separate peaks with retention indices of 620 and 725, respectively (at 140 "C), 1-pentene and the 2-pentenes produced a single peak with a retention index of 700 and considerable tailing and 1-hexene provided a peak with a retention index of only 760, also with substantial tailing. It appeared that the samples were undergoing a reaction catalyzed by the packing material. Stopped-flow experiments with the 2-butenes, shown in Figure 3, confirmed this. The chromatograms show that the initially pure isomers have isomerized to a significant extent while they were stopped in contact with the resin. The larger olefins may well isomerize to the most readily eluted form, providing a single peak with a tail caused by further reaction of the more strongly retained isomers taking place gradually on the column. Cadmium ion is known to catalyze these reactions (12). The partially-sulfonated porous polymer packings are easy to prepare and retain the high separating efficiency of the parent material. They would appear to offer a great deal of flexibility in meeting the selectivity requirements of a specific sample. Further work is under way to determine more completely the advantages and limitations of these packings in gas chromatography.
Table I. Retention of Unsaturated Compounds on Sulfonated Porapak Q Kovats Retention Index on Resin I Resin I11 (15.5% Ag) Resin I1 (9% Ag) (5.5% Ag) 225°C 230°C 1 4 0 ° C 1 4 0 ° C trans- 2-Butene cis-2-Butene 1-Pentene trans-2-Pentene cis-2-Pentene Benzene Toluene
... ...
620 720 800 720 830 830 960
900 830 930 920 1070
680 750
...
780 860 810
...
570 650 730 680 750 700 830
LITERATURE CITED (1) (2) (3) (4) (5) (6) (7) (8)
Is for pure trans-2-butene, bottom for pure cis-2-butene injected into column
K. Ohzeki and T. Kambara, J. Chromatogr., 55, 319 (1971). R. F. Hirsch et al., Anal. Chem., 45, 2100 (1973). K. Fujimura and T. Ando, J . Chromatogr., 114, 15 (1975). S. Allulli et al., Anal. Chem., 48, 1259 (1976). P. Magidman et al., Anal. Chem., 48, 44 (1976). J. S. Fritz and J. N. Stary, Anal. Chem., 46, 825 (1974). L. C. b n s e n and T. W. Gilbert, J. Chromatogr. Sci., 12, 458, 464 (1974). T. S. Stevens and H. Small (Dow Chemical Company), U S Patent 3,966,596 (1976). (9) H. Small, J. Inorg. Nucl. Chem., 18, 232 (1961). (IO) R. Lane, E. Lane, and C. S. G. Phillips, J. Catal., 18, 281 (1970). (1 1) D. G. Howery and S. Tada, J. Macromol. Sci., Chem., 3, 297 (1969). (12) I. Hadzistelios, F. Lawton, and C. S. G. Phillips, J. Chem. Soc., Dalton Trans., 2159 (1973).
is shown in Table I. In all cases they are fully resolved from the saturated hydrocarbons, but the enhancement of retention is different for each resin. The cadmium form of resin I1 gave quite different results for the olefins. While trans-2-butene and cis-2-butene gave
RECEIVED for review March 18,1977. Accepted June 27,1977. Acknowledgement is made to the donors of the Petroleum Research Fund, administered by the American Chemical Society, for support of this research.
I
TIME
Figure 3. Stopped-flow studies of the 2-butenes. Cd2+ form of resin 11; 140 OC; 45 mL/min carrier gas flow; (A) flow stopped, (B) flow restarted, (C) trans-2-butene, (D) cis-2-butene. Top chromatogram
Determination of Ethyl and Methyl Parathion in Runoff Water with High Performance Liquid Chromatography Daniel C. Paschal, * Richard Bicknell, and David Dresbach
Department of Chemistry, Illinois State University, Normal, Illinois 6 176 1
High performance liquid chromatography with variable wavelength detection is described for the determination of methyl and ethyl parathion at the part per billion level in runoff water. The macroreticular resin XAD-2 was used as an adsorption medium for preconcentrationof trace organics in water by a factor of 100. Linear relationships between peak height or area and concentration were obtained in the range 0 to 120 ppb of methyl and ethyl parathion, with a lower detectlon limit of 2 to 3 ppb ( S / N = 2). Relative standard deviations In this range were 1 to 6%, with an average recovery of 99 %. Only 30 mln is required for the complete determination, and as little as 2 ng of methyl or ethyl parathion can be quantified with a 10-pL injection.
Organophosphorous insecticides enjoy wide use due to their relatively rapid decomposition and low accumulation in biological food chains. For these reasons, the organophosphorous insecticides are rapidly replacing the more persistent organochlorine agents. In fact, recent EPA restrictions have curtailed the use of several of the once widely used organochlorine pesticides such as DDT, aldrin, dieldrin, and heptachlor (1). Among the more popular replacements for these organochlorine compounds are ethyl and methyl parathion. Ethyl parathion (diethyl p-nitrophenyl phosphorothionate) and methyl parathion (dimethyl p-nitrophenyl phosphorothionate) were introduced in the 1940s. Their high, wideANALYTICAL CHEMISTRY, VOL. 49, NO. 11, SEPTEMBER 1977
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spectrum insecticidal activity makes them useful in a number of applications. Both methyl and ethyl parathion are subject to hydrolysis, with a strong dependence on pH and temperature. In general, half life decreases at elevated temperature and pH. For example, at pH 7.4 and 20 "C, ethyl parathion has a half life of 2594 h, while at the same temperature and pH 5.0, the half life is 3670 h. At pH 5.0 and 70 "C, the half life decreases to 19.5 h (2). Under field application conditions, about 1-10 lb (0.45 t o 4.5 kg)/acre are normally applied (31, which could lead to entry of the parathion into runoff water. The presence of ethyl or methyl parathion in runoff water presents a potential hazard due to the high mammalian toxicity of ethyl and methyl parathion as well as that of their major hydrolysis product, p-nitrophenol. Under environmental conditions both ethyl and methyl parathion could persist a t the sub-ppm level in water for a number of days or weeks, depending on temperature and pH
(4). Most methods for the determination of methyl or ethyl parathion involve an extraction followed by chromatographic separation and quantification. Thin-layer chromatography methods are generally slow and difficult to quantify; while gas chromatographic methods can give unreliable results due to the thermal lability of ethyl and methyl parathion. High performance liquid chromatography offers a nearly ideal system for determination of parathions due in part to the gentleness of the technique in which separations are accomplished at ambient temperature. A recent review article (5)described the usefulness of liquid chromatography for ethyl parathion and other thermally labile compounds such as the carbamates. Variable wavelength detectors offer much in the way of increased selectivity, a factor particularly important in environmental samples in which a large number of potentially interfering compounds are often present. As has been suggested in recent articles (6, 7) the variable wavelength spectrophotometer also permits the optimization of sensitivity, a function of source intensity and detector response as well as the absorption maximum of the species determined. With the use of such a detector, even such weak UV absorbers as lindane can be determined at the microgram level. Extraction procedures have been developed recently which involve the use of macroreticular resins which offer much in the way of pre-concentration (8). The XAD materials (Rohm and Haas) are particularly useful in this regard. Organics in water can be sorbed on a small column of macroreticular resin, and the sorbed organics then eluted by diethyl ether. After evaporation of the eluate, the concentrated organics can be determined by chromatography. In addition to the obvious benefit of 100- to 1000-fold concentration, this method offers the possibility for on-site sampling, avoiding the necessity to transport, store, and preserve large volumes of water (9, IO). A procedure has been developed for the determination of ethyl and methyl parathion using an XAD resin for sampling and preconcentration, followed by chromatographic separation and quantification by high performance liquid chromatography on a reverse phase microparticle column, with detection by variable wavelength UV-vis detector. The method is simple, rapid, and free from most interferences. EXPERIMENTAL Apparatus. A modular chromatographic system was used consisting of a Spectra-Physics Model 740B pump, a Glenco 7000 PSI six-port valve injector, a Whatman (Reeve Angel) prepacked microparticle reverse phase column (Partisil ODS), and a Perkin-Elmer Model LC-55 variable wavelength detector. The pump was used in the analytical range (0-4 mL/min) at a flow rate of 2.4 mL/min. Injections were 10 pL and were accomplished with fixed volume sampling loop for maximum precision. The detector was operated at 270 nm for the determination of methyl and ethyl parathion, or at an absorption maximum of potentially interfering 1552
ANALYTICAL CHEMISTRY, VOL. 49, NO. 11, SEPTEMBER 1977
compounds in the interference study. A 10-mV recorder was used in the 5-mV range with scale expansion of 5X from the detector, which provides a sensitivity of 0.02 aufs. Reagents and Materials. The macroreticular resin XAD-2 was obtained from Rohm and Haas and was purified by soxhlet extraction as described by Junk et al. (8), and stored under AR methanol. Pesticide grade acetonitrile was used as received; diethyl ether was glass distilled before use. Glass distilled water was used throughout. Parathions were "analytical standard grade (99+ % by GC analysis) obtained from Monsanto Agricultural Products (ethyl) or Research Triangle Health Effects Research Lab (methyl). Both parathions were stored in the dark at 4 "C, and fresh 100-ppm methanolic stock solutions were made from the standard materials at least once a week. The methanolic standards were also stored at 4 "C in the dark. Interference studies were conducted using analytical reference standards obtained from Research Triangle Health Effects Research Lab, used as received. Precautions similar to the above were taken with organophosphorous and carbamate pesticides. Procedure. Preparation of Standards. Microliter amounts of 100-ppm stock solutions of organophosphorous insecticides made up in methanol were diluted volumetrically with glass distilled water to 100 mL. The diluted standards were then passed through a 10-cm column of purified XAD-2 resin, prepared according to the method of Junk et al. (8),at a rate of 4-6 mL/min. After the last of the dilute aqueous standards were passed through the column, most of the water clinging to the resin was removed by gentle vacuum aspiration. Thirty mL of glass distilled diethyl ether was then passed through the column at 2-3 mL/min, after which the last of the ether was removed by passing dry purified nitrogen through the column. The ether was dried by shaking with 2 g of anhydrous sodium sulfate, and evaporated to dryness using a rotary evaporator at temperatures not exceeding 35 "C. The residue was then dissolved in 1.00 mL of nanograde acetonitrile, and the resulting solution chromatographed on a Partisil-ODS reverse phase column at 2.40 mL/min with 50% acetonitrile-water mobile phase. Recovery Studies. Standards made as described were compared with volumetric dilutions of methanolic stock solutions in nanograde acetonitrile to evaluate the recoveries obtained by the described method. Comparison of the peak areas and heights of the extracted and volumetrically diluted standards showed a 9&101% recovery of methyl and ethyl parathion, with an average value of 99% recovery. Extraction of Runoff Water. Grab samples of 2-L volume were obtained from a nearby drainage stream which removes runoff water from a large agriculturalarea. Samples were either analyzed immediately or stored for no more than 24 h at 4 "C in the dark. Since ethyl and methyl parathion were not found in the samples at levels above the limit of detection of the procedure (2 ppb), microliter amounts of the methanolic parathion standards were diluted with runoff water to evaluate the chromatographic behavior of extracts of these spiked samples and to evaluate recoverability of the parathions in this matrix. Extracts were prepared as in the above procedure, and chromatograms were evaluated to establish calibration curves for the parathions. R E S U L T S A N D DISCUSSION In order to evaluate the efficiency of extraction of XAD-2 for trace organics in natural water samples, several different types of water were analyzed. Well water, spring water, and runoff water were all examined by the described procedure. A large number of peaks were present in the 0-2 min region of the chromatogram in all three water samples. An increase in the number of peaks and peak areas was observed in runoff water as compared to either spring or well water. This result suggests a greater variety and larger number of dissolved organics in the runoff water, This is consistent with previous work (II), in which similar observations were made. The chromatograph of runoff water used in this study is shown in Figure 1. A number of relatively polar compounds elute early in the chromatogram, with relatively few peaks in the 3-10 min region of the chromatogram. On changing from 50% acetonitrile to 100% acetonitrile to regenerate the column,
Table I. Reproducibility of Method Methyl Parathion A 270 nm
Taken pg/L
Found lJglLa
SD
RSD, %
15.0 37.5 75.0 112.5
14.8 37.1 75.9 112.7
0.45 1.07 0.73 2.56
3.0 2.8 1.0 2.3
Ethyl Parathion Figure 1. Chromatogram of runoff water extract. Separation of organics in runoff water. Eluent; 5050 (v/v) acetonkrile water, Partisil ODS 4.6 mm X 25 cm; detector at 270 nm, 0.02 aufs
ItTHYL A 270 nm
a
Taken wg/L
Found lJg/La
10.0 25.0 50.0 75.0
9.9 24.6 49.3 75.0
Figure 2. Chromatogram of spiked runoff water extract. Conditions
several more peaks were eluted, apparently consisting of less polar materials strongly adsorbed under the conditions of the procedure. No interference was obtained from these strongly adsorbed compounds, although it was found to be useful to regenerate the ODS column with 100%acetonitrile after every five to six runs to insure reproducibility. The retention times for methyl and ethyl parathion, obtained from volumetric dilutions of methanolic standards with acetonitrile, were 3.45 and 4.65 min, indicating no interference from naturally occurring organics in the runoff water. Spiked samples of runoff water were prepared containing ethyl and methyl parathion. A typical chromatogram for such a spiked sample is shown in Figure 2. The parathions are well-separated, with no observed interference from organics already present in the water. Retention times obtained for the parathions were in agreement with those of standards. Preparation of Calibration Curves. Calibration curves were prepared from a set of standards made up by volumetric dilution of methanolic stock solutions in runoff water. The concentration range of parathions in the resulting solutions was from 10 to 120 ppb. Atrazine was added as an internal standard to the concentrated extracts, on the basis of examination of the chromatogram of the runoff water for the absence of any eluted material with retention time identical to atrazine. Since no material naturally occurring in the water samples determined was found that behaved like atrazine chromatographically under the conditions described, it was determined to be a suitable internal standard. Ratios of peak heights or areas of parathions to those of atrazine were plotted vs. concentration. Good linearity was obtained over the range of concentration examined for both parathions. In order to evaluate the accuracy and reproducibility of the method, a series of solutions was prepared in runoff water with concentrations of parathions in the range of the calibration curve. The results of this study are given in Table I. The lower limit of detection, defined as that amount giving a reproducible signal a t least twice that of noise, was calculated from these data to be 3.1 and 2.9 ng for methyl and ethyl parathion, respectively.
0.37 1.40 0.97 2.40
3.7 5.6 1.9 3.2
Table 11. Interference Study
Compound
1
RSD, %
Average of six determinations.
Aroclor 1260 as in Figure
SD
Relative retention (Methyl Parathion = 1.00)
length measured, nm
3.94-5.88
2 25
Wave-
multiple peaks Atrazine Azinphos Ethyl Alachlor Carbaryl (Sevin) Carbofuran Chloramben Chlo rp yrif os p,p'-DDT DEHP Dialifor Diazinon Dyfonate (Fonofos) Fenitrothion Methoxychlor p-nitro phenol
Phosmet Phorate Propachlor 2,3,5-T
Trifluralin
0.75 1.14 0.89 0.69 0.61 0.26 2.01 2.78 1.59 1.61 1.30 1.36 1.08 1.72 0.72 0.93 1.30 0.67 0.28 0.58
265 285 235 280 27 0 240 290 235 235 290 245 240 265 225 310 230 220 260 250 270
Interference Studies. Potential interference by other agricultural chemicals and organics commonly occurring in natural water were examined, the results of which are given in Table 11. All compounds were examined under the conditions described for analysis, and relative retention times were calculated in comparison with methyl parathion. Wavelengths chosen for measurement were a t or near the absorbance maxima for the compounds as determined by UV scans from 350-200 nm. If a potentially interfering compound showed a retention time near one of the parathions, then chromatography was performed with detection a t 270 nm. Many of the pesticides and herbicides are in common use in Central Illinois (22), a somewhat representative agricultural area. Of the compounds investigated, only Fonofos (Ethyl 5'-phenyl ethyl phosphonothiolothionate) interferes. All others are either separated chromatographically or only weakly absorb at 270 nm. A 10-ppm solution of Fonofos elutes at 4.7 min and absorbs at 270 nm to the same extent as 2 ppm ethyl parathion. However, if the wavelength of detection is changed to 280 nm, the interference is overcome. Only a slight loss of sensitivity is observed for the parathions at this wavelength, so that if Fonofos (Dyfonate) is known to be present, then ANALYTICAL CHEMISTRY, VOL. 49, NO. 11, SEPTEMBER 1977
1553
analysis can be performed at this wavelength.
LITERATURE CITED (1) Chem. f n g . News, 53 (30),19-21 (1975). (2) S. D. Faust and H. M. Gomaa, fnvlron. Lett., 3 (3), 171-201 (1972). (3) M. Eto, “Organophosphorous Insecticides, Organic and Biological Chemistry”, CRC Press, Cleveland, Ohio, 1974, p 241. (4) S. D. Faust and H. M. Gomaa, Environ. Lett., 3 (3) 171-201 (1972). (5) H. A. Moye, J . Chromatogr. Sci., 13, 266-279 (1975). (6) D. H. Rodgers, Am. Lab., 9 (2), 133-138 (1977). (7) C. M. Sparacino and J. W. Gines, J. Chromatogr. Sci., 14, 546-556 (1976).
(8) (9) (10) (11) (12)
G. A. Junk et al., J. Chromatogr., 99, 745-762 (1974). A. K. Burnham et al., Anal. Chem., 44, 139-142 (1972). Chem. Eng. News, 54 (IS), 35-36 (1976). C. G. Creed, Res./Dev., 27 (9),40-44 (1976). Eugene Mossbacker, extension advisor for agrlculture, McLean County Cooperative Extenslon Service, Private Comrnunicatlon.
RECEIVED for review April 11, 1977. Accepted June 23,1977. Work supported in part by an institutional grant from Illinois State University (No. 75-32).
Laser Two-Photon Excited Fluorescence Detection for High Pressure Liquid Chromatography Mlchael J. Sepaniak and Edward S. Yeung” Ames Laboratory-ERDA and Department of Chemistry, Iowa State University, Ames, Iowa 500 11
A laser two-photon excited fluorometric detector for high pressure liquid chromatography Is described and characterized for the separation of the oxadiazoies PPD, PBD, and BBD. Excitation is provlded by the absorption of two photons of radiatlon at 5145 A from an argon ion laser. The detectlon limits, linearity of response, precision, and selectivity are reported and are found to compare favorably with other UV detection methods.
While the fluorometric high pressure liquid chromatography (HPLC) detector is not as commonly used as the UV absorbance detector (1, 2 ) ) it does possess some definite advantages, namely higher sensitivity for those compounds with an appreciable fluorescence quantum efficiency and greater selectivity since relatively few of the molecules that absorb UV radiation actually fluorescence. Selectivity is also enhanced by the fact that fluorescent molecules have both an excitation and emission spectrum that can be scanned ( 3 , 4 ) . The present paper describes a fluorometric detection method that has two unique features. First, excitation is provided by an argon ion laser capable of 4 W of radiation at 5145 A. Second, the excitation process is the result of the absorption of two photons of the 5145-A light. In 1931 Maria Goppert-Mayer realized that a molecule could absorb two photons simultaneously to achieve a change in its quantum level (5). The process involves some distinctive selection rules and represents a way for spectroscopists to find and describe new molecular states (6). The value of the two-photon process in fluorometric HPLC detection lies in its improved selectivity. The fact that two-photon absorption involves different selection rules than one-photon absorption results in different one-photon and two-photon absorption spectra, and this produces an additional variant in the selective detection of fluorescent molecules. Two-photon fluorescence detection is somewhat limited by the small size of the twophoton absorption strength (6) and it is only with the high output power of a laser that measurable fluorescence signals can be obtained. The two-photon absorption strength is defined by the relationship
AP = P’CLA-‘ 6 1554
ANALYTICAL CHEMISTRY, VOL. 49, NO. 11, SEPTEMBER 1977
where AP is the absorbed optical power, P is the optical power, C is the solute concentration, L is the path length, and A is the optical beam cross-sectional area (6). Typical values for 6 are 110-4scm4 s photon-I molecule-’. The fraction of the absorbed optical power that is actually detected as fluorescence ( R ) can be calculated from Equation 2
R
= Qkq
where Q is the solute fluorescence quantum efficiency, k is the optical collection efficiency, and q is the detector quantum efficiency. This report characterizes a laser two-photon excited fluorometric (LTPEF) detector used in the two-photon detection of PPD, PBD, and BBD. These oxadiazoles have the general structure N-N
R-c,
II
II
0
,C-R
,
where R and R’ are either phenyl or biphenyl groups. A UV absorbance detector is used for the comparison of detection limits, linearity of response, and selectivity.
EXPERIMENTAL Chromatographic System. The liquid chromatographic system was composed of a LDC, Riviera Beach, Fla., minipump capable of delivering 16-160 mL/h of eluent at pressures up to 5000 psi, Rheodyne, Berkeley, Calif., injection valve with a 100-pL sample loop, and a Waters Associates, Milford, Mass., p-Bondapak CIS column (30 cm long X 3.9 mm id.). The eluent used for all separations was 60/40 UV grade tetrahydrofuran/water. The oxadiazoles were from Pfaltz and Bauer, Inc. Separations were all at ambient temperature with a flow rate of 2.0 mL/min and an injection volume of 100 pL. UV Absorbance Detector. The UV detector used for comparison purposes was a Chromatronixs,Berkeley, Calif., Model 230 mixed wavelength detector. The detector was operated at 280 nm where background noise was smallest and oxadiazole absorptivities greatest. LTPEF Detector. The fluorometric detector (see Figure 1) is composed of a light-tight cubic metal box containing a 1-mm i.d. X 3-mm 0.d. quartz flow cell. The 5145-A laser radiation of a Control Laser model 553 argon ion laser passes through a 0.5-inch aperture, then two Corning 3-71 sharp cut-off filters, and one Corning 4-96 wide bandpass filter, before being focused on the center of the flow cell by a 50-mm focal length X 25-mm diameter
