Anal. Chem. 1980, 52, 1379-1381 1100,000
e
900,000
700,000
e 300,000
as well as the corresponding hydrocarbons, is shown in Figure 3. It appears from Figure 3 that as the number of carbons increase, the difference in the detector relative response between the fluorocarbons and the corresponding hydrocarbon increases. Evaluating HID response to perfluorocarbons according to their ionization potentials is not possible because such data are not available. The parent ions of these molecules (M+) are not stable enough to allow actual measurement of ionization potentials, and can only be estimated for some molecules. The ionization potential for CF4 has been estimated to be 14.9 to 15.1 eV (8). This is higher than the ionization potential of methane (12.6 eV), arid hence the response to methane should be higher than the response to tetrafluoromethane. There are no published data on estimated ionization potentials for higher perfluoroalkanes (9). However, one may conclude from Figure 3 that the ionization potentials of C2 to C5 perfluoroalkanes are higher than those of the corresponding hydrocarbons, with the difference increasing as the number of carbon atoms increases. The possibility of forming negative perfluorocarbon ions may add to the complexity of the ionization mechanism; however, verifying the mechanisms of the HID mechanisms to perfluorocarbons is beyond the scope of this work. The helium ionization detector is a simple and sensitive device; and in the saturation region of the detector field intensity, the detector operation is reliable. Besides being a sensitive and universal detector, it is the only detector that is capable of detecting trace amounts of certain classes of compounds such as those investigated here.
LITERATURE CITED
PERFLUOROALKANES
100.000
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(1) C. A. Clemons and A. P. Altshuller, Anai. Chem., 38, 113 (1966). (2) W. C. Askew and K . D. Maduskar, J . Chromatcgr. Sa.,9, 702 (1971). (3) A. T. Blades, J . Chromatogr Sci., 1 1 , 267 (1973). (4) M. A. Pringuer, J. Porter, andT. A. Gough, J . Chromatogr. Sci., 17, 387
.- . - .
11979\ , ,
1
2
3
4
5
C A R B O N NUMBER
Figure 3. Detector relative response to C,-C5 n-perfluorocarbons and the corresponding hydrocarbons (response to 100 ppm)
detectable amount was about 0.3% and above this concentration the detector response is deformed to the M-shaped peak (6). T h e magnitude of the detector response reported here is far better than that reported for FID or ECD. This is particularly significant for the lower members of this perfluorocarbon group where both FID and ECD provide extremely small responses. The detector relative response for C1 to C5 perfluorocarbons,
(5) E. Proksch, P. Gehringer, and W. Szinovatz, J . Chromatogr. Sci, 17, 568 (1979). (6) F. F. Andrawes. R. S. Brazell. and E. K. Gibson. Jr.. Anai. Chem.. 52. 891 (1980). (7) T. R. Majer, “Advances in Fluorine Chemistry”, Vol. 2, M. Stacy, T. C. Tatlow. and A. G. Sharpe, Eds., Butterworth, London, 1961, p 58. (8) R. W. Kiser and D. L. Hobrock, J . Am. Chem. Soc., 87,922 (1965). (9) T. L. Frankline, J. G. Dilbrd, H. Rosenstock, J. T. Heron, and F. H. Field, ~I
“Ionlzatlon Potentials, Appearance Potentials, and Heat of Formation of Gaseous Positive Ions”, National Standard Reference Data Service, National Bureau of Standards, Gaithersburg, Md. 1969,p 26.
RECEIVED for review March 3,1980. Accepted April 21,1980. This work was performed in part under the auspices of the National Aeronautics and Space Administration, Contract NAS9-15800 to Lockheed Engineering and Management Services Company, Incorporated.
Determination of Hydrogen Cyanide in Blood Using Gas Chromatography with Alkali Thermionic Detection R. W. Darr,” T. L. Capson, and F. D. Hileman2 Flammability Research Center, University of Utah, Salt Lake City, Utah 84 708
The possible production of hydrogen cyanide (HCN) in fire situations coupled with the uncertainty of the toxic effects of HCN in combination with other toxic gases (I) produced in fires (e.g., carbon monoxide) has led this laboratory to carry
out animal exposures involving these toxic gases. As an initial measure of the uptake of HCN by the exposed animals, a method was sought to determine the blood cyanide levels. While several methods are available (2-4), they are either semiauantitative (2) or reauire relativelv large volumes of blood ( 3 , 4 ) (Le., greater than 1 mL:l with extensive work-up exposure studies, where procedures* Carrying Out repeated samples of blood are withdrawn from several labo“
‘Present address: Allied Chemical Corporation, CMA Box 1020R, Morristown, N.J. 07960. 2 Present address: Monsanto Research Corporation, p.0. Box 8, Station B, Dayton, Ohio 45407.
0003-2700/80/0352-1379$01.00/0 0 1980 American Chemical Society
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ANALYTICAL CHEMISTRY, VOL. 52, NO. 8, JULY 1980
ratory animals, required a simple, highly quantitative blood cyanide analysis using only small amounts (-0.2 mL) of blood. In devising such an analytical procedure, it was noted that most of the methods for the analysis of cyanide in biological samples involve acidification of the sample followed by extended collection of the evolved HCN in some suitable trap. The trapped HCN is then analyzed using a variety of techniques including colorimetry ( 2 , 3 )or conversion to a material which may be chromatographed such as cyanogen chloride ( 4 ) . If a sensitive method were available for the detection of the HCN in the headspace above acidified blood, then the necessity for trapping of the HCN could be eliminated. Such a method was reported by Donike ( 5 ) in which he was able t o detect picogram quantities of HCN using an alkali flame ionization detector. Recent advances in alkali thermonic detector design have improved their sensitivity and reliability (6). Thus the combination of improvements in detector performance along with the need for a small scale method for the determination of blood cyanide has resulted in the procedure described in this paper.
EXPERIMENTAL Chemicals. Standard cyanide solutions were prepared with KCN (Mallinckrodt, Reagent Grade) using distilled water made basic with 1 mg NaOH (Baker, Reagent Grade) per liter of water. Concentrated (85%)phosphoric acid used for the acidification of the blood samples was obtained from Alfa Division, Ventron Corporation, Danvers, Mass. Apparatus. Two-mL Pyrex hypovials with Teflon lined compression sealing septum lids (Wheaton Scientific) were used to contain the acid, blood, and HCN-containing headspace. A Blue M Model MSB-1122A agitating water bath was used for sample temperature equilibration. Samples of the headspace were taken using a Unimetrics Model 4001 1-mL gas-tight syringe with holding valve. Chromatographic analysis of the headspace was carried out on a Hewlett-Packard Model 7620 gas chromatograph equipped with a 6-foot, l/s-inch 0.d. Teflon column packed with Porapak QS maintained at 80 "C. Helium was used as a carrier gas at a flow rate of 30 mL/min. HCN detection was achieved using a Tracor Model 702 thermionic detector. Procedure. Standard aqueous cyanide (CN-) solutions containing 2.5,5, and 25 pg/mL CN- were prepared by adding 1part of these aqueous CN- solutions to 4 parts of whole blood. These "spiked" blood solutions were used to calibrate the actual concentrations of cyanide in the blood of animals exposed to inhaled HCN. The analysis of blood cyanide was carried out by introducing 0.1 mL of concentrated H3P04into a glass 2-mL hypovial and sealing the vial with a septum lid. Two tenths of a milliliter of whole blood from either the spiked blood or the blood from an experimental animal was then injected through the septum lid into the sealed vial, shaken on a vortex mixer, and incubated for 60 min at 60 "C in the agitated water bath. After incubation, 1 mL of the headspace above the sample was withdrawn with the gas-tight syringe and injected into the gas chromatograph for analysis. An example of the chromatographic trace for the analysis of blood cyanide of an exposed animal is given in Figure 1. This trace shows the relative blood cyanide values before and after an exposure to HCN as compared to a prepared standard. Either electronically integrated peak areas or manually measured peak heights were used for the preparation of calibration curves and for quantitative analysis for all samples.
RESULTS A N D DISCUSSION T h e selection of an acidifying agent was based on the necessity of the acid being strong enough to release the HCN and yet having no significant vapor pressure which might cause interferences in subsequent chromatographic analyses. Concentrated phosphoric acid was used in this study; however, concentrated sulfuric acid has been shown to work as well. No detailed study was carried out to determine the lowest concentration of acid necessary to cause conversion t o HCN,
iOpg/ml i t on da r d s
0 Minute xposure iarnplrs
I
-
Controls h
A
J
Figure 1. Chromatographic traces for the analysis of blood cyanide
in standards and animals exposed to gaseous HCN
IC
.. . . . 0
6 -
4l
..
I
3r
..
0
.
.
2 1
4
8
12 16 20
32
40
60
80
TIME (Minutes)
Figure 2. A plot of HCN liberation from blood as a function of time while incubated at 60 O C
and thus concentrated acid was used in all cases. A study was performed to determine the optimum temperature for rapid equilibration of HCN into the headspace for blood spiked a t a level of 1pg/mL. At room temperature, a partition ratio ( K ) ,defined as: mg of HCN per liter gas
K=
mg of HCN per liter of solution of approximately 7.0 X was observed which increased only slightly on raising the temperature to 60 "C. This deviation from applicable thermodynamic considerations could result from the complex set of reactions that can occur between cyanide and the various components of blood. Equilibration temperatures higher than 60 "C resulted in water condensing in the gas-tight syringe. This made reproducible injections of HCN impossible, and thus an optimum equilibration temperature of 60 "C was used. At 60 "C, approximately 60 min was required for the concentration of HCN in the headspace to reach to a constant value as shown by the plot of Figure 2. At lower temperatures, the equilibration time was longer and thus 60 "C was used throughout this study. In this work, the samples were kept
Anal. Chem. 1980, 52, 1381-1383
in the constant temperature bath for a t least 1 h to ensure complete equilibration of all samples. Using these conditions, a plot of the integrated HCN peak areas vs. spiked blood cyanide levels was linear over a range of 0.1 t o 5 pg/mL blood cyanide. The lower detection limit taken from this plot is 0.05 pg/mL, an amount typical of control levels of cyanide in unexposed laboratory animals used in this study. Using the acidification and equilibration procedure described above, no interferences were detected in any of the unspiked blood samples (Le., no spurious high cyanide levels were ever detected). The only suspected interfering species might be blood thiocyanate which could be converted to HCN under the experimental conditions. However, examination of blood spiked with thiocyanate a t the 1 pg/mL level gave a HCN response comparable to background levels present in unspiked blood, a result in agreement with the results of Valentour et al. ( 4 ) . T h e percent standard deviation of spiked blood samples was typically on the order of 3-5%. This error could be attributed in part to the injection error (found to be approximately 2%) and the measuring of the blood into the hypovials (approximately 1% error). Some difficulty accompanied the measurement of small volumes of the viscous concentrated phosphoric acid into the hypovials. Since the volume of the headspace must be constant and is in part determined by the volume of fluids added to the vials, this difficulty in pipetting the phosphoric acid contributed to the error in the analyses. When animals were exposed to gaseous HCN and actual blood cyanide levels determined, some increase in the error of analysis was observed as compared to the spiked blood. This error is believed to be due to a clot partition formed in the vial during extended incubation times which can occur when a large number of samples from the animal exposures
1381
were being analyzed. The physical clot barrier could possibly prevent the release of HCN into the headspace. When care was taken to break any clots prior to analysis, the margin of error was substantially reduced. In spite of these difficulties, the range (only two replicates could be made for each sample of withdrawn blood) in these values was typically less than 10% of the level of cyanide analyzed. This technique seems to be extremely versatile in that preliminary investigations have shown that not only may HCN be detected, but also carbon monoxide, carbon dioxide, and oxygen. These gases are also displaced into the headspace above the blood and thus could be quantitated using chromatographic separation and thermal conductivity detection. Thus, a wide variety of blood gases may be easily detected using this methodology.
ACKNOWLEDGMENT The authors thank W. A. Galster and B. M. Hughes for their assistance in carrying out this project. They also gratefully acknowledge the many suggestions given by M. M. Birky and M. Paabo. LITERATURE CITED (1) R. H. Moss, C. F. Jackson, and J. Saberlick, Arch. ind. Hyg. Occup. Med., 4, 53 (1951). (2) C. N. Carducci, P. Luis, and A. Mascaro, Mikrochim Acta, 339 (1972). (3) R. Shanahan, J. Forensic Sci., 18, 26 (1972). (4)J. C. Valentour, V. Aggarwal, and I. Sunshine, Anal Chem., 46, 924 ( 1974). (5) M. Donike, 2.Nafurforsch., 28, 533 (1973). (6) M. J. Hartigan, J. E. Purcell, M. Novotny, M. L. McConnel, M. L. Lee, J . Chromatogr., 99,339 (1974).
RECEIVED for review April 16, 1979. Resubmitted April 21, 1980. This work was supported by the Center for Fire Research, National Bureau of Standards.
Determination of Oryzalin and Prosulfalin Residues in Soil by High Pressure Liquid Chromatography Thomas D. Macy and Andrew Loh' Agricultural Analytical Chemistry, Lilly Research Laboratories, Division of Eli Lilly and Company, Greenfield, Indiana 46 140
Oryzalin (Figure 1) [3,5-dinitro-N4,N4-di(rz-propy1)sulf- to inject a larger sample onto the LC column and by optianilamide] is a selective, pre-emergence herbicide for the mizing the HPLC system for trace analysis. Kennedy (8) control of certain annual grasses and broadleaf weeds in reported the use of HPLC for analyzing oryzalin; however, soybeans and other selected crops (I,2 ) . Prosulfalin (Figure his work was conducted for formulated and technical material. 1) [N*-dimethylsulfilene-3,5-dinitro-N4,N4-di(rz-propyl)sulf- In this work, analytical procedures are described for the anilamide] is an experimental pre-emergence herbicide for use trace analysis of oryzalin and prosulfalin residues in soil using on established turfgrass species ( 3 ) . an optimized HPLC system. The method requires no deThe reported method for analyzing oryzalin in soil and crops rivatization and the two compounds are purified and separated involves an overnight methylation with methyl iodide prior by florisil column chromatography. to gas chromatographic analysis ( 4 ) . The gas chromatographic EXPERIMENTAL analysis of prosulfalin requires that the compound be hydrolyzed to oryzalin, then methylated overnight as with oryApparatus. The liquid chromatographic systeni is comprised zalin. The disadvantages of derivatization methods are that of a Waters Model 6000A pump, Waters Model 440 fixed wavelength detector, a 30 cm X 3.9 mm i.d. pBondapak C18reverse extra time is required for derivative formation, the derivaphase column (Waters Associates, Milford, Mass.), a variable input tization step is usually not quantitative, and the probability recorder, Model B5117-5 (Houston Instrument, Austin, Texas), of error increases because of the extra sample manipulation. and Sample Injector, Model 7120 (Rheodyne, Berkeley, Calif.). High pressure liquid chromatography (HPLC) has recently The precolumn consisted of an 8 cm X 1/16 inch i.d. column packed been shown to be useful for the trace analysis of a number with Bondapak CIS37-50 pm (Waters Associates). The in-line of pesticide residues (5-7). The UV detectors commonly used filter was a 2-pm filter with '/,,-inch tubing (Alltech Associates, for HPLC often lack sensitivity and selectivity when compared Arlington Heights, Ill.). The variable wavelength detector was to the commonly used gas chromatographic detectors. These a Waters Model 450. A Sola constant voltage transformer, type disadvantages can be partially offset by the inherent ability CVN (Sola Electric Co., Elk Grove, Ill.) was used for all analyses. 0003-2700/80/0352-1381$01 .OO/O
1980 American Chemical Society
