removing the gas by boiling in a stream of COz and titrating with 0.02AV Ce(SO&. Chemical determinations were made on stock solutions 1000 times more concentrated than t'he diluted solutions used for the radio-release measurements. Interferences. Chloride ion proved to be a serious interference and a major part of this work was expended in finding a suitable complexing agent for XgC1. Many complexes dissolved AgC1, but most of them caused release of silver from the column probably through oxidation by dissolved oxygen. Three suitable silver complexes not exhibiting this property were found in a family of niercaptoalkylamine hydrochlorides synthesized in a separate work by Carroll, White, and Wall (I 2 ) . These were 2-niet'hyl2-mercaptobutylamine hydrochloride, 3-mercapto-2-aminoheptane hydrochloride, and 3-mercapto-2-aminohexane hydrochloride. Xone of these was available commercially or from custom synthesis services. Further work led to the discovery of two thiourea derivatives that complexed silver chloride without causing high blanks. These were 1,3-diethyl-2-thiourea and thiosinamine. The first is preferred and is purified from the technical grade by three recrystallizations in water. Some background increase is introduced by the recrystallized compound so that additions are made in moderation. The concentration of 50 mg./liter customarily used introduces a background corresponding to about 0.1 p.p.m. of vanadate. This material is sufficient to complex silver released by ~
about 20 p.p.m. of vanadium(V). If higher vanadium levels are contemplated, more 1,3-diethyl-2-thiourea is required. Iron(II1) interference has not been completely removed. With columns containing rather large silver particles consisting of silver-plated platinum chips or silver chips irradiated in the Oak Ridge reactor iron(III), interference was insignificant and often not detectable. However, with these columns, the release of silver by vanadate ion was very slow and the reaction nonstoichiometric for usable flow rates. Even with carefully regulated flow rates through the column reproducibility was poor. Iron(II1) did interfere when columns of finely divided silver were used and in which the vanadate reaction was complete and reproducible. In some of these columns iron(II1) interference could be masked completel> by the addition of 200 mg. each of (ethylenedinitrilo) tetraacetic acid and either sodium fluoride or phosphate. For other colunins the coniplexing agents suppressed the iron(II1) activity by a factor of about 10. The need for a complexing agent in addition to EDTh is not fully understood; however, the combination does suppress iron(II1) in the range of pH = 3. Xo single component masking agent was found that would prevent or suppress silver oxidation by iron in the presence of 1,3diethyl-2-thiourea. From the present data it appears that the iron(II1) reaction with silver 1s slower than the vanadate reaction.
For moderate flow rates (1 to 3 nil,/ minute) through a column of coarse silver particles (small surface ar-a), the iron(II1) reaction rate is too slow to produce a measurable activity level although the vanadate reaction goes partially to completion. When columns containing finely divided silver particles (large area) are used, t,he vanadate reaction with silver goes to completion and the iron(II1) reaction activity is about of its stoichiometric value. It' would appear that an optimum flow rate exists for a particular column wherein the vanadate reaction is stoichiometric and the iron(II1) interference is a minimum. This has not been investigated in the present work. Dissolved oxygen does not interfere with this specific technique in spite of the fact that thermodynamically it should do so. hcids, except for powerful oxidizing acids such as HSOa, do not interfere. Nitrate ion does interfere at higher concentrations but not in the range to 10 p.p.m. LITERATURE CITED
( 1 ) Carroll, F. I., White, J. D., Wall, XI. E., J . Org. Chem. 28, 1236 (1963). ( 2 ) Ibid.,p. 1240. 13) GillesDie. A. S.. Jr.. Richter. H. G..
RECEIVED for review September 10, 1964. Accepted September 30, 1964. This work was carried out under Atomic Energy Commission, L3ivision of Isotopes Development, Contract No. AT-(40-1)-2513. Presented at the Pittsburgh Conference on hnalytical Chemistry and Applied Spectroscopy, IIarch 1964.
Titration of Aromatic SuIfinic Acids in Nonaqueous So Ivents DAVID
L. WETZEL'
and CLIFTON E. MELOAN
Department of Chemistry, Kansas State University, Manhattan, Kan.
b Acid-base titrations of sulfinic acids and mixtures of sulfinic and sulfonic acids with quaternary ammonium titrants in nonaqueous solvents is possible. Titrants investigated were tetramethyl, tetraethyl, tetra-n-butyl, trimethyl benzyl, trimethylphenyl, and cetyldimethylethyl ammonium hydroxides. The following solvents were suitable for titrating milligram quantities of aromatic sulfinic acids, producing from 400- to 600-mv. changes: ethyl acetate, dimethyl sulfoxide, chlorobenzene, nitrobenzene, acetone, acetonitrile, diethyl ether, tetrahydrofuran, benzene-methanol, n-butyl alcohol, t-butyl alcohol, pyridine, dimethylformamide, and dimethyl aniline. In addition, the following sol-
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ANALYTICAL CHEMISTRY
vents produced at least 400-mv. changes when sulfinic-sulfonic acid mixtures were titrated with tetra-nbutyl ammonium hydroxide: benzenemethanol, t-butyl alcohol, tetrahydrofuran, dimethylformamide, and pyridine, In general, the relative standard deviation was within 70.2570 and the relative error within 70.4%.
S
ACIDS (R-SO2H) are notorious for their instability and the ease with which they undergo autoxidation. The corresponding sulfonic acid which is a final product of both the decomposition and autoxidation is a potential source of interference in the quantitative study of the acidic properties of sulfinic acids.
ULFINIC
There are a t present four areas of sulfinic acid analysis. These include the iron(1II) salt procedure (6, 7 ) , the nitrite titration (11, 13, f 4 ) , oxidation methods (1, 2, I O ) , and aqueous neutralization reactions. The need for the use of large samples, prior separations, temperature control, unstable titrants, or external indicators imposes limitations on these methods. The strength of sulfinic acids can generally be described as being greater than that of corresponding carboxylic acids and less than that of corresponding sulfonic acids. The relative acidity of the sulfinic acids has not been extensively used for analytical purposes, 1 Present address, Kansas State Teachers College, Emporia, Kan.
Comparison of Use of Different Titrants n-Toluenesulfiiiic Benzenesulfinic acid ~ _ _ _ ~ _ _ _acid _ _____ Taken, Found, Taken, Found, Titrant mg. mg. /c mg. mg. 10 TSIRAHn 88 76 87 97 99 1 77 66 76 87 99 1 7 2 84 73 15 100 4 81 54 79 93 98 2 8-
1953.
( 7 ) Feiyel, F., “Chemistry of Specific,
(14) Ponjini, S., Farm. Sci. e. Tec. (Pavia) 2, 198 (1947); C . A . 42, 3701. (15) Samuelson, Olof, “Ion Exchangers in Analytical Chemistry,” Wiley, New York, 1953. (16) Smiles, S., Bere, C., “Organic Synthesis,” Vol. I, p. 7, Wiley, New York, 1946. (17) Tan der Heijde, H. B., Dahmen, E. A,, Anal. Chim.Acta 16, 378 (1957). (18) Whitmore, F. C., Hamilton, F. H., “Organic Synthesis,” Vol. I, p. 492, Wiley, New York, 1946. RECEIVEDfor review March 4, 1963. Resubmitted June 29, 1964. Accepted September 23, 1964.
A n Automatic Spectrophotometric Method for the Specific Enzymatic Determination of Galactose in Whole Blood and Plasma CHRISTOPHER S. FRINGS and HARRY
1.
PARDUE
Department o f Chemistry, Purdue University, lafayette, Ind.
An automatic enzymatic method is described for the determination of galactose in blood. The method is based on the catalysis by galactose oxidase of the oxidation of galactose. Hydrogen peroxide produced by the enzymatic reaction oxidizes o-dionisidine in the presence of peroxidase to a species which absorbs a t 440 mp. The formation of the colored species is detected spectrophotometrically. Commercially available automatic control equipment provides direct readout of the time required for a predetermined decrease in per cent transmittance to occur. The reciprocal of the measured time interval when plotted against the galactose concentration provides a linear working curve. Automatic results for aqueous samples show relative standard deviotions within 2% over a range of 33-500 p.p.m. galactose. Results for galactose in blood between 99 and 1500 mg. per 100 ml. show similar limits of precision and accuracy.
T
XEED for good methods for determining galactose quantitatively in blood for the purpose of diagnosing galactosemia has been discussed in recent papers (1, 2 ) . It has been suggested that a laboratory test for the disease should be available in every laboratory ( 1 ) . h symptom of galactosemia is the presence of galactose in the blood. Difficulties associated with conventional methods for galactose in blood have been discussed ( 3 ) . The present work was undertaken to provide a simple, rapid, and selective method for this analysis. This has been accomplished utilizing the enzyme galactose oxidase.
HE
Galactose oxidase catalyzes the oxidation of galactose to the dialdose producing an equivalent amount of hydrogen peroxide. h method for galactose in aqueous solutions utilizing the iodineiodide redox couple to detect the formation of peroxide was described recently ( 3 ) . Attempts to adapt this method for galactose in blood were only moderately succeqsful. The coupled enzyme reaction using horseradish peroxidase and o-dianisidine to detect the hydrogen peroxide was superior to iodide in this reaction. The present method is baqed on the measurement at 440 rnp of the rate of formation of the colored product resulting from the coupled reaction. Automatic control equipment measures the time required for the transmittance to decrease over a small predetermined interval. The reciprocal of the measured time is a linear function of galactose concentration. Results for aqueous galactose solutions between 33 and 500 p.1j.m. and galactose in blood between 100 and 1500 mg. per 100 ml. are precise and accurate to within 2%. Measurement times range from a few seconds a t higher concentrations to about 5 minutes a t the lower concentrations reported here. EXPERIMENTAL
Reagents. A11 solutions are prepared in water which has been passed through a mixed cation anion exchange resin bed. All solutions are stored at 4’ C. GALACTOSESTANDARDS. Standard galactose solutions are prepared by dilution of a 2000 p.p.m. solution prepared by dissolving 2.000 grams of D ( + ) galactose (Sigma Chemical Company,
St. Louis, Mo.) in water and diluting to 1 liter. PHOSPHATE BUFFER. Potassium dihydrogen phosphate (1.4 grams) is dissolved in 900 ml. of water. The solution is adjusted to pH 7.0 and 0.75N KaOH and diluted to 1 liter. GALACTOSEOXIDASE. Five milligrams of galactose oxidase dry powder (Worthington Biochemical Corp., Freehold, N. J., 5 to 7 units per milligram) is triturated in a mortar, diluted to 50 ml. with buffer solution and filtered through Whatman No. 1 filter paper. This gives an enzyme concentration of about 0.5 Worthington unit per milliliter. This solution was stable for several days a t 4” C. COLORREAGENT. Thirty milligrams of o-dianisidine (Eastman, Rochester, N. Y.) in 3 ml. of methanol and 12 m g . of horseradish peroxidase (Worthington Biochemical Corp., Freehold, N . J.) are dissolved in 500 ml. of buffer. DEPROTEINIZATION REAGENTS. A zinc sulfate solution is prepared by dissolving 5 grams of ZnS01.7H,0 in 100 ml. of water. =\ barium hydroxide solution is prepared by dissolving 4.5 grams of 13a(OH)?.8H20in 100 ml. of water. These solutions are titrated to the phenolphthalein end pqint. The more concentrated solution is diluted with water until the acid and base strengths of the two are the same. Instrumentation. Changes in transmittance are observed using a regulated Bausch & Lomb Spectronic 20 spectrophotometer. l‘he unregulated model is not satisfactory. l’he instrument meter is disconnected and external hookup leads are connected to the amplifier output leads (points G and H on t h e instrument diagram). I t should be noted t h a t both points G and H are about 100 volts above instrument ground. Caution must be exercised in handling these leads and in connecting them to measurement VOL. 3 6 , NO. 13, DECEMBER 1 9 6 4
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