Supercritical Fluid Technology - American Chemical Society

Chapter 13

Supercritical Fluid Extraction of Polar Analytes Using Modified C O and In Situ Chemical Derivation Supercritical Fluid Technology Downloaded from pubs.acs.org by NANYANG TECHNOLOGICAL UNIV on 09/20/17. For personal use only.

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Steven B. Hawthorne, David J. Miller, and John J . Langenfeld Energy and Environmental Research Center, University of North Dakota, Box 8213, University Station, Grand Forks, ND 58202

Polar and ionic analytes can be extracted from solid samples using supercritical CO containing organic modifiers, or alternatively, by performing in-situ chemical derivatization during the S F E step. Static S F E performed with the addition of chemical derivatizing reagents can be used to reduce the polarity of target analytes, which makes the analytes easier to extract and prepares them for direct analysis using capillary G C . Following derivatization, the analytes are extracted using dynamic S F E . Quantitative derivatization (to the methyl ester) and extraction of polar analytes such as 2,4-dichlorophenoxyacetic acid (2,4-D) have been achieved. The use of modified CO and in-situ chemical derivatization for the extraction of polar pesticides, bacterial lipids, ionic surfactants, and wastewater phenolics from real-world samples is discussed. 2

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Analytical-scale supercritical fluid extraction (SFE) has recently been demonstrated to be a rapid and quantitative method for extracting many relatively non-polar organics (e.g., "GC-able" organics) from a variety of sample matrices. However, reports of quantitative extractions of polar, high molecular weight, and ionic analytes have been less frequent, and such extractions have generally required the addition of organic modifiers to C 0 (l-4\ or fluids (e.g., Freon-22) which are less acceptable for routine applications (5). After analytes are extracted, many analytical schemes require that polar and high molecular weight analytes be derivatized so that they can be determined by gas chromatography. For example, E P A methods for acid herbicides require methylation using diazomethane after extraction so that the herbicides can be analyzed using G C with E C D detection. This paper will discuss the development of S F E techniques for polar and ionic analytes based on two different approaches. The first approach uses the 2

0097-6156/92/0488-0165$06.00/0 © 1992 American Chemical Society

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addition of organic modifiers to increase the solubility (and extractability) of polar analytes. The second approach uses in-situ chemical derivatization under S F E conditions to decrease the analyte's polarity, and thus to increase its extractability and ease of analysis by conventional G C techniques.

Supercritical Fluid Technology Downloaded from pubs.acs.org by NANYANG TECHNOLOGICAL UNIV on 09/20/17. For personal use only.

Experimental S F E extractions were performed using an ISCO model 260D syringe pump (for pure fluids). Organic modifiers were mixed with C 0 using two of these pumps according to the manufacturer's directions, except for the linear alkylbenzenesulfonate (LAS) extractions which were performed as previously described (6). In-situ chemical derivatizations were performed by adding 0.5 to 2 m L of the derivatization reagent, trimethylphenyl ammonium hydroxide ( T M P A ) in methanol (Eastman Kodak Company, Rochester, N Y ) directly to the sample cell of an ISCO model S F X extraction unit. The sample and reagent were pressurized with C 0 (typically 400 to 500 atm), and heated to an appropriate temperature (typically 80°C). The derivatization was performed in the static S F E mode for 5 to 15 minutes, then the derivatized analytes were recovered by dynamic S F E using a typical flow rate of 0.6 to 1.0 m L / m i n (measured as liquid C 0 at the pump). No other sample preparation of the derivatized extracts was performed prior to G C analysis. A l l G C analyses were performed using Hewlett-Packard 5890 G C s with appropriate detectors and HP-5 columns (25 m X 250 i.d., 0.17 nm film thickness). 2

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The steps in the SFE/derivatization/extraction were: 1.

The sample was weighed into the extraction cell, and the derivatizing reagent was added.

2.

The cell was placed into the extraction unit (which was pre-heated to the extraction temperature) and immediately pressurized with 400 to 500 atm C 0 . 2

3.

The sample was derivatized and extracted under static S F E conditions.

4.

The outlet valve (restrictor end) of the extractor was opened and the derivatized analytes were extracted using dynamic S F E (400 to 500 atm) and collected in ca. 3 m L of methanol.

SFE and Derivatization/SFE of Linear Alkylbenzenesulfonates (LAS) Development of extraction conditions for L A S was performed using sludge from a municipal wastewater treatment plant, and agricultural soil from a field which had been used for disposal of sewage sludge several months prior to

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sample collection. The structure of L A S is shown in Figure 1. As an ionic surfactant, L A S has very low solubility in pure supercritical C O ^ and no detectable amounts of L A S could be extracted with the pure fluid. Supercritical N 0 has previously been reported to yield better extraction efficiencies than C 0 for some analytes from environmental matrices (2,2), but also showed no detectable extraction of L A S . However, when organic modifiers were added to the C O ^ extraction efficiencies increased dramatically, and the extraction with methanol-modified C 0 (ca. 40 mole %) at 125°C gave good recoveries as shown in Figure 2 (6). A n alternate approach for extracting L A S from solid samples is now being developed using in-situ chemical derivatization/SFE with the methylating reagent, trimethylphenylammonium hydroxide ( T M P A ) in methanol. A 500-mg sample of sludge was placed in a 2.5-mL extraction cell, 1 m L of 5% (wt) T M P A in methanol was added, and the cell was pressurized to 500 atm C 0 and heated to 80°C. The static derivatization/extraction step was performed for 10 minutes followed by 5 minutes of dynamic extraction at ca. 0.9 m L / m i n with trapping of the extracted analytes in 3 m L of methanol. The extract was then analyzed using G C without any further treatment. As shown in Figure 3, derivatization of the sewage sludge results in a very complex extract. However, a comparison of an L A S standard (spiked on sand and derivatized in the same manner) with the sludge extract shows good agreement for peaks eluting between ca. 15 and 20 minutes, although the sludge L A S peaks had a slightly higher molecular weight distribution than the commercial L A S peaks. The identity of the methylated L A S species in the sludge extract has been confirmed by G C / M S analysis of the derivatized sludge extract and the derivatized L A S standard. Although only a few of the non-LAS peaks in the sludge extract have been identified, G C / M S analysis shows that many of the major species are methyl esters of biological carboxylic acids. Since the G C / F I D chromatogram of the derivatized sludge extract appears too complex to allow reliable quantitation of the individual I A S homologs (Figure 3), G C with atomic emission detection ( G C / A E D ) in the sulfur-selective mode was also used to analyze the sludge extract (note again that the extract was analyzed without any additional preparation after the derivatization/SFE procedure). As shown in Figure 4, the use of G C / A E D in the sulfur mode virtually eliminates the detection of non-LAS peaks in their retention time window, and thus makes quantitation of the L A S derivatives practical. As was shown by G C / M S analysis, the L A S in the sludge sample was shifted to higher molecular weight homologs compared to the standard L A S . Only preliminary quantitative evaluations for the derivatization/SFE of L A S have been performed, and it appears that the conditions described above yield only ca. 30 to 40% recovery of the native L A S with one derivatization/extraction step. It is apparent from the G C / F T D chromatogram that the matrix contains a very high concentration of materials that react with the T M P A and it is likely that the reaction is reagent limited. This idea is further supported since three derivatization/SFE steps on one sample reduces the L A S to undetectable levels. 2


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Figure 2. Extraction of I A S from municipal wastewater treatment sludge using pure C 0 , pure N 0 , and organically-modified C 0 . Each sample (50 mg) was extracted for 15 minutes at 380 atm and a flow rate of ca. 0.5 m L / m i n . Recoveries are based on the average of two extractions. Results are adapted from reference 6. 2

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HAWTHORNE E T AL.



13.

Retention Time (min)

Figure 3. G C / F T D separation of the derivatization/SFE extract from municipal wastewater treatment sludge (bottom) and commercial L A S (top).

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Bacterial Phospholipids Phospholipid-derived fatty acids are often used to identify bacteria by capillary G C analysis after liquid solvent extraction, concentration steps, and chemical derivatization to their methyl esters. Our initial investigations attempted to extract the intact phospholipids, but no significant recoveries were achieved using pure C 0 . Even if S F E conditions were developed that could extract intact phospholipids, an additional derivatization step would be required before G C analysis of the fatty acid components. For these reasons, chemical derivatization/SFE was investigated in an effort to eliminate the lengthy conventional liquid solvent extractions as well as to combine (and shorten) the extraction and derivatization steps. The derivatization/SFE procedure was performed on samples of whole bacteria using 0.5 m L of 1.5% T M P A in methanol. The static derivatization step was performed for 10 minutes at 80°C and 400 atm C 0 , followed by dynamic S F E for 15 minutes at a flow rate of ca. 0.5 m L / m i n of the pressurized C 0 . Extracts were collected in ca. 3 m L of methanol and immediately analyzed by capillary G C without any further sample preparation. Typical results of the derivatization/SFE procedure are shown in Figure 5 for a 20-mg sample of the bacteria Bacillus subtilis. The chromatogram shows the typical distribution of the fatty acid methyl esters expected for this bacteria (back chromatogram). A second derivatization/SFE extraction was performed in an identical manner on the same sample to determine the completeness of the first extraction. Based on the lack of chromatographic peaks for the 2nd extraction (front chromatogram), the first 25-minute derivatization/extraction procedure appeared to be quantitative. Preliminary comparisons of the SFE/derivatization method with the conventional liquid solvent extraction method have also shown good quantitative agreement (7).


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Chlorinated Acid Pesticides SFE/derivatization of several chlorinated acid pesticides (those listed in E P A method 515.1) have been performed using conditions similar to those used for the bacterial phospholipids. The derivatized products from the S F E procedure for several representative organics are shown in Figure 6. As would be expected using the TMPA/methanol reagent, the carboxylic acids form the methyl esters (2,4-D and dicamba) while the phenols form the methyl ethers (pentachlorophenol). Esters of the carboxylic acids (e.g., the di-isopropyl amine ester of 2,4-D) also form the methyl esters. For ethers, two derivatized products resulted since the ether linkage could be cleaved on either side of the oxygen and methylated as shown by acifluorfen. Quantitative recovery of the pesticides using the SFE/derivatization procedure was found to be matrix dependent, with matrices that contained higher organic content requiring longer derivatization time and higher concentration of the T M P A reagent to obtain good spike recoveries. For example, 2,4-D acid was efficiently derivatized and extracted from a 2-gram


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Figure 6. SFE/derivatization products of representative pesticides spiked on sand using the TMPA/methanol reagent.


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sample of sand using a 5-minute derivatization procedure (400 atm, 80°C) followed by a 10-minute dynamic extraction (Table I) with collection into 3 m L of methanol. However, when the same extraction conditions were used to derivatize and extract 2,4-D from river sediment, the extraction efficiencies dropped dramatically. The extracts from the river sediment were bright yellow while the extracts from the sand were colorless, and we expect that the higher organic content in the sediment (ca. 4%) was responsible for reacting with and using up the T M P A reagent, as was previously discussed for the sewage sludge. Fortunately, when the concentration of T M P A was increased to 20%, and the derivatization time was increased to 15 minutes, reasonably good recoveries of the 2,4-D were achieved from the sediment sample (Table I). Phenolic Wastewaters from "Empore" Sorbent Discs S F E and SFE/derivatization for the extraction of phenols from a coal gasification wastewater have been performed by first collecting the wastewater organics on "Empore" C-18 sorbent discs (8). The sorbent discs were first prepared by washing with methanol and water as per the manufacturer's instructions. A 100-mL sample of wastewater was acidified to a p H is shown in Figure 7c. Figure 7d shows the methyl ethers from the S F E / T M P A derivatization extract.


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static S F E step reduces the polarity of the analytes making them easier to extract by S F E and analyze using conventional G C techniques. The derivatization and extraction steps are completed in < 30 minutes, and many extracts can be analyzed without further treatment. With the use of the T M P A reagent, quantitative recoveries of target analytes can be dependent on the amount of reactive matrix components, so careful evaluation of the reaction/extraction conditions is necessary. Fortunately, a wide range of potentially useful derivatizing reagents is available that have potential to both reduce the matrix dependence of the derivatization step, as well as to increase analytical sensitivity and selectivity of the SFE/derivatization procedure. Acknowledgements The authors would like to thank the U.S. Environmental Protection Agency, E M S L , Cincinnati, for partial financial support. Instrument loans from ISCO are also gratefully acknowledged. References 1. 2. 3. 4. 5. 6. 7.

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Hawthorne, S. B . Anal. Chem. 1990, 62, 633A. Proceedings of the International Symposium on Supercritical Fluid Chromatography and Extraction; Park City, Utah, January 1991. Wheeler, J. R.; McNally, M. E . J. Chromatogr. Sci. 1989, 27, 534. Ramsey, E . D . ; Perkins, J. R.; Games, D . E . ; Startin, J. R . J. Chromatogr. 1989, 464, 353 L i , S. F. Y.; Ong, C. P.; Lee, M. L.; Lee, H. K . J. Chromatogr., 1990, 515, 515. Hawthorne, S. B.; Miller, D . J.; Walker, D . D . ; Whittington, D . E . ; Moore, B. L . J. Chromatogr. 1991, 541, 185. White, D . C.; Nivens, D . E.; Ringelberg, D . B.; Hedrick, D. B . Proceedings of the International Symposium on Supercritical Fluid Chromatography and Extraction; Park City, Utah, January 1991, p 43. Markell, C.; Hagen, D . F.; Bunnelle, V. A. LC-GC 1991, 9, 331.

R E C E I V E D December 2, 1991

Supercritical Fluid Technology - American Chemical Society

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