TERBIUM METHOD
Table 11. Effect of Foreign Ions Tolerable level, ppm Eurooium Terbium method method
Ion NOaNO, ICr 3~ 1 3 +
BaZ+ Na+ K+
Pb2+ Hgz+
Fe3+ SiO3ICNSMn2+ Brco2cu2Nil+ Y3f SOa2S2-
F-
1 1 1 1 1 1 1 1 1 0.5 0.3 0.3 0.2 0.2 0.1 0.1 0.1 0.1 0.1 0.06 0.06 0.00
1 1 1 1 1
... ... ... 0.25 ... 0.25 0.1 * .
0.5 0.1 1 0.1
... ... ... ...
Chelates prepared in 95% ethanol gave more intense fluorescence than those in methanol, acetone, water, and EtOHH20mixtures less than 95% in ethanol. No significant increase in sensitivity resulted from using 100 % ethanol. Measure the fluorescence intensity a t 614 nm, with the excitation monochromator at 312 nm and a 430-nm cutoff filter in the emission path.
Procedure. Similar variable studies were performed on the terbium chelate system as on the europium. The resulting procedure for T b differs only slightly from that for Eu. The T b chelate forms more readily and stabilizes more rapidly so the solution need stand only 5 minutes prior to dilution and may be read immediately thereafter. The T b chelate solutions are read at emission wavelength of 544 nm and excitation wavelength of 312 nm. DISCUSSION AND CONCLUSIONS
The 1,1,1,5,5,5-hexafluoro-2,4-pentanedionechelates of europium and terbium show intense fluorescence at wavelengths characteristic of the particular ion. This fluorescence, when used as the basis for the selective analysis of nanogram quantities of Eu and Tb, produces a more sensitive method than any previously reported for a trifluoro-P-diketone. Calibration curves for both ions were prepared according to the methods outlined. Both methods have a sensitivity of 1 ppb. The europium calibration curve is linear between 1 and 50 ppb and is analytically useful to 80 ppb. The foreign ions considered are listed in Tables I and 11. The interferences of these ions were considered tolerable at the concentration level given if their presence altered the fluorescence intensity of a 4-ppb solution of Eu(Tb) by less than 6%. The data reported in Table I indicate that Eu and T b can be selectively determined in the presence of large excesses (at least 15-fold) of other rare earth ions without separation procedure.
RECEIVED for review August 5, 1970. Accepted October 12, 1970.
Spectrophotometric Determination of Sulfide Ion with Organic Disulfides Ray E. Humphrey, Willie Hinze, and William M. Jenkines I11 Department of Chemistry, Sam Houston State University, Huntsville, Texas 77340 FEWSPECTROPHOTOMETRIC METHODS have been reported for the determination of hydrogen sulfide or sulfide ion. The procedure which appears to be the most widely used for this analysis involves the reaction of hydrogen sulfide with p amino-N,N-dimethylanilinein the presence of ferric chloride to form methylene blue (I). This method is quite sensitive, the product having a molar absorptivity of about 35,000. In order to measure hydrogen sulfide in the parts per billion range, a n indirect fluorescence method involving fluorescein mercuric acetate has been used (2). The reaction of organic disulfides with sulfide ion was reported by Otto and Rgssing (3). The following equation for this interaction was given: (1) G. D. Patterson in “Colorimetric Determination of Nan-
Metals,” D. F. Boltz, Ed., Tnterscience, New York, N. Y . , 1958, pp 273-277. (2) H.D. Axelrod, J. H.
Cary, J. E. Bonelli, and J. P. Lodge, Jr.,
ANAL.CHEW, 41, 1856 (1969). (3) R. Otto and A. Rossing, Chem. Ber., 19, 3129 (1886). 140
RSSR
+ 2K2S
+
2RSK f KzSz
(1)
This reaction has apparently not been studied very extensively. We have found that a number of water-soluble organic disulfides, 2,2’-dithiodipyridine (2-PDS), 4,4’-dithiodipyridine (4-PDS), 2,2’-dithiodipyrimidine (2-PyDS), and 5,5 ’-dithiobis(2-nitrobenzoic acid) (DTNB), react with sulfide ion rapidly at room temperature t o produce the thiol anion and elemental sulfur. This reaction is represented by the following equation : RSSR
+ S-2
+
2RS-
+ So
This interaction has been found to be useful for the spectroDhotometric determination of sulfide in the very low microgram per milliliter range. The disulfides DTNB, “Ellman’s reagent,” ( 4 ) , 2-PDS and 4-PDS (5), and 2-PyDS (6) are used (4) G. L. Ellman, Arch. Biochem. Biophys., 82, 70 (1959). ( 5 ) D. R. Grassetti and J. F. Murray, Jr., ibid.,119,41 (1967).
(6) D. R. Grassetti and J. F. Murray, Jr., Anal. Chim. Acta, 46, 139 (1969).
ANALYTICAL CHEMISTRY, VOL. 43, NO. 1, JANUARY 1971
Table I. Spectral Data for Thiol Anions h max Thiol Molar Disulfide anion, nma absorptivityb 2-PDS 340 7,000 (7060) 4-PDS 324 21,500(19,800) 2-PyDS 27 5 16,000 (16,000) DTNB 412 15,500 (13,600) for the anion of 4-PDS shifts to 285 nm at pH 9 with a The X shoulder appearing at 324 nm. The h max for the 2-PyDS anion is at 268 nm at pH 9 and at 278 nm in distilled water. b Literature values in parenthesis, reference (5) for 2-PDS, reference (6) for 2-PyDS, and reference (8) for DTNB.
09
0 8
0 7
E
* 0 N
0 6
Q
E 0 5
34
0 3
0 2
for the spectrophotometric determination of sulfhydryl groups. Sulfite ion has also been determined with 4-PDS and DTNB (7).
M o l e F r a c t i o n 5'
Figure 1. Continuous variations plot for the reaction of 4,4'dithiodipyridine with sulfide ion
EXPERIMENTAL
Apparatus. Absorption measurements were made with a Beckman DB-G spectrophotometer and spectra recorded using a Beckman DK-2A instrument. Polarograms were recorded with a Sargent Model XVI Polarograph. Reagents. The disulfides 2-PDS, 4-PDS, and DTNB were obtained from Aldrich Chemical Co., Milwaukee, Wis., and were used without further purification. The disulfide 2-PyDS was obtained by iodine oxidation of 2mercaptopyrimidine, also obtained from Aldrich. Stock solutions of the disulfides, approximately O.OOlM, were generally made up in 95% ethanol. These solutions appeared to be quite stable. Stock solutions of sulfide ion, also about 0.001M, were prepared from Na2S.9H20by weighing crystals which had been washed with distilled water, dried, and dissolved in distilled water or a 0.001M solution of disodium ethylenediamine tetraacetate (EDTA) using a volumetric flask. The compound Na2S.9Hz0 has been found to be a good working standard ( 2 ) . We found that the concentrations as determined by an iodate-iodide procedure with a thiosulfate titration were essentially the same as those calculated from the weight and volume. Buffer solutions were prepared from 0.025M Na2HP04 and K H 2 P 0 4 ,pH 6.86, 0.01M Na2B40,,pH 9.18, and 0.05M KHCsH404, pH 4.01. All other chemicals used were the best available reagent grade. Procedure. Solutions were prepared by measuring the required volumes of the stock solutions of sulfide ion or disulfide by pipet and diluting to the final volume with a buffer solution or distilled water. For establishing or using a Beer's law plot, the disulfide was present in excess. All reactions were run at room temperature. RESULTS AND DISCUSSION
Spectra. Spectral data for the disulfides and the corresponding thiol anions are presented in Table I. Spectra have been published for 2-PDS (5), 4-PDS (5, 7), and DTNB (7, 8). The oxidation product of sulfide ion, elemental sulfur, does not contribute to the absorption at the wavelength maximum for any of the thiol anions. The spectrum of every reaction solution using excess sulfide ion was essentially that of the respective thiol anion. The only disulfide which absorbs significantly at the wavelength maximum for the corresponding thiol anion at every p H used is 2-PyDS. It is desirable to make a correction for unreacted disulfide when using this com-
pound. The wavelength maxima for all the thiol anions, except 4-thiopyridone, are not affected significantly by a change in pH. The compound 4-PDS is not as useful at pH 9, as the maximum for the thiol anion is shifted to shorter wavelengths and the interference of the disulfide is quite large. A shoulder at 324 nm can be used with a considerable decrease in sensit ivit y . Stoichiometry. Mole ratio and continuous variations plots for the reaction of sulfide ion with these disulfides indicate that the stoichiometry in every case is 1:1 as shown in Equation 2. A continuous variations plot for the reaction of sulfide ion with 4-PDS is shown in Figure 1. There was little curvature in any of these plots indicating that the reactions were essentially complete when equimolar amounts of sulfide and disulfide were present. The reactions were quite fast and appeared to be practically complete within 5 minutes, and probably less, after adding the sulfide solution. Polarography of Reaction Solutions. These disulfides all show a reduction wave at the dropping mercury electrode with a half-wave potential, Eli2,in the region of -0.4 to -0.5 volts us. the saturated calomel electrode (SCE). Sulfide ion shows an oxidation wave with Ed2 of about -0.6 volt US. SCE while elemental sulfur shows a reduction wave in the same region. These potential values are for solutions of pH 5 or slightly higher. Polarograms of reaction solutions with approximately equimolar reactants showed the loss or decrease of the reduction current for the disulfide group and the appearance of the reduction wave for elemental sulfur, characterized by a very steep slope. Oxidation current was also present at the less negative potentials due to any unreacted sulfide ion and the thiol anion produced by the reaction. Polarography confirmed that elemental sulfur is the oxidation product of sulfide ion and that the reaction is close to completion at 1 :1 reactant ratios in many instances. Effect of pH on the Reaction. Sulfide ion reacts readily with 2-PDS, 4-PDS, and 2-PyDS in distilled water and in buffers of pH 4, 7, and 9. The compound DTNB does not react in water or a pH 4 buffer but does react readily at p H 7 or 9. At pH values higher than 10, the disulfides are converted to the thiol anion, presumably by reaction with hydroxide ion
(9). (7) R. E. Humphrey, M. H. Ward, and W. Hinze, ANAL.CHEM., 42 698 (1970). (8) G. L. Ellman, Arch. Biochem. Biophys., 74,443 (1958).
(9) A. J. Parker and N. Kharasch, Chem. Rev., 59,608 (1959).
ANALYTICAL CHEMISTRY, VOL. 43, NO. 1, JANUARY 1971
141
Table 11. Beer’s Law Data for Sulfide Ion Determination Sulfide, gg/ml 0.23 0.46 0.92 1.38 1.84 a
~ _
_ 2-PDS _ _
A 340 nm 0.17 0.32 0.51 0.67
_ _ _ ea
11,500 11,Ooo 11,500 11,300
4-PDS A 324 nrn 0.29 0.56 1.15
2-PyDS A 275 nm
€
39,000 38,000 39,000
0.20 0.38 0.76 1.12
DTNB e
26,000 25,500 25,500 25,000
-_
A 412 nrn 0.19
25,500
0.78 1.15 1.50
26,500 26,000 25,500
E
These values are for a pH 7 buffer and are based on the molar concentration of sulfide ion.
Table 111. Recovery of Sulfide from Synthetic Samples a t pH 7 Sulfide present, Sulfide found, gg/rnl pg/rnl DTNB 4-PDS 0.32 0.44 0.57 0.70 0.83
0.35 0.44 0.55 0.72 0.84
0.31 0.43 0.56 0.68 0.82
Sensitivity. Molar absorptivity values for the determination of sulfide ion are listed in Table 11. These values are based on the concentration of sulfide and confirm that two thiol anions are formed for every sulfide ion since the molar absorptivity values are quite close to twice that of the thiol anions. The molar absorptivity value using 4-PDS is somewhat higher than the methylene blue procedure while with the other disulfides the value is lower. Beer’s law is followed in every case. The molar absorptivities d o not vary appreciably a t the different p H values. These compounds should be useful for the determination of sulfide ion over the range of approximately 0.05 t o 2.0 pgiml, the exact range depending on the disulfide used. Recovery of sulfide from synthetic samples is reasonably good, as shown in Table 111. Stability of Solutions. Disulfide solutions in 95% ethanol appear to be stable indefinitely. Solutions of DTNB are also quite stable in a p H 7 buffer. Solutions of sulfide ion in distilled water oxidize quite rapidly. A 0.001M solution of sulfide in distilled water was oxidized about 25% in 4 hours and 50% in 24 hours. Very dilute solutions, about 10-7 M, of NasS were reported t o be oxidized t o the extent of 3 % in 6 hours when made up in 0.1N NaOH (2). We also found that sulfide solutions in 0.1N NaOH were reasonably stable, suffering less than 10% loss in 24 hours. However, sulfide solutions in NaOH were not used in most of this work t o avoid adding hydroxide ion to the reaction solutions. In this work EDTA was found t o stabilize the sulfide solutions considerably, the results being similar t o those for sulfite solutions (7). Sulfide solutions which were 0.001M in EDTA were approximately 5 % oxidized after 4 hours and slightly less than 1 5 % after 24 hours. The extent of loss of sulfide had not increased appreciably from this after 10 days.
142
The thiol anions produced in the reaction are reasonably stable (7). There was little decrease in absorbance of these solutions on standing for 2-3 hours. Absorbance readings could probably be made anytime from 5 minutes to several hours after mixing the solutions and still give approximately the same result. Interferences. These disulfides have been found to react with sulfite ion (7) and cyanide ion (IO) and are useful for the determination of low concentrations of these substances. These ions could interfere in the analysis for sulfide ion. However, it seems likely that these interferences could possibly be avoided by choice of disulfide and p H to be used. Cyanide reacts very slowly a t p H 8 or below unless present in much higher concentration than the disulfide. Sulfite will not react at p H 4 or below so that it might be possible to determine sulfide in the presence of sulfite using unbuffered solutions and either 2-PDS, 4-PDS, or 2-PyDS. These disulfides will also react with base at high p H values so that such solutions must be avoided. Obviously any metal ion which will precipitate sulfide will be a serious interference. Determinations of sulfide at a level of 0.32 pg/ml were attempted using 4-PDS and having a metal ion, Cd(II), Cu(II), Fe(II), Hg(II), Ni(II), Pb(II), or Zn(II), present at 20 pg/ml. All of these ions interfered when no buffer was used for the reaction. Having EDTA present at 0.001M prevented the interference of Cd(II), Zn(II), Pb(II), and Ni(I1) under these conditions. At p H 7, Cd(IJ), Cu(II), Hg(II), and Zn(I1) interfered, while at p H 4, Fe(II1, Hg(II), and Cu(I1) showed interference. The presence of EDTA had n o effect on the interferences in the buffer systems. In SUIfide determinations using DTNB, all of the metal ions except Fe(II), Ni(II), and Pb(I1) interfered. Results were the same at both p H 7 and 9 and with or without EDTA in the solution. RECEIVED for review August 31, 1970. Accepted October 14, 1970. The authors express their appreciation to the Robert A. Welch Foundation of Houston, Texas for support of this research (10) R. E. Humphrey and W. Hinze, Tuluntu, in press.
ANALYTICAL CHEMISTRY, VOL. 43, NO. 1, JANUARY 1971
