precipitation, and subsequent calcination. I n the three separation techniques there always remain traces of uranium that interfere by imparting a violet color to the chloroform glyoxal extract. Color development experiments were made which showed that when 100 to 500 mg. of uranium were used, 0.2 to 5.0 mg. remained after separation by the three methods suggested. When hydroxylamine was added, the same experiments showed no interference. The experiments were made with or without calcium present and uranium was detected in the organic phase, chloroform, after evapora-
tion and destruction of organic matter with HN03-HCl and evaporation to dryness, addition of 3-4 drops of acetic acid and 2 drops of 3y0 potassium ferrocyanide solution. In those experiments in which hydroxylamine was present no uranium was detected. It has been observed that the chloroform extract of calcium-glyoxal complex starts fading after 5-10 minutes and for a series of determinations the last readings give systematically lower results. To avoid this, the color development and extraction are carried out only on two samples at a time. Calcium is usually and routinely determined by flame photometry,
which is a relatively insensitive method compared with the spectrophotometric one. Beer’s law is obeyed from 0 to 18 pg. of calcium. LITERATURE CITED
(1) Goldstein, D., Stark-Mayer, C., Anal. Chim. Acta, 1 9 , 437 (1958). ( 2 ) Rodden, Clement J., “Analytical Chemistry of the Manhattan Project,” Vol. VIII-1, p. 18 McGraw-Hill, Yew
York, 1950.
( 3 ) Williams, T. Kenneth, Wilson, R. John, ANAL.CHEM.33, 244 (1961). ALcfDIo ABRXO
Radiochemistry Division Instituto de Energia AtBmica Sao Paulo, Brazil
Rapid Gas Chromatographic Analysis of Fractional Microgram Concentrations of Lebaycid SIR: In the past year, Hirano and Tamura (4) published a spectrophotometric method for the determination of { O-dirnet*hyl-O-[4-(methylLebaycid thio) - m - tolyl]phosphorothioate}; the reported method obeying Beer’s law a t concentration levels ranging from 10 to 100 pg. In their procedure, the phosphorothioate compound is hydrolyzed with an alcoholic sodium hydroxide solution and the 4-methylthio-m-cresol obtained is condensed with 4-aminoantipyrine by the modified
4
method of Emerson (2) in an alkaline sodium carbonate buffered medium in the presence of sodium periodate. The orange-colored condensation product is extracted with chloroform and determined spectrophotometrically a t 458 mp with a relative error of 3% in the 10- to 100-pg. range. The authors substituted sodium periodate for potassium ferricyanide in order to. obtain lower blanks. To circumvent the time-consuming hydrolysis by refluxing, buffering with COz gas and sodium carbonate, coupling or condensation reaction and chloroform extraction steps specified in this colorimetric procedure, this communication presents a direct, rapid gas chromatographic method for the determination of fractional microgram concentrations of Lebaycid.
EXPERIMENTAL
The Jarrell-Ash Model 28-710 equipped with electron affinity and flame ionization detectors and the Bristol Dynamaster Model 1P12H560, 11-inch strip-chart recorder, 0- to lO-mv., was used for this study. The chromatographic column was made of borosilicate glass (&foot by 3/16-in~h0.d.) packed with 5% by weight Dow-Corning QF-1 (trifluoro propyl methyl silicone polymer) on 80/90 mesh Anakrom AS (Analabs, Inc., Hamden, Conn.). Prior to use, the column was conditioned a t 250’ C. for 48 hours to minimize column bleed. Flame ionization detector sensitivity settings of 1 X and 1 X 10-lo amperes were used for this study in addition to the following chromatographic operating conditions : hydrogen gas pressure, 7.0 psi.; air flowrate, 1.25
2 0 RETENTION TIME, MINS.
Figure 1. Gas chromatograms of Lebaycid at column temperatures of 177” and 190” C. and sensitivity and 1 X settings of 1 X amperes (flame ionization detector)
0.0 0.0
0.4 0.8 1.2 1.6 0. I 0.2 0.3 0.4 MICROGRAMS PER ONE MICROLITER INJECTION
2.0 0.5
Figure 2. Plot of peak height vs. micrograms of Lebaycid per one microliter injection (flame ionization detector) VOL. 37, NO. 3, MARCH 1965
439
c.f.h.; column temperature] 177' and 190' C.; detector temperature] 250' C.; injector temperature, 280' C.; nitrogen carrier gas flowrate, 71.5 cc./minute. To compare the area response per microgram of Lebaycid with both the electron affinity and flame ionization detectors, the electron affinity detector's senistivity was optimum when operated at 12 volts a t a 1 x 10-9 ampere gain setting. With this detector, the 6-foot column was packed with 5y0 SE-30 (General Electric's methyl silicone gum rubber) on 80/90 mesh -4nakrom AS. This liquid phase was used in view of the high background and noise level resulting from the QF-1 phase. At a column temperature of 177' C., the nitrogen carrier gas flowrate was increased to 150 cc./minute in order to obtain equivalent retention times with both liquid stationary phases. All other operating parameters were maintained similar to those noted above for the flame detector with the exception of the detector temperature which was held at 210' C.; this was limited by the detector's tritium source. One-microliter Hamilton microsyringe injections of varying Lebaycid concentrations dissolved in benzene as solvent were made directly onto the coated column solid support; thereby elimi-
nating the possibility of catalytic degradation a t metallic surfaces. DISCUSSION
OF
RESULTS
Using the conditions cited in the experimental section for the flame detector, typical chromatograms obtained for Lebaycid at 177' and 190' C. are shown in Figure 1. Chromatogram A compares the peaks obtained a t two different sensitivity and column temperature settings for known amounts of the phosphorothioate compound injected, whereas Chromatogram l3 shows superimposed gas chromatographic peaks resulting from different concentrations of Lebaycid a t 177" C. and 1 x ampere. By plotting peak-heights us. concentration of Lebaycid a t gain settings and 1 X 10-10 ampere, of 1 x nearly linear relationships are obtained as shown in Figure 2. At 1 x 10-lo ampere and a column temperature of 177' C., as little as 22 nanograms of this compound is easily detected; thus showing the gas chromatographic procedure to be about 500 times more sensitive than the spectrophotometric analysis.
This study has also shown that the area response in square millimeters per microgram of compound at 1 x 10-9 ampere with the flame detector (1517 mm*) is approximately 1.53 times greater than that obtained with the electron affinity detector (995 mm.2).
S
//
This indicates that the -P-0-group-
l
ing in Lebaycid has a poor affinity for electrons and confirms and is consistent with the findings of Cook et al. ( I ) and Gudzinowicz et al. (3. LITERATURE CITED
( 1 ) Cook, C. E., Stanley, C. W., Barpey,
J. E. 11, Abstracts, Fifteenth Pittsburgh Conference o n Analytical Chemistry and Applied Spectroscopy, X a r c h
1964, p. 53. (2) Emerson, E., J . Org. Chem. 8, 417 (1943 ). (3) Gudzinowicz, B. J., Clark, S. J., J . Gas Chromatog. 2, 335 (1964). ( 4 ) Hirano, Y., Tamura, T., ANAL. CHEM.36, 800 (1964).
Jarrell-Ash Co. 590 Lincoln Street Waltham, Maas.
B. J. GUDZINOWICZ
Quartz Window Cuvette Prepared by Sealing Quartz to Borosilicate Glass Wm. T, Roubal, U. S. Bureau of Commercial Fisheries and The Department of Food Science and Technology, University of California, Davis, Calif.
of commercially automated liquid flow analytical systems, many laborious and time-consuming analyses can now be run a t the rate of 60 per hour. The modular approach, extensively used for these analyses, also allows the operator to incorporate a wide variety of other laboratory instruments into an analytical scheme. The relatively low liquid flow rate of such systems coupled with the fact that air bubbles often find their way into optical systems, either by accident or intentionally] places severe limitations on existing small volume flow cuvettes. A variety of commercial flow cuvettes designated as microcuvettes were tried as cells for a commercial spectrophotometer. These cells ranged from rectangular quartz cells and small diameter cylindrically shaped cells to the recently available microaperture flow cell. At low flow rates (several milliliters per hour), bubbles either I T H T H E ADVENT
Mi available
440
e
ANALYTICAL CHEMISTRY
lodged within the optical path of the cell or peak distortion was observed. At higher flow rates (several milliliters per minute), channeling was a problem in cells with large cross sectional area. This paper describes in detail the fabrication of a streamlined, liquid flow, small volume, deep path, UV transmitting cuvette for the Beckman DB or similar spectrophotometer. Air bubbles do not lodge within the cell and peak distortion is no problem a t low or high flow rates.
MORGANITE FORM
a,
3
b.
PROCEDURE
Prepare a heat resistant form, the dimensions of which correspond to the desired internal width and depth of the cell, from Morganite (Morganite] Inc., Canoga Park, Calif.). A form with dimensions of 5/u inch x '/a inch x 11/4inch gives a finished cell with an active volume of about 0.3 ml. Push the form into the middle of a 12- to 14-inch length of borosilicate glass
C.
Figure 1 . Steps in the fabrication of a quartz window flow cell ( a ) Cell body molded over Morganite form ( b ) Shoulders drawn down to form Inlet and exit tubes. (c) Quartz windows a r e sealed onto narrow faces after borosilicate glass portion has been cut a w a y
