Determination of Dissolved Oxygen Using Photoreduced Leuco Phenothiazine Dyes Philip A. Hamlin and Jack L. Lambert Department of Chemistry, Kansas State University, Manhattan, Kan. 66502
CHEMICALLY REDUCED leuco dyes such as indigo disulfonate (indigo carmine), safranine red T , and methylene blue have been used for the colorimetric determination of oxygen (1-12). We have studied five phenothiazine dyes photoreduced to the leuco form in the presence of aminopolycarboxylate salts and found them to be oxidized quantitatively to the colored form by dissolved oxygen without intermediate colored species being formed. The process is reversible and repeated determinations may be made a t the expense of the oxidizable aminopolycarboxylate substrate, which may be present in very large excess. Aminopolycarboxylate salts such as ethylenediaminetetraacetate, EDTA, have the added advantage of sequestering oxidizing cations that otherwise might interfere with the oxygen determination. All the phenothiazine dyes are cations, of which thionine (Lauth’s violet) is the simplest. Related dyes include azure A, azure B, azure C, and methylene blue. These dyes differ from thionine only in the number of methyl groups attached to the two amino nitrogen atoms. The number of methyl groups are as follows: thionine, 0; azure C, 1; azure A, 2 (unsymmetrical); azure B, 3; and methylene blue, 4. Methylene blue has been shown by Tzung and by Diehl to possess the sulfoxide structure in aqueous solution at room temperature (13, 14). Presumably, the other phenothiazine dyes have similar structures. Merkel and Nickerson (15) observed that methylene blue, in the presence of disodium ethylenediaminetetraacetate, is reduced by light in the visible region to the leuco form, and is regenerated when the solution is aerated. Since that time, several investigators have studied the photoreduction of phenothiazine dyes in the presence of EDTA, amines, and related compounds (16-18), and the oxidation of the leuco (1) V. V. Efimov, Biochem. Z . , 155, 371 (1925). (2) L. Buchoff, N. M. Ingber, and J. H. Brady, ANAL.CHEM.,27, 1401 (1955). (3) K. Wichert and E. Jaap, Z. Anal. Chem., 145, 338 (1955). (4) W. F. Loomis, ANAL.CHEM., 26, 402 (1954). (5) Zbid., 28, 1347 (1956). (6) G. Alcock and K. Coates, Chem. Znd. (London), 1958, 554. (7) L. Buchoff and N. Ingber, U. S. Patent 2,967,092, January 3, 1961. (8) A. H. Meyling and G. H. Frank, Analyst, 87,60 (1962). (9) P. A. St. John, J. D. Winefordner, and W. S. Silver, Anal. Chim. Acta, 30, 49 (1964). (10) V. B. Aleskovskii, V. A. Koval’tsov, I. N. Fedorov, and G. P. Tsyplyatnikov, Zuuod. Lab., 28, 1440 (1962). (11) Zbid., 30, 105 (1964). (12) I. V. Devdariani and G. N. Shmal’tsel, Prib. Sist. Upr., 1969, 42.; ChEm. Abstr., 12, 82834~. (13) C. Tzung, ANAL.CHEM.,39,390 (1967). (14) H. Diehl, Iowa State University, Ames, Iowa, personal communication, 1970. (15) J. Merkel and W. Nickerson, Biochim. Biophys. Acta, 14, 303 (1954). (16) G. Oster and N. Wotherspoon, J. Amer. Chem. SOC.,79, 4836 (1957). (17) H. Martin, S. Price, and B. J. Gudzinowicz, Arch. Biochem., Biophys., 103, 196 (1963). (18) J. Joussot-Dubien and J. Faure, J. C/iim.Phys., 60, 1214 (1963). 618
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form by molecular oxygen (19). N o analytical use of this type of reaction has been reported in the literature. JoussotDubien and coworkers (20, 21) reported direct complexometric titration methods for aluminum, and iron(II1) in the presence of aluminum, in which the end point is observed as the photoreduction of phenothiazine dyes to the leuco upon the addition of a minute excess of uncomplexed aminopolycarboxylate titrant.
EXPERIMENTAL All solutions were prepared in deionized water. All chemicals were of the purest available grade and were used as obtained from the supplier, except purified methylene blue prepared according t o the procedure of Nerenburg and Fischer (22). All dye solutions were 5 X 10-5M. Weights of dyes required for solution preparation were corrected for the reported dye content.
RESULTS pH and Mole Ratio Effect on Photosensitivity. Optimum conditions for the photoreduction of thionine and methylene blue were determined for the following 12 compounds which contain tertiary nitrogen atoms: triethylamine, triethylenetetraamine, triethanolamine, N,N-diethylglycine, iminodiacetate, N,N-diethylhydroxylamine, nitrilotriacetate (NTA), ethylenediamine-N,N-diacetate, ethylenediaminetetraacetate (EDTA), 1,2-cyclohexylenediaminetetraacetate, ethylenebis(oxyethylene)diaminetetraacetate, and diethylenetriaminepentaacetate (DPTA). The mole ratio of substrate to dye varied from 1 :1 t o 100: 1 or 1000:1, generally in exponential steps. The optimum p H for maximum photosensitivity was determined by plotting time to decolorization us. pH. Exposures were carried out in open 100-ml beakers, using 50 ml of solution. The light source was a 250-watt General Electric reflector infrared heat lamp located about 18 inches above the solutions. p H values were determined before exposure, using glass us. saturated calomel electrodes. The exposure area was surrounded on four sides and bottom by aluminum foil, and six solutions were exposed simultaneously. N o temperature control was exercised. On six selected dyesubstrate combinations, minumum mole ratios were determined at the optimum p H values, as reported in Table I. Small initial shifts of several tenths of a p H unit were observed upon photoreduction followed by air-reoxidation. These shifts tended t o be smaller when higher mole ratios of substrate to dye were used. Shifts in wavelength of maximum absorption of azure B, azure C, and methylene blue were observed on the initial photoreduction-reoxidation cycle, as reported in Table 11. Subsequent cycles had no effect on the new absorption maxima. No such shift was observed for thionine and azure A.
(19) H. Obata and M. Koizumi, Chem. SOC.Japan Bulletin, 30, 136 ( 1957). (20) J. Joussot-Dubien and G. Oster, Bull. SOC.Chim. Fr. 1960, 343. (21) J. Joussot-Dubien and J. Faure, Bull. SOC.Chim. Belges, 71,877 (1962). (22) C. Nerenberg and R. Fischer, Stain Technol., 38,75 (1963).
Table I. Minimum Mole Ratio of Substrate to Dye at Optimum pH Methylene Thion in e blue5 Azure A Azure B (pH = 9.5) (pH = 11.2) (pH = 10.0) (pH = 11.2) Triethanolamine 20:l 20:l ... ... N,N-Diethylglycine 20:l 1O:l ... ... N,N-Diethylhydroxylamine 20:l 20:l ... ... Nitrilotriacetate 1O:l 1O:l ... ... Ethylenediaminetetraacetate 4:l 3:l 1O:l 5:l ... Diethylenetriaminepentaacetate 4:l 3:l ... and purified methylene blue. a Identical results were obtained with both commercially available 87
From the results obtained on the minimum mole ratio study, DPTA appeared to produce the most rapid photoreduction, However, as EDTA gave comparable results and can be obtained readily in reagent grade, the following methods for dissolved oxygen were based on the use of EDTA. Dissolved Oxygen Determination. The photochemically generated leuco form of each of the phenothiazine dyes in EDTA solution was studied as a reagent for dissolved oxygen. A 1OO:l Na2EDTA:dye solution, 5 X 10-5M in dye, is degassed with nitrogen until it becomes colorless under the photoreducing effects of ordinary laboratory fluorescent lighting. Degassing can be accomplished most easily in a buret equipped with a three-way stopcock. Nitrogen gas is admitted through one arm of the stopcock and purged through a hypodermic needle inserted through a rubber septum in the top of the buret. A second hypodermic needle is attached to the other outlet of the stopcock to facilitate filling of the reagent tubes. Five milliliters of the photoreduced solution were then introduced into a 100 X 13 mm B-D Vacutainer tube (Becton-Dickinson Co., obtained from E. H. Sargent and Co.). These tubes contain 9 mg of Na2EDTA and are supplied with reduced internal pressure, which aids in drawing the solution into the tube. The resulting solution is 197:l in Na2EDTA :dye. Each tube is then degassed individually with nitrogen gas for 10 to 15 minutes until diffuse laboratory lighting effects total decolorization. The nitrogen is passed in through a long hypodermic needle and purged through a short needle, both inserted through the septum. Photoreduction of the reagent for reuse following each determination is accomplished by exposure to strong artificial light or sunlight. Following injection of a sample, the reagent tube should be protected from light until the absorbance has been determined. Calibration curves are prepared by injecting volumes of 10 to 250 pl of air-saturated water into a series of reagent tubes and measuring the absorbance after a minimum development time of 5 minutes at the absorption maximum shown in Table I1 for the dye used. A control tube photoreduced at the same time is used as the blank, The concentration of dissolved oxygen in the sample tube is calculated from the equation
where CI is the concentration of oxygen in air-saturated water, and u is the volume in pl of air-saturated water injected into the reagent tube. Air-saturated water at 25 “C contains 8.1 ppm of oxygen; at other temperatures, the concentration of oxygen can be calculated from the Equation (23) C(ppm) = 14.161
- 0.3943t + 0.007714t2 - 0.0000646t3 (2)
(23) A. Siedel, “Solubilities of Inorganic and Metal Organic Compounds,” W. F. Linke, Ed., Vol. 11, 4th ed., D. Van Nostrand and Co., New York, N. y., 1958, p 1228.
Azure C (pH = 10.4)
... ... ... ...
1O:l ...
Table 11. Shifts in Wavelength of Maximum Absorbance of Phenothiazine Dyes upon Photoreduction at Optimum pHa Final, Initial, nm PH nm Dye cation 595 9.5 595 Thionine 610 10.4 620 Azure C 625 10.0 625 Azure A 625 11.2 645 Azure B 610 11.2 658 Methylene blue Methylene blue 610 11.2 658 (purified) a Mole ratio of EDTA:dye = 197:l. Table 111. Absorbances and Standard Deviations for Calibration Curve Prepared with Methylene Blue-EDTA Reagenta Time,c Concentrationsb min 17 PPb 167 ppb 328 ppb0.49 f 0.024 3 0 . 0 4 i 0.007 0 . 2 9 f 0.022 0 . 5 0 f 0.046 10 0 . 0 4 i 0.007 0.28 f 0.026 0.23 f 0.026 0.57 f 0.060 60 0.04 i 0.007 a Results based on five determinations at 610 nm. b Concentrations are those after dilution of the sample in the reagent solution. c Time of color development in the dark. Straight line Beer’s law plots passing through the origin are obtained with each of the phenothiazine dyes for dissolved oxygen in the range 16 to 385 ppb, with upper absorption limits in the range 0.65 to 0.80. Absorbances measured for higher concentrations fall below the straight line projection. For direct analysis of water samples containing less than the saturation concentration of oxygen, the size of the injected sample should be selected to give a n absorbance between 0.3 and 0.6. To convert from concentration of oxygen in the reagent tube, C’, to concentration in the injected sample, the following equation may be used
where Zu’s is the total volume of previously injected samples (assuming reuse of the reagent tubes). If a calibration curve is prepared to read directly in parts per million of dissolved oxygen in the original water sample, and samples of constant volume are injected into the reagent tube, the concentration of oxygen can be calculated from the Equation [(5000
C(pprn)
=
C”(ppm)
+ u ) + Xu’s]
X (5000
+ u)
(4)
where C is the actual concentration and C” is the apparent concentration read from the calibration curve. The most extensive studies were made with the photoreduced methylene blue-EDTA reagent. The rates of color ANALYTICAL CHEMISTRY, VOL. 43, NO. 4, APRIL 1971
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Table IV.
Color Stability of Regenerated Methylene Blue in EDTA. Minutes (in dark): 3 10 60 100 240 480 Colorstability 0.295 0.285 0.245 0.195 0.175 0.145 0 Minutes (in light): 5 10 15 Color stability 0.210 0.095 0.055 0.040 aConcentration of oxygen was 167 ppb after dilution of the sample in the reagent solution. Table V. Summary of Interference Effects on Methylene Blue-EDTA S y s t e m Effect on Concn, absorbance, Ion Salt PPm units Nitrate NaNOs 50 $0.05 Nitrite NaN02 10 +0.01 Sulfate Na2SOa 250 +0.01 Bicarbonate NaHC03 100 +O. 03 Sulfide Na2S 5 +0.07 Iron(I1) FeCI2.4H20 10 -0.03 Iron(II1) Fe(NO& .9H20 10 +0.01 Manganese(I1) MnC12'4H20 10 +o. 01 a Mole ratio of EDTA :dye = 197 : 1; concentration O2 167 ppb in sample tube; absorbance 0.37.
formation in the dark at three concentrations of oxygen are shown in Table 111. The stability of the color formed by the reaction of oxygen with the reagent when developed in the dark and in diffuse laboratory light is shown in Table IV. The molar absorptivity of the reagent is approximately 46,000 in terms of oxygen equivalents, i.e., the absorptivity produced by oxygen on reaction with the photoreduced reagent. The absorbances were measured at 610 nm, which is the wavelength of maximum absorbance for the regenerated methylene blue. A Hellige color comparator, modified to prevent excess light from entering, was found satisfactory for rapid visual estimation of dissolved oxygen using methylene blue. The standard color disk for methylene blue in the ABS (alkyl benzene sulfonate) determination is used. Dilution effects would limit reuse of the reagent tubes. Interferences. Ions found in natural waters, which might possibly affect the determination of oxygen by leuco dyes, were tested for interference using air-saturated samples of the ions as samples. The concentrations of ions listed in Table V are those in the undiluted, air-saturated samples, 100 pl of which were injected into the sample tubes. The oxygen concentration was approximately 167 ppb. Possible interferences not studied included hydrogen peroxide and ozone, which react in the same manner as oxygen. DISCUSSION
All five of the phenothiazine dyes tested were found capable of undergoing reversible photoreduction-reoxidation in the
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presence of compounds containing tertiary amino groups. Presumably the tertiary amino group is oxidized to the Noxide. Aminopolycarboxylate anions produced the most rapid photoreduction, but the reduction is not so rapid as to affect use of the reagent in diffuse laboratory lighting or in spectrophotometric analysis. Aminopolycarboxylate anions have the added advantage of chelating oxidizing cations which might interfere with the oxidation reaction. Thionine and azure A apparently undergo the photoreduction and reoxidation cycle unaltered, while azure B, azure C, and methylene blue show some chemical change during the first cycle. Following the initial spectral shift, the absorption maxima of azure B, azure C , and methylene blue remain constant through repeated cycles. The spectral shifts may be due to dimerization of the dye cations (24), or to demethylation, but this has not been elucidated. No intermediate colored species were observed during either the photoreduction or oxidation steps. The minimum mole ratio of aminopolycarboxylate to dye necessary for rapid photoreduction to the colorless leuco form is so well defined as to suggest some sort of complex formation. The time for complete photoreduction of methylene blue with EDTA or DPTA initially increases from about 2 hours in their absence to nearly 3 hours a t 2:1 mole ratio, then abruptly decreases to the minimum mole ratios indicated in Table I. This was not observed in the case of thionine. Above a mole ratio of lO:l, the photoreduction time is constant. Methylene blue is recommended because of its stability and ready availability in purified form. One methylene blue-EDTA solution has undergone more than 150 photoreduction-air oxidation cycles without apparent degradation of the dye, The capacity of the methylene blue-EDTA reagent solution is limited only by the amount of EDTA. Thionine and azure A reagent solutions with EDTA appear to lose some sensitivity on storage. The method is comparable in sensitivity to other methods now in use. It has the advantages of photochemical regeneration of a reagent, and a large reserve of oxidizable substrate which permits repeated use of the reagent solution. These properties recommend its use for analysis in the field, and offer opportunities for the development of new types of automated procedures.
RECEIVED for review August 10, 1970. Accepted December 2, 1970. P. A. Hamlin was a National Science Foundation Faculty Science Fellow, 1969-70, on leave from Oklahoma Panhandle State College, Goodwell, Okla. This work was supported by National Science Foundation Fellowship No. 69077, and NSF grants GY-5633 and GP-8629. (24) S. R. Palit and G. K. Saxena, Nature, 209, 1127 (1966).
