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Anal. Chem. 1991, 63, 1672-1675
(33) Mason, R. G.; Skmer, R. W.; Zucker, W. H.; Elston, R. C.; Blackwelder, W. C. J . Blomed. Mter. Res. 1974, 8 , 341-356. (34) Maloney, J. V., Jr.; Rher, D.; Roth, E.; Latta, W. A. Surgery(Sf.Lwls) 1989, 66, 275-283. (35) Andrade, J. D.; Hledy, V. A&. W m . Sd. 1988, 79, 1-83. (36) HLbogefs h Msdk/ne and phemrecy; Pappas, N. A., Ed.; CRC Press: Boca Raton, FL, 1988, 1987; Vols. 1-111. (37) Diirselen, L. F. J.; Wegmenn, D.; May, K.; Oesch, U.; Simon, W. Anal. Chem. 1988, 60, 1455-1458. (38) PrObebilHy and StaHstlcs tor Engneerhg end the Sciences; Devote, J. L., Ed.; Brodts-Cole Publlshlng: Pacific Qrove, CA, 1991. (39) S M , M. D.; Oenshaw. M. A.; Qreyson, J. Anal. Chem. 1973, 4 5 , 1782- 1784. (40) Van den Vlekkert, H.; Francis, C.; Gtlsel, A.; de Roolj, N. Analyst 1988, 713, 1029-1033. (41) Dlaz, C.; VMal, J. C.; Galban, J.; Urarte, M. L.; Lanaja, J. Microchem.
J . 1989, 39, 289-297.
RECEIVED for review December 13,1990. Accepted May 16, 1991. We gratefully acknowledge the support of this work by Fleck Controls, Inc., Brookfield, WI, through Grant No. UM389466, and by the US. National Science Foundation, through Grant No. ECS-8915497. We also acknowledge the Standards and Reagents Core facility, Reproductive Sciences Program, for the preparation of iodinated proteins. The Core is supported by the National Institutes of Health through P30 Center Grant No. P30-HD-18258-06.
Flow-Through Sensor for the Direct Determination of Pesticide Mixtures without Chromatographic Separation B. Fernlndez-Band,' P. Linares, M. D. Luque de Castro,* and M. Valclrcel Department of Analytical Chemistry, Faculty of Sciences, University of Cbrdoba, E-14004 Cbrdoba, Spain
A flow-through sensor for determination of carbamate compounds (carbofuran, propoxur, and carbaryl) based on retentlon of the products on C,, bonded phase beads packed In a flow-cdi was developed. The assodated determlnatlve procodwe reikron the hyddysisdthe analytes and couplkr(l to diazotized sulfanilic acid to yield the corresponding dyes. A conventknal Row lnJectbn(FI) method was also devekped for comparison. A diode array spectrophotometer was used In both cases to monitor the reaction products at nine wavelengths, resulting in maximal dmerences between the absorption spectra. Linear calibration curves over the ng/mL to pg/mL range (determinatkn IWt 50 times lower than that of the conventional FI method) and excellent resuits on applkatlon to mixture resolution in water samples from dtfferent sources testify to the usefulness of the proposed sensor.
INTRODUCTION Pesticides of the carbamate family have become increasingly popular in recent years on account of their broad biological activity spectrum. They are used as insecticides, miticides, fungicides, nematocides, and molluscicides (I). Owing to their usually toxic and frequently chronic character, assessment of water and soil pollution calls for the concentration of carbamates to be determined. Their different toxicity requires discrimination capabilities for them and their metabolites, which are also usually toxic. A host of methods have been developed in the last few years for determination of carbamates; most of them are based on a separation by gas or high-performance liquid chromatography (2),if two or more analytes are to be determined, and on simpler techniques if individual carbamates (plus their metabolites) need to be assayed. Such is the case with the determination of carbaryl and ita hydrolysis product by stopped-flow (3) or by synchronous first- or second-derivative fluorometry (4). Also, two carbamates used as insecticides (carbofuran and propoxur) ',Permanent address: Departamento de Quimica e Ingenieria Quimica, Universidad Nacional del Sur, Bahia Blanca, Argentina. 0003-2700/91/0383-1872$02.50/0
were recently assayed by a straightforward photometric method (5). However, no simple automatic flow methods for the simultaneous determination of carbamates requiring no separation technique has so far been reported. In this work we developed two straightforward automatic methods for the simultaneous determination of three carbamates u e d as insectides, namely carbofuran (CBF), propoxur (PPX), and carbaryl (RYL). Both are based on the use of a flow injection (6, 7)manifold where these three compounds are hydrolyzed to their corresponding phenols (carbofuranphenol, 2-isopropoxyphenol, and 1-naphthol,respectively) and subsequently coupled to diazotized sulfanilic acid. The reaction products can be monitored in two ways, which result in two methods with different features: (a) The dyes formed can be directly driven to the flow-cell of a diode array spectrophotometer (DAS) to be simultaneously monitored at several wavelengths, which also allows the simultaneous determination of the analytes. This determinative method has moderately good features. (b) A special flow cell packed with suitable support (e.g. CISbonded silica beads) can be used to integrate retention (concentration) and detection by using the same detector (DAS). The simultaneous in situ concentration and detection thereby achieved endows the resulting method with excellent features.
EXPERIMENTAL SECTION Instruments and Apparatus. A Hewlett-Packard 8415
diode-array spectrophotometer equipped with an HP 9121 floppy disk drive, an HP 98155 keyboard, and an HP 7470 plotter was used. A G h n Minipuls-2 and an Ismatec 5-840 peristaltic pump, two Rheodyne 5041 injection valves (one of which acted as a selecting valve), and Hellma 178.5268 (10" optical path) and 138 OS (1-mm optical path) flow cells were also used. The 138 OS cell was packed with the support by aspirating it in an ethanol suspension with the aid of a peristaltic pump. Reagents. Standards solutions of carbofuran, propoxur, and carbaryl (from Chemical Service) were prepared in 1:4 dioxane. Working strength solutions were prepared from these by diluting with distilled water. Aqueous solutions of 0.2% NaNOzand 0.2% sulfanilic acid in 30% acetic acid were used. The carrier solution was a 1:9 ethanol-water mixture. The eluent was a 1:l ethanol-HN03 (2 M)solution. The solid support in the flow cell was 0 1991 American Chemical Soclety
ANALYTICAL CHEMISTRY, VOL. 63,NO. 17, SEPTEMBER 1, 1991
A) al 0
n
0.400
L
0
VI n 0.200
a
I
0.600
U
C
1673
1 I
I
60
120
Time
I
180 (5)
B) CARRIER
~
No OH
q2
No NO2
q3
Sulphanilic acid
qb
ELUENT
qs 1
30
60
90
Time (s)
Figure 1. Manifolds used for the detennlnatlon of carbamate compounds based on hydrolysis of the analytes and dye formatlon and recordings obtalned by using each manlfokl: (A) Normal FIA conflguratbn; (8)configuration with integrated retention/detection of the products (q = flow-rate, R = reactor, SV = selectlng valve, DAS = diode array spectrophotometer, W = waste).
Cls bonded silica of 60-100 pm from SepPak C18cartridges (from Waters). Manifolds. The two manifolds shown in Figure 1 were used to develop two methods for the determination of carbamate pesticides. Manifold A, used to develop the normal FIA method, was a four-channel configuration where the sample was inserted into an ethanol-water carrier merging with a basic stream to favor hydrolysis of the analytes along reactor R1. The other two channels were also merged to facilitate formation of diazotized sulfanilic acid along reactor R2.The subsequent confluence of R1and & resdted in the formation of the corresponding dyes along R* Product formation was monitored at the preset wavelengths on passage of the reacting plug through the conventional flow cell used. Hydrolysis and derivative reactions were boasted by immersing the reactors in a thermostated bath at 60 O C . The first part of manifold B was similar to that of A, as the chemical steps involved were the same. Thus, a short reador (%) led the mixture to the special flow cell packed with chromatographic sorbent to retain the nascent dyes formed. After recordinga at several wavelengths had been collected, valve SV was switched to the eluent stream, which removed the dyes from the support. The flow cell was then regenerated and readied for insertion of a fresh sample. The nine wavelength selected for the simultaneousmonitoring, viz. 350,360,372,380,4~,430,460,476, and 480 nm, resulted in dt" d i f € e r e n between ~ the a ~ o ~ t i 8-ao n Of*e three andytes. A calculation program based on the establishment of nine equations with three unknowns (MIXFIA program (8))was used. The recordingsobtained with these configurations were rather different (see Figure 1).
RESULTS AND DISCUSSION As the aim of the work was to develop a sensor for determination of pesticides improving on the features of normal FIA methods, parallel preliminary assays were performed on the configurations depicted in Figure 1. Figure 2 shows the absorption spectra of the reaction products of the three analytes. An analyte concentration of 1pg/mL yielded between 0.3 and 0.5 absorbance units by the in situ concentration method, but near-zero absorbance by the conventional manifold; thus, the sensitivity improvement
5' PPX
300
350
LOO
L50
so0
550
600
WAVELENGTH l n m l
Figure 2. Absorption spectra of carbofuran (CBF) (..), propoxur (PPX) (-), and carbaryl (RYL) (-- -), obtalned by uslng the sensor (1) and the conventional system (2) (concentration: 1 pg/mL).
achieved by using the sensor was clear from the very early steps of development of the methods. To check for similarities between the spectra of the dissolved and retained products, the normal FIA manifold required analyte concentrations 20 times higher to obtain recordings comparable to those provided by the 8en90r. No appreciable changes in the absorption spectra of the dyes were found to result from binding to the chromatographic support. Optimization of Variables. We first optimized the normal FIA method. Then, the optimal values found for common variables were used to optimize the sensor system. Normal FIA Manifold. By plotting the analytical signal (maximum of the FIA recording) against the value of the variable concerned we obtained maxima at the values listed as optimal in Table I for the reactor lengths and flow rate. Shorter lengths or faster flow rates resulted in a smaller extent of development of the reaction step taking place in the reactor, while longer lengths and slower flow rates gave rise to the prevalence of dispersion over reaction development. The
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ANALYTICAL CHEMISTRY, VOL. 63, NO. 17, SEPTEMBER 1, 1991
Table I. Optimization of Variables variable
optimum value normal FIA system FIA sensor system
range studied
R1: cm R2,0 cm R3,(1cm flow rate, mL/min (ql, 92, q3) flow rate, mL/min (q,) flow rate, mL/min (qa) Vi, mL NaOH, M sulfanilic acid, w/v % NaOH ethanol, % (in carrier) eluent temp, "C
60-310 60-210 98-400 0.45-300
160 180 250 0.97 0.55
0.50-3.00 0.1-1.5 0.5-3.0 0.05-0.30 0.05-0.30 1.0-40.0 3070 ethanol-water-3070 ethanol-HNO, (1-3 M) 20-70
95 90 70 0.97 0.55 2.00
1.3 1.3 2.0 2.0 0.20 0.20 0.20 0.20 10.0 10.0 1:l ethanol-HN03 (2 M)
60
60
"Inner diameter 0.7 mm.
Table 11. Figures of Merit of the Methods method n-FIA flow-through sensor (7.0mm) flow-through sensor (5.5mm)
analyte CBF PPX RYL CBF PPX RYL CBF PPX RYL
eq" y = 0.029
y y y y y y y y
+ + +
0.0181: = 0.027 + 0.0191: = 0.025 0.0321: = 0.076 + 0.0471: = 0.065 0.065~ = 0.038 + 0.0611: = 0.072 + 0.2711: = 0.078 + 0.3511: = 0.078 + 0.4331:
13
linear rangeb
rad, %'
0.9918 0.9957 0.9993 0.9961 0.9981 0.9962 0.9987 0.9881 0.9910
1.0-40.0 1.0-40.0 1.0-40.0 0.2-30.0 0.1-15.0 0.1-15.0 0.05-2.0 0.05-2.0 0.05-2.0
3.61 3.50 2.26 3.20 2.86 2.38 2.88 3.36 1.82
"y = absorbance (maximum of the FIA recording), 3c = concentration in mg/mL. injection.
analytical signal also increased on increasing the injected sample volume up to 1.3 mL, above which the absorbance remained constant. As the analytical reaction involved is well-known, the optimization of the chemical variables was predictable and warrants further comment. The prior hydrolysis step required by the analytes were favored by the presence of ethanol; a 10% content was found to be optimal. Increased temperatures favored both the hydrolysis and the diazotization-dye formation steps. However, the analytical signal decreased above 60 "C,possibly because of product instabilization. Sensor System. Retention of the reaction products on the support located in the flow cell called for an extra channel and a selecting valve to alternate retention and elution. An 1:l ethanol-HN03 (2 M)solution was found to be the most appropriate for eluting the products from the support. The flow rate of this stream was not critical, as the action of the eluent was highly efficient. A flow rate of 2.00 mL/min was selected. A sampling frequency of 40 h-l was achieved under these working conditions with no cross-contamination between successively injected samples (complete return to the baseline), which proves that the products were rapidly eluted (Figure 1). It must be emphasized that the signal obtained by using the optimal reactor length for the normal FIA configuration was smaller than that recorded at shorter lengths. This was not a result of the sequential retention of the reagents and analytes because sequential passage of these through the flow cell gave rise to no detectable signal. Thus, a plausible explanation could be that only the nascent or an intermediate product is retained. Typical variables of the flow-throughsensor such as the type of cell and the packing level of the support in the flow cell
sampling frequency, h-'
60 40 40
pg/mL. c5pg/mL, 11 different samples in triplicate
were optimized. In previous work (9,lO)we found a Hellma 138 OS cell of 1-mm optical path to be the most appropriate for the support in question. The packing level of the support in the flow cell was a key variable because if the solid phase did not reach the optical path, product measurements were actually made on the solution, and if the packing top was far above the light beam, the support area with the highest product concentration (that closest to the surface, i.e. the Area on which the incoming flow impinged) fell outside the sensed area. Thus, two assayed levels resulted in different sensitivity (5.5 and 7.0 mm from the bottom). The top of the former was closer to the light path, so it resulted in higher sensitivity. Features of the Methods. Individual calibration curves were run from standards of each pesticide by the normal and FIA sensor method (in this latter case a t the two selected support packing levels). The equations of the linear segments obtained in each case, the linear concentration range, regression coefficient,precision (expressed as nd), and sampling frequency are listed in Table II. As can be seen, the sensitivity (slope of the calibration curves) was dramatically higher for the flow sensor system. The slope was between 10- and =fold higher for the flow 8 e m r with a 5.5" support packing level. The determination limit was 50-fold lower, and the reg-ression coefficients and repeatability were similar in a l l instances. The features of the method implemented with a 7.0-mm packing level were intermediate between those of the normal FIA method and those obtained by using a 5.5-mm packing level. Application of the Method. Once the excellent features of the flow-through sensor compared to the normal FIA systems were checked, its performance was tested by application to the resolution of mixtures of the three analytes. First, the absence of synergisticeffects between the products of the three analytes was checked. For this purpose, mixtures of carbofuran, propoxur, and carbaryl standards in different
ANALYTICAL CHEMISTRY, VOL. 63, NO. 17, SEPTEMBER 1, 1991
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Table 111. Resolution of Standard Mixtures of Carbofuran, Propoxur, and Carbaryl CBF
conc in the mixtureso PPX RYL
CBF
PPX
RYL
CBF
PPX
RYL
1.0 1.0 3.0 1.0 1.0 1.0 1.8 1.0 1.2 6.0 1.3 0.1 0.2 0.3 0.2 0.6
1.57 1.83 1.05 0.85 0.57 0.56 2.51 3.68 0.87 1.85 2.77 0.56 0.36 0.14 0.22 0.44
1.46 2.90 1.77 0.88 0.77 0.53 0.71 2.77 3.85 0.86 1.45 0.23 0.38 0.53 0.20 0.72
1.33 1.12 2.95 1.05 1.10 1.14 1.93 0.93 1.28 6.10 1.35 0.10 0.20 0.30 0.20 0.60
104 91.5 105 85 114 112 100.4 92 87 92.5 92.3 93.3 120 140 110 110
107 96.7 88.5 88 77 106 71 92.3 96.3 86 96.7 115 95 106 100 102.8
133 112 98.3 105 110 114 107 93 106.7 101.6 103.8 100 100 100 100 100
1.5 2.0 1.0 1.o 0.5 0.5 2.5 4.0 1.0 2.0 3.0 0.6 0.3 0.1 0.2 0.4 O
1.5 3.0 2.0 1.0 1.0 0.5 1.0 3.0 4.0 1.0 1.5 0.2 0.4 0.5 0.2 0.7
found concg
In pg/mL.
Table IV. Determination of Carbofuran, Propoxur, and Carbaryl in Different Proportions in Spiked Water Samples water samples hP
well, river,
river2
pond1 pond2 welll
well3 well, (I
% recovery
CBF 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 1.0 0.4 0.4 1.0 0.4 0.4 1.0 0.4 0.4
added conP PPX RYL 1.0 2.0 1.0 2.0 1.0 2.0 1.0 2.0 1.0 2.0 1.0 2.0 0.4 1.0 0.4 0.4 1.0 0.4 0.4 1.0 0.4
3.0 2.0 3.0 2.0 3.0 2.0 3.0 2.0 3.0 2.0 3.0 2.0 0.4 0.4 1.0 0.4 0.4 1.0 0.4 0.4 1.0
% recovery
CBF
PPX
RYL
84.0 9715 116.5 100.0 87.0 107.5 105.5 101.5 98.5 78.5 78.5 76.5 96.0 115.0 115.0 99.0 100.0 90.0 99.0 100.0 87.5
113.0 104.0 103.0 93.5 129.0 93.0 116.0 104.0 76.0 89.0 94.0 100.0 105.0 108.0 120.0 97.5 101.0 120.0 105.0 93.0 100.0
100.0 103.5 90.3 93.0 101.0 106.5 106.6 98.0 88.0 100.5 92.3 89.0 107.5 102.5 104.0 112.0 112.0 108.0 125.0 112.0 112.0
Concentration in rcdmL.
proportions were asaayed. Table III lists the added and found concentrations and the percent recoveries achieved. The results clearly reveal the absence of synergistic effects. Next, water samples from different sources (tap, pools,wells, etc.) were spiked with the analytes because of the inavailability of real samples containing them. Both support levels were randomly used, and the results obtained, as would have been predicted from the values found in the precision study, were very similar (see Table IV).
FINAL REMARKS The performance of the proposed flow-through sensor for the determination of carbamate compounds compares favorably with that of a conventional FIA method also reported here for the first time. Considerably improved sensitivity was achieved thanks to the in situ concentration process derived from retention of
the products on the chromatographic sorbent placed in the flow cell. The features of the method developed by using this sensor are quite similar to those provided by a chromatographic technique: The concentration step is enacted by the chromatographic material packed in the flow cell and discrimination is made by the features of the detector. The maximum number of components that can be determined with the proposed sensor is limited by the spectral differences between the corresponding reaction produds and by the discriminating power of chemometric techniques for multicomponents. The determination limits achieved by using the proposed sensor are quite similar to those afforded by HPLC techniques (2, 111, while the resolution obtained is adequate for these compounds. More complex mixtures would possibly require a more powerful technique such as HPLC, which also features higher purchase and maintenance (solvents, columns) costs. Registry No. Carbofuran, 1563-66-2;propoxur, 114-26-1; carbaryl, 63-25-2;water, 7732-18-5.
LITERATURE CITED Ruricka. J. H. Roc.Soc. Anal. Chem. 1973, 10, 32-34. Sharp, G. J.; Brayan, J. G.; Haddad, P. R. Analyst 1988, 713, 1493- 1507.
Quintero, M. C.; Silva, M.; Pbrez-Bendlto, D. Talents 1888, 35,
943-948.
Gar&-Snchez. F.; Cruces Blanco, C. Talents 1990, 37,573-578. Naidu, D. V.; Naidu, P. R. Talents 1990,37,629-631. Valdrcel, M.; Luque de Castro, M. D. Flow Injectbn Analysb: M n ciples and Applications; Ellis Horwood: Chlchester, U. K., 1987. Valdrcel, M.; Luque de Castro, M. D. Automatic Methods of Ana&sb; Elsevier: Amsterdam, 1988. Lkaro, F.; Rbs, A.; Luque de Castro. M. D.; Valdrcel, M. Anal.
m i m . ~ c t 1988, s 179,279-287. Fednder-Band. B.; Unares, P.; Luque de Castro, M. D.; VaGrcel, M. Anal. Chim. Acts 1990, 229, 177-182. Linares, P.; Luque de Castro, M. D.; Valdrcel. M. Anal. Chim. Acta 1990, 230, 199-202. Macherey-Nagel Catalog, Germany, 1987.
RECEIVED for review January 11, 1991. Accepted April 26, 1991. Financial support (Grant No. PA86-0_146) from Comisidn Interministerial de Ciencia y Technologia (CICfl) is gratefully acknowledged. B.F.B. wishes to express her gratitude to the Consejo Nacional de Investigaciones Cientificas y TBcnicas de la Repdblica Argentina for award of a grant covering the expenses incurred during her stay in Spain.
