Anal. Chem. 1987, 59,2914-2917
2914
by improving the phase-separator design in order to handle higher flow rates. A unique feature of the system is the ability to carry out several washing steps on the organic phase trapped in the loop to remove interferences and/or to strip the analyte, without the need for extra phase separators. Extraction in a closed system has the advantage of avoiding exposure to hazardous solvents, minimizing evaporation, and decreasing cross contamination (26). It would be ideally suited for radiochemical separations. The system can be used only when a relatively large sample volume is available. However, compared to the limitations encountered in batch extraction procedures (2), factors such as organic solvent loss due to adherence on the wall of the extraction vessel, emulsion formation, and time of analysis are decreased. With the present method, the small loop surface area decreases the relative volume of organic solvent adherence, plus it is washed along the tube, and the absence of mechanical mixing minimizes emulsion formation. In addition, the method can be easily automated.
LITERATURE CITED (1) Minczewski, J.; Chwastowska, J.; Dybczynskh R. Separation and Preconcentration Methods in Inorganic Trace Analysk ; Ellis Harwood: Chichester, U.K., 1982. (2) Leyden, D.; Wegscheider, W. Anal. Chem. 1981, 53,1059A-1065A. (3) Cresser, M. S. Solvent Extraction in Name Spectroscopic Analysis: Butterworths: London, 1978. (4) Lazne, M. Radiochemical Separation Methods ; Elsevier: Amsterdam, 1975.
( 5 ) Charlot, G. Colorimetric Determination of Nements ; Elsevier: New York, 1964. (6) Foreman, J. K., Stockwell, P. B. Automatic Chemical Analysis; Ellis Harwocd: Chlchester, U.K., 1975. (7) Furman, J. K. Continuous Now Analysis Theory and Practice; Dekker: New York, 1976. (8) Snyder, L. R.; Levine, J., Soy, R.; Conetta, A. Anal. Chem. 1976, 48, 942A-956A. (9) Ruzicka, J.; Hansen, E. H. Anal. Chim. Acta 1980, 714, 19-44. (10) Burns, D. A. Anal. Chem. 1981, 53, 1403A-1417A. (11) Ruzicka, J.; Hansen, E. H. Now Injection Analysis; Wiley: New York, 1961. (12) Snyder, L. R. Anal. Chim. Acta 1980, 174, 3-18. (13) Ruzicka, J.; Hansen, E. H. Anal. Chim. Acta 1988, 779, 1-58. (14) Fang, 2 . ; Ruzicka, J.; Hansen, E. H. Anal. Chim. Acta 1984, 764, 23-29. (15) Hartenstein, S. D.; Ruzicka, J.; Christian, G. D. Anal. Chem. 1985, 5 7 , 21-25. (16) Kraak, J. C. TrAC Trends Anal. Chem. (Pers. Ed.) 1983, 2 , 183-167. (17) Karlberg, B.;Thelander, S.Anal. Chim. Acta 1978, 9 8 , 1-7. (16) De Castro, L. J. Autom. Chem. 1986, 8 , 56-62. (19) Shelly, D. C.; Rossi, T. M., Warner, I. M. Anal. Chem. 1982, 5 4 , 87-9 1. (20) Bengtsson, M.; Johansson, G. Anal. Chim. Acta 1984, 758, 147-151. (21) Ogiolda, K. Chem. Anal. (Warsaw) 1985, 10, 611-618 (Chem. Abstr. 1966, 6 4 , 5738f). (22) Atallah, R. H. Ph.D. Thesis, Unlverslty of Washington, 1987. (23) Nord, L.; Karlberg, B. Anal. Chim. Acta 1984, 764, 233-249. (24) Nord, L. Ph.D. Thesis, The Royal Institute of Technology, Stockholm, Sweden, 1984. (25) Fossey, L.; Cantwell, F. F. Anal. Chem. 1982, 5 4 , 1693-1697. (26) Fogelqvist, E.; Krysell, M.; Danielsson, L. Anal. Chem. 1986. 5 8 , 15 16- 1520.
RECEIVED for review March 31, 1987. Accepted August 10, 1987.
Gas Chromatographic Determination of Sodium Monofluoroacetate in Water by Derivatization with Dicyclohexy Icarbodiimide Hideaki Ozawa* and Tadashi Tsukioka Nagano Research Institute for Health and Pollution, Nagano-shi, Nagano 380, J a p a n
A method b described for the determlnatlon of trace amounts of sodlum monofluoroacetate (MFA-Ne) In water. MFA-Na was converted to the dlchloroanlllde derlvatlve In a water sample acidifled wlth hydrochloric acld by uslng N,N'-dlcyclohexykarbocHhnMe (DCC) and 2,4dkhloroanlllne ( M A ) and the derlvatlve was extracted from the sample water and cleaned up by silka gel column chromatography. The derlvatlve was quantified by gas chromatography wlth electron capture detectlon (GC-ECD). The llmlt of detectlon was 0.0006 pg/mL, wlth a 50-mL water sample. The recoverles from environmental waters splked at concentratlons of 0.005-0.01 hg/mL were 93-97% wlth less than 4 % relatlve standard devlatlon.
Sodium monofluoroacetate (MFA-Na) is used as a rodenticide for the control of field mice. Since this rodenticide is highly toxic, the potential environmental exposure of nontarget animals to MFA-Na requires a method for determining MFA-Na in the environment at very low levels. Several methods have been reported for the determination of MFA-Na mainly in biological samples, related to its poisoning. Chromatographic methods with sensitivity require
certain types of derivatization of MFA-Na prior to instrumental analysis because of a high polarity of monofluoroacetic acid (MFA). Conversion of MFA-Na to alkyl esters such as methyl (1, 2), ethyl ( 3 ) ,or n-propyl esters (3) or to pentafluorobenzyl ester (4-6) has been performed for gas chromatography with flame ionization, electron capture, or mass spectrometric detection. For high-performance liquid chromatography, MFA-Na has been converted to p-nitrobenzyl ester (7) or derivatized with p-bromophenacyl bromide (8) or 4-bromomethyl-7-methoxycoumarin(9) and analyzed with ultraviolet or fluorescence detection. MFA is a small volatile molecule and has a high water solubility and polarity. These properties make it difficult to separate MFA from water which interferes with the later derivatization reaction, that is, esterification, and often cause low recoveries, and time-consuming extracting or drying steps are required. Vartianinen et al. have extracted MFA from an aqueous extract into diethyl ether for no less than 72 h by using a continuous liquid extraction apparatus (6). One of the methods to derivatize carboxylic acids is activating the carboxyl group with carbodiimides. Carbodiimides are useful coupling reagents for the amide synthesis, which are available in aqueous solution, and N,N'-dicyclohexylcarbodiimide (DCC) has been used for peptide synthesis (IO).
0003-2700/87/0359-2914$01.50/0 0 1987 American Chemlcal Society
ANALYTICAL CHEMISTRY, VOL. 59, NO. 24, DECEMBER 15, 1987 2815 Derivatization with carbodiimides has been applied to the spectrophotometric determination of carboxylic acids in aqueous solutions (11-14). For example, they have been coupled with hydroxylamine by using DCC to hydroxamic acids and determined as ferric hydroxamates (11). But these methods are generally lacking in selectivity. In this study, a new derivatization with DCC has been investigated for the gas chromatographic determination of traces of MFA-Na in water sample, and a method of determination has been developed in which MFA-Na is converted to the dichloroanilide derivative in aqueous solution with 2,4-dichloroaniline (DCA) and analyzed by gas chromatography with electron capture detection (GC-ECD).
EXPERIMENTAL SECTION Apparatus. Analyses were carried out on a Shimadzu Model GC-3BE gas chromatograph equipped with a e3Nielectron capture detector. The mass spectra were recorded on a JMS-D-300 mass spectrometer (JEOL, Ltd. Tokyo, Japan). Reagents and Materials. Sodium monofluoroacetate (MFA-Na) was obtained from Wako Pure Chemicals Co., Ltd. (Osaka, Japan). Standard solutions were prepared by dissolving MFA-Na in distilled water and appropriately diluting the solution with distilled water. N,"-dicyclohexylcarbodiimide (DCC) was obtained from Kanto Chemical Co., Ltd. (Tokyo, Japan). 2,4Dichloroaniline (DCA) was obtained from Tokyo Kasei Kogyo Co., Ltd. (Tokyo, Japan). DCC and DCA were dissolved in ethanol prior to use. These chemicals were used without further purification. Organic solvents (ethanol, ethyl acetate, diethyl ether, benzene, n-hexane) and anhydrous sodium sulfate were of the grade suitable for pesticide residue analysis. Silica gel, Wako-gel S-1from Wako was activated prior to use by heating at 130 "C for 3 h and keeping in a desiccator for 1 h. Other chemicals were of commercially available reagent grade. Analytical Procedure. A 50-mL portion of the sample water was placed in a 1WmL seperatory funnel, to which 1 g of sodium chloride and 0.25 mL of 10 N hydrochloric acid were added. To the acidified sample, 2 mL of 0.5 M DCA in ethanol and 0.8 mL of 1 M DCC in ethanol were added and the mixture was shaken with 15 mL of ethyl acetate for 1 h. The aqueous layer separated after adding 5 g of sodium chloride was extracted with 5 mL of ethyl acetate for 10 min. The ethyl acetate extracts containing the insoluble materials were combined, washed with 5 mL of 3 N hydrochloric acid, saturated sodium hydrogencarbonate solution, and saturated sodium chloride solution, and dehydrated with anhydrous sodium sulfate. The extract was rotary evaporated at about 30 OC to dryness and the residue was dissolved in about 2 mL of benzene. The insoluble materials were removed by filtration and washed with a small volume of benzene. The filtrate combined with the washings was transferred onto a column of silica gel (3 g of silica gel, 10 mm id.) that had been slurry-packed with benzene, washed with 50 mL of benzene, and eluted from the column with 100 mL of a mixture of diethyl ether and nhexane (1/19). The eluate was concentrated in the KudernaDanish (KD) concentrator to less than 5 mL and diluted appropriately with benzene, and a 5 WLaliquot of this solution was injected onto a gas chromatographic column. The quantitation was performed by peak height. A blank test was conducted with 50 mL of distilled water according to the procedure mentioned above. Gas Chromatographic Conditions. The analysis was performed on a 3 mm i.d. X 2.1 m glass column packed with DEGS-H3P04 (5 + 1%) on Chromosorb W 60-80 mesh and Apiezone grease L.-H3p04 (5 + 2%)on Chromosorb W 60-80 mesh in equal length. The former was packed into half of the column in the injector side and the latter in the detector side. The column and detector temperature were 175 O C , and the injector temperature was 195 O C . Nitrogen was used as the carrier gas at a flow rate of 20 mL/min. Five-microliter volumes were injected onto the column for analysis. RESULTS AND DISCUSSION Derivatization and Extraction of the Derivative. As the derivatization of the gas chromatographic determination of trace concentrations of MFA-Na, conversion of MFA-Na
I
I
0
0.2
0.1
0.3
05
0.4
Volume of acid added ( m l )
Flgure 1. Effects of the volume of acids added on the derivatizatlon. Five micrograms of MFA-Na In 50 mL of distilled water was treated according to the analyHcal procedure described here without lnltlal NaCl addition, in which the volume of acid was changed (0, 10 N HCI; 0 , 10 N-H,SO,; DCA, 1 mmol; DCC, 0.8 mmoi).
o
a2
04
oa
06
Amount of DCC
(
mmol
IO )
Figure 2. Effect of the amount of DCC on the derivatization. Five
micrograms of MFA-Na in 50 mL of distllled water was treated according to the analytical procedure without initial NaCl addltlon, in whlch various amounts of DCC were used (0, 1 mmol of DCA; 0, 0.3 mmol of DCA; HCI, 2.5 mmol).
in an aqueous sample to the anilide derivative was attempted. MFA-Na was allowed to react with DCA in aqueous medium by using DCC, forming 2,4-dichloromonofluoroacetanilide (MFA-DCA). In our preliminary investigations, the derivatization of MFA-Na and extraction of MFA-DCA formed could be carried out simultaneously in one step. The derivatization procedure used in the present study was based on the result, and experiments were performed to determine the suitable reaction conditions. The derivatization reaction did not proceed in cases where no acid was added to the sample water. The effect of the volume of acid added on the derivatization reaction was investigated with 5 pg of MFA-Na in 50 mL of distilled water following the analytical procedure. Initially no sodium chloride was added to the sample water, and the volume of 10 N acids added was changed in the range of 0.1-0.5 mL. A constant yield was obtained when 0.2-0.4 mL of 10 N hydrochloric acid was added to the sample water. Addition of more than 0.4 mL resulted in a decrease in yield (Figure 1). In contrast with hydrochloric acid, addition of 10 N sulfuric acid resulted in an extremely low yield (Figure 1). The volume of acid added was determined to be 0.25 mL of 10 N hydrochloric acid. The amounts of reactants required for the derivatization reaction were investigated. Five micrograms of MFA-Na in 50 mL of distilled water was treated with various amounts of DCC or DCA dissolved in ethanol following the analytical procedure. The yield increased with an increase in the amount of DCC until it reached a constant value at about 0.8 mmol (Figure 2). On the other hand, a constant yield was obtained when more than 0.9 mmol of DAC was added to the sample water (Figure 3). The use of too large an excess of reactants gave disturbing influences on the later steps. Therefore, additions of 0.8 mmol of DCC and 1 mmol of DCA were chosen for the derivatization conditions. Preliminary experiments revealed that the derivatization reaction with DCC proceeded at room temperature, and hence the derivatization could be carried out in seperatory funnels. Into seperatory funnels in which water samples were placed
2916
ANALYTICAL CHEMISTRY, VOL. 59, NO. 24, DECEMBER 15, 1987
0
05 Amount Of OCA
IO 15 (mmol )
Flgure 3. Effect of the amount of DCA on the derivatlzatlon. Five micrograms of MFA-Na in 50 mL of dlstilled water was treated according to the analytical procedure without Inltlel NaCl addltkm, in whlch various amounts of DCA were used (HCI, 2.5 mmoi; DCC, 0.8 mmol).
and reactants were added, extracting solvent was added and the mixture was shaken in a shaker at room temperature to promote the reaction and to extract the derivative formed. The time course of the yield of MFA-DCA was investigated with 5 pg of MFA-Na in 50 mL of distilled water following the analytical procedure. Shaking time varied in the range of 2-90 min. The yield increased with an increase in shaking time until it reached a constant value at about 60 min. The extraction efficiency of the derivative was investigated by using several organic solvents. MFA-DCA was effectively extracted with ethyl acetate. Diethyl ether was inferior to ethyl acetate in efficiency. Little of the MFA-DCA was extracted with n-hexane or benzene. The influence of sample volume on the derivatization reaction was investigated with 5 pg of MFA-Na in the range of 10-100 mL of distilled water under the same conditions in the analytical procedure. The volume of 10 N hydrochloric acid added was kept a t 0.25 mL. A constant yield was obtained in the range of 40-60 mL volume of sample water. Sample volumes out of this range reduced the yield of MFA-DCA. The same experiment was further performed under the conditions in which hydrochloric acid concentration added was kept constant (0.05 mL of 10 N hydrochloric acid per 10 mL of volume of sample water). The yield was constant in the range of 10-50 mL and was reduced at volumes of more than 50 mL. To examine the influence of salt concentration in sample water on the recovery of MFA-DCA, 5 pg of MFA-Na in 50 mL of distilled water was subjected to the analytical procedure in which the concentration of sodium chloride initially added was changed. Sodium chloride concentrations in the range of about 1-4% (w/v) gave constant recoveries, which were 10% higher than that obtained with no initial addition of sodium chloride. Taking into account the original occurrence of salts in sample water, it was decided to add 1g of sodium chloride to a 50-mL volume of the sample water in the first step of the analytical procedure. Cleanup by Silica Gel Column Chromatography. A large excess of DCC and DCA used for the derivatization reaction, products of side reaction, etc. were also extracted with ethyl acetate, in which MFA-DCA was extracted, and interfered with the analysis of MFA-DCA with GC-ECD. A method was required for cleanup of the ethyl acetate extract, and a cleanup method by silica gel column chromatography was examined. The ethyl acetate extract of the derivatization reaction mixture with 5 pg of MFA-Na in aqueous solution was evaporated to dryness under reduced pressure and dissolved in about 2 mL of benzene. The benzene solution was loaded onto the silical gel column (3 g of silica gel, 10 mm i.d.) slurry-packed with benzene. Fifty milliliters of benzene was allowed to flow through the column. The major interfering substances were eluted, and no MFA-DCA was found in the eluate. MFA-DCA was completely eluted from the column with less than 80 mL of a mixture of diethyl ether and n-
rn/Z
Figure 4. E1 mass spectrum of the MFA derivative (MFA-DCA): column, 2 m, Apiezone grease L-H,PO, (5 2%); column temperature, 200 O C ; ion-source temperature, 250 O C ; ionization voltage, 70 eV; carrier gas, He at a flow rate of 40 mLlmin.
+
Table I. Recovery of MFA-Na from Spiked River Watera amt spiked, rg
recovery, %
av, %
% RSDb
0.25 0.5
102, 101, 96, 98, 95, 96, 94 93, 89, 97, 92, 95, 92
97 93
3.1 3.0
" A 50-mL volume of river water was used for the recovery test.
Relative standard deviation hexane (1/19) from the column. Accordingly, a method for cleanup of the derivative was decided as follows. A silica gel column on which the derivatization mixture in benzene was loaded was washed with 50 mL of benzene, and subsequently MFA-DCA was eluted with 100 mL of a mixture of diethyl ether and n-hexane (1/19). To obtain the derivatization mixture in benzene, ethyl acetate was rotary evaporated under reduced pressure. Keeping the derivatization mixture under reduced pressure after the solvent had disappeared diminished the recovery of MFA-DCA. A careful removal of ethyl acetate from the ethyl acetate extract is required to prevent a loss of MFA-DCA. Gas Chromatographic Analysis and Identification of the Derivative. Several gas chromatographic column packings were tried for the analysis of MFA-DCA with GC-ECD. Of the column packings tried, DEGS-H3P04, Apiezone grease L-H3P04 showed excellent characteristics. A comparison was done, in which the former showed worse separation, but inhibited the derivative peak from tailing, and the latter showed a disadvantage in the appearance of peaks other than the MFA-DCA peak. Other column packings such as 5% XE-60, 2% OV-17, and 5% DEGS gave an unsatisfactory peak for MFA-DCA. Consequently, a gas chromatographic analysis was carried out on the double column packed with DEGSH3P04and Apiezone grease L-H3P04. The column and the detector were operated at 175 O C without detector contamination. The interference from low molecular weight fatty acids (such as formic, acetic, propionic, and butyric acid) which were expected to be derivatized in this derivatization reaction with DCC was examined. Contamination of the extracted derivatives by the acetic acid derivative was unavoidable because of the use of ethyl acetate as an extractant in the analytical procedure. The derivatives of those acids were removed to a certain extent in the cleanup step by silica gel column chromatography. In addition, their gas chromatographic retention times were different from that of MFA-DCA. Relative retention times ( r ( ) were about 1.4, 1.8, 1.8, and 2.3 for the acetic, propionic, formic, and butyric acid derivatives, respectively. The presence of those acids at 300-fold concen-
ANALYTICAL CHEMISTRY, VOL. 59,
%
8
a
Time
(min)
Figure 5. Gas chromatogram of MFA-Na as MFA-DCA (10 ng/mL of
+
+
MFA-Na): column, 2.1 m, DEGS-H,PO, (5 1%) Apiezone grease L-H,PO, (5 2%); cdumn and detector temperature, 175 OC; injector temperature, 195 OC; carrier gas, N, 20 mL/min.
+
NO. 24, DECEMBER 15, 1987 2917
observed at mlz 186, which corresponded to the fragment ion [M - Cl]+. Calibration Graph and Recovery Experiment. Amounts of 0.05-1.0 c ~ gof MFA-Na in 50 mL of distilled water were subjected to the analytical procedure described here and the chromatograms were obtained (Figure 5). The calibration graph was prepared with the amounts of MFA-Na against the peak heights of MFA-DCA. Excellent linearity was obtained for calibration over the range examined. The limit of detection was about 0.03 ng, corresponding to a concentration of O.OOO6 Fg/mL of MFA-Na in a 50-mL water sample. The recovery was investigated by using 50 mL of environmental water spiked with a given amout of MFA-Na. MFANa was recovered from river water at 9347% with less than 4% relative standard deviation (Table I). Chromatogram obtained from river water spiked with MFA-Na shows an usefulness of the proposed method for determination of traces of MFA-Na in environmental water (Figure 6). Further, applications of this method to soil and biological samples are under investigation. In addition, this method is expected to be applicable to the analysis of low molecular weight carboxylic acids with high solubility in water.
ACKNOWLEDGMENT The authors are grateful to Y. Koseki and A. Momose for helpful advice. Registry No. DCC, 538-75-0; MFA-Na, 62-74-8; H20, 773218-5. LITERATURE CITED
Time
- (min )
Flgure 6. Gas chromatogram of MFA-Na as MFA-DCA obtained from a river water spiked at 0.005 pg/mL under the conditions given in Figure 5.
tration of MFA-Na caused no interference on the determination. The mass spectrum of the MFA derivative prepared under the conditions of the analytical procedure showed an ion peak a t m / z 221, which corresponded to the molecular ion (M') of 2,4-dichloromonofluoroacetanilide(MFA-DCA), and thus the formation of MFA-DCA from MFA-Na and DCA was confirmed (Figure 4). The base peak of MFA-DCA was
(1) Stahr, H. M. J. Assoc. Off. Anal. Chem. 1877, 6 0 , 1434-1435. (2) Stevens, H. M.; Moffat, A. C.; Drayton, J. V. Forenslc Scl. 1976, 8 , 131-137. (3) Peterson, J. E. Bull. Mvlron. Contam. Toxlcol. 1975. 13, 751-757. (4) Okuno, I.; Meeker, D. L. J. Assoc. Off. Anal. Chem. W80, 63, 49-55. (5) Okuno, I.; Meeker, D. L.; Feiton, R. R. J. Assoc. Off. Anal. Chem. 1982, 65, 1102-1105. (6) Vartiainen. T.; Kauranen, P. Anal. Chim. Acta 1984, 157, 91-97. (7) Ray, A. C.; Post, L. 0.; Reagor, J. C. J. Assoc. Off. Anal. chem. 1981, 64, 19-24. (8) Kramer, H. L. J. Assoc. Off. Anal. Chem. 1984, 6 7 , 1058-1061. (9) Collins, D. M.; Fawcett, J. P.; Rammel, C. G. Bull. Envlron. Contam. TOXICOI.lS81, 26, 669-673. (10) Sheehan, J. C.; Hess, G. P. J . Am. Chem. SOC. 1955, 7 7 , 1067-1068. (11) Kasai, Y.; Tanimura, T.; Tamura, 2. Anal. Chem. 1075, 4 7 , 34-37. (12) . , Kasai. Y.: Tanimura. T.: Tamura, 2.; Ozawa. Y. Anal. Chem. 1977. 49, 655-658. (13) Takeuchi, T.; Horikawa, R.; Tanimura, T. Anal. Leff. 1980, 73(A7), 603-609. (14) Horikawa, R.; Tanlmura, T. Anal. Left. 1982, 15(A20), 1629-1642.
RECEIVED for review March 11, 1987. Accepted August 14, 1987.
