.m. Vinyl sulfonyl ethanol and acrylonitrile present in quantities as large as 20 times the divinyl sulfone content do not affect the amount of the precipitate. ACKNOWLEDGMENT
The author thanks H. B. Freyerniuth, D. I. Randall, and H. J. Stolten for
their helpful suggestions during this work. LITERATURE CITED
(1) Alexander, J. R., McCombie, H., J. Chem. SOC.1931,1913-18. (2)Beesing, D. W., Tyler, W. P., Jurtz, n.M.. Harrison. S. A.. -4N.4L. CHEW Z,1073-6(1949 j. (3) Ford-Moore, 9.H., J . Chem. SOC. 1949, 2433-40. (4) Ford-Moore, A. H., Peters, R. A., Wakelin. R. W.. Zbid.. D. 1754-7. ( 5 ) Stahmann, 31. A.;* Golumbic, C., Stein, W. H., Fruton, J. S., J . Org. Chern. 11, 719-35 (1946).
RECEIVEDfor review March 1, 1962. Accepted May 10, 1962.
Determination of Isotope Effects by "Double Labeling" Oxidation of D-Glucose with Iodine HORACE S. ISBELL, LORNA T. SNIEGOSKI, and HARRIET L. FRUSH Division of Physical Chemisfry, National Bureau of Standards, Washington, D. C.
b A simple method is presented for determining isotope effects by the simultaneous use of two different isotopes in a compound undergoing reaction. Mixed with the compound containing the functional isotope (at or near the center of reaction) i s the same compound bearing the second (reference) isotope placed a t a point, remote from the reaction center, where it will have little or no effect on the rate of reaction. The effect of the functional isotope on the reaction rate i s measured b y changes in the ratio of the two isotopes as the reaction proceeds. Carriers can be used, and quantitative separation of the materials i s unnecessary because the determination depends on th- ratio of the isotopes. The method is applicable to a variety of chemical and biological reactions. It i s illustrated b y a study of the oxidation, b y iodine in alkalinz solution, of isotopically labeled D-glucose-1 -t, 6-CI.I and D-glucose-1 -C1', 6-t.
A
s
effect, that is, a difference in the rates of reaction of labeled and unlabeled molecules, provides a versatile means for studying reaction mechanisms; it is associated with the difference in mass of the isotopic and nonisotopic atom. When the isotope is directly involved in the rate determining step of a reaction, a relatively large difference (primary isotope effect) is found. K h e n the isotope is not directly involved, hut is suitably located with respect to the reaction center there may lie smaller (secondary) isotope
982
ISOTOPE
ANALYTICAL CHEMISTRY
effects. An isotopic atom remote from the reaction center has little or no effect on the reaction rate. Because the magnitude of the isotope effect depends on the role of the isotope in the reaction, isotope effects can be used for probing reaction mechanisms (2, 11). This paper reports a double-label technique in the study of isotope effects of carbon-14 and tritium. Elegant analytical methods involving derivatization by the use of two radioisotopes (S3*and n-ere first elaborated b y Keston, Udenfriend, et al. (8, 17, 18) for determining and identifying amino acids. More recently. the double isotope derivatization procedure with carbon-14 and tritium has been applied in the analysis of steroids (.9,12)and gibberellic acids ( 1 ) . I n the present kinetic study of isotope effects, both isotopes are present in the compound before reaction. The functional isotope (whose effect is to be determined) is placed a t or near the reaction center, and a reference isotope is placed nhere it has little or no effect on the reaction rate. I f an isotope effect exists in the reaction of a doubly labeled compound, the ratio of the tlyo isotopes in both the residual reactant and the product changes progressively as the reaction proceeds. The method has the advantage that nonradioactive carriers can be used in the separation of either the r e d u a l reactant or the product, because the dilution of radioactivity has no effect on the ratio of the isotopes; furthermole, quantitative separations are unnecessary. The magnitude of the isotope effect is usually eupressed b y the ratio k * / k , in
which k* and k are, respectively, the rate constants for the labeled and the unlabeled forms of the reactant' molecule. When k* is less than k , the labeled reactant is consumed more s l o ~ d ythan the unlabeled, and the concentration of the labeled form in the residual reactant gradually increases. At the beginning of the reaction, the molar concentration of the isotope in the product is ion-er than that in the initial react'ant,, but it increases as t'he reaction proceeds. If the reaction goes to completion nit'hout side reactions, the molar concentration of the isotope in the total product becomes the same as that in the iiiit'ial reactant. For a reaction that is first order (or pseudo first order) with respect to the reactant, the value of k*, k can be calculated by the equations of Stevens and Attree (16) as modified by Ropp ( I S ): k*/k
=
k*/k
1 =
+
[log r ' l l o g (1 - ,f)] (1)
log (1 - ?f),!log (1
-
,f) ( 2 )
I n these equations. f is the fraction of starting material that has reacted. K h e n the equations are applied t o radioactive compounds, r is the ratio of t'he molar specific activity of the accumulated product to that of the initial reactant; and r' is the ratio of the molar specific activity of the residual reactant t o that' of the initial reactant. (The value for the molar specific activity of the accumulated product of 10070 reaction may be used instead of that for the initial reactant.) Determination o f f , r , and r' by convent,ional met'hods is difficult, because
