The described valves have been used satisfactorily for over two years in various analyses systems and have proven durable and dependable. Precision of repeated calibration gas sample analyses by flame ionization gas chromatography has been within 0.7%. No evidence of absorption or contamination by outgassing has been observed using either Viton A or silicone rubber O-ring seals. In addition to
satisfying the stated requirements, the valves are easy to disassemble for OCCaSiOnal cleaning Or for infrequent O-ring replacement.
LITERATURE CITED
(1) dperstein, M., Bradow, R. L., Rev. Sci. Instr. 36, 1028 (1965). (2) van de craats, F., Chim. A ~ 14, 136 (1956).
(3) Fredericks, E. M., Brooks, F. R., ANAL.CHEM.28, 297 (1956). (4) Hausdorf, H. H., “Vapor Phase Chromatography,” p. 377, Butterworths, London, 1957. (5) Hill, D. W., Hook, J. R., J. Sci. Instr. 37, 253 (1960). (6) Percival, W. C., ANAL.CHEM.29, 20 (1957). (7) Pratt, G. L., Purnell, J. H., ANAL. CHEM.32, 1213 (1960). (8) Watson, E. S., Bresky, D. R. (to ~ Perkin-Elmer ~ . Corp.), U. S. Patent 2,757,541 (Aug. 7, 1956).
Quinhydrone as Quantitative Standard for Electron Spin Resonance Spectrometry of Biological Systems Giuseppi Narni,
A
H. S.
Mason1, and lsao Yamazaki2 University of Oregon Medical School, Portland, Ore.
of substances such as peroxylamine disulfonate, diphenyl-picryl-hydrazyl, powdered coal, charred dextrose, copper sulfate, manganese sulfate, vanadyl sulfate, and synthetic ruby, have been used as standards of unpaired electron concentration in electron spin resonance spectrometry. None has proved altogether satisfactory for several reasons (2,s), and the general problem of determination of spin concentration by ESR for paramagnetic substances in solid, liquid, and gaseous states, through a broad temperature range, remains to be solved. I n a relatively narrow aspect of the problemdetermination of free radical concentration in aqueous solutions of neutral pH a t room temperature, as in the case of free radical generation from substrates by oxidative enzymes-it is essential that the unknown and standard solutions occupy identical space in the spectrometer cavity, that the free radical character of the unknown arise from a structure similar to that of the standard, that the dielectric constants of the standard and unknown solutions be similar, and that pH and ionic strength of the unknown be similar to that of the standard so that the replacement of one by the other in the sample cell by a flow method cannot produce residual changes. Solutions of peroxylamine disulfonate in mildly alkaline solution are useful as standards of spin concentration under these conditions because the compound is readily estimated by optical spectrometry; however, the peak-to-peak width of the ESR absorption lines is very narrow and difficult to integrate. I n this study, we have employed peroxylNUMBER
‘To whom inquiries should be addressed. * Present address, Departplent of Biophysics, Hokkaido University, Sapporo, Japan.
PEROXYAMINE DISULFONATE
p-SEMIOUINONE
Figure 1. Integrated signals of peroxylamine disulfonate in 0.01 N Na2C03, and quinhydrone in 0.2M phosphate buffer, pH 7.5; each solution approximately 5 m M
amine disulfonate as a primary standard, against which we have determined the concentration of p-benzosemiquinone in equilibrium with quinhydrone. This has given us an estimate of the reliability of quantitative ESR spectrometry. I n addition, the concentrations of p-semiquinone a t neutrality proved to be at convenient levels for ESR spectrometry of aqueous solutions, and this system appears to offer a readily available, easily prepared, and reasonably stable aqueous standard for enzymological work. EXPERIMENTAL
Apparatus. A Varian V-4500 xband ESR spectrometer was used with 100-kc. field modulation. The Varian quartz flat cell, 0.25 mm. internal thickness, containing 0.075 ml., was mounted in a fixed position in the cavity for optimal signal strength and maximum stability of the cavity and sample geometry. Sample solutions were placed in the cell by means of a syringe attached a t the bottom, and, with each replacement, the cell was very carefully flushed in
order to avoid contamination. All experiments were done with a field sweep speed of 4.3 gauss per minute, a t a field modulation amplitude of 0.2 gauss. The spectrometer response time was 0.3 second, and the full scale recorder response time was 1.0 second. Field strengths were monitored with a Varian F-8 fluxmeter and a Hewlett-Packard 524C electronic frequency counter and klystron frequencies with a HewlettPackard K532B frequency meter. Reagents. Potassium peroxylamine disulfonate prepared by the procedure of Harvey and Hollingshead ( I ) was twice recrystallized, dried with methanol, and stored in a freezer a t -2OO C. in sealed, evacuated tubes. The compound was standardized by iodometric titration, and used immediately for ESR measurements as a solution in 0.01N Na2C0a;only freshly standardized solutions were used for this purpose. Quinhydrone (Eastman Co.) was recrystallized to m.p. 170-1°, and finely ground in a mortar before making solutions. It was observed that oxygenfree buffer solutions gave rise to relatively stable concentrations of free radicals, and all solutions were accordingly prepared by shaking the powdered VOL. 38, NO. 2, FEBRUARY 1966
367
20-
P H 7.51
15p n 7.38
.
c
h 0
IO-
.
pH 7 . 2 1
D
D,
L,
5pH 6.78
pH 6 . 6 5
I -
I 0
1
2
3
4
5
Quinhydrone Concentration
(mM)
Figure 2. Height of the central (derivative) line of the p-benzosemiquinone signal plotted against quinhydrone concentrations at five pH's (0.2M phosphate buffers)
quinhydrone with buffers made anaerobic by bubbling with purified nitrogen gas. Over a period of an hour, the color of the quinhydrone solutions, especially at high pH, changed; although the effect of free radical concentration was small, quinhydrone standards were never used for longer than one-half hour. The quinhydrone powder dissolved somewhat slowly, but 5mJf solutions were readily prepared within five minutes a t room temperature with the aid of a magnetic stirrer. RESULTS
Peroxylamine disulfonate and pbenzosemiquinone gave the signals already reported for them; the integrated shapes of one peroxylamine line, and of the total p-benzosemiquinone signal are shown in Figure 1. The measured line separation for the latter was 2.349 gauss; this compares with the value, 2.368 gauss reported earlier (4). It was not possible, with our instrumentation, to obtain a perfectly flat base line a t the instrumental gain used, nor did the forward and reverse field sweeps produce identical superposable signals. The reasons for this are obscure, but certainly every effort n-as made throughout this work to obtain optimal sample cell placement, cavity placement, and instrumental tuning. If the published examples of free radical signals through-
368
ANALYTICAL CHEMISTRY
out the literature are examined closely, it may be seen that absolute symmetry at higher amplifications is very rare. For this reason, the problem of spin concentration, which is estimated by the double integral of the recorded signal (a derivative of the actual spectrum) requires multiple determinations from both standard and unknown. This will be dealt with further, below. We have sought the conditions which would produce an easily measured, stable concentration of p-benzosemiquinone, relatively insensitive to small changes in the medium. In Figure 2 is shown the effect of quinhydrone concentration a t five different pH's on pbenzosemiquinone concentration as measured by the height of central line (derivative). Both pH and quinhydrone concentrations have marked effects. The effect of pH is shown more clearly in Figure 3, in which signal height is plotted against pH a t three quinhydrone concentrations. The concentration of p-benzosemiquinone as measured by signal height appears t o be very sensitive to small changes in pH in the region of neutrality. For this reason, it is particularly important when using quinhydrone as an ESR standard to reproduce the desired pH exactly. The effect of buffer concentration is small. The effect of temperature change on the signal height, using 5 mM quinhydrone a t pH 7, 0,2X, was also observed, and it was found that for every degree rise between 0" and 25' C., the signal height increased by 0.7% of its original height. In order to develop a quantitative standard from the p-benzosemiquinone signal, pH 7.20 was chosen for the medium. This value provides a readily observable signal but does not lie on the steepest portion of the pH-concentration curve. It was shown that signal height increased linearly with electronic amplification (gain) from 1x to 200 X within the limits of accuracy of the subsequent integrations, and the signal characteristics of the primary standard, peroxylamine disulfonate, were observed a t gain = lox, with lO-5;M solutions, while the signal characteristics of the secondary standard, p-benzosemiquinone, were determined with 5mM quinhydrone solutions, pH 7.20, 0.2X phosphate buffer, ambient temperature, at gain = 2 0 0 x . Cnder these conditions, the values of double integrals of three increasing (forward) field sweeps, for the first peroxylamine disulfonate line observed, only, were: 826, 821, and
PI
5,O m M
OH
2.5 m M O H
1.25mM OH
6.5
7.0
7.5
PH-0.2 M phosphate
Figure 3. Signal height plotted against pH in 0.2M phosphate buffers for three quinhydrone concentrations Instrumental conditions are given in the text
806 mma2;and the corresponding values for three decreasing (reverse) field sweeps were: 841, 819, and 777 mm.2. Thus, the maximum deviation was 5% of the average value (815 mrns2),but the standard deviation was very much better (22 mm.2). The corresponding values for the p-benzosemiquinone standard (five lines) were: two forward (increasing field) sweeps, 6931 and 6424 mm.2; and three reverse (decreasing field) sweeps, 6586, 6267, and 6535 mm.2. The maximum deviation for a single sweep was 6% of the mean value; the standard deviation was 260 mm.2. On this basis, the measured concentration of p-benzosemiquinone, calculated by comparing the mean values of the second integrals of ESR signa's measured under otherwise identical conditions, was 6.4 x 10-6X, and the standard deviation was 4% of this value. LITERATURE CITED
(1) Harvey, G., Hollingshead, R. G. W., Chem. & Ind. (London) 1953, 244. (2) Vanngard, T., Aasa, R., Proc. 7th International Conference Coordination Chemjstry, Stockholm, 1962, . 137. (3) .Vanan Associates, TechnicayInformation Publication 87-100-074, Palo Alto,
Calif.
(4) Venkataran, B., Segal, B. G., Fraenkel, G. K., J . Chem. Phys., 30, 1006 (1959).
