the clay soils. In fact, the reproducibility of the recoveries at fortification levels from 5 to 50 ppm is quite precise in all types of samples, as is demonstrated by the low percents of average relative error shown in Tables I and 111. Samples fortified with fewer than 5 ppm of arsenic standard showed a slightly greater percent average relative error. The influence of acid strength on the reduction of As(V) to As(II1) was also studied by varying the acid concentration in the reduction step. For this study, 1000 wg of As(V) as As205 were reduced with 50% SnCl2 and 15% KI in various concentrations of HC1. The amount of As(II1) found as shown by readings on the AA spectrophotometer was compared directly to the readings given by 1000 pg of As(II1) as As203 The re-
sults are listed in Table IV. I t is observed that efficient reduction takes place a t normalities below 7, whereas the reduction efficiency drastically drops at normalities greater than 7.
LITERATURE CITED (1) G. R. Kingsley and R . R. Schaffert, Anal. Chem., 23, 914 (1951). (2) H. C. Beard and L. A . Lyerly, Anal. Chem., 33, 1781 (1961). (3) W. Slavin, S.Sprague, and D. C. Manning, At. Absorp. News/., 3 (18), 1 (1964). (4) H. L. Kahn and J. E. Schallis, At. Absorp News/.,7 (1). 5 (1968).
RECEIVEDfor review August 21,1974. Resubmitted May 13, 1975. Accepted February 18,1976.
Colorimetric Determination of Sodium 2-Pyridinethiol I-Oxide with Ferric Ammonium Sulfate S. Oliveri-Vigh' and H. L. Karageorian G. S. Herbert Laboratories, Allergan Pharmaceuticals, 2525 DuPont Drive, Irvine, Calif. 927 13
A colorimetric method for determining sodium 2-pyridinethiol I-oxide was developed In order to gain greater specificity over existing methods. Linearity was demonstrated between 0.002-0.01 YO.The relative standard deviation (RSD) for this method is f0.4% with a precision ( t s / f i ) of f0.3% for 9 degrees of freedom at the 95 % confidence level.
Methods currently available for the analysis of sodium 2pyridinethiol 1-oxide include an ultraviolet method, an iodimetric method ( I ) and N-oxide determination with titanous chloride ( 2 ) ,and polarography ( 3 ) .Outside of polarography, none of these methods are very specific for sodium 2-pyridinethiol 1-oxide. The ultraviolet curves for sodium 2-pyridinethiol 1-oxide and analogous compounds such as; 2,2'-dithiobis(pyridine 1-oxide),2-mercaptopyridine, pyridine disulfide, and pyridine N-oxide 2-sulfonic acid are shown in Figure 1. From these curves it is clear that if sodium 2-pyridinethiol 1-oxide is mixed with any of these analogous compounds, an accurate determination of sodium 2-pyridinethiol 1-oxide would be impossible. The iodimetric method, which reacts with the thiol group in the pyridinethiol moiety, is also sensitive to possible impurities such as 2-mercaptopyridine and antioxidants such as cysteine, both of which are drastically different substances in terms of structure. The N-oxide titration with excess titanous ion is a back titration, a limitation in itself, which has the drawback of not distinguishing the difference between 2,2'-dithiobis(pyridine 1-oxide), the chief oxidation by-product of sodium 2-pyridinethiol 1-oxide, and sodium 2-pyridinethiol 1-oxide itself. Specificity is greater in the colorimetric method described in this paper since it requires not only the thiol group, but also the presence of an adjacent N-oxide. Both structurally and stoichiometrically the reaction is hypothesized to proceed as in Equation 1.
n T o lend support to this hypothesis, a sample of the ferric pyrithione (the ferric chelate of 2-pyridinethiol 1-oxide) supplied by Olin Chemicals was placed under the same conditions as described in the assay prodecure (i.e., the pH 0 and there was excess ferric ammonium sulfate present). The visible absorbance spectrum of ferric pyrithione appeared identical to that produced in the assay procedure.
-
EXPERIMENTAL Apparatus. Absorbance measurementswere recorded with a Cary 15 spectrophotometer. Matched quartz cells (Coleman) with 1-cm path legnth were used to hold all solutions for measurement. Reagents. Reagents used without further purification are 2,2'dithiobispyridine 1-oxide), 2-mercaptopyridine, pyridine disulfide, pyridine N-oxide 2-sulfonic acid, and sodium 2-pyridinethiol1-oxide ( O h Corporation, Rochester, N.Y.); ferric ammonium sulfate, hydrochloric acid, and methanol were reagent grade (Mallinckrodt Chemical Works). Deionized water was used in preparing all solutions. Procedure. Stock sodium 2-pyridinethiol 1-oxide solution was prepared by dissolving 0.100 g of sodium 2-pyridinethiol 1-oxidein a 100-ml volumetric flask with 1N hydrochloric acid. Aliquots of stock solution were taken to prepare 0.002, 0.004, 0.006, 0.008 and 0.01% solution in a 25-ml volumetric flask. Exactly 1 ml of a 2% aqueous solution of ferric ammonium sulfate was added with thorough mixing, and then the solution volume was adjusted to the mark with 1 N hydrochloric acid. These solutions were then read on the spectrophotometer at 610 nm against a blank consisting of 1ml of ferric ammonium sulfate and 24 ml of 1 N hydrochloric acid. The resulting pH of all solutions is approximately zero.
RESULTS The absorbance was plotted vs. concentration and observed to be linear. Using linear regression analysis the best straight ANALYTICAL CHEMISTRY, VOL. 48, NO. 7, JUNE 1976
1001
0.6
0.5
I
0.4
P
I 0.3
0.2
0 . 0 1 1 1 1
I
I
I
I
I
/
i
1
I
1
l
I
/
l
I
I
I
600 nm
500 nm
kA'X
I
I
700 nm
LENGTH
Figure 2. Visible spectra for sodium 2-pyridinethiol l-oxide complexed with iron in the presence of related compounds WAVE
LENGTH
Figure 1. Ultraviolet curves for sodium 1-pyridinethiol 1-oxide and
(A) 2,2'dithiobis(pyridine loxide), (6) Pmercaptopyridine, (c) pyridine disulfide, ( D )pyridine Koxide-2-sulfonicacid
analogous compounds. (A) 5 X 2,2'-dithiobis(pyridine 1-oxide), (6) 5X 2 mercaptopyridine, (C) 5 X pyridine disulfide, (0) 5X pyridine Noxide2-sulfonic acid, ( E ) 5 X sodium 1-pyridinethiol 1-oxide.The solvent in each case was water
(1) 2, 2 DITHIC-BIS (PYRIDINE-1-OXIDE1
t 0
t
0
Table I. Determination of Sodium 2-Pyridinethiol I-Oxide in Aqueous Solution. Sample No. 1 2
3 4 5 6 7 8 9 10
Absorbance at 610 nm 0.695 0.698 0.699 0.699 0.698 0.701 0.691 0.699 0.698 0.698 8.07 x 10-3
8.04 8.01 8.08 8.08 8.04 8.11 7.99 8.08 8.07 8.07
line is described as follows:
+ 0.000206
The molar absorptivity a t 610 nm is 3850. Both sensitivity and reproducibility of the method were checked by taking 2-ml aliquots from a 0.100% solution of sodium 2-pyridinethiol 1-oxide and transferring to 25-ml volumetric flasks (resulting concentration 0.00800%). The average value for 10 runs was 0.00807% with a standard deviation of f3.21 X (f0.4%, RSD). The precision ( t s l m ) for 9 degree of freedom a t the 95%confidence level was f2.42 X lov5 (f0.396 with respect t o RSD). See Table I. 1002
QISH
% (X
Average: Standard deviation 3.21 X lov5 (0.4%) (RSD) (0.3) Precisionn (with h2.42 X respect to RSD) a t s / T N for 9 degrees of freedom at the 95th confidence level.
Concentration (%) = 0.01127 Absorbance
121 2 MERCAPTO PYRIDINE
ANALYTICAL CHEMISTRY, VOL. 48, NO. 7, JUNE 1976
(3) PYRIDINE DISULFIDE
QLs-.q-)
(4) PYRIDINE N-OXIDE -2- SULFONIC ACID
O
S
O
s
H
D
t 0
Flgure 3. Structures of possible impurities and degradation products
The visible spectra for sodium 2-pyridinethiol 1-oxide complexed with iron is shown in Figure 2. Although the absorptivity is lower than that of direct ultraviolet readings, there is no chance for color formation with similarly related compounds. This was shown by adding a known aliquot of sodium 2-pyridinethiol 1-oxide to a flask with an equal amount of the following impurities and degradation products, Figure 3. The reaction with ferric ion showed no increase in absorbance (Figure 2). Pyridine disulfide shows a decrease indicating a possible competing reaction for ferric ions. The exact nature of this reaction is left for a further study. Other sources of interferences have been noted with the following compounds that are not intermediates or degradation products but could possibly be in a formulation or solu-
tion to be assayed. These anions were checked under the assay conditions described (oxalate ions, pyrophosphate ions, sulfite ions, thiosulfate ions, cupric ions, and dichromate ions) and were found to cause interferences by either forming a more stable complex with pyrithione, by reducing the ferric to ferrous, or by their ability to form a colorless compound with iron, thus reducing the intensity of the ferric pyrithione complex if sufficient excess iron were not present ( 4 ) . It should be mentioned that although sodium 2-pyridinethiol 1-oxide is not stable for long periods of time in acid solution, the ferric pyrithione complex showed neglible loss after 1hour which is more than sufficient time for numerable 0) was assays of this kind. The condition of low pH (pH chosen because of the solubility of the ferric complex. T o ensure that sufficient ferric ion was added, a spectrophotometric titration was performed using a known aliquot of sodium 2-pyridinethiol 1-oxide and varying the concentration of ferric ion. The amount of 1 ml of a 2% solution is -+
0.020 g of Fe(NH4)(S04)2-12 HzO which ensures that the reaction goes to completion instead of the theoretical alhount of 0.00215 g (based on Equation 1).
ACKNOWLEDGMENT The authors express their appreciation to John Wedig of Olin Chemicals for donating pure samples of the degradation and reaction products as well as impurities of sodium 2-pyridinethiol 1-oxide. Special thanks also goes to Sally Fraser for her drawings of the graphs and figures.
LITERATURE CITED (1) Olin Technical Bulletins, Olin Corporation, Rochester, N.Y. (2) R. T. Brooks and P. 0. Sternglanz, Anal. Chem., 31, 561 (1959). (3) A. F. Krivis, E. S.Gazan, G. R . Supp, and M. A. Robinson, Anal. Chern.,35, 966 (1963). (4) J. P. Mehlig, Anal. Chem. 10, 136 (1938).
RECEIVEDfor review October 20, 1975. Accepted February 17, 1976.
Evaluation of Peroxyoxalate Chemiluminescence for Determination of Enzyme Generated Peroxide David C. Williams
Glenn F. Huff, and W. Rudolf Seitz*
Department of Chemistry, University of Georgia, Athens, Ga. 30602
The chemiluminescence (CL) generating reaction of bis(2,4,6-trichlorophenyi) oxalate (TCPO) with hydrogen peroxide In the presence of perylene has been evaluated as a means of determining hydrogen peroxide. in a mixed ethyiacetatemethanol-aqueous buffer solvent system, performlng the reaction in a flow system, CL intensity is linearly proportlonai to peroxide from 7 X M, the detection limit, up to M, the highest concentration tested. The CL intensity is increased both by adding small amounts of triethylamine and by decreasing the concentration of the aqueous buffer. The pH of the aqueous buffer affects CL intensity; however, sensitive analysis for peroxide is possible from pH 4 to 10. Using immobilized glucose oxidase to convert glucose to hydrogen peroxide, the CL reaction of TCPO can be used to determine glucose in urine without interference from uric acid.
Recently, the chemiluminescence of 5-amino-2,3-dihydrophthalazine (luminol) in the presence of ferricyanide has been used for the determination of low concentrations of peroxide (1-4). The utility of this reaction was extended by coupling the enzymatic generation of peroxide from glucose to the CL from luminol(1-4). The luminol system is sensitive to concentrations of peroxide as low as 8-10-9M and is linear over four orders of magnitude. The glucose oxidase (E.C. 1.1.3.4) coupled CL method for glucose is extremely sensitive and specific. In spite of these advantages, two problems, a high background level of light emission and interference problems, have been encountered with luminol-CL analyses. The background light emission arises from the reaction of luminol-ferricyanide with oxygen. As a result of this high background it is necessary that the luminol-ferricyanide mix well, before entering the CL flow cell (I).If this requirement is not met, then the del Present address, Clinical Labarory, H a r t f o r d Hospital, Hartford, Conn.
tection limit is diminished because of a noisy background. Furthermore, with a high level of background light emission, achieving minimum detection limits requires constant flow. If a peristaltic pump is used with the luminol system, the limit of detection is diminished to M H202 because of surges in the flow system. The interference problem was observed when attempting to apply the CL method to the determination of glucose in urine. Uric acid, which may be present in urine a t concentrations as high as 75 mgldl, readily reduces peroxide and oxygen at the basic pH’s required for luminol CL. This causes a decrease of backgroundlpeak height and interferes with quantitation of urine glucose. We have reported a method for removing this interference involving the addition of equimolar portions of Ba(OH)2 and ZnSOI in order to determine urine glucose ( 5 ) .The resulting filtrate, the Somogi filtrate, is free of reducing substances other than glucose (6) and is, therefore, useful for glucose analyses. However, this pretreatment is time consuming. In order to overcome this problem, efforts were made to alter the pH to a value less favorable to the oxidation of uric acid. Since the CL of luminol occurs only in basic solutions a t pH’s between 9 and 12, it was, therefore, decided to replace luminol with another CL reaction Peroxyoxalate CL has been investigated by several workers (7-9). A simplified version of the proposed chemistry for peroxyoxalate CL is given by the reaction sequence shown in Figure 1 (8). Reaction 3 is an energy transfer from the strained ring to a fluorescer. Any fluorescer having a first excited singlet with an appropriate energy level can be excited by the reaction. The aromatic oxalate that was used in this work is bis(2,4,6-trichlorophenyl) oxalate (TCPO). TCPO gives efficient CL, is stable as a solid and in solution and is easy to prepare (7). Perylene was chosen as the fluorescer because it is stable and fluoresces efficiently over a wavelength range where our photomultiplier tube responds sensitively. In addition to the goal of eliminating the interference by ANALYTICAL CHEMISTRY, VOL. 48, NO. 7, JUNE 1976
1003
