Anal. Chem. 1990, 62, 1495-1498
1495
Automated High-Performance Liquid Chromatography for the Determination of Pesticides in Water Using Solid Phase Extraction C h r i s H. Marvin a n d Ian D. Brindle
Department of Chemistry, Brock University, St. Catharines, Ontario L2S 3A1, Canada C. David Hall
Ontario Ministry of the Environment, Rexdale, Ontario M 9 W 5L1, Canada Mikio Chiba*
Research Station, Agriculture Canada, Vineland Station, Ontario LOR 2E0, Canada
An automated solid phase extraction/hlgh-performance liquid chromatography (SPE/HPLC) method has been developed to determine trace concentrations of Propoxur, Carbofuran, Carbaryl, Propham, Captan, Chloropropham, Barban, and Butylate In water. One hundred mlillllters of sample water Is passed through a dlsposable SPE Cartridge packed wlth 90pm sorbent at 10 mL/mln. The concentrated analytes are eluted from the cartridge wlth acetonltrile. The resultlng eluate ls blowndown under nitrogen, made up In water, and injected Into the HPLC. The analytes are separated on a 2J-cm C, analytkai column and determined by UV absorption at 220 nm. The total analytlcal t h e Is 90 mln a d the lowest detectable concentrations are In the range of 0.02--0.92 pg/L for the eight pesticldes. Recoveries for the eight pesticides ranged from 84 % to 93 % . The procedure Is totally automated and can analyze 30 samples consecutively and unattended.
INTRODUCTION The use of a solid sorbent for the extraction and preconcentration of trace organic pollutants from water has been widely investigated and the advantages of such a technique over a conventional liquid-liquid extraction have been well documented (1-3). One of the greatest of these advantages is the possibility of more efficient and reproducible recoveries. On-line preconcentration (or trace enrichment) and solid phase extraction (SPE) are two of the most popular solid sorbent extraction techniques. On-line preconcentration usually employs a precolumn containing an appropriate stationary phase while S P E techniques involve the use of commercially available cartridges. On-line preconcentration techniques are relatively easier to automate and several papers have been published (4-6). Many SPE procedures require off-line manual manipulation of the cartridges since the techniques require the collection of eluate from the cartridge and the evaporation of solvent (7,8). In our study, a completely automated method has been developed for the determination of trace quantities of selected pesticides in water using S P E and high-performance liquid chromatography (HPLC). SPE is performed with the aid of the Waters Millilab Workstation resulting in complete automation of cartridge conditioning, sample loading, cartridge air-drying, analyte elution, eluate blow-down, sample dilution,
* To whom correspondence should be addressed. 0003-2700/90/0362-1495$02.50/0
and sample injection steps. With the addition of multiple intake accessories (MIA's), total automation for the analysis of 30 samples is possible. The study involves the extraction and preconcentration of eight selected pesticides from water and their subsequent determination by HPLC. These eight pesticides were chosen as they are among the organic pollutants of concern in Ontario waters. Among the experimental parameters that have been investigated in the study are (1) the flow rate of sample through the SPE cartridge, (2) the amount of sorbent needed in the cartridge to adequately retain the analytes, and (3) the sorbent packing particle diameter. At each stage of the SPE procedure, variable operating conditions were investigated to optimize the method. EXPERIMENTAL S E C T I O N Solvents. Acetonitrile was of HPLC grade from Fisher Scientific (Fairlawn, NJ) and Caledon Laboratories (Georgetown, Ontario, Canada). Water used for preparation of standards was distilled in glass in the laboratory. HPLC grade water from Fisher Scientific (Fairlawn,NJ) was used to provide the cleanest possible chromatograms. Pesticides. Solid pesticide standards were obtained from the United States Environmental Protection Agency, Research Triangle Park, NC. Purities of the individual standards ranged from 97.5% to 100%. The pesticides, listed in the order in which they appear in the chromatograms, are Propoxur, Carbofuran, Carbaryl, Propham, Captan, Chloropropham, Barban, and Butylate. Safety Considerations. The toxicities of the pesticides used in this study range from 11to 9000 mg/kg (oral LD, for rats). Normal laboratory safety procedures should be followed when handling these pesticides. Kuhr and Dorough (9) provide an excellent overview of the toxicities and modes of action of most of the pesticides used in this study. Preparation of Standard Solutions. Solid standards were dissolved in acetonitrile and diluted in acetonitrile. These individual standard solutions were combined at different concentrations because of varying sensitivities to ultraviolet (UV) detection. Water Samples. The combined standard concentration of each pesticide is listed in Table I. Standard water samples were prepared by adding 1 mL of the combined standard to 1000 mL of distilled water from the laboratory unless otherwise noted. Several tap and untreated surface waters were collected from local sources and analyzed. HPLC Apparatus. The HPLC system consisted of a Waters Millilab Workstation, a 600E Powerline Multisolvent Delivery System, a 484 tunable absorbance UV detector, four Multiple Intake Accessories (for use with the Millilab), and an 815 or 840 Baseline chromatography software package (Waters Associates Millford, MA). 0 1990 American Chemical Society
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ANALYTICAL CHEMISTRY, VOL. 62, NO. 14, JULY 15, 1990 4
Table I. Selected Pesticides, Their Retention Times (RT), Average Recoveries from Five Replicate Measurements, Sample Concentrations, and Minimum Detectable Concentrations for a 100-mL Sample sample no."
RT,
recovery,
concn,
compound
min
70
rg/L
Propoxur Carbofuran Carbaryl Propham Captan C1-Propham Barban Butylate
10.80 11.40 12.80 15.20 17.40 18.70 19.40 24.90
92 f 0.3 91 f 0.3 93 f 1.0 92 f 3.6 88 f 3.6 89 f 4.9 89 f 1.7 84 f 1.2
3.84 4.35 0.42 3.17 9.70 0.98 1.08 4.07
min detectable concn, @g/L
NO-3
130 140 20 100 920 60 80 300
The pesticides are numbered to coincide with those in the figIIPPR
SPE Cartridges and Analytical Column. Three types of SPE cartridges were evaluated in the study: (1)the Sep-Pak Plus C-18 cartridges containing 330 mg of 90-pm sorbent, (2) the Sep-Pak Light (2-18 cartridges containing 120 mg of 90-pm sorbent, and (3) the Sep-Pak Custom C-18 cartridges containing 330 mg of 40-pm sorbent (Waters Associates, Millford, MA). The analytical column was a Supelcosil LC-8 5 pm 25 cm X 4.6 mm i.d. (Supelco Inc., Bellefonte, PA). HPLC Operating Conditions. The HPLC operating conditions were as follows: wavelength, 220 nm; flow rate, 1.5 mL/min; chart speed, 0.5 cm/min; detector sensitivity, 0.075 AUFS; recorder range, 10 mV F.S.;column temperature, ambient. Gradient Elution Program. The gradient elution program was as follows (elapsed time, composition of mobile phase): initial, 30% acetonitrile and 70% water; 5 min, 30% acetonitrile and 70% water; 15 min, 60% acetonitrile and 40% water; 25 min, 60% acetonitrile and 40% water; 30 min, 30% acetonitrile and 70% water; 35 min, 30% acetonitrile and 70% water. Changes in the percentage of organic solvent in the mobile phase throughout the gradient program occurred linearly. The final 10 min of the gradient program serves to return the system to the initial conditions to enable another analysis run. S P E Procedure. (1) Pass 1 mL of acetonitrile through a Sep-Pak Light cartridge as a conditioning step. (2) Pass 2.5 mL of water through the cartridge in preparation for sample loading. (3) Pass 100 mL of sample through the cartridge at a flow rate of 10 mL/min. (4) Air-dry the cartridge for a period of 15 s. (5) Elute the analytes from the cartridge by passing 0.750 mL (3 X 0.250 mL) of acetonitrile through the cartridge at 2.0 mL/min. (6) Gently blow-down the eluate with nitrogen at 5 psi for 15 rnin to 0.20 A 0.01 mL. (7) Make up the sample solution to 0.35 mL with water. (8) Inject 0.175 mL of the prepared solution into the HPLC via the Millilab. Note: The prepared samples should be analyzed as soon as possible after preparation as the concentrated Captan is very insoluble in aqueous media (solubility in water is approximately 0.5 ppm) and adsorbs readily on glass. R E S U L T S AND DISCUSSION In the development of the methodology, several experimental parameters were investigated. When loading sample water, a flow rate of 10 mL/min through the cartridge was found to be optimum. At a flow rate of 20 mL/min, breakthrough of the early eluting compounds (Propoxur, Carbofuran, and Carbaryl) was evident. This was confirmed by the analysis of the sample water, collected after passing a sample through the cartridge, by the previously described method (5). A comparison of the retention capabilities of the Sep-Pak Custom C-18 (40 pm) cartridges and the Sep-Pak Plus C-18 (90 pm) cartridges revealed no significant differences in the retention of any of the analytes (Figure 1). Acetonitrile, chosen as an elution solvent as it eluted all of the analytes from the cartridge, is water miscible and is suitable for reversed-phase HPLC analysis. Other solvents
I 5
10
15
20
25
30
35
MINUTES
Figure 1. A comparison of the retention of analytes by (A) 40-pm C,, (SepPak Custom)cartridge and (B) 90-pm C,* (SepPak Plus) cartridge. Chromatograms were produced by injection of 0.175 mL of eluate without a blowdown step. Chromatograms are plotted at 25 mV. may have greater eluting power in reversed-phase chromatography but are not water miscible (with the exception of methanol). To evaporate the eluate, a 15-min blow-down time with a gentle stream of nitrogen was chosen. Under these conditions, reproducible results were obtained; there was essentially no loss of analytes owing to splashing or coevaporation. The Sep-Pak Plus and Sep-Pak Light cartridges were compared in this study to determine the weight of solid sorbent needed to retain the analytes. Our results suggest that the Sep-Pak Light cartridge not only is adequate to retain all the analytes under the conditions described but also is superior to the Sep-Pak Plus cartridge. The air-drying step did not remove the majority of the residual sample water from the larger Sep-Pak plus cartridge. Consequently, when acetonitrile was passed through the Sep-Pak Plus cartridge, it became sufficiently diluted with water that a fraction of the eluate could be injected directly into the HPLC without furthur dilution with water for analysis of the majority of the pesticides. The resolution between Propoxur and Carbofuran, however, was not as good as with a straight injection of the concentrated standard which was prepared at 30% acetonitrile in water (the same percentage composition of the mobile phase at the starting of the gradient program). This suggested that the percentage of acetonitrile in the eluate was somewhat higher than 30%. This solvent effect is well explained in a paper published recently (IO). Our studies have shown that all of the residual sample water on the Sep-Pak Plus cartridge is displaced by the first 0.50 mL of acetonitrile. The residual water on the Sep-Pak Plus cartridge is a disadvantage because it lowers the volatility of the eluted solvent under the nitrogen stream. Forcing the evaporation of this eluate resulted in the loss of analytes, particularly Butylate. In contrast with the Sep-Pak Plus cartridge, the air-drying step eliminated most of the residual sample water from the Sep-Pak Light cartridge. We have estimated that approximately 0.10 mL of water remained on the sorbent after a brief 15-s air-drying step. A longer period of air-drying may have eliminated more of the residual water, but this was not necessary. A gentle 15-min blow-down of the eluate with nitrogen a t 5 psi results in a 0.20 i 0.01 mL sample that can be diluted to 0.35 mL with water (resulting in a sample composition of not greater than 30% acetonitrile) of which 0.175 mL can be injected. A sample solution of at least 0.30 mL is needed to adequately flush the sample injector and sample loop assembly with sample to attain a reproducible injection. The methodology employing the Sep-Pak Light cartridges is judged to be superior as the analytes can be retained and
ANALYTICAL CHEMISTRY, VOL. 62, NO. 14, JULY 15, 1990 4
Table 11. Average Recovery of Butylate in Relation to the Final Volume of the Eluate after the Blow-Down Step under Nitrogen (Average of Five Replicate Measurements) final vol of
recovery of
eluate, mL
butylate, 70
final vol of eluate, mL
butylate, %
0.25
80 82
0.15 0.10
79 30
0.20
1407
recovery of
I
1
U ! VUTES
Figure 3. Chromatograms resulting from three successive injections of the combined standard sample solution by following the described method. Chromatograms were plotted at 50 mV and gradient correction was made.
? I
c CJ
0 50
1 00
1 50 x
2 00 rr
2 50
7
ni.tss
Flgure 2. Chromatogram of a combined standard solution sample prepared in the best commercially available HPLC grade water The analysis was done by the described method. collected with half the volume of eluant needed with the Sep-Pak Plus cartridges (0.75 mL vs 1.50 mL, respectively). The eluate from the Sep-Pak Light is sufficiently volatile for evaporation under nitrogen. It should be stressed that the flow of nitrogen onto the eluate in the evaporation step must be gentle to avoid losses of analytes. Originally, we attempted to blow-down the eluate from the Sep-Pak Light to a volume of approximately 0.10 mL. An addition of 0.25 mL of water made up the solution to the required volume of 0.35 mL (resulting in a sample composition of less than 30% acetonitrile if the original eluate is 100% organic solvent). This procedure was found to be unsatisfactory as Butylate recoveries averaged only 30%. Since the approximately 0.10 mL of water remaining on the cartridge becomes part of the eluate, any attempt to blow-down the eluate to 0.10 mL neccessitates the evaporation of all of the acetonitrile. It took an extended period of time to reduce the volume of eluate from 0.15 to 0.10 mL, and during this period a large percentage of Butylate was lost (Table 11). In the described method, the volume of the blown-down eluate is set at 0.20 f 0.01 mL and then made up to a final volume of 0.35 mL with water. With this procedure, the percentage of acetonitrile in the sample solution remained lower than 30% and there was no sign of peak shape deterioration as shown in Figure 2. Figure 2 is a chromatogram of the eight pesticides in the combined standard prepared in the purest water found among those analyzed. All pesticides are completely resolved and the peak shapes are very good. Gradient correction can be used to improve chromatographic profile. Background subtraction is impractical for the analysis of real samples because of the absence of an appropriate blank. Figure 3 shows good reproducibility of the chromatograms resulting from three consecutive sample analyses by using the described method. With the described method, the recoveries of the pesticides from five replicate samples ranged from 84% to 93% (Table I). In addition to recoveries, Table I includes standard deviations, prepared sample concentrations, and minimum detectable concentrations. The minimum detectable concentrations were derived
0. 0 0
0. 50
1. 00
I . 50 x
2. 00 101
2 . SO
3. 00
rnl"UtPS
Figure 4. Chromatogram resulting from the analysis of an untreated lake water. The sample was analyzed by the described method. None of the peaks corresponds to pesticides of interest. from a 3 to 1 signal-to-noise ratio. Ultimately, the choice of cartridge size will depend upon the desired application. Our studies of municipal tap waters have shown that there is breakthrough of the early eluting analytes present in the sample matrix. This was confirmed by the analysis of the sample water collected after passing a sample through the cartridge by the method previously described (5). An untreated lake water sample was also analyzed (Figure 4). These results indicate a great potential to apply the described method to the analysis of not only drinking water but also other types of water samples. As demonstrated, the totally automated SPE/HPLC system developed is accurate, efficient, economical, and sensitive. The method can be employed in the unattended analysis of 30 or more samples. Registry No. Propoxur, 114-26-1; carbofuran, 1563-66-2; carbaryl, 63-25-2;propham, 122-42-9;captan, 133-06-2;Cl-propham, 101-21-3; barban, 101-27-9; butylate, 2008-41-5; water, 7732-18-5.
LITERATURE CITED (1) (2) (3) (4)
(5) (6) (7)
(8)
Junk, G. A.; Richard, J. J. Anal. Cbem. 1988, 6 0 , 451. Bushway, R. J. J . Chrornatogr. 1981, 277, 135. Andrews, J. S.;Good, T. J. Am. Lab. 1982, 74,7 0 . Ramsteiner, K. A. J. Chromatogr. 1989, 465, 410. Marvin, C. H.; Brindle, I. D.; Hall, C. D.; Chiba, M. J . Chromatogr. 1990, 503, 167. Chaput, D. J. Assoc. Off. Anal. Cbem. 1988, 69 (6), 985. Rostad, C. E.; Pereira. W. E.; Ratcliff, S. M. Anal. Cbem. 1984. 56, 2856. Chladek, E.; Maranaro, R. S. J. Chrornarogr. Sci. 1984, 22, 313.
Anal. Chem. 1990, 62, 1498-1501
1498
(9) Kuhr, R. J.; Dorough, H. W. Carbemate Insectkkjes: Chemistry, Biochemistry and Toxlcobgy; CRC Press: Cleveland, OH, 1976; Chapters 3 and 5. (10) Vukmanic, D.; Chiba. M. J . Chromatogr. 1989, 483, 189.
RECEIVED for review October 16,1989. Accepted April 2,1990.
The authors thank the Research and Technology Branch of the ontario of fie vir^^^^^ for financial support, Use of the equipment herein described does not represent endorsement by the Ontario Ministry of the Environment or by Agriculture Canada.
Microhole Array Electrode as a Glucose Sensor Yoshihiro Shimizu* a n d Ken-ichi Morital
Basic Research Laboratories, Toray Industries, Inc., 11 11 Tebiro, Kamakura 248, Japan
Mkrohole array enzyme electrodes (1000 mkrohdes of 7 pm diameter and 50-450 pm depth) were fabricated for the measurement of glucose. Enzyme was Immobilized on the surface of the platlnlzed mlcrohole array electrode. The enzyme electrode of hydrogen peroxide detection type was sensltlve to glucose concentration over a wide range. The sensitivity of the 50-450 pm depth microhole array enzyme electrode was hlgher than that of a nonetched one. The linearity of the electrode current response with glucose concentration was extended by lncreaslng the depth of the mlcrohoies. Increaslng the hole depth also increased the stabillty of the enzyme electrodes.
INTRODUCTION Microhole array electrodes can be fabricated by the electrochemical etching of carbon fibers embedded in an epoxy resin. They have proved to be promising as oxygen sensors ( I ) . The linear concentration gradient of the electroactive species in the diffusion layer is stably established in the steady state. The thickness of the diffusion layer for the reduction of dissolved oxygen is found to be the s u m of the depth of the microhole and the thickness of the solution boundary layer (1). Cyclic voltammograms obtained with the microhole array electrode are quite similar in shape to those for rotating disk or ultramicrodisk electrodes. In other words, the voltammograms obtained at slow potential sweep rates are sigmoidal (2). Other features of the microhole array electrode include its small size and being moderately flow-rate-insensitive and being discriminative against certain poisons ( I ) . In this paper we report on an amperometric biosensor utilizing a 0.5 mm diameter microhole array electrode. Amperometric glucose electrodes are based on the use of the enzyme glucose oxidase
+
-
+
+
D-glucose H 2 0 O2 D-gluconic acid H202 (1) This reaction has been followed by the amperometric detection of hydrogen peroxide at an electrode polarized a t +0.6 V (vs SCE)
-
Hz02
O2 + 2H+ + 2e-
(2) We have also pursued the current response for the oxygen reduction a t -0.6 V since oxygen is consumed by the reaction (1)
O2 + 2Hz0+ 4e-
-
40H-
(3) Glucose oxidase was immobilized on platinized carbon fibers Present address: Tbin University of Yokohama, 1614 Kuroganecho, Midori-ku, Yokohama 227, Japan 0003-2700/90/0362-1498$02.50/0
inside the microholes. I t was found that the hydrogen peroxide detection electrode (H202electrode) responded better electrode) and the than the oxygen detection electrode (02 sensitivity of the microhole array enzyme electrode for the hydrogen peroxide detection was higher than that of a nonetched array enzyme electrode. EXPERIMENTAL SECTION Chemicals and Reagents. The glucose oxidase used (100 X lo4 units/g) was obtained from Nagase Biochemicals, Ltd. The l-cyclohexyl-3(2-morpholinoethyl)carbodiimide metho-ptoluenesulfonate and glucose (P-D-glUCOSe) were purchased from Tokyo-Kasei Japan. Phosphate buffer solutions (Na2HP02(0.033 mol/dm3) + NaH2P0, (0.033mol/dm3)) were used in all experiments. Preparation of Microhole Array Enzyme Electrode. Microhole array electrodes with a diameter of 0.5 mm were prepared from high-strength carbon fibers (TORAYCA T-300, the number of fibers in a single electrode, 1000, 6.93 pm in diameter) using fabrication techniques described in the previous paper ( I ) . The platinized electrode was immersed in a phosphate buffer solution for a few minutes and then placed in 1 mL of a slowly stirred phosphate buffer solution containing 5 mg of glucose oxidase and 25 mg of l-cyclohexyl-3-(2-morpholinoethyl)carbodiimide metho-p-toluenesulfonate at room temperature for 15 h. The microhole array enzyme electrodes thus prepared were stored in a phosphate buffer solution. Electrochemical Measurement. Amperometric measurements were carried out with a Nikko Keisoku potentiostat (Model N-POT 2501). The electrochemical cell (three-electrode system) consisted of a 1WmLglass beaker with a silicone rubber iid having four holes. Three of these holes were used for the electrodes and the other was used for the injection of concentrated glucose solution into the buffer solution. A saturated calomel electrode (SCE) and a platinum wire served as reference and the auxiliary electrodes. Potential values are referred to SCE. The electrodes were placed in air-saturated buffer solutions (100mL), stirred and thermostated at 37 "C,and then poised at +0.6 V for the hydrogen peroxide measurement or -0.6 V for the oxygen measurement. A solution of low oxygen concentration was prepared by purging one volume of air and three volumes of nitrogen gas into the solution. After background current decayed to a steady-state value, aliquots of a concentrated glucose solution were stepwise added. The steady-state current was recorded for each addition of aliquots. Each point shown in Figures 2, 4, 5, 6,and 7 corresponds to a single measurement. Definitions. Response time (tW)was defined as the time it took to reach 90% of the steady-state current after the glucose injection. Sensitivity was defined as an absolute value of the slope in the linear range of the calibration curve divided by the total cross-sectional area of the microholes. RESULTS AND DISCUSSION Immobilization. Glucose oxidase enzyme was immobilized by immersing the platinized microhole array electrode in the solution containing glucose oxidase and the soluble carbo0 1990 American Chemical Society
