Anal. Chem. 1994,66, 4459-4465
Simultaneous Extraction and Methylation of Chlorophenoxyacetic Acids from Aqueous Solution Using Supercritical Carbon Dioxide as a Phase Transfer Solvent Marguerite Y. Croft, E. John Murby, and Robert J. Wells* Australian Government Analytical Laboratory, P.0. Box 385 1, Pymble, NS W 2073, Australia
The rapid and efficient methylation and extraction of organic acids from aqueous solution using supercritical carbon dioxide containing methyl iodide and tetrahexylammonium hydrogen sulfate was shown to be analogous to similar reactions in conventional liquid-liquid extractive alkylation systems. The test analytes 2,4-dichlorophenoxyacetic acid (2,4-D) and 2,4,5-trichlorophenoxyacetic acid (2,4,5-T) were only extracted as their methyl esters by unmodified carbon dioxide at moderate temperature and pressure when methyl iodide was present in the extraction solvent, Conversion to these derivatives from 1 ppm solutions was complete in 30 min with yields of 89%and 103%for 2,4-Dand 2,4,5-T, respectively, and relative standard deviations better than 10%.The presence of methanol modifier in the supercritical carbon dioxide was deleterious to recovery of the methyl esters. A method in which the derivatizingreagent was continually supplied to the extraction vessel was found to be more demanding of reagents and equipment and gave less repeatable results than a simpler static reaction system. Supercriticalfluid extraction (SFE) has been, for some years, an area of analytical chemistry with great potential which is yet to be fully realized. It is not only more rapid than traditional solvent extraction methods but has environmentaland economic advantages over, for instance, the use of halogenated solvents under Soxhlet conditions. Carbon dioxide, by far the most commonly used fluid, is inexpensive and nontoxic and is recycled into the atmosphere, thereby avoiding disposal problems. For all practical purposes, it is still the only real contender as a supercritical extraction fluid. As a solvent, supercritical carbon dioxide at accessible conditions of temperature and pressure is most comparable to nonpolar organic solvents ranging from hexane to toluene. Consequently, the potential of SFE as a universal extraction technique is limited by the inefficient extraction of many more polar substances using pure carbon dioxide. This may be overcome to some extent by the incorporation of up to 10%polar organic modifiers in the supercritical fluid or, less conveniently, by the use of very high temperatures and pressures. The use of solvent-modified COZ not only increases analyte solubilities but also can interrupt matrix effects during the extraction process.' An alternative to the use of solvent modifiers in overcoming problems with solvent strength and matrix interactions, first (1)Hills, J. W.;Hill, H. H., Jr.; Maeda, T.Anal. Chem. 1991,63, 2152-2155. 0003-2700/94/0366-4459$04.50/0 0 1994 American Chemical Society
brought to prominence by Hawthorne and co-workers? involves the derivatization of analytes during the extraction process. They proposed two alternative mechanisms for the methylation of 2,4 dichlorophenoxyacetic acid when extracted using SFE in the presence of phenyltrimethylammonium hydroxide: in situ methylation in the extraction vessel or, more likely, methylation in the injection port of the GC after extraction of the analyte as an ion pair with the derivatizing agent. This use of a single reagent to promote both extraction and derivatization is obviously very attractive; however, care in the choice of derivatizing agents would be required to avoid a gradual degradation of GC columns by tertiary amine salts3.4 in routine use of such a procedure. In an alternative approach,acidic analytes were methylated in situ using BF3/methanol.2 The methyl esters were then readily extracted by SFE prior to GC quantitation. It was found, however, that only a limited number of the acidic analytes tested were efficiently derivatized using this reagent system. A comparison of these methylation techniques, as well as in situ silylation, demonstrated the importance of overcoming matrix interactions in analysis of real sample^.^ The theme of combined supercritical extraction and derivatization has been continued by a number of authors to include the butylation of aromatic sulfonic acids by tetrabutylammonium salts? the extraction of oxalic acid, dicarboxylic acids, and alcohols from roasted coffee by silylation,' the in situ derivatization of resin and fatty acids from sediments with a-bromo-2,3,4,5,&pentanuorotoluene and triethylamine? and the acetylation of phenolic compounds with acetic anhydride in the presence of triethylamine under static SFE conditions.* More recently, the alkylation of chlorophenoxy acid herbicides with a series of quaternary ammonium salts and, in better yield, with tetrabutylammoniumhydroxide and methyl No mechanism was presented for methyl iodide was de~cribed.~ iodide methylation in the latter study. Alkylation under conditions involving phase transfer between water and a water immiscible organic solvent has been established (2) Hawthorne, S. B.; Miller, D. J.; Nivens, D. E.; White, D. C . And. Chem. 1992,64,405-412. (3) Lisi, A M.; Trout, G. J.; Kazlauskas, R J. Chromafogr. 1991,563, 257270. (4)Lisi, A M.; Kazlauskas, R; Trout, G. J.J Chmmufogr. 1992,581.57-63. (5)Rochette, E.A;Harsh, J. B.; Hill, H. H. Tulanfa 1993,40,147-155. (6)Field, J. A;Miller, D. J.; Field, T. M.; Hawthome, S. B.; Giqer, W. Anal. Chem. 1992,64,3161-3167. (7) Lee, H.-B.; Peart, T. E. J Chromufogr. 1992,594,309-315. (8) Lee, H.-B.; Peart, T.E.; Hong-You, R L. J Chromufogr. 1992,605, 109113. (9) Lopez-Ada, V.; Dodhiwala, N. S.; Beckert, W. F.]. Agric. Food Chem. 1993, 41,2038-2044.
Analytical Chemistry, Vd. 66, No. 24, December 15, 1994 4459
as a convenient and high-yielding method to convert acidic substances to their less polar alkylated derivatives.1° Dichloromethane and toluene have proven to be suitable organic solvents for these reactions, and the high-yield methylation of a series of acidic diuretics in a dichloromethane-urine system3j4exemplities the use of this technique. At moderate pressures and temperatures, supercritical carbon dioxide not only has solvating properties comparable to those of an aromatic solvent such as toluene but also is immiscible with water." It should therefore be a suitable solvent for phase transfer reactions from aqueous solutions. This paper reports on the use of supercritical C02 as one of the phases in the phase transfer methylation of acidic s u b stances. The application and mechanism of supercritical fluid extractive methylation was studied using 2,4dichlorophenoxyacetic acid (2,4D) and 2,4,5trichlorophenoxyaceticacid (2,4,5T) as trial analytes in aqueous solution. EXPERIMENTAL SECTION
Standards and Reagents. Nanograde toluene and ChromAR HPLC grade methanol were obtained from Mallinckrodt Australia (Clayton, Victoria, Australia). Methyl iodide, AR grade, was supplied by Mallinckrodt Speciality Chemical Corp. Paris, KY). Tetrahexylammonium hydrogen sulfate (lXA) was Purum grade from Fluka (Castle Hill, NSW, Australia). Carbon dioxide (Purity > 99.5%) was from Linde Gas Co. (Fairfield, NSW, Australia), supplied pressurized to 12 MPa with helium (99.999% purity). Herbicides (including 2,4D and 2,4,5T, >98%purity) were provided by the Curator of Standards, Australian Government Analytical Laboratories, Pymble, NSW, Australia. The methyl esters of 2,4D and 2,4,5-T were prepared in this laboratory by methylation of 5 g of the appropriate acid with 20 mL of methanol containing 10%by weight of concentrated sulfuric acid at 60 "C for 1h followed by cooling to 4 "C for 30 min and filtration under vacuum. They were twice recrystallized from hot methanol followed by storage at 4 "C for 30 min. Solutions of the prepared material gave a single peak by GC/MSD. Spiking solutions of the acidic herbicides were prepared in 0.2 M sodium bicarbonate buffer solution. Solutions of the methylated standards were prepared in toluene. Resins and SFE Support Materials. Bio-beads SM-7 resin (200-400 mesh), a neutral macroporous acrylic ester, was obtained from Bio-Rad Labs (Sydney, Australia). Prior to use, it was washed by suspension in methanol several times to remove fines. Extrelut was purchased from Merck (Darmstadt, Germany), Hydromatrix from Varian (Harbor City, CA), and Kenite Diatomite from Witco Co. (New York, NY). Preparation of Sample Solutions for Methylation and Fktraction. The majority of this work was carried out on the methylation and extraction of 2,4D and 2,4,5T from aqueous standard solutions prepared in 0.2 M sodium bicarbonate buffer (PH 10). Before extraction, samples were mixed with 0.2 M tetrahexylammonium hydrogen sulfate (50-600 pL), dissolved in the same bicarbonate buffer, to give a final maximum volume of up to 3 mL. This mixture was then evenly distributed on 3 g of Extrelut using a vortex mixer. The resulting relatively dry sample was packed into a 10 mL stainless steel SFE extraction vessel. (10)Persson, B. A; Schill, G. In Handbook of Derivatives for Chromatography, 2nd ed.; Blau, K, Halket, J. H., Eds.; John Wiley and Sons: Chichester, England, 1993. (11) Thiebauf D.; Chervet, J.-P.; Vannoort, R W.; DeJong G. J.; Brinkman, U. A Th.; Frei, R W. J. Chromatogr. 1989,477,151-159.
4460 Analytical Chemistry, Vol. 66, No. 24, December 15, 1994
This was the maximum workable charge for the ISCO extraction vessels used throughout this work. Spiked urine solutions were prepared by adding 100 pL of herbicide standard solution to 2 mL of urine to give a final concentration of 1.3 mg/mL 2,4D and 0.36 mg/mL 2,4,5T. These samples were then used directly or made basic by the addition of 300 pL of aqueous bicarbonate buffer solution (1.3 M) and processed as above. To ensure that the availability of phase transfer reagent did not limit the reaction, 600 pL of THA solution was used. To prepare spiked rice and soil samples, 100 pL of an aqueous herbicide standard solution was added to 1.5 g of soil or rice in individual vials. This gave final spiking concentrations of 1.3ppm 2,4D and 0.5 ppm 2,4,5T. Acetone (2 mL) was added to the sample vial to assist distribution of the spike throughout the sample. The vial was vortexed and left uncovered until the acetone had evaporated, and then the vial was sealed and the sample aged for approximately 1month prior to extraction in the static SFE methylation system described below using 600 pL of the THA solution and 75 pL of methyl iodide. Supercritical Fluid Extraction. An ISCO Model SFX 2-10 extraction unit and two ISCO Model 260D (Lincoln, NE) syringe pumps were used throughout this work. Fused silica restrictor tubing of 50 pm i.d. and approximately 25 cm in length was used to control the flow of pressurized C02 to 0.9-1.5 mL/min. The quantity of pressurized C02 consumed was used to monitor the length of the extraction. This was the simplest way to ensure comparability of results in the presence of minor variations in flow rate over the course of a run. Extractions were typically performed at a constant pressure of 200 bar with temperatures from 60-90 "C. Blockages of the SFE restrictor capillary were a common occurrence when 0.1 M sodium hydroxide was used to ensure basic conditions for ionization of the analytes. These blockages may have been due to deposition of silica eroded from the Extrelut support at high pH. The use of 0.2 M bicarbonate (PH 10) to maintain a basic reaction environment eliminated the problem. Dynamic SFE Methylation. Dynamic methylative extraction was implemented by supplying methyl iodide and C02 to the extraction vessel from separate syringe pumps connected via a T-junction. A Rheodyne (Cotati, CA) 7125 injector was inserted between one pump containing methanol and the T-junction, and a continuous metered supply of methylating agent was delivered to the extraction vessel from the injector loop by displacement with methanol. Methyl iodide solutions (5-75% in methanol) were mixed into the extraction fluid during the continuous (dynamic) methylation and extraction procedure using 0.36, 1.64, or 5 mL loops on the injector inserted in the modifier supply line. The concentration of the methyl iodide in the extraction vessel was controlled by varying the contribution of the secondary pump containing methanol to the total flow of the fluid stream (1-10%). The primary pump contained carbon dioxide. Such continuous extractions were allowed to proceed until one to two loop volumes of modifier had been consumed. Static SFE Methylation. For the static methylation technique, only a single syringe pump containing C02 was required. Methyl iodide (25-100 pL) was pipetted onto prepared samples after they had been distributed on the inert support matrix and placed in the extraction vessel. The extraction chamber was then pressurized with COz, and the reaction was allowed to continue
under pressure for 10 min with no flow. This was followed by extraction with 20 mL of pressurized COZ. Collection and Cleanup of Extracts. The carbon dioxide effluent from the SFE restrictor was bubbled through 5 mL of toluene in a 15 mL glass screw-cap test tube to collect extracted analytes. To minimize the release of methyl iodide and toluene, the collection tube was fitted with a septum, and waste extraction fluid was vented via Teflon tubing through a trap consisting of pelletized charcoal and glass wool in a large disposable syringe barrel. Previous work334 showed that in a water-organic phase transfer system there was some retention of the phase transfer catalyst in the water-immiscible layer and that it was necessary to remove this catalyst by passage through a short bed of SM-7 resin in a Pasteur pipet in order to prolong GC column lifetime. Similar partitioning into the COZlayer leading to coextraction of some of the phase transfer catalyst was also noted in the present work by the presence of trihexylamine in GC/MS chromatograms where SM-7 was not used. Phase transfer reagent which had carried over into the collection solvent during extractions was therefore removed on a 2-3 cm bed of Biebeads SM-7 retained in a Pasteur pipet by a small glass wool plug. This resin column was regenerated between extractions with 5-10 mL of methanol and treated with 5 mL of toluene before use. Purifled extracts were diluted to a final volume of 10 mL with toluene prior to GC analysis. liquid-liquid Extractive Alkylation. In determining the extent of extraction of underivatized chlorophenoxyacetic acids, a mixture of 5 mL of methanol and 0.15 mL of 6 M aqueous sodium hydroxide was used as the SFE collection solvent rather than toluene. The use of this solvent prevented losses of the analytes during evaporation prior to liquid-liquid extractive alkylation and quantitation by GC/ECD. After evaporation of the methanol used for collection, 1 mL of water, 0.15 mL of 0.2 M THA, and 5 mL of 0.5 M methyl iodide in toluene were added to the sample, and extractive alkylation was performed for 30 min on a rotating shaker at low speed. The toluene layer was prepared for GC using SM-7 resin as previously described. Gas Chromatography. Yields of methyl ester derivatives were determined using a Hewlett-Packard Model 5890 GC with electron capture detector (ECD) in comparison to external standards of the methyl ester. Separationswere carried out using 1 pL splitless injections on an HP Ultra 1 column (12 m x 0.2 mm i.d. x 0.33 mm kthickness). Helium (1mL/min) was used as the carrier gas and nitrogen (50 mL/min) as the makeup gas. The injector temperature was held at 230 "C and the detector temperature at 300 "C. The initial oven temperature of 90 "C was held for 1 min, increased to 110 "C at 10 deg/min and then to 260 "C at 20 deg/min, where it was held for 7 min. For identification and confirmation of components in SFE extracts, GC/MS analyses were performed in full scan mode on a HewlettPackard 5890 (ID with a 5971 mass selective detector (MSD) using similar chromatographic conditions. RESULTS AND DISCUSSION
The methylation of 2,4D and 2,4,5T in aqueous solutions in the presence of a phase transfer reagent catalyst and supercritical carbon dioxide containing methyl iodide was found to be an efficient and reliable process. Table 1shows the yields of methyl esters obtained using either the dynamic technique, in which derivatization and extraction proceeded simultaneously, or the
Table 1. Efficiency and Repeatabilityof SFE Phase Transfer Methylation of 24-D and 2,4,S-Tmunder Static and Dynamlc Reaction and Extraction Conditions*
dynamic techniqueC yield (%) CVe (%)
analyte 2,4-D methyl 2,4,5T methyl
90
static techniqued yield (%) CVe (%) 89 103
9 6
92
8 9
Solutions of 1 pg/mL 2,4D and 0.25 pg/mL 2,4,5T in 0.2 M bicarbonate buffer contamng 13 mM tetrahexylammoniumhydrogen sulfate. * Extractions using supercritical COz at 90 "C and 200 bar. cMod%er flow was 0.5%methanol, 0.5% methyl iodide (180 L consumed in 30 min). Static reaction, 10 min with 75 p L of medyl iodide under supercritical conditions. e Coefficient of variation.
2,4,5-T
0
2
4
6
8
10
12
14
lime (min.)
Figure 1. Static methylative extraction of 2,4-D and 2,4,5-T (1.2 and 0.4pg/mL, respectively) from 0.2 M bicarbonatesolution as their methyl esters using supercritical carbon dioxide. SFE at 90 "C,200 bar using 75 pL of methyl iodide and 13 mM THA.
static one, where reaction under supercritical conditions was followed by collection of the products. Optimized conditions for these methylative extractions are indicated in the table. A typical chromatogram obtained using the electron capture detector after a static methylation and extraction is shown in Figure 1. Using either methylation technique, recoveries were essentially quantitative and had coefficients of variation of less than 10%. These recoveries were maintained for solutions containing 2,4D and 2,4,5T in the ranges 0.2-2 and 0.06-0.3 pglmL, respectively. Four dynamic SFE methylations of the analytes at various concentrations in these ranges gave ECD response curves which were h e a r (12 > 0.99) and passed through the origin. The dynamic technique was employed in our initial investigations of phase transfer SFE methylation as it was expected to allow maximum flexibility in altering the reaction and extraction conditions. The constant supply of methylating reagent and continuous removal of any product as the reaction progressed was expected to minimize any yield limitationswhich may result from unfavorable reaction equilibria. Once optimized, the technique gave excellent results as shown in Table 1; however, it was ultimately found that dynamic introduction of methylating reagent was unnecessary, and the more simple and rugged system using a static reaction step was developed. Nevertheless, it was investigations of the dynamic system which yielded the mechanistic evidence leading to an understanding of this simple process for in situ methylation. Dynamic Two Phase Extradive Methylation with Supercritical Carbon Dioxide. To obtain the maximum flexibility, our Analytical Chemistty, Vol. 66, No. 24, December 15, 1994
4461
initial investigations were conducted in a manner which allowed adjustment of the solvating properties of the fluid with a more polar momer (methanol). A continuous flow of derivatizing agent in methanol and supercritical carbon dioxide was utilized with no static reaction period. Exploratory extractions using the dynamic SFE system commenced with reagent concentrations comparable to those employed in liquid-liquid phase transfer methylations. To minimize the amount of toxic methyl iodide used, it was mixed with the extraction fluid as a dilute solution in methanol. The initial flow chosen for this modifer was 5%,and a final concentration of methyl iodide of about 0.5%by volume in the supercritical extracting fluid gave the most promising results. During the process of optimizing the SFE conditions to maximize methylation yields, it was found that this overall methyl iodide concentration worked well but that minimizing the methanol in the extraction fluid was advantageous (vide infra). A number of variables in the methylation process were studied, including the nature of the support matrix for the aqueous phase, the concentration and type of phase transfer reagent, the concentration of methanol, the concentration of methyl iodide, and the effects of temperature and pressure on methylation yields. Support Matrix for Aqueous Solutions. Three support matrices, all diatomaceous earth derived, were studied. Two of these, developed for laboratory use, were pelletized, while the third was an inexpensive Celite powder used in the filtration system of swimming pools. All proved equally effective in supporting aqueous solutions, and reaction yields were essentially identical regardless of the diatomaceous earth support used. Although the densities of each support matrix limited the quantity which could be packed into the 10 mL extraction vessels used (Hydromatrix, 2 g; Celite, 2.5 g; Extrelut, 3 g), each was capable of accepting up to 3 mL of aqueous sample distributed on its surface while retaining a free-flowing consistency. Any sample loading up to this maximum value did not s@cantly affect derivatization yields under constant reaction conditions. Extrelut was chosen as the support matrix for all subsequent experiments as it produced the cleanest chromatograms with the electron capture detector utilized in all quantitation work. Effect of Temperature and Pressure. A pressure of 200 bar has been used throughout this study. Previous studies on these analytes2aghave employed extraction at twice this pressure, where COz exhibits greater solvating power. However, movement to higher pressures had little effect on the yield, which, as shown in Table 1, was excellent in both the optimized dynamic and static SFE methylation systems. By contrast, raising the temperature had a beneficial effect on yields, particularly in the dynamic system, where yields shown in Table 1 were almost halved by operating the extractor at 60 "C instead of 90 "C. Concentration and lfipe of Phase Transfer Reagent. Heniot and PickerI2 have examined the catalytic effects of 21 quaternary ammonium and phosphonium salts in a liquid-liquid phase transfer reaction. They found that catalytic efficiency increased with chain length in the quaternary ammonium salts and that symmetrical salts were superior to those with a single long hydrocarbon chain. Based on this work and previous phase transfer methylation methods,3s4J3methyltrioctylammonium chloride and tetrahexylammonium hydrogen sulfate were chosen as phase transfer catalysts in this SFE study. However, the low water (12) Heniot, A. W.; Picker, D.J. Am. Chem. SOC.1975, 97, 2345-2349. (13) Ervik, M.: Gustavii, K Anal. Chem. 1974, 46, 320-323.
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Analytical Chemistry, Vol. 66,No. 24, December 15, 1994
100 T
80
T
0 0
10
20
30
40
50
THA Concentration (mM)
Figure 2. Relationship of methylation efficiency to phase transfer reagent (THA) concentration, methanol concentration, and methyl iodide concentration. All analyses performed at 90 "C,200 bar using dynamic SFE methylative extraction. Methylation efficiency is shown as an average of 2,4-D and 2,4,5-T methyl ester yields (means of 2-6 replicates with coefficient of variation of 6-14%). (-) Average of 2,4-D and 2,4,5-T recovery when extracted as methyl esters with Con containing 0.5% methanol and 0 ,0.05%; x , 0.25%; A, 0.5%; or +, 0.75% methyl iodide. (- -) Average of 2,4-D and 2,4,5-T recovery when extracted as methyl esters with COZcontaining 4.5% methanol and 0.5% methyl iodide, A.
-
solubility of methyltrioctylammonium chloride made this salt unsuitable for routine use, and all subsequent studies were carried out using tetrahexylammonium hydrogen sulfate as the phase transfer catalyst. The effect of THA concentration on yields in the optimized dynamic system is clearly seen as the solid line in Figure 2. The data in the figure demonstrated that 13mM tetrahexylammonium hydrogen sulfate was sufficient for efficient derivatization under favorable reaction conditions. Methanol Concentration. Investigation of the effect of phase transfer catalyst concentration on methylation yields strongly suggested that high methanol concentrations were deleterious to derivatization in an SFE system. This was established by comparing extractive methylations using COz modifed with 5% of a 9O:lO methanol/methyl iodide mixture rather than 1%of a 50:50 mixture, resulting in a %fold variation in the amount of methanol present in the extraction fluid. As can be seen by comparison of the curves for 0.5%and 4.5%methanol in Figure 2, methylation efficiency was vastly improved at all THA concentrations when the lower methanol concentration was employed. The effect of methanol on derivatization yield was also very marked in the static derivatization procedure discussed later. Methyl Iodide Concentration. The results in Figure 2 demonstrate the effect on methylation yields obtained when, with a total modifier flow of 1%and 13 mM THA, the concentration of methyl iodide in the supercritical extraction fluid was varied over a range of 0.05-0.75%. The effects of the different methyl iodide concentrations are shown by plotting them as individual points for comparison with the curve for 0.5%methyl iodide and 0.5% methanol. Yields increase dramatically until 0.5%methyl iodide is reached. The use of neat methyl iodide (delivered as a 1% m o d ~ eflow) r for methylation tended to cause restrictor blockage, so, to minimize this and ease solvent handling, a 1%flow of 50:50 methyl iodide-methanol mixture was used routinely. With 5050 methyl iodide-methanol mixtures delivered at 1% of the total flow in the optimized dynamic system, complete conversion to the methyl esters consumed 180pL of methyl iodide itself. As discussed below, as little as 25 pL of methyl iodide achieved similar results in a static SFE derivatization system.
Table 2. Effect of Methanol on Recovery of the Methyl Esters of Chlorophenoxyacetlc Acid Herbicides Using Static System of SFE Extractive Methylation'
Table 3. Effect of Methyl lodlde on tlre Supercritical Fluid Extraction of 2,4D and 2,4,5T from Aqueous Soiutlon on Extrelut.
recovery (CV, %) of spiked analytes (%) 5%methanol 10%methanol
spiked analyte recovered (%)
0%methanol
2,4D 2,4,5-T no. of replicates
89 (8) 103 (9)
5
80 (13) 100 (13)
5
40 (22) 50 (18) 4
Solutions of 2,4D (0.7pg/mL) and 2,4,5T (0.24 pg/mL) in 0.2 M bicarbonate @H 10) containin the indicated concentrationof methanol derivatized with 75 pL of mekyl iodide and extracted with COz at 90 "C and 200 bar. (2
Static Phase Transfer Methylation in Supercritical COz. The improvementsin yield and in repeatability of results obtained at low methanol concentrations led to the investigation of a procedure which eliminated methanol from the system. Only a single pump was required to deliver pressurized COZ. Excellent yields of both 2,4D and 2,4,5T methyl esters were achieved (see Table l), and restrictor blockage was not observed. Apart from maximizing the yield of methyl esters, one of the benefits of performing the reaction in the absence of methanol was an improvement in the ruggedness of the procedure. In the absence of methanol, factors such as temperature (between 70 "C and 100 "C) and volume of methyl iodide (25-100 pL) had only minor effects on yields of the methylated products. This insensitivity to variations in reaction conditions in the absence of methanol is reflected in the data in Table 2, where repeatability of results can be seen to decrease with increasing methanol concentration. With the static system the reaction and extraction processes were very rapid. The yield of methylated 2,4D and 2,4,5T after 2 min of reaction in the supercritical extractor was equal to that obtained after a 10 min reaction time. Mechanism of Extractionand Methylation. There has been some discussion in reports of previous studies on derivatization SFE about whether methylation occurs at the time of extraction or in the hot injector port of the GC.2v5 Some methylating agents such as trimethylphenylammonium (TMPA) salts are now generally believed to react in the injector after first effecting extraction of acidic analytes into the hydrophobic supercritical fluid via their ion-pairingabilities.9~'~A recently reported investigation by LopezAvila et al. into ion-pairing methylation reagents in SFEgincluded a description of the extraction of chlorophenoxy acid analytes using methyl iodide and tetrabutylammonium hydroxide. The location and mechanism of the methylation reactions observed was not elucidated. By analogy to their investigations into reactive ion-pairing reagents such as TMPA, the authors suggested that methylationwith methyl iodide probably occurred in the injector port of the GC. To investigate whether methylation could be an injection port phenomenon rather than a true phase transfer reaction in the supercritical fluid environment, we therefore determined whether the phenoxyacid herbicides could be extracted by ion-pairing alone (in the absence of methyl iodide). We also examined whether the underivatized compounds can be methylated outside the SFE system by methyl iodide and phase transfer catalyst, be it in the GC or in the SFE collection solvent. Supercritical Fluid Extraction of Acids. To determine under what conditions the acids can be extracted by SFE, aqueous (14)Amijee, M.;Wells, R J. /. Chroniutop. 1994, 662,123-137.
0% MeOH
1%MeOH
10%MeOH
First Extractionb(without CH3I) 2,4D
2,4,5T 2,4D
2,4,5-T
20 15 Second Extractionc(50 pL of CH3I Added) 85 85
76 74
66 58 4 5
Solutions of 2,4-D (0.7pg/mL) and 2,4,5T (0.24 p /mL) in 0.2 M bicarbonate @H 10) c o n m THA and the indicated concentration of methanol. Average of dupfcate analyses under static extraction conditions with COz at 90 "C and 200 bar. Initial SFE extraction without any methyl iodide (analytes quantitated by GC/ECD after liquid-liquid extraction alkylation). Second SFE extraction of the same samples after addition of 50 pL of methyl iodide.
solutions of 2,4D and 2,4,5T were spiked with THA, distributed on Extrelut, and subjected to two sequentialextractionsusing COz with varying amounts of methanol modifer. During the first extraction no methyl iodide was used. Any free acid eluted was then quantitated after liquid-liquid extractive alkylation of the supercritical fluid extract. The second extraction was performed after the addition of methyl iodide to the cartridge along with further THA to replace that lost during the first extraction. Previous studies have shown that unmodified COZis a poor extraction solvent for the free chlorophenoxyaceticacids.g This is corroborated by our findings. As shown in Table 3, little or no extraction occurred with use of pure COZat 90 "C and 200 bar, even in the presence of phase transfer reagent. However, addition of methanol as a modifier facilitated elution of the underivatized acids, which were quantitated after external extractive alkylation. Using 10%methanol-modified COZ,around 60%of each analyte was extracted without methylation. By contrast, when methyl iodide and THA were present in the extraction vessel, 2,4D and 2,4,5T were recovered in reproducibly high yield (as their methyl esters) even when COZalone was used for extraction. As seen in Table 3, when samples which had been once extracted with pure or methanol-modified COZwere subject to a second extraction, this time after addition of methyl iodide and THA, excellent recoveries of the methyl esters were obtained (when allowance is made for analyte lost in the first extraction). It was found that 2,4D and 2,4,5T can be extracted using 10% methanol-modified COZ (Table 3). This may provide an insight into the poor yields of methyl esters obtained by extractive SFE alkylation in this study when increasing amounts of methanol modifier were used. One of the roles of the phase transfer reagent in liquid-liquid extractive alkylation is to effectively increase the partition of the basic (charged) form of the analyte into the hydrophobic phase, for which it has little affinity. Increasing the concentration of the anionic form of the analyte in the phase containing the methyl iodide obviously accelerates the methylation reaction. M o d ~ e dsupercritical fluids with significant ability to dissolve the free non-ionized form of the analyte can be expected to proportionately reduce the rate of methylation. Consequently, the low yields of methyl esters obtained using 5-10% methanol may be due to competitive direct extraction of the analytes without methylation. As is shown by the data in Table 3, approximately 60% of the 2,4D and 2,4,5T was directly extracted by 10% Analytical Chemistry, Vol. 66, No. 24, December 15, 1994
4463
2,4,5-T
Table 4. Effect of Phase Transfer Reagent Concentration and Reaction Time on Yield of 2,4-D and 2,4,5-T Methyl Esters Using Dynamlc SFE Methylation'
concn
THA (mM)
analyte
3.3 3.3 10 10 20 20
2,4D methyl 2,4,ST methyl 2,4D methyl 2,4,5T methyl 2,4D methyl 2,4,5Tmethyl
yield (%) 30 min reaction 100 min reaction
15 22 26 23 50 58
43 52 58 49 57 62
Urine
Jz//\
Dynamic SFE methylation of 0.2 M bicarbonate solution containing 2,4D (7 g/mL) and 2,4,5T (2.5 pg/mL). .Au extractions at 60 "C and 200 {ar. Extrachon fluid was COz, modded with 4.5%methanol and 0.5%methyl iodide.
methanol-mod~edCOz. These free chlorophenoxy acids would not be detectable by gas chromatography. This explanation may also apply to observations on the methylation of other compounds using this SFE extractive methylation technique. Closely related compounds with the readily ionized carboxylic functional group, such as 2,4DB, dicamba, and fluazifop, all produced signiscant quantities of methyl ester product. These were identified by GC/MS but require authentic standards for quantitation. By contrast, sulfadimidine, a less acidic compound which nonetheless methylates well using on-column methods with tetraalkylammonium reagents,14 gave only a very weak trace of methylated product using the SFE method described here. This compound is possibly extracted more readily without methylation by the supercritical carbon dioxide than the carboxylic acid analytes.
Mode of Methylation in SFE with Methyl Iodide and Phase Transfer Catalyst. It is clear from the data presented here that significant extraction of 2,4D and 2,4,5T from basic solutions containingTHA occurred only when methyl iodide was present in the extraction vessel. This could conceivably be due to methyl iodide simply acting as a modifer in the extractionfluid and facilitating extraction of the free acids either by altering the solvent strength or overcoming matrix-analyte interactions. If such was the case, the methylation reaction would have to be occurring subsequent to extraction, either in the collection solvent or in the GC. However, repeated attempts to methylate 2,4D and 2,4,5T standards outside the supercritical fluid environmentwith methyl iodide and THA at the concentrations used in this work never produced more than trace quantities of the methyl esters. This was true whether the analytes and reagents were added to the collecting solvent prior to a blank extraction and subjected to a normal GC workup or were added directly to a GC vial prior to injection. The elimination of these other possible sites of reaction verifies that methylation takes place in the extractionvessel prior to extraction, as would be expected if this system mimics the liquid-liquid extractive alkylation procedure. Herriot and Picker12 showed that the rate of phase transfer methylations catalyzed by tetraalkylammonium salts is dependent on the concentration of the phase transfer reagent. Results obtained using inefficient reaction conditions (before the dynamic SFE methylation system had been fully optimized) demonstrate that the rate of derivatization here also depends on the THA concentration. Table 4 shows the effect of varying THA concentration in the sample on the SFE extraction yields of methylated 4464
Analytical Chemistry, Vol. 66, No. 24, December 15, 1994
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8
10
Time (min.)
Figure 3. Dynamic methylative extraction of chlorophenoxy acids from urine as methyl esters with supercritical carbon dioxide. SFE at 90 "C,200 bar, using 0.5% methanol, 0.5% methyl iodide, and 40 mM THA (a) 1.3 ppm 2,4-D and 0.4 ppm 2,4,5-T in urine, (b) 1.3 ppm 2,4-D and 0.4 ppm 2,4,5-T in pH 10 bicarbonate solution, and (c) methyl esters standard in toluene.
2,4D and 2,4,5T. When a limited time was allowed for reaction (supplying 85 pL of methyl iodide over 30 min) yields increased with increasing phase transfer reagent concentration up to the maximum (50-60%) typically obtainable under the conditions used (60 "Cand 4.5%methanol). Given suf6cient time with the same reagent concentrations (when 250 y L of methyl iodide was delivered over 100 min),yields were constant even with the lowest THA concentration used. The latter observation clearly shows that the reduction of methylation yield noted at low phase transfer reagent concentration is a consequence of a decreased reaction rate rather than the alternative possibility that THA was being stripped to below some effective level during the course of the extraction process. Our observations show conclusively that methylation with tetrahexylammoniumhydrogen sulfate and methyl iodide takes place rapidly in the extraction vessel and can therefore be regarded as a true phase transfer process in which supercritical COZfulfils the role of the organic solvent in "traditional" phase transfer reactions. Methylative Supercritical Fluid Extraction from Other Sample Matrices. Preliminary investigation of the extraction of 2,4D and 2,4,5T from several sample matrices was attempted using the SFE extractive methylation techniques described here. Extracts from urine, rice, and soil which had been spiked near the 1 ppm level were suf6ciently clean for quantitation of the methyl esters by GC/ECD (see Figures 3 and 4). The urine sample was extracted using the dynamic SFE methylation technique, while the static procedure was used for the rice and soil samples. There was generally little difference in the quality of extracts obtained from the two procedures (variations in retention
from the 10 mL used here and by cleaning-up the samples before methylation via a preextraction with supercritical COZ in the absence of methyl iodide. These modifications will be pursued in future adaptation of the methodology to analysis of real samples. We have also found quaternaryammonium ionexchange resins to be highly effective combined solid support/reaction media for the methylation of absorbed organic acid anions by methyl iodide in supercritical carbon dioxide. A detailed description of this work will appear separately.
1 2,4-D
I
s1 aI n
b?
h
6
8
10
lime (min.)
Flgure 4. Static methylative extraction of 2,4-D and 2,4,5-T (1.3 and 0.5 ppm, respectively)as methyl esters with supercriticalcarbon dioxide from (a) rice and (b) soil. SFE at 90 "C,200 bar using 100 pL of methyl iodide and 13 mM THA. (c) Methyl esters standard in
toluene. time between Figures 3 and 4 are due to the effects of column maintenance over the 6 month period between performance of these experiments). Both analytes could be recovered as their methyl esters from urine and rice in yields of 80-90%. Lower recoveries (60-70%) were achieved from spiked soil, which is known to be a difficult matrix for the extraction of 2,4D.5 It is expected that the sensitivity of the procedure could be signihntly improved by reducing the final volume of the extract
CONCLUSIONS It has been clearly demonstrated in this work that chlorophenoxyacetic acids can be methylated and extracted by supercritical fluids in a process analogous to liquid-liquid phase transfer extractive alkylation. Two methods of achieving this result were examined. The continuous flow method minimizes sample handling and has the potential to overcome any equilibrium limitations on the methylation reaction. The static derivatization system is, however, more rugged as it requires less equipment, allows the elimination of methanol, thereby reducing the effects of any variability in the other reaction parameters, and gives comparable results for the analytes used here. Using this type of system and the optimized conditions described, near quantitative yields of the methyl esters of 2,4-D and 2,4,5T were obtained. The extraction of these analytes has been shown to be dependent on their derivatization by methyl iodide withii the extraction vessel. This indicates that the SFE system described emulates the commonly used liquid-liquid phase transfer extractive alkylation procedure with carbon dioxide replacing the organic solvent phase. Received for review June 15, 1994. Accepted September
19,1994.B
@Abstractpublished in Advance ACS Abstracts, October 15, 1994.
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