AuRomat ic Measurements of Hydrogen Peroxide Utilizing a Xylenol Orange-Titanium System Carleton D. Nordschow and Arnold R. Tammes Division of Clinical Laboratories, College of Medicine, University of Iowa, Iowa City, Iowa
THEUSE OF a xylenol orange-hydrogen peroxide system to measure titanium as the third reactant of a trimeric chelate, has been described by Otomo ( I ) . Available spectrophotometric evidence (1-3) suggests the chelate to be equimolar in each constituent. The system, in principle, should be equally adaptable to hydrogen peroxide assay. We required a rapid selective and repetitive analysis of hydrogen peroxide, at micromole concentrations, in several protein-containing complex biological solutions, e.g., animal serums, plasmas, and Eagles' modified and unmodified culture media in which were suspended metabolizing cell colonies. We were unable to use certain procedures for automatic continuous flow analysis because of their lack of specificity ( 4 ) or their dependence upon concentrated reagents where the constraint of specificity has been satisfactorily shown (56). We therefore modified the titanium assay method described by Otomo ( I ) and adapted it to a continuous flow system. In this system dialysis is required to process protein-containing solutions but would be unnecessary where this requisite does not obtain. EXPERIMENTAL
Apparatus. In the system illustrated in Figure 1, standard AutoAnalyzer components are used. Complete descriptions of these components are available in the literature (7-9). Samples are diluted with an air-segmented stream of 0.9% sodium chloride and the resultant stream is mixed and then dialyzed against an air-segmented stream of 0.9 % sodium chloride. A xylenol orange-titanium sulfate mixture is added to the dialystate, and the combined stream is mixed and delayed in a reaction coil. The absorbance of the solution is measured at 550 mp in a flow cuvette with a 15-mm light path. Samples are processed at a rate of 50 per hour. The sample time is 20 seconds with a wash time of 52 seconds. The sampler cam was constructed from a standard fifty per hour cam supplied by Technicon Instruments Corp. by reducing the sample cogs so that each subtends an angle of 20" and has a rim length of 0.365 inch. This results in cog wash recesses which each subtend an angle of 52". The sampler cog was modified in this fashion in order to obtain a maximum rate of analysis while maintaining adequate sample separation; hence, the need to increase the wash time. This decrease in sampling time results in but a slight reduction of sensitivity. Standards are used at the beginning and end of each analytic sequence in concentrations of 5 , 10, 15, and 20 p M / ml. The medias analyzed were screened prior to use for HzOzor cation interferents by ordinary operation of the procedure for the former and by replacement of titanium by (1) &I. Otomo, Bull. Chem. SOC.Japan, 36, 1341 (1963). (2) P. B. Sweetser and C. E. Bricker, ANAL.CHEM.,26, 195 (1954). (3) S. Musha and K. Ogawa, J. Chem. SOC.Japan, Pure Chem. Sec., 78, 1686 (1957). (4) E. K. Dukes and 11.L. Hyder, ANAL.CHEM.,36, 1689 (1964). (5) W. Pilz and I. Johann, 2.Anal. Chem., 210,358 (1965). (6) W. C. Wolfe, ANAL.CHEM., 34,1328 (1962). (7) L. T. Skeggs, Am. J. Clin. Parhol., 28, 114(1957). (8) W. H. Marsh, "Automation In Clinical Chemistry," Charles C Thomas, Springfield, 1963. (9) L. T. Skeggs, Jr. Ed. "Automation In Analytical Chemistry," Mediad Incorporated, New York, 1966.
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Figure 1. Instrument flow schematic The instrument was assembled from standard AutoAnalyzer parts except for modification of the timer cog of the Sampler 11. The composition of the diluent can be varied dependent upon the characteristics of the samples or may be omitted. For biological fluids 0.9% NaCl has been used. H I is the Technicon Instrument Corporations designation for a glass cactus stream bubbler with attached capillary side arm
HzOz in the composite color reagent for the latter. The lines carrying the Ti-XO reagent were cleansed after use with 6N aspirated for 15 minutes. Reagents. TITANIUM : XYLENOL ORANGE. These were constituted as 1 X lO-IM aqueous solutions. Titanium (IV) was prepared from 0.0799 gram of TiOz, J. T. Baker Chemical Co., by heating in the presence of 0.5 gram of (NH4)&04and 2.5 ml of concentrated with subsequent dilution to 1 liter. Xylenol orange was prepared from 0.7606 gram of the reagent supplied as the tetrasodium salt, Fisher Scientific Co. The composite reagent was prepared from 400 ml of 1 x 10e3M titanium stock, 400 ml of 1 X 1O-I xylenol orange stock, 200 ml of 2N sulfuric acid, and 0.5 ml of Brij-35. Brij-35 is the trademark name for the wetting agent polyoxyethylene lauryl alcohol obtained from Technicon Instruments Corp. Titanium and xylenol orange complex at low hydrogen ion concentrations (IO), hence, it is advantageous to add one to the acidified solution of the other particularly if the reagent is to be used immediately. DILUENT.Sodium chloride of 0.9 concentration is used when H z 0 2originating from physiological fluids is measured. Otherwise water, other diluent mixtures, or diluent omission may be employed. Brij-35,0.5 mliliter is added to the chosen diluent . HYDRGGENPEROXIDE.Approximately 0.2M H 2 0 2 was prepared from3 0 % stock (Fisher Scientific Co.) and standardized with KMn04. This was used to prepare standard solutions containing from 1 to 50 pM/ml. RESULTS AND DISCUSSION
The absorbance characteristics of an acidic titanium-xylenol orange mixture, and the change in its characteristics subsequent to hydrogen peroxide addition, are shown in (10) M. Otomo, Bull. Chem. SOC.Japan, 36,1577 (1963). VOL. 40, NO. 2, FEBRUARY 1968
465
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Table I. Summary of Precision Data of Standards
H202 in standardsa
Absorbance meanb
Std. dev:
5
0.1000 0.2012 0.2973 0.3838
0.0010 0.0021 0.0025 0.0023
10 15 20 a
Standardized by KMnOc titration. Means of ten measurements of absorbance. Standard deviation(s)of absorbance mean.
Figure 2. The figure shows the absorption of undissociated xylenol orange (11) at pH 1.20 i 0.05 vs. water. The curve set shows the subsequent absorption of Ti-XO-H202 measured vs. Ti-XO reagent as a blank. The values shown are in micromoles per milliliters of H202 in standard samples. The analytic range of absorbance-concentration linearity extends to 20 pM/ml Hz02. Above this, a close approximation of linearity results but the slope of the absorbance-concentration curve, measured at 550 mM, decreases. This shift occurs between 20 and 25 pM/ml. Concentrations can be determined to 1 X 10-I micromole. In typical sets of standard samples of 10, measured to 1 X 10-l pM/ml H202 the following results, Table I, illustrate the performance of the method. Sensitivity can be enhanced approximately tenfold by omission of the sample diluent and receipt of dialyzed components (11) B. gehak and J. Korbl, Collection Czech. Chem. Commun., 25, 797 (1960).
WAVELENGTH IN MILLIMICRONS
0
Figure 2. Absorbance characteristics of Ti-XO and Ti-XO HzOz The spectra were recorded on a Beckman DB-G spectrophotometer. The Ti-XO curve shows the absorption of undissociated xylenol orange in the composite reagent. The curve set shows the absorpsamples through 50 pM/ml. The tion produced by standard HZO2 aliquots used for spectral recording were obtained from the flow cell of the AutoAnalyzer
from the sample stream into a smaller volume in the recipient stream of the dialyzer. Simple aqueous solutions not requiring separation of proteins may be processed directly, with omission of the dilutional effects which attend preparatory dialysis. We have not encountered interferents in the biological medias listed in this report. Otomo ( I ) has specified a variety of cations and anions, with comments on inhibitory concentrations, as encountered when this procedure is used to measure titanium(1V). The selectivity shown by xylenol orangetitanium(1V) toward hydrogen peroxide and the favorable absorption maxima of the complex makes possible the accurate measurement of hydrogen peroxide in complex biological solutions.
RECEIVED for review September 14, 1967. Accepted November 24,1967.
Micro Test for Thermooxidative Stability of Fluids John Mann Butler, Guthrie Wheeler, Jr., and William D. Ross Monsanto Research Corporation, Dayton Laboratory, Dayton, Ohio 45407
IN THE DEVELOPMENT of oxidatively and thermally stable fluids and lubricants and in the use of prototype and model compounds in the development of oxidatively and the thermally stable polymers, a thermooxidation test is needed that can be applied to materials that are too volatile to use in standard high temperature oxidation tests for lubricants. A test that can be used when only small quantities of materials are available is needed also. The objective of this work was to develop and demonstrate the basis for such a test. The approach employed uses melting point capillaries as micro "reactors" to contain a few milligrams of the fluid in an air atmosphere. Such sealed reactors are exposed at the desired temperatures and times. The oxygen/nitrogen ratio in the residual gas is then determined by gas chromatography.
temperatures (between 200 and 500" C). Individual capillaries containing the fluids were removed at 10-minute intervals, usually covering a time span of 90 minutes. The residual gases in the capsules were released into a gas chromatographic system using an F & M SI-4 solid sample injector to crush the capsules. The ratios of oxygen to nitrogen were determined using an F & M Scientific Corp. Gas Chromatograph, Model 700. The detector used was a thermal conductivity sensor which is very sensitive to changes in concentration of oxygen in relation to the nitrogen internal standard. Conditions of the analyses were: column, inch stainless steel tubing packed with 150-200 16 feet X mesh Porapak Q (Waters Assoc.); column temperature, -76" C ; detector temperature, 160" C ; injection port temperature, 110" C ; flow rate, 43 ml/minute. The oxygen and nitrogen components are well resolved (Figure 1) using these conditions.
EXPERIMENTAL
Quantities of 2 to 3 mg of a fluid to be tested were charged to 4 cm, size D (1.5 mm o.d., 1.0 mm i.d.) melting point capillaries using a microsyringe. The capillaries were sealed so as to entrap as nearly as possible the same amount of air in each. The sealed capillaries were placed in aluminum block holders and heated in an electric furnace a fixed 466
ANALYTICAL CHEMISTRY
RESULTS AND DISCUSSION
Using the procedure outlined above, two well characterized fluid base stocks and one exploratory compound were tested. These materials were: a five-ring polyphenyl ether c&(OCeH4)a-meHr (Monsanto Research Corp.); a diester of a
