N,N Filter Sticks. Two glass filter sticks, the first having a porous tip 5 mm in diameter, 10 mm in height and the second having a porous tip 10 mm in diameter and 2 mm in height (Ace Glass, Vineland, N.Y., product numbers 9255-10 and 9256-10, respectively) were reacted as per Method 11, except that reaction and washing solutions were pumped through the filter sticks. Because the pore diameter and volume is much smaller in these N,N glasses (largest pore diameter 40-80 A), p H change took place a t more extreme pH's: deep-red to orange as p H was raised from 1.5 to 2.3. Using flow-through systems, N,N glass was also prepared from various fritted disks. F u r t h e r Indicators (Azo Linkage). Arylamine silica glass fragments (average pore diameter 550 A) 0.25 gram, were prepared and diazotized under suction for 20 minutes a t 0 OC, in 70 mg NaNOz and 5 ml 2M HC1. This diazo glass was washed several times with cold H20 and added immediately to 0.05 gram of bromocresol purple in HzO. The indicator was in its purple, basic form and the solution p H was 7.0. T h e reaction mixture was left overnight under suction a t 0 "C. T h e bound indicator was washed clean with H20 and acetone. The same indicator was prepared in a flow-through system from two arylamine fritted disks and an arylamine filter stick (prepared from Ace Glass product 9255-10). A bifunctional glass was prepared from 0.25 gram arylamine silica glass fragments and an arylamine filter stick, method as per bromocresol purple but with the following modification: the diazo glasses were washed for 10-15 seconds with cold N,N- dimethylaniline before addition to the bromocresol purple solution. Arylamine fragments (0.25-gram) were diazotized and bound t o each of crystal violet, methyl red, and bromocresol green, method as per bromocresol purple. Arylamine fragments (0.25-gram) and 0.25 gram Zr-clad arylamine fragments were each bound to phenol red, to phenolphthalein, and to thymosulfonphthalein, method as per bromocresol purple, with the modification that the aqueous dye solutions were 8.6, 10.0. and 9.6, respectively. These solution pH's were chosen to maximize dye solubility and to have the dyes in anionic form. Results using Zr-clad fragments and non-Zr-clad fragments were identical. The phenolphthalein experiments were repeated in a one-step reaction. The mixtures each contained 0.25 gram of the same arylamine glasses, 1 gram of phenolphthalein, and 0.25 gram of sodium nitrite in 10 ml of glacial acetic acid, under evacuation, at 0 OC. The mixtures were left overnight and the products were washed clean with aqueous base, p H 10-11. The results were identical t o those obtained by the first method. Amino Linkage. While azo linkage allows binding of virtually any indicator, it has two obvious disadvantages over other binding modes. T h e leuco form is colored and, hence, the intensity of color changes is not maximized. Also, the site of binding onto indicators is non-specific and may inactivate some or all of the indicator mol-
ecules. In fact, all anthraquinoid indicators tested were totally inactivated by azo linkage. Hence, the feasibility of the binding of reactive chloro-s- triazinyl indicators to alkylamine glass was shown by binding non-indicator textile dyes, Colour Index numbers C.I. 61210 and C.I. 17912 (gifts from Bill Ottney, Ciba-Geigy, Toronto) to alkylamine glass. Solutions containing 10 ml CHC13, 0.1 gram dye, and 0.25 gram alkylamine fragments or an alkylamine filter stick were refluxed 15 min and washed with boiling water. Covalently-bound, colored glasses were obtained. The dyes did not bind t o control solutions containing non-activated glass.
DISCUSSION Further development is leading to superior indicators. Reusable chelating indicators (ion detectors), also prepared by azo linkage, will be reported in a further paper. The binding of indicators to the chloro-s- triazinyl glass derivative (11) and other binding modes are currently being investigated. Results for p H indicators, prepared by azo linkage, are shown in Table I. All dyes used in this work have been in long use, since the chemical identities of p H indicators produced in recent years are generally industrial secrets. In all cases, the colors of the bound indicators differ from those of the free indicators. ACKNOWLEDGMENT The author thanks C. A. Sankey, P. H. Bickart, and A. J.
S.Ball of Brock University for their advice and encouragement. LITERATURE CITED Mellan, "Corrosion Resistant Materials Handbook," Noyes Development Corp., Park Ridge, N.J.. 1966, p 118. Modified from G. M. Cameron and J. G. Marsden, Chem. Brit., 8, 381. 1972. K. Venkataraman, "The Chemistry of Synthetic Dyes," Vol. 3, Academic Press, New York, N.Y., 1960, p 24. "Colour Index," 3rd ed., Vol. 3, Lund Humphries, Bradford and London, London, 1971, D 3391. I.
RECEIVEDfor review June 20, 1974. Accepted September 9, 1974. This work is included in U.S. Patent Application 409,87611973 and British Patent Application 22811174. Brock University and Resources Research Co. provided financial support.
Calibration of Oxygen Polarographs by Catalase-Catalyzed Decomposition of Hydrogen Peroxide W. J. Wingo and G. M. Emerson Department of Biochemistry, University of Alabama in Birmingham Medical Center, Birmingham, Ala. 35294
The earlier manometric method for determination of oxygen uptake by various biological systems has in recent times been largely supplanted by the oxygen polarographic method. This later development was given much impetus by Clark's introduction of small "oxygen electrodes" ( I ) which enabled measurement in small volumes. Oxygen polarographs are usually calibrated by measuring the current generated in an air-saturated medium and in an anaerobic medium; the solubility coefficient of oxygen in the medium used is assumed to be the same as that for pure water. Alternatively, various known mixtures of oxygen and nitrogen are equilibrated with the medium, and
the currents for each mixture are determined (2). Submitochondrial electron transport particles which catalyze the oxidation of NADH by oxygen, have also been used to calibrate polarographs; the change in current associated with the oxidation of a known amount of NADH is measured ( 2 ) . For use in connection with measurements of oxidative phosphorylation by mitochondrial suspensions, we have developed a simple and convenient chemical method for calibrating oxygen polarographs based on the decomposition of hydrogen peroxide by catalase (E.C. 1.11.1.6, hydrogen-per0xide:hydrogen-peroxideoxidoreductase).
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P
'Ornm-Cl
f
t i J Q
*ot
70t time
/
-
M
2Osec Figure 1. Representative recording made during calibration of oxygen electrode
,r/
a: addition of peroxide: b: addition of catalase: a,,&intersections of extrapolated lines: A; deflection due to oxygen evolution from H202. This recording is made on a Gilson Medical Electronics Oxygen Polarograph Model K using a temperature-controlled chamber fitted with Yellow Springs Instrument Go., Clark type oxygen electrode. Ten millimeters deflection of ordinate corresponds approximately to 0,117-pA current change
EXPERIMENTAL Reagent 30% (w/w) hydrogen peroxide is diluted to about 0.160.17M with deionized water. The diluted peroxide is stored in the refrigerator in a polyethylene bottle. The peroxide is reasonably stable under these conditions, losing only about 0.03% of its strength per month, but a t least weekly before it is used in standardizing the oxygen polarograph, its concentration is determined iodometrically ( 3 - 5 ) . For calibration of the oxygen polarograph, 0.20 ml of the stock dilute peroxide is diluted to 10.0 ml with ice-cold deionized water, and the twice-diluted peroxide is stored in an ice bath. About 15 ml of the medium for the oxygen polarograph chamber are evacuated and shaken to reduce the oxygen content to 15-20% of that at air saturation. The chamber of the polarograph is rinsed three times and then filled with the deaerated medium; the chamber stirrer is started, and a base line is recorded for about a minute. A measured sample (usually 50 p1 of the twice-diluted peroxide, containing from 0.16-0.17 pmoles Hz02) is then added from a 50-pl syringe. The oxygen concentration of the medium in the chamber increases very rapidly for a few seconds because of the free oxygen content of the added solution; then it continues to increase much more slowly because of the nonenzymatic decomposition of the peroxide. After 1-2 minutes, 5 to 10 pl of a dilute solution of catalase (about 0.1 ml of a 3% suspension of crystalline catalase diluted with 3 ml of water [Sigma Chemical (2-30. This batch = 23,000 Sigma Units (pmoles H202 degraded/min) per mg catalase.]) are added to the chamber; the hydrogen peroxide is rapidly decomposed.
RESULTS A typical recording for a calibration is shown in Figure 1. The initial rapid rise, the slower secondary rise, the rapid
0
io
20
30
40
50
Microliters H202 added Figure 2. Relationship between the hydrogen peroxide added (x axis) and the response of the oxygen electrode (y axis)
Ten millimeters deflection of ordinate corresponds to 0.11 7-pA current change. Correlation coefficient = 0.99, slope = 1.4, y intercept = 2.4. One standard deviation of the points from the line is 2.16 mm in deflection
rise following the addition of catalase, and the final steady state tracing are all extrapolated as shown in Figure 1. The difference in ordinate (A) between the intersections ( a and 8) of these pairs of extrapolations represents the change in current due to the oxygen evolved from the known quantity of peroxide added to the polarograph chamber. Figure 2 shows the response of the polarograph as a function of the quantity of hydrogen peroxide added. The method given here is very convenient and rapid, and requires a minimum of specialized apparatus or reagents. The principle might also be applied to the assay of enzymes which catalyze reactions producing hydrogen peroxide.
LITERATURE CITED (1) L. C. Clark, Jr., Trans. Amer. SOC.Artif. Intern. Organs, 2, 41 (1956). (2) R . W. Estabrook, "Methods in Enzymology," Voi. X, S. P. Colowick and N. 0. Kaplan. Ed, Academic Press, New York, N.Y., 1967, p 41 (3) C. T. Kingzett, J. Chem. Soc., 37, 792 (1880). (4) C. T. Kingzett's Method, in "Scott's Standard Methods of Chemical Analysis," 5th ed.. Vol. 2, N. H. Furman, Ed., D. Van Nostrand. New York, N.Y.. 1939, p 2180. (5) I. M. Kolthoff, E . B. Sandell, E. J. Meehan. and S . Bruckenstein, "Quantitative Chemical Analysis," 4th ed., The Macmillan Go., London, 1969, p 854.
RECEIVEDfor review July 8, 1974. Accepted September 20, 1974.
Reduction of Quantization Effects by Time Averaging with Added Random Noise Gary Horlick Department of Chemistry, University of Alberta, Edmonton, Alberta, Canada T6G 2G2
Quantization may be defined as the conversion of the magnitude of a continuously varying analog signal to a number. In modern experience, quantization is most frequently associated with electronic analog-to-digital conversion but it is also present in any human scale reading operation involving devices such as meters, strip chart recorders, or even burets ( I ) . Quantization should not be con352
fused with digitization. Digitization refers to the overall operation of converting a continuous analog signal into a set of numbers and consists of two basic steps: sampling and quantization ( 2 ) . Two main parameters characterize quantization: dynamic range and quantization level. The dynamic range is the maximum range of input signal that can be quantized and
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