Table I. Estimation of Nitrogenase Activity in Field Peas (Pisum sativum) on a Farm near Alysham, Saskatchewan mmol C,H, Date h- ' plant- ' July 1 5 (first flower) July 22 (flowering) July 29 (pod filling)
1.91 4.80 4.17
which CzHz(1%v/v) was added. After 1 h, 1-mL samples of the gas phase were removed and assayed for CzH4 using the portable gas chromatograph. Plant roots do not produce gaseous products with column retention times similar to ethylene. The data show the typical increase of nitrogen fixing activity till flowering, followed by the decrease in activity during pod fill. The total cost of components, excluding column packing, is less than $50. The gas chromatograph (Figure 5), in a sturdy metal box, weighs less than 2 kg. It is not necessary to carry heavy tanks of carrier gas, for a convenient and inexpensive source of compressed air can be obtained by fitting an ac-
tivated carbon filter and two-stage pressure regulator to a pump-tank commonly sold for spraying pesticides (Figure 6). The apparatus described thus meets the requirements of low cost and portability. Changes in column packing would permit the separation and measurement of gases other than acetylene and ethylene. The identification and quantitation of carbon monoxide or ethanol are obvious useful examples. Because the instrument is easily made, inexpensive, has simple circuitry, and can be readily dissembled for inspection, it should make a suitable instructional aid for demonstrating to students the theory and principles of gas chromatography.
LITERATURE CITED (1) K. J. Skinner, Chem. Eng. News, 54 (41), 22 (1976). (2) J. G. Criswell, R. W. F. Hardy, and U. D. Havelka, World Soybean Res.,
108 (1976). (3) T. A. LaRue and W. G. W. Kurz, Plant Physiol., 51, 1074 (1973). (4) C. R. Lewart, Popular Nectronics, August 1976, p 46.
RECEIVED for review December 20,1976. Accepted March 28, 1977.
Electron Paramagnetic Resonance Sample Cell for Lossy Samples Sandra S. Eaton Department of Chemistry, University of Colorado at Denver, Denver, Colorado 80202
Gareth R. Eaton" Department of Chemistry, University of Denver, Denver, Colorado 80208
In many cases where electron paramagnetic resonance (EPR) spectroscopy is the analytical method of choice, the sample has such a high dielectric loss that the standard 3- or 4-mm 0.d. cylindrical sample configuration cannot be used. Freezing the sample may be inconsistent with the goals of the experiment; and since most investigators have access only to X-band (9.5 GHz) or Q-band (35 GHz) spectrometers, it is not realistic to propose selecting a frequency at which the loss tangent for the solvent is a minimum. The usual experimental solution to this problem is to minimize extension of the sample into the microwave electric field, either by using a very small diameter cylindrical sample, or by using a flat sample cell (1-3). (The TMllo cavity is now available as the Varian E-238 cavity.) Both theoretical considerations and experimental results indicate that with X-band TElozcavities, the Varian aqueous flat cell is close to optimum. (Varian Instrument Division E-248-3, Aqueous Solution Sample Cell; Scanco S-812, EPR Aqueous Cell). Unfortunately the current cost for this cell is in excess of $100. Consequently for routine measurements it is common to use a small diameter cylindrical tube, such as a melting point capillary (e.g., Kimble No. 34507, Tubes, Capillary Melting Point, U.S.P.) to inexpensively minimize the extension of the sample into the microwave electric field. In this paper we report an improved inexpensive aqueous sample cell using flat glass tubing which recently has become commercially available.
EXPERIMENTAL Rectangular cross section (Figure la) glass tubing obtained from Vitro Dynamics Inc. (114 Beach Street, Rockaway, N.J. 07866. The 0.4 by 4 mm tubing is catalog No. 2540.) was used as the sample container. This tubing is available in quantity with internal cell thicknesses, a, of 0.05,0.1,0.2,0.3,0.4 mm at a cost of less than $0.50 for a 50-mm long piece. The standard tubing with a = 0.4 mm has a b dimension of 4 mm. The tubing can
be supported in the cavity in any low-loss material, such as standard quartz cylindrical tubing. However, it was of interest to design a system in which a controlled atmosphere could readily be maintained. A cylindrical 11-mm 0.d. Teflon container with internal diameter machined to accept the 0.4 by 4 mm glass tubing M aqueous solutions of was fabricated. Samples were 2.6 X a commercially available nitroxyl radical, 4-hydroxy-2,2,6,6tetramethylpiperidinooxy. Microslides were sealed with Parafilm. The sample tubes can be filled in a Glove-bag (Instruments for Research and Industry, 108 Franklin Ave., Cheltenham, Pa. 19102) and an air-tight seal on the Teflon container accomplished with a piece of Teflon tape between the cylindrical container and its cap. Comparison samples in Kimble 34507 melting point capillaries were filled with aliquots of the same nitroxyl solution and placed in standard 3-mm 0.d. quartz tubing before inserting in the cavity. EPR spectra were run on a Varian E-9 EPR spectrometer with a dual cavity (Varian E-232 TEIM). Only one sample was inserted in the cavity at a time, because the Teflon holder distorts the power distribution in the other half of the cavity. The spectrometer was stabilized with an E-272B FieldIFrequency Lock Accessory. Spectra were obtained in digital format using a Varian 620L computer, and double integrations were conducted using the CLASSlanguage (4). Correction was made for signal amplitude differences between the two cavities, determined by using Varian strong pitch to be 7%. Three Microslides were sandwiched together as shown in Figure lb, one 0.3-mm slide on each side of a 0.4-mm slide. This assembly was placed in the same Teflon holder as was used for the 0.4-mm slide by itself.
RESULTS AND DISCUSSION Linewidths and power saturation behavior were the same for samples in Microslides and capillary tubing. Consistent with this, integrated area ratios were the same as peak height ratios. Therefore the results presented are based on peak height measurements. ANALYTICAL CHEMISTRY, VOL. 49, NO. 8, JULY 1977
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A
B
Figure 1. (a) Sketch of Microslide cross-section indicating internal dimensions; (b) Cross-section sketch of “sandwich” arrangement of Microslides
The signal to noise ratio was 2.5 f 0.2 times as good for the Microslide in the Teflon holder as for the melting point capillary in the quartz tube. This ratio is based on measurements on each of the three peaks in the spectrum for eight capillary tubes and eight Microslides. Average peak heights for the sample in the capillaries were 60 f 2,60 f 2,50 f 2, and in the Microslides were 154 f 11, 152 f 11, and 144 f 11 (in arbitrary units). The major uncertainty appeared to be a systematic bias introduced by differences in the two Teflon sample holders used. Thus it appears that even though the procedure is proposed as a “quick and dirty” survey technique, it will easily provide results quantitatively useful to better than 10%. For comparison, the same sample solution in the Varian flat cell yielded peak heights of 159,154, and 145. Thus the Microslide gives essentially the same results as the expensive flat cell. The Microslides are easier to use than either capillary tubes or the flat cell. The Microslides can be filled by dipping the end in the sample solution; they fill by capillary action.
A rather striking result was obtained with the three Microslide “sandwich” assembly. With the Microslides parallel to the nodal plane of zero rf electric field, the spectrometer could not be tuned up. However, when the Microslides were rotated by 90’ so that they were perpendicular to the nodal plane of the rf electric field, the spectrometer tuned quite easily and yielded a signal 3.8 f 0.4 times as good as a capillary and 1.5 times as good as a single 0.4-mm Microslide or the Varian flat cell in the usual (parallel to node) orientation. This experiment was stimulated by Hyde’s similar finding with specially machined Rexolite sample cells in a TMllo cavity ( 2 ) . Hyde obtained a 4.9 improvement relative to a 1.1-mm capillary, using larger cells. The TEloz cavity has only a 11-mm opening so the larger flat cell for which Hyde obtained a 6.25 improvement relative to a capillary cannot be used (2). Thus for the cavity we used, the Microslides in the perpendicular orientation give the best improvement relative to the capillary tube and the flat cell that has been observed to date. The Microslide exhibits the signals expected for glass-a strong resonance at -1600 G and a weak broad resonance near 3400 G-but these do not obtrude significantly for most spectra. The peak near 3400 G increased above noise level when the power was increased to 20 mW and the modulation amplitude was increased to 2 G. These are much higher settings than would be used with organic radicals such as the nitroxyl radical used in this study.
LITERATURE CITED (1) R. S. Aiger, “Electron Paramagnetic Resonance:
Techniques and Applications”, Interscience, New York, 1968, p 504. (2) J. S. Hyde, Rev. Sci. Instrum., 43, 629 (1972). (3) G. Brown, J. fhys. E, Sci. Instrum., 7, 635 (1974). (4) C. Klopfenstein, P. Jost, and 0. H. Griffith, Comput. Chem. Biochem. Res., 1, 175 (1972).
RECEIVED for review February 28, 1977. Accepted April 8, 1977. Acknowledgement is made to the Donors of the Petroleum Research Fund, administered by the American Chemical Society, the Research Corporation, and the National Institutes of Health (GM 21156) for partial support of this research.
Advances in Assembling Permeation Tubes Andrew E. O’Keeffe Environmental Sciences Research Laboratory, U S . Environmental Protection Agency, Research Triangle Park, North Carolina 277 1 1
Since publication of our initial article (I) on permeation tubes, these simple devices have attained wide acceptance as accurate primary standards for the calibration of air pollutant measurement methods and instruments. Concurrently, certain improvements have been made in the method of assembling permeation tubes, resulting in a simpler procedure and a more reliable product, Several of these are described below: 1. T u b e Seals. In place of the steel ball originally used, we now recommend FEP Teflon rod (or other polymer rod to match the tubing used) slightly larger than the lumen of the tubing used. Figure 1is a dimensional sketch of a typical tube in a size having broad application. 2. Filling. The valving technique originally described for steel ball seals and later ( 2 , 3 )for rod-shaped seals is difficult to apply in the latter case. An alternate technique that is both simple and virtually free of difficulty due to spillage consists of the following steps (also shown in Figure 1): (a). Assemble 1278
ANALYTICAL CHEMISTRY, VOL. 49, NO. 8 , JULY 1977
tube with a rod seal a t each end. (b). Place a 1-cm collar of gum rubber tubing (3-mm wall) around the rod at one end, its center aligned with the inboard end of rod. (c). Place a worm-gear tubing clamp around above assembly, tightening moderately. It is important that this clamp engage some portion of the length of the rod seal. (d). Fill through a hypodermic needle [No. 27, 0.018-in. (0.046-cm) diameter] inserted just inboard of rod. (e). Turn off gas supply and withdraw needle; rubber collar forms a temporary seal. (f). Push rod inward 1cm to final position (Figure 2) (g). Remove hose clamp and rubber collar. This will be easier if, prior to assembly, the collar is slit from end to end along a line approximately opposite the point a t which needle will be inserted. 3. Reinforcing Ferrules. It is recommended that the seals of permeation tubes made as described above be reinforced with Type 304 stainless steel ferrules ( 2 , 3 )as otherwise some
