Anal. Chem. 1989, 61,1363-1367 (11) Goodwin, J. F. Clin. Chem. 1968, 14, 1080-1090. (12) Poole, N. F.; Meyer, A. E. Proc. SOC. Exp. Biol. Med. 1958, 98, 375-377. (13) Ryan, J. A. J. Pharm. Sci. 1984, 73, 1301-1302. (14) Lehmann, P. A. Anal. Chlm. Acta 1971, 5 4 , 321-336. (15) Chen, P. R.; Dauterman, W. C. Anal. Blochem. 1970, 38, 224-228. (16) Garratt, D. C.; Johnson, C. A.; Lloyd, C. J. J. pharm. Pharmacol. 1957, 9 , 914-928. (17) Zahn, H.; Wurz, A. 2.Anal. Chem. 1951, 134, 183-187. (18) Cohen, J. C.; Norcup, J.; Ruzicka, J. H. A.; Wheals, B. B. J. Chromatog. 1989, 4 4 , 251-255. (19) Timbreil, J. A,; Wright, J. M.; Smith, C. M. J. Chromatog. 1977, 138, 165-172. (20) Lawrence, J. F.; Frei, R. W. Chemical Derivatization in Li9uM Chromatography; Elsevier; Amsterdam, 1976. (21) Barends. D. M.; Blauw, J. S.; Mijnsbergen. C. W.; Govers, C. J. L. R.; Hulshoff, A. J. Chromatogr. 1985, 322, 321-331. (22) Sadee, W.; Beeien, G. C. M. Drug LevelMonitmhg; J. Wiiey and Sons: New York, 1980; p 251. (23) Zeman, A. Wirotama, J. P. G. Z . Anal. Chem. 1969, 247, 155-156. (24) Efstathiou, C. E.; Koupparis, M. A.; Hadjiioannou, T. P. Ion-Sel. Electrode Rev. 1985. 7 . 203-259. (25) Siddigi, I. W. Cllh. Chem. 1982, 2 8 , 1962-1967. (26) Skaltsa, H. D.; Koupparis, M. A.; Philianos, S. M. J. Assoc. Off. Anal. Chem. 1988, 69, 1006-1008.
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(27) Bunnett, J. F.; Garbisch, E. W.; Pruitt, K. M. J. Am. Chem. SOC. 1957, 79, 385-391. (28) Bunnett, J. F.; Randall, J. J. Am. Chem. SOC. 1958, 80, 6020-6030. (29) Bunnett, J. F.; Garst, R. H. J. Am. Chem. SOC. 1965, 8 7 , 38375-3878. - -. - - - . -. (30) Ross, S. D. Tetrahedron 1989, 2 5 , 4427-4436. (31) Bunton, C. A.; Robinson, L. J. Org. Chem. 1970, 35, 733-736. (32) Kavaiek, J.; Haasova, J.; Sterba, V. Collect. Czech. Chem. Commun. 1972, 37, 3333-3338. (33) Kavaiek, J.; Kubias, J.; Sterba, V. Collect. Czech. Chem. Commun. 1972, 3 7 , 4041-4045. (34) Kavaiek, J.: Sterba, V. Collect. Czech. Chem. Commun. 1972, 38, 884-69 1. (35) de Rossi. R. H.; Pierini, A. B.; Rossi, R. A. J. Org. Chem. 1976, 4 3 , 2982-2986. (36) Forlani, L. J. Chem. Res., Synop. 1984, 260-261. (37) Wong, M. P.; Connors, K. A. J . Pharm. Sci. 1983, 72, 146-150. (38) Bunnett, J. F.; Hermann, D. H. Bbchemisfry 1970, 9 , 816-822. (39) Connors, K. A. Reaction Mechanisms in Organic Ana/ytlcal Chemistry; Wiley: New York, 1972; p 274. (40) Athanasiou-Malaki, E.; Koupparis, M. A. Anal. Chim. Acta. in press.
RECEIVED for review July 28,1988. Accepted March 14,1989.
Extraction and Isolation of Phenoxy Acid Herbicides in Environmental Waters Using Two Adsorbents in One Minicartridge Antonio Di C o d a , * Marcello Marchetti, and Roberto Samperi
Dipartimento di Chimica, Uniuersitci La Sapienza di Rorna, Piazzale Aldo Moro 5, 00185 Roma, Italy
Selectlve solid-phase extraction from environmental waters of nlne popular acldlc herblcldes was accompllshed by using one mlniaturlzed cartridge contalning in the top slde 50 mg of a nonspecific adsorbent, that Is graphitized carbon black (Carbopack B), and In the lower side 70 mg of a slllca-based strong anlon exchanger (SAX). After sample percolation, the SAX materlai was actlvated by passlng through the trap sodlum acetate, l moi/L. A 3-mL solvent mlxture of CH,Ci,/ CH,OH (80:20, by volume) badfled with 1mmol/L NaOH was then allowed to flow through the cartridge, and phenoxy acids were removed from the Carbopack surface and selectively readsorbed on the exchanger surface. After washing, the nlne herblcides were desorbed from the SAX surface wkh 0.0 mL of water/methanol (50:50, by volume) contalning trlfluoroacetlc acid and potasslum chloride. A 100-pL portion of thls solution was directly injected into the HPLC column, operatlng in the Ion suppression-reversed-phase mode. As ion suppressor, the substitutlon of the commonly used acetlc acld with trlfluoroacetlc acid allowed sensitive detectlon at 230 nm. Recovery of the nlne acidic herblcides ranged between 94% and loo%, lrrespectlve of the sample type considered. By thls extractlon method, bask, neutral, and weakly acldic compounds do not interfere with the analysis. Interestlngly, thls cartridge Is suitable for fleld sampllng, as Carbopack is able to adsorb phenoxy aclds from water at whatever pH value. The llmlts of detectlon of the nlne herbicides were well below 0.1 pg/L. The effectiveness In terms of recovery and selectivity of the two-adsorbent tandem system was compared with that of a C-18 disposable cartrldge.
* To whom correspondence should be addressed.
In the last decade, as an alternative to liquid partitioning, the method of combined extraction and preconcentration of organic compounds in water by adsorption on proper solid materials followed by desorption with a small quantity of an organic solvent has been employed extensively for trace determination of contaminants in environmental waters (1-8). The recent availability of sorbents in small, inexpensive cartridges has contributed t o the dramatic expansion of the use of solid-phase extraction (SPE), as evidenced by some selected publications (9-13). Besides solving well-known problems associated with the classical solvent extractionsolvent removal method, the S P E technique is particularly attractive as it lends itself to coupling with chromatographic systems for on-line applications (14). Another impressive feature of the S P E technique is that small sorbent traps can be deployed in the field by using newly available submersible instrumentation (15, 16). In this way, combined sampling, extraction, and preconcentration are done at the sampling site, thus eliminating most contamination and handling problems connected to the sample collection. In addition, immediate isolation of organics from the aqueous matrix by an adsorbing material can preserve analytes from bacterial attack occurring between the time of sample collection and analysis (17, 18). The small-volume column could be sealed and shipped to the laboratory for elution and chromatographic analysis. Phenoxy acid herbicides have become widely used because of their relative cheapness and effectiveness in controlling the presence of unwanted broad-leaf weeds in crops. For the extraction of this class of herbicides from water, various procedures involving the S P E technique by an anion exchanger (19) and C-18 bonded silica (20-22) have been proposed. None of these methods, however, seems to be sufficiently sensitive to detect phenoxy acids at concentrations lower than 1 Fg/L. Moreover, compared to the three-step
0003-2700/89/0361-1363$01.50/00 1969 American Chemical Society
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ANALYTICAL CHEMISTRY, VOL. 61, NO. 13, JULY 1 , 1989
liquid extraction procedure, i.e. solvent extraction, back-extraction with basified water, and reextraction with an organic solvent (23), the proposed SPE methods are certainly less tedious, but also less selective. When one is analyzing largely contaminated water samples and selective detection cannot be used, a rapid and simple procedure for cleanup of the extract prior to chromatographic analysis is desirable as, at least, it can provide added evidence for peak identity. Very recently, we succeeded in developing a selective high-performance liquid chromatography (HPLC) assay for eight triazine herbicides in water and vegetables by coupling a graphitized carbon black (Carbopack) cartridge (extraction column) t o another one filled with a strong cation exchanger (isolation column) (24). The object of this work was that of developing a sensitive and specific HPLC assay for monitoring phenoxy acid herbicides in environmental aqueous samples. The extraction and purification of the sample were performed by one single miniaturized cartridge containing in the upper part Carbopack and in the lower part a silica-based strong anion exchanger (SAX). Concentration factors higher than 500 were achieved by suitably sizing the trap. Direct injection of a large fraction of the final sample extract into the HPLC column operating in the reversed -phase mode was possible, as the eluotropic strength of the solvent system for elution of phenoxy acids from the trap was lower than that of the mobile phase for HPLC. Possible interferences by other acidic pollutants, such as phenols, were considered. EXPERIMENTAL SECTION Reagents and Chemicals. Authentic phenoxy acids and one benzoic acid derivative were purchased from Eurobase (Milan, Italy). The purity grade was equal or more than 99%. They are as follows: 2-methoxy-3,6-dichlorobenzoic acid (Dicamba); 2,4dichlorophenoxyacetic acid (2,4-D); 4-chloro-2-methylphenoxyacetic acid (MCPA); 2-(2,4-dichlorophenoxy)propionicacid (2,4-DP); 2,4,5-trichlorophenoxyaceticacid (2,4,5-T); 2-(4chloro-2-methy1phenoxy)propionic acid (MCPP); 4-(2,4-dich1orophenoxy)butyric acid (2,4-DB);4-(4-chloro-2-methylphenoxy)butyric acid (MCPB); 2-(2,4,5-trichlorophenoxy)propionicacid (2,4,5-TP). A standard solution was prepared by dissolving 1 g of each herbicide in 1 L of methanol. This solution was further diluted to obtain a working standard solution of 10 mg/L. For HPLC, distilled water was further purified by passing it through a Norganic cartridge (Millipore, Bedford, MA). Methanol of HPLC grade was from Carlo Erba, Milan, Italy. Trifluoroacetic acid was supplied by Fluka AG, Buchs, Switzerland. All other chemicals were of analytical reagent grade (Carlo Erba). Apparatus. Both Carbopack B and the silica-based anion exchanger had a particle size between 37 and 74 pm. As supplied, chloride was the counterion of the exchanger. They were packed in polypropylene tubes, 6 X 0.5 cm i.d. Polyethylene frits, 20-pm pore size, were located above and below each sorbent bed. All the materials cited above were kindly supplied by Supelco, Bellefonte, PA. The extraction/purification cartridge was prepared by introducing first 70 mg of the SAX material and then 50 mg of Carbopack B, with one frit interposed between the two sorbent beds. Before water samples were processed, no prewashing of the cartridge was necessary. The trap was fitted into a side-arm filtering flask, and liquids were forced to pass through the cartridge by vacuum done by a water pump. Procedure. Aqueous samples were fortified with the nine herbicides by adding known volumes of the working standard solution. Water samples were then introduced in a polypropylene sample reservoir that was connected to the trap through an adapter. Suspended sediments contained in river and seawater samples, which can obstruct the cartridge, were removed by interposing between the reservoir and the cartridge a 6 cm X 1cm i.d. plastic tube containing only two polyethylene frits (20-pm pore size). Samples were percolated through the cartridge a t a flow rate of 9-10 mL/min, which was the maximum flow rate attainable with the apparatus used. After the sample was passed through the two-adsorbent trap, vacuum was reduced to give a flow rate
Table 1. Recovery of Herbicides at Increasing Drinking Water Volumes Sampled" 7~recoveryb
Dicam ba 2,4-D MCPA 2,4-DP MCPP 2,4,5-T 2,4-DB MCPB 2,4,5-TP
100 mL
200 mL
-100 mL
98.4 97.3 98.2 98.0 96.7 94.5 99.3 96.7 94.7
96.6 99.0 97.9 97.3 96.8 94.9 100.3 96.3 95.3
98.6 98.7 98.5 98.7 96.2 96.5 98.9 97.2 96.0
'Water was spiked with 0.5 bg/L of each compound. *Mean values were calculated from three determinations. of about 2 mL/min, as measured for water. An 8-mL aliquot of 1 mol/L sodium acetate in water was percolated through the cartridge to displace inorganic anions from the ion-exchange sites of the SAX material and convert it to the acetate form. The salt excess was eliminated by 2 mL of distilled water, which was in turn removed from the trap by 0.5 mL of acetonitrile. Then, the vacuum was further decreased, and phenoxy acids and Dicamba trapped by the Carbopack column were removed from it to the SAX surface by passing slowly through the trap 3 mL of methylene chloride/methanol (80:20, by volume) basified with sodium hydroxyde, 1 mmol/L, at a flow rate of about 1 mL/min. This solvent mixture was freshly prepared every 3 days, since it lost gradually its extraction efficiency on aging. Residual amounts of neutral and basic organic compounds remaining in the cartridge were drained with 0.5 mL of acetonitrile. Thereafter, the analytes of interest were eluted from the SAX column by passing through the cartridge at a flow rate of about 0.3 mL/min water/methanol (50:50, by volume) containing trifluoroacetic acid (0.5%, by volume) and KCl (0.16 mol/L) and collecting the first 600 pL of this solution. A 100-pL aliquot of this was injected into the HPLC column. HPLC Apparatus. A Model 5000 liquid chromatograph (Varian, Walnut Creek, CA) equipped with a Rheodyne Model 7125 injector having a 100-pL loop and with a Model 2050 UV detector (Varian) was used. A 25 cm X 4.6 mm i.d. column filled with 5-pm LC-18 reversed-phase packing (Supelco) was used. The mobile phase was a premixed water/methanol (4159, by volume) solution containing 0.08% (v/v) trifluoroacetic acid. The flow rate was 1.5 mL/min. The herbicides considered were monitored with the detector set at 230 nm. The concentrations of the herbicides in water were calculated by measuring the peak height of each herbicide and comparing them with those obtained with a standard solution. The latter was prepared by adding an appropriate volume of the herbicide working standard solution to 600 pL of the same aqueous solution used for elution of the herbicides from the trap. RESULTS AND DISCUSSION Recovery Studies. For the herbicides considered, the extraction efficiency of the Carbopack/SAX cartridge at increasing volumes of a drinking water specimen (pH 7.1) was evaluated. Water samples were spiked with the nine analytes a t an individual concentration of 0.5 wg/L. No p H adjustment of the sample was done prior to extraction. Data are reported in Table I. Under the conditions mentioned above, the acidic herbicides considered were present in water virtually as anions. To first view, it is surprising that the small cartridge has the ability to trap such very water soluble analytes from relatively large sample volumes. Carbopack B was responsible for this effect, as, when in the chloride form, the low-capacity anion exchanger material located below the Carbopack particles has almost no affinity for organic anions. In two previous papers (25,26) it was reported that some anomalous effects displayed by Carbopack particles immersed in water could be explained by assuming that some carbon-oxygen complexes naturally contaminating its graphitic surface framework are rearranged
ANALYTICAL CHEMISTRY, VOL. 61, NO. 13, JULY 1, 1989
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Table 11. Recovery of Herbicides at Increasing River and Seawater Volumes Sampled" % recoveryb
river water, mL Dicamba 2,4-D
MCPA 2,4-DP
MCPP 2,4,5-T 2,4-DB
MCPB 2,4,5-TP
seawater, mL
50
200
400
50
200
400
98.5 97.6 98.4 98.2 96.6 94.7 98.9 97.3 94.8
31.6 (96.8)c 96.8 98.0 97.5 96.4 94.9 98.1 97.3 95.1
7.9 (97.4) 96.1 98.9 99.0 95.7 95.6 98.2 97.0 94.3
98.7 96.6 98.1 97.7 96.4 95.0 98.8 96.5 94.3
48.9 (97.8) 97.9 98.5 98.5 95.2 94.6 98.5 97.3 95.8
22.7 (98.1) 98.6 99.0 97.5 96.1 96.5 99.1 96.1 96.7
OWater was spiked with 2 pg/L of each compound. *Mean values were calculated from three determinations. cIn parentheses are reDorted recoverv values of Dicamba after samDle acidification. to form chemical groups bearing a positive charge. This surface modification enables Carbopack to strongly but reversibly adsorb organic anions; this is likely due to the added contributions of electrostatic and van der Waals forces. By taking advantage of this feature, workers have successfully employed Carbopack for the extraction of very polar phenols (7) and for the class separation of conjugated estrogens (27). The effect that the matrix can have on the breakthrough volumes of the phenyl acetic derivative (Dicamba) and phenoxy acids was assessed by assaying increasing volumes of water samples from two different sources, that is river and seawaters. Likewise with solvent extraction methods, when the sorbent trap technique is used with nonspecific materials, such as chemically bonded silica (20-22), ion suppression by suitable pH adjustment is usually an unavoidable preliminary step in order to retain ionogenic substances. Obviously, this precludes field sampling of water by the use of extraction columns. On purpose, the addition of the herbicides considered a t the individual concentration of 2 bg/L was the only modification we made on the environmental samples. As measured, the apparent pH values of the river and seawater specimens were respectively 8.1 and 7.8. Both the river and seawater samples, the latter being collected very close to the outlet of the river considered (Tevere), contained a lot of unknown acidic organic compounds, as evidenced by a large initial front appearing on chromatographing the relative sample extracts. In spite of this, recovery data (Table 11) of the eight phenoxy acids were very comparable to those obtained for drinking water extraction. Evidently, no significant adverse saturation effect took place on the Carbopack surface, within the range of water volumes considered. On the contrary, a dramatic loss of the phenylacetic derivative, that is Dicamba, occurred by sampling water volumes larger than 100 mL. Among the acidic herbicides considered, Dicamba was observed to have by far the highest mobility on the Carbopack column. Competition for the relatively few, positively charged active centers of the Carbopack surface mentioned above by the other organic anions naturally contaminating the two environmental aqueous samples considered was supposedly responsible for the loss of Dicamba. This hypothesis was substantiated by the fact that no significant loss of Dicamba was further observed by acidifying the water samples (pH 2). In such ambient, the phenyl acetic derivative is virtually un-ionized, and as such, its adsorption takes place on the predominant, normal adsorption sites of the Carbopack surface, which are far from saturation. Anyway, an accurate determination of Dicamba at concentrations lower than 1bg/L could be still performed without sample manipulation by submitting to the extraction procedure only 50 mL of a surface water specimen. Finally, another interesting feature of the combined use of a nonspecific adsorbent and an ion exchanger is that selective,
Table 111. Accuracy and Precision of the Method with High and Low Herbicide Contents in 200-mL Groundwater Samples % recovery f SD"
0.1 FLg/L
Dicamba 2,4-D
MCPA 2,4-DP
MCPP 2,4,5-T 2,4-DB
MCPB 2,4,5-TP
98.8 f 98.2 f 99.3 f 98.7 f 96.3 f 95.4 f 99.0 f 96.6 f 94.9 f
5.7 2.6 2.4 2.1 2.7 2.9 1.8 2.9 3.2
50 Ng/L 98.3 f 98.9 f 97.9 f 98.8 f 96.7 f 96.1 f 98.0 f 97.4 f 95.6 f
2.0 1.6 1.3 1.4 1.6 2.1 1.1 1.5 2.3
Standard deviation calculated from six determinations. quantitative extraction of ionogenic organic compounds can still be accomplished from high-ionic-strength aqueous samples, such as seawater, where an ion exchanger alone fails. Precision. The recovery and the within-run precision of this method at low and high concentrations of the nine herbicides considered were assessed. A sample of groundwater was divided into two portions that were supplemented with the analytes respectively at the levels of 0.1 and 50 bg/L. Each portion was divided into six 200-mL aliquots, which were analyzed by this procedure. Quantitative results are reported in Table 111. As can be seen, the recovery efficiency was independent of the herbicide concentration, thus demonstrating the absence of any adverse effect of both irreversible adsorption by the materials composing the extraction apparatus and saturation of the two adsorbents. Method Comparison. In terms of recovery and selectivity, the effectiveness of the extraction procedure by the two-adsorbent tandem assembly was compared with those obtained by using a 1-g C-18 disposable extraction column (Baker Chemical Co., Phillipsburg, NJ) (20, 22) and a homemade cartridge containing 50 mg of a high-capacity resin-based strong anion exchanger, such as Amberlite CG-400-11(Fluka). This material was converted to the hydroxide form prior to use. For these experiments, 50-mL aliquots of a Tevere River water sample were supplemented with the nine herbicides at the individual concentration of 2 Fg/L. The extraction of the analytes with the C-18 cartridge was performed from both unacidified and acidified (pH 2) water samples. According to the method of Hoke et al. (ZO), the herbicides were eluted from the C-18 cartridge with two 1-mL portions of methanol that were suitably diluted with water prior to HPLC quantification. Removal of the analytes from the anion exchanger material was performed first by washing it with 1 mL of acidified water (pH 1)and then desorbing the analytes with 1mL of water/methanol(50:50, by volume) acidified with HC1,
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ANALYTICAL CHEMISTRY, VOL. 61, NO. 13, JULY 1, 1989 ,n
A,B,c-*F
I
G
1
I
+ I z3
I
0
1 L
L
4
8
12
16
20
24 28 tirne(rnin)
l
0
'a
I
d\ l
4
I
l
l
a
l
12
l
l
'
m
l
20
l
I
l
l
24 20 t i m e (rnin)
Figure 1. Chromatograms obtained on sampling 50 mL of river water (Tevere,June 1988)spiked with 2 Fg/L concentrations of the nine herbicides considered and 4 FgIL concentrations of the 11 priority pollutant phenols by this extraction procedure (A) and the 1-g, C-18 extraction column (B): 1, Dicamba; 2, 2 . 4 4 ; 3, MCPA; 4, 2,4-DP 5, MCPP; 6, 2,4,5T; 7, 2,4-DB;8, MCPB; 9, 2.4,5-TP; A, phenol; B, p-nitrophenol; C, o-chlorophenol; D, 2,4-dinitrophenol; E, o-nitrophenol; F, 2,4dimethylphenol; G, 4,6-dinitro-o-cresol; H, 4-chloro-m-cresol; I, 2,4dichlorophenol; J, 2,4,6-trichlorophenol; U. unknown compounds contaminating the water specimen. Pentachlorophenol was eluted with a retention time greater than 50
min.
0.1 mol/L. Recovery data are reported in Table IV. A large loss of Dicamba and incomplete recovery of phenoxy acids were obtained by using the resin-based exchanger material. Moreover, the breakthrough volumes of the herbicides considered on the exchanger column were strictly dependent upon the ionic strength of the water sample percolating through it, as observed by adding moderate amounts of an inorganic salt to the aqueous sample considered. A good extraction efficiency for the nine herbicides from acidified water was obtained with the C-18 cartridge, but this material was ineffective for use in field extraction of ionogenic compounds since the herbicides considered passed almost unretained along it when the preliminary acidification step of the water sample was omitted. As designed, the Carbopack/SAX cartridge is able to trap selectively only compounds acidic in nature. Among these, we considered the 11 priority pollutant phenols as compounds that may interfere with the analysis of the herbicides considered. In terms of selectivity, the performance of the two-adsorbent tandem assembly was assessed and compared with that of the 1-g, C-18 extraction cartridge by sampling surface water spiked with both the nine acidic herbicides and the 11 phenols. Figure 1 shows typical chromatograms obtained by the two extraction procedures. As can be seen, the selective extraction procedure developed by us was able to eliminate not only two unknown neutral compounds present in the environmental water sample that interfere with the
Table IV. Recovery of Herbicides from 200-mL Groundwater Samples by the Proposed Method Compared with That from Two Other Extraction Methods %
c-18 pH 2 pH 7.9
Dicamba 2,4-D
MCPA 2,4-DP
MCPP 2,4,5-T 2,4-DB
MCPB 2,4,5-T
94.2 96.1 96.4 97.7 94.4 93.5 96.0 95.8 93.1
