pheric pressure n-ould be required to obtain a similar spectrum in a 10-meter gas cell. The spectrum of dimethylamine illustrates an extreme example of a disadvantage associated with condensed samples. Although reference spectra of the common gases are readily obtained (S), few curves have been published for condensed gases. The spectra of many frozen gases resemble the spectra of the corresponding vapors closely enough to permit identification. Dimethylamine, however, s h o m marked spectral differences in different states. The strong
absorption at 882 cm.-’ in the solid, for example, is found at 735 crn.-’ in the vapor.
of the Department of Health, Education, and Welfare as part of a broad air pollution study authorized by Public Law 159, enacted by the 84th Congress.
ACKNOWLEDGMENT LITERATURE CITED
The author expresses his appreciation to J. D. Michaelsen for assistance during the design and use of this device and to E. I. Klein and H. W. Bailey, who constructed the glass and metal portions of the cell, respectively. This research was supported in part b y funds administered by the Public Health Service
.,
(1) Anderson. D. H.. Miller. 0. E..’ J . Opt . ~ O C Am. . 43, 77T (i953). (2) Bnderson, D. H., ANAL.CHEM.25, 1906 (19 (3) Pierson, R. H., Gantz, E. St. C., Ibid.,‘ (1956j. (4) White, J. U., J . Opt. SOC.Am. 32, 285 (1942).
Chemical Method for Calibration of Warburg Manometers C. R. Scott and W. B. Dandliker, Department of Biochemistry, University of Washington, Seattle, Wash. HE
use of the Warburg manometer
Tin tissue respiration studies or in other measurements of gas exchange requires a calibration equivalent to a determination of the volume of the system under consideration. There are several methods by \T hich this volume may be determined (1). The most widely used involves filling the system IT ith mercury and weighing the mercury. This method, although accurate, is laborious and involves unn ieldy operations in getting the manometers filled. The calibration procedure described here is based upon gas evolution from a quantitative chemical reaction and affords an accuracy comparable to that of the mercury method. besides being superior in several respects: -4 large number of manometers can be calibrated mor? or less simultancou4y; this type of procedure utilizes only the simplest manipulations of volumetric anal!-si‘; and the flask constant is obtained directly a t the desired temperature. The authors have studied the catalytic decomposition of hydrogen peroxide n i t h catalase in neutral solution and n ith osmium tetroxide ( 2 ) in bahic solution and have found the oxygen evolution to be quantitative and reproducible. The hydrogen peroxide solution )vas stabilized with urea and standardized by titration n ith ceric ammonium nitrate ( 3 ) ,using this salt as a primary standard. L-rea does not interfere in this titration, but n ould be unsuitable if standardization n ere accomplished by pcrnianganate ( 2 ) . Table I.
Manometer 1 2 3 4
METHOD
T o 10 ml. of hydrogen peroxide (10-5M) in a flask are added 3 ml. of perchloric acid (Sly). This is titrated n-ith the ceric solution, using one drop of o-phenanthroline ferrous sulfate or nitrophenanthroline ferrous sulfate as the indicator (S). Results are reproducible to within 0.2%. For use in the calibration of the Karburg manometers, 2 ml. of standard hydrogen peroxide are placed in the Xarburg flask n-ith 0.1 ml. of osmium tetroxide in the side arm. After equilibration, the osmium tetroxide is tipped in, and after 10 minutes the preswre change is recorded. Total volumes were calculated, using the usual equations and corrections for manometric measurements ( 4 ) . RESULTS A N D DISCUSSION
Table I gives a typical set of results. The greatest deviation of 3 single meas-
urement from the average was found to be 1.6%, and the unbiased estimate of the standard deviation for all the volume measurements was 0.12 ml., or 0.55%. The last column of Table I gives the total volumes as measured b y filling the manometers with mercury and weighing the mercury. The agreement between these results and those of the chemical method is extremely good, indicating that the latter method is capable of adequate accuracy for biochemical work. Catalase (30 units in 0.1 nil.) can be used in place of the 0.1 ml. of osmium tetroxide v i t h the same results. The reaction in this case was carried out in dilute phosphate buffer at p H 7.4, using a commercial preparation of crystalline catalase. ACKNOWLEDGMENT
The authors wish to acknonledge financial support from a grant of the National Heart Institute (H-2217) and from Initiative 171 Fund, State of Vashington. LITERATURE CITED
(1)Dixon, RI., “Rlanometric Methods,” 2nd ed., Rlacmillan, New York, 1943. (2)Huckaba, C.E.,Keyes, F. G., J . Am. Chem. SOC.70, 1640 (1948). (3) Hurdis, E. C., Romeyn, H., AKAL. CHEX.26,320 (1954). (4) Umbreit, W. K., Burris, R. H., Stauffer, J. F., “RIanometric Tech-
niques and Tissue Metabolism,” Burgess Publishing Co., RIinneapolis, Minn., 1951.
Calibration of Warburg Manometers b y Oxygen Evolution from Hydrogen Peroxide and Osmium Tetroxide T‘ol. by Filling
Total Volume of Manometer, M I . 21.05 20:h 19.54
21.18 21.10 20.96 22.00 21.90 21.90 20.59 20.50 20.55 19.60 19.60 19.60
i
2074
REAGENTS
Osmium tetroxide (4 X lO-’M). Osmium tetroxide (1 gram) is dissolved in 50 ml. of water. T o 0.2 ml. of this solution is added 5 ml. of 1M sodium hydroxide, and the volume is made up to 100 ml. with water. Catalase (30 units in 0.1 ml.), from the St70rthington Biochemical Corp., Freehold, N. J. o-Phenanthroline ferrous sulfate or nitrophenanthroline ferrous sulfate (0.025M). o-Phenanthroline (0.125 gram) is mixed n-ith 0.060 gram of ferrous sulfate and diluted to 25 ml. Time is allov-ed for complexing and dissolving before use as an indicator.
ANALYTICAL CHEMISTRY
20.80 21.50 ,
lj.54
21.13 21.94 20.47
19.67
21.30 21.20 21.9; 21.82 20.50 20.42 19.72 19.68
Std. Dev.
.IV.
21.28 21.93 20.53 19.78
21.13 21.85 20.60 19.70
21.11 21.85. 20.52 19.64
0.15 0.15 0.059 = 0.079
with Mercury
u =
= u = u
21:84 20.40 19.60
