Spectrophotometric Determinations Based on the Formation of Tris-1,lO-PhenanthrolineIron(l1) T. J. Bydalek, J. E. Poldoski, and D. Bagenda John Department of Chemistry, University of Minnesota-Duluth,
Duluth, Minn. 55812
IN A RECENT publication ( I ) , it was shown that the reaction of nitrilotriacetatoferrate(II1) [Fe(III)NTA] with cysteine in the presence of 1,lO-phenanthroline formed the basis for the quantitative determination of cysteine in the concentration Tris-1 ,lo-phenanthroline iron(I1) is range of lO-6-lO-‘M. produced in stoichiometric amounts and spectrophotometrically determined at 510 nm. It appeared that the reaction sequence could be used for the determination of other species if they possessed sufficient reducing capability and favorable rates of reaction. I n order to determine the usefulness and limitations of the above reaction sequence, seven additional reducing species have been examined and the formal reduction potential of the Fe(II1)NTA-Fe(I1)NTA couple has been determined under specified conditions. The species investigated include two aliphatic acid thiols, thiosuccinic acid and 2,3-dithiosuccinic acid; an aliphatic amine thiol, 2-thioethylamine ; a thiophenol, thiosalicylic acid ; a thiol containing the peptide linkage, glutathione (2-glutamylcystenylglycine); and two inorganic ions, cobalt(I1) and ferrocyanide. EXPERIMENTAL
Reagents and Solutions. The preparation of reagent solutions is given in a previous work ( I ) . Tris (hydroxymethyl) aminomethane-HC1 mixtures (tris) were used as the buffer for the pH range 7.0-8.5, and sodium acetate-acetic acid mixtures were used for the pH range 4.0-5.3. Solutions (lO-aM) of thiosuccinic acid, 2,3-dithiosuccinic acid, and thiosalicylic acid were prepared by dissolution of the appropriate weight of the thiol in a minimum amount of base, and diluting with water. In the case of thiosuccinic acid and 2,3-dithiosuccinic acid, the pH was adjusted to 7.2 with tris buffer. The percent purity of thiosuccinic acid and of 2,3-dithiosuccinic acid was determined by iodimetric titration (2), 98.5Z (4 detn, standard deviation 0.36Z) and 96.8 % (3 detn, standard deviation 0.30Z), respectively. Thiosalicylic acid was not analyzed. In the case of 2-thioethylamine, approximately 10-IM solutions were prepared by dissolution of 2-thioethylamine hydrochloride in water. These solutions were standardized by iodimetric titration ( 2 ) (4 detn, standard deviation 4 ppt) and diluted for use. Solutions of thiosuccinic acid, 2,3-dithiosuccinic acid, and 2-thioethylamine were shown to be stable for 2 hours. Glutathione (99.87 Z , Cal. Biochem.) solutions (10-3M) were prepared by dissolution of the solid in water and were used immediately after preparation. Cobalt(I1) solutions (0.005M) were prepared by dissolution of cobalt(I1) nitrate hexahydrate in water and diluted for use. The stock solutions were analyzed by a direct titration (3)
with standard EDTA using murexide as indicator. The standard deviation of 4 determinations was 3 ppt. Solutions (lo- 3M) of potassium ferrocyanide were prepared by dissolution of anhydrous potassium ferrocyanide in water. The anhydrous salt was prepared ( 4 ) by drying the recrystallized trihydrate at 100-110 “C. An average percent purity of 100.0% (3 detn, standard deviation 0.13 %) was obtained for the anhydrous salt by titration with standard cerium(1V). For the measurement of the formal Fe(II1)NTA-Fe(I1)NTA potential, Fe(I1) solutions were prepared from Fe(NH4)?S04. 6 H 2 0 (Mallinckrodt). The Fe(I1) concentration was determined spectrophotometrically with 1,lO-phenanthroline. Fe(I1)NTA solutions were prepared by adding an aliquot of the freshly prepared Fe(I1) solution to a known amount of NTA (buffered with tris) in the absence of oxygen. Apparatus. Absorbance measurements were made on a Beckman D U spectrophotometer using 1.OO-cm. cells. Solutions to be measured spectrophotometrically were prepared in Pyrex brand red glass flasks. A Corning Model 10 p H meter was used for all pH and potentiometric (platinum indicator electrode) measurements. Formal Potential. The formal electrode potential of the Fe(II1)NTA-Fe(I1)NTA couple was evaluated from potential measurements of Fe(II1)NTA-Fe(I1)NTA mixtures at a platinum electrode. The Fe(II1)NTA concentration was 1.00 x 10-3M. All solutions were maintained at pH 7.6 =t 0.1 with 0.1M tris buffer. The total chloride ion concentration was 0.15 =t 0.03M. Oxygen was removed from these solutions by purging with nitrogen. The potential measurements at a platinum electrode followed the Nernst equation as the Fe(III)NTA/Fe(II)NTA ratio was varied from 1/3 to 9jl. The formal reduction potential of the couple was evaluated to be $0.06 i 0.01 V cs. the standard hydrogen electrode. Procedure. The procedure is similar to that reported for cysteine ( I ) . Buffer components (final concentration approximately 0.1M) and 7.00 ml of 10+M NTA [l-3 percent excess with respect to Fe(III)] are added to a 100-ml red glass volumetric flask. Fe(III), 7.00 ml of 10-2M, is then added and the walls of the flask are washed with distilled water. After allowing 2 minutes for Fe(II1)NTA formation, 7.0 ml of 5 X 10-3M phenanthroline [for cobalt(II), 6 ml of lO-*M] are added and immediately followed by the addition of the appropriate amount of sample. Absorbance measurements are made at 510 nm after allowing sufficient time for color development. A similar procedure is used for the blank determination. Table I gives the details of pH and times of color development. The concentration range is 10-j-10-4M for all species except 2,3-dithiosuccinic acid where the range is 5 X 10-6-5 X 10-5M. Glutathione and thiosalicylic acid cannot be quantitatively determined. The molar absorptivity (1.10 X 10f4M-l cm-l) of tris-1,lOphenanthroline iron(I1) was determined as given inReference 1. RESULTS AND DISCUSSION
(1) T. J. Bydalek aid J. E. Poldoski, ANAL.C H E M . 1878 , ~ ~ ,(1968)
(corr. 2133). (2) S. Siggia, “Quantitative Organic Analysis via Functional Groups,” 3rd ed., Wiley, New York, 1963, p 578. (3) F. J. Welcher, “The Analytical Uses of Ethylenediaminetetraacetic Acid,” D. Van Nostrand, Princeton, N. J., 1958, p 230.
The reaction of each species with Fe(II1)NTA in the presence of 1,lO-phenanthroline was investigated under condi(4) I. M. Kolthoff and E. B. Sandell, “Textbook of Quantitative Inorganic Analysis,’’ 3rd ed., Macmillan, New York, 1952, p 563. ANALYTICAL CHEMISTRY, VOL. 42, NO. 8, JULY 1970
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Table 1. Spectrophotometric Conditions Absorbance measurement Minimum Maximum time, time, Species pH range minutes minutes Thiosuccinic acid 7.2-7.8 10 30 2,3-Dithiosuccinicacid 7.2-7.6 10 30 2-Thioethylamine 7.6-8.1 15 35 Cobalt(1I) 4.0-4.9 2 45 Ferrocyanide 4.0-4.8 2 45 7.0-7.8 15 45
tions similar to those used in the cysteine study. The concentration of Fe(II1)NTA was maintained at 7.0 x lO-4M. An excess of 1 - 3 z NTA was used. The concentration of 1,lO-phenanthroline was 3.5 X 10-4M in all cases, except in the analysis of cobalt(II), where the concentration was 6.0 x 10-4MM. Preliminary results showed that for quantitative reaction with cobalt(II), the 1,lo-phenanthroline concentration must be maintained at a level sufficient to complex both the iron(I1) and cobalt(II1) produced. The effect of pH on the percent reaction at a specified reaction time after the sample addition was investigated. In these studies, the concentration of the species being investigated was maintained at 7.0 X lO-SM. In the case of 2,3dithiosuccinic acid, both thiol groups undergo oxidation and the concentration was maintained at 3.5 X 10-5M. The pH range in which the percent reaction is 100 i 1 is given in Table I. In general, 10 minutes were allowed for color development, but in the case of 2-thioethylamine and ferrocyanide (pH 7-8), somewhat longer times (15 min) were needed. For ferrocyanide and cobalt(I1) in the pH range 4-5, color development was found to be complete in shorter times (2-3 min). For the thiols in Table I, the absorbance values are stable for 30-35 minutes. At longer times (60 min), the values tend to be high by 1-2 For ferrocyanide and cobalt(II), the absorbance values are stable for 45 minutes, the longest time studied. Data for glutathione and thiosalicylic acid are not included in Table I. In the pH range 7-8, glutathione reacts at a very slow rate, approaching 100 percent reaction in 7 hours. The blank is not stable over this time interval and analysis is not possible. Thiosalicylic acid reacts at a faster rate but proceeds beyond 100 percent reaction in approximately 1 hour. Stable and reproducible readings could not be obtained. Using the conditions given in Table I, the concentration
z.
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of each species was varied from 1 X 10-6M to 1 x lO-4M (5 X 10-6Mto 5 X 10-5M for 2,3-dithio succinic acid). The agreement between the concentration taken and that found is generally within f 1 relative and the typical range for six determinations is 1 %. Data for 2-thioethylamine are typical and values x l O + 5 (taken, found, range of 6 detn.) are: 0.98, 0.96, 0.02; 1.96, 1.94, 0.01; 2.94, 2.94, 0.02; 4.90, 4.93, 0.02; 6.86,6.89,0.03; 9.80,9.85,0.10. The reaction conditions vary somewhat among the compounds studied but not sufficiently to determine one species in the presence of another. It is expected that other thiols would behave similarly. In the systems studied, the reaction pH is the most important variable, influencing both the rate and the extent of the reaction. In all cases studied, no interference is observed for complexing species such as glycine and phosphate at the 0.005M concentration level. In the pH range 4-5, ethylenediaminetetraacetic acid (EDTA) showed no interference at concentrations as high as 0.001M. In the pH range 7-8, lod4M E D T A shows no interference; 5 x 10-4M gives a 10% negative error. Metal ions, copper(II), nickel(II), that form phenanthroline complexes are serious interferences, causing very slow color development. In the pH range 4-5, nickel(I1) ( 5 X 10-4M) but not copper(I1) can be easily masked by the addition of EDTA. In the determination of ferrocyanide, at pH 4-5, concentrations of ferricyanide as high as 10-3M have no effect. When the Fe(II1)NTA-Fe(I1)NTA potential is compared to the F~(CN)G-~-F~(CN reduction ) ~ - ~ potential of approximately f0.4 volt (9,the reaction between Fe(III)NTAandFe(CN)6-4 is not favored. However, an examination of the stability constants (6) for Fe(I1)NTA (lof8.*)and Fe(II)phenr clearly indicates that the reaction between Fe(I1)NTA and 1,lo-phenanthroline is highly favored and sufficient to drive the overall reaction to completion even in the presence of excess Fe(CN)6-3. It appears possible that methods can be devised using excess Fe(CN)6-3 as oxidizing agent and completed by the spectrophotometric determination of the Fe(CN)6-4 produced. RECEIVED for review December 8, 1969. Accepted April 16, 1970. This work was supported by the National Science Foundation, Grants GP-7319 and GP-12168. (5) I. M. Kolthoff and W. J. Tomsicek, J. Phys. Chem., 39, 945 ( 1935). (6) L. G. Sillen and A. E. Martell, “Stability Constants of MetalIon Complexes,” The Chemical Society, London, 1964.
