1120 LCGC VOLUME 19 NUMBER 11 NOVEMBER 2001 Sample Prep Perspectives Guest Author Greg LeBlanc Collecting, preserving, and preparing samples are critical to producing accurate and reliable results in the analysis of organic compounds. This “Sample Prep Perspectives” column reviews sample preparation techniques that are available to organic laboratories under SW-846 regulations for the analysis of nonand semivolatile compounds from environmental samples. Ronald E. Majors Sample Prep Perspectives Editor www.chromatographyonline.com A Review of EPA Sample Preparation Techniques for Organic Compound Analysis of Liquid and Solid Samples E nvironmental analysis often involves analytes in a wide variety of matrices, ranging from air to sewage water to polluted soil samples. Proper sample preparation procedures are necessary to achieve optimum analytical results. The U.S. Environmental Protection Agency (EPA) is the government body responsible for the definition, development, and enforcement of analytical measurements for specific pollutants that are deemed to be harmful to the environment. The EPA’s Test Methods for Evaluating Solid Waste — SW-846 — provides a comprehensive source of information about sampling, sample preparation, analysis, and reporting for compliance with the Resource Conservation and Recovery Act. Furthermore, SW-846 outlines test procedures used to characterize solid waste in accordance with 40 Code of Federal Regulations (CFR) Part 261, Identification and Listing of Hazardous Waste. The sample preparation and analytical procedures or determinative steps are categorized by the analyte, either inorganic or organic. Inorganic analyte procedures are characterized by acid digestion steps using conventional or microwave heating (3000 series methods), followed by atomic absorption or emission spectroscopy (6000 and 7000 series methods). Inorganic analysts are concerned with approximately 30 analytes or elements for environmental analysis. Organic analyte procedures are characterized by solvent extraction steps for nonvolatile and semivolatile analytes (3500 series methods) and postextraction cleanup (3600 series methods). Sample preparation methods for volatile compounds define methodologies such as purge-and-trap, distillation, headspace, or dilution in the 5000 series methods. The analytical steps are gas chromatography (GC) (8000–8200 series methods), high performance liquid chromatography (HPLC) (8300 series methods), and GC–Fourier transform infrared (8400 series methods). Organic analysts are concerned with more than 450 analytes. Compared with inorganic analytical laboratories, organic laboratories face a major challenge to cost-effectively prepare and analyze the wide variety of analytes from environmental samples. As with any process, the primary focus is on the determinative step that produces the result. However, the front-end work of sampling, preservation, and sample preparation is critical to producing accurate and reliable results. In this report, I will review the sample preparation techniques — 3500 and 3600 series methods — that are available to organic laboratories under SW-846 for the analysis of non- and semivolatile compounds from environmental samples. The 3500 series methods cover the extraction steps, and the 3600 series methods include the cleanup steps. Extraction Techniques The sample matrix and analytes define the 3500 series sample extraction methods. The matrix is aqueous, solid, an air sampling train, or nonaqueous soluble. The analytes are characterized as either non- or semivolatile organic compounds. All samples analyzed for nonvolatile or semivolatile organic compounds require a solvent extraction step, with the exception of nonaqueous solvent–soluble samples. The solvent-soluble samples use a simple solvent dilution step, a so-called dilute-and-shoot method. Because both solid and liquid samples are injected as an extracted liquid, I first will discuss sample preparation techniques for solid samples and later those for aqueous samples. NOVEMBER 2001 LCGC VOLUME 19 NUMBER 11 www.chromatographyonline.com Solid samples: The technologies used for the extraction of non- and semivolatile organic compounds from solid samples are more diverse than for water and other liquid samples. The techniques vary by the (a) (b) (c) Figure 1: Three-step extraction procedure using the Foss-Tecator Soxtec Avanti automated extraction system. Shown are (a) the solubilization of extractable matter from sample immersed in boiling solvent, (b) rinsing of extracted solvent (similar to conventional Soxhlet extraction), and (c) concentration of the extracted sample by evaporation and collection of distilled solvent for reuse or disposal. During evaporation, solvent is blocked from returning to the extraction cup and flows into a collection tank. (Courtesy of Foss North America, Eden Prairie, Minnesota.) method used to enhance the action of the solvent for the extraction. They range from classic Soxhlet extraction to modern microwave extraction. For this discussion, I will define a solid sample as clay, soil, sludge, sediment, or waste. Soxhlet extraction (EPA Method 3540C): Analytical chemists have used Soxhlet extraction for more than 100 years (1). This method is the classic approach to extracting solid samples for a spectrum of non- and semivolatile organic compounds. It works in a manner analogous to continuous liquid–liquid extraction, except the sample is solid instead of liquid. The sample, held in a porous cellulose thimble, is extracted continuously with a fresh aliquot of distilled and condensed solvent. Thus, the extraction is performed at temperatures below the solvent’s boiling point. In practice, the method is simple to perform. The technique is time consuming but can be automated, and it has a low acquisition cost. Typically, the extraction step requires 16–24 h at 4–6 cycles/h. Automated Soxhlet extraction (EPA Method 3541): This technique is an automated version of the classic Soxhlet approach to extracting solid samples, with two modifications (Figure 1). This approach initially immerses the thimble that contains the sample directly into the boiling solvent. Then, the thimble is moved above the solvent to mimic the rinse-extraction step of Soxhlet extraction. Finally, a concentration step using modern automated equipment reduces the final vol- Load sample into cell Fill cell with solvent Pump Heat and pressurize cell Hold sample at pressure and temperature Pump clean solvent into sample cell Purge solvent from cell with nitrogen gas Solvent Oven Extraction cell Vent Nitrogen Collection vial Extract ready for analysis Figure 2: Schematic diagram of a pressurized-fluid extraction system. (Courtesy of Dionex Corp., Sunnyvale, California.) 1123 ume to 1–2 mL. This three-stage approach shortens the extraction step to 2 h, because it provides direct contact between the sample and solvent at the solvent’s boiling point. It also reduces the consumption of solvent. For more details about automated Soxhlet extraction, please see Arment’s review (1). Pressurized-fluid extraction (EPA Method 3545A): Pressurized-fluid extraction is one of the latest technologies to be approved for solid-sample extraction. The method performs extractions at elevated solvent temperatures and pressures to achieve performance comparable to the Soxhlet technique with a significant reduction in time and solvent consumption. The instrumentation to perform pressurized-fluid extraction, more commonly known by its trade name of accelerated solvent extraction, is semiautomated (see Figure 2). After loading a sample into the extraction cell and sealing it, the instrument performs the extraction, separation, and collection steps automatically. Samples are processed sequentially in batches of as many as 24 samples. Equipment is available that will perform the extraction of six samples simultaneously (2). The principle of pressurized-fluid extraction is simple. The sample (or a sample mixed with a drying agent) is loaded into a high-pressure, high-temperature extraction cell, which is sealed. The cell is heated to the extraction temperature, which often is two- to threefold the atmospheric boiling point of the solvent; the extracting solvent is added and held in contact with the sample for 5–10 min; the extract then is flushed from the cell into the collection vessel with a volume equal to 60–75% of the cell volume; and finally the extract is purged with nitrogen. In pressurized-fluid extraction, the sample is diluted by the volume of extraction solvent and must be concentrated before analysis. For more details about the pressurized-fluid extraction technique, please see the review by Richter (3). Microwave extraction (EPA Method 3546): Microwave extraction is the latest technique to be included in SW-846. The microwave extraction method is the process of heating solid sample-solvent mixtures in a sealed (closed) vessel with microwave energy under temperature-controlled conditions. Although used less frequently, the extraction also can be performed in an open vessel at atmospheric pressure. Figure 3 depicts a typical microwave extraction cell used in a closed extraction system. This system provides significant temperature elevation above the atmospheric boiling point of 1124 LCGC VOLUME 19 NUMBER 11 NOVEMBER 2001 www.chromatographyonline.com the solvent, accelerates the extraction process, and yields performance comparable to the standard Soxhlet method. Samples are processed in batches of as many as 14 samples per run. The microwave energy provides very rapid heating of the sample batch to the elevated temperatures, which shortens the extraction time to 10–20 min per batch. Solvent consumption is only 25–50 mL per sample. After the heating cycle is complete, the samples are cooled and the sample is filtered to separate the sample from the extract for the analytical step. The technique was reviewed in LCGC (4). Ultrasonic extraction (EPA Method 3550C): This method uses mechanical energy in the form of a shearing action, which is produced by a low-frequency sound wave. The sample is immersed in an ultrasonic bath with solvent and subjected to ultrasonic radiation for 2–3 min. The sample is separated from the extract by vacuum filtration or centrifugation. The process is repeated 2–3 times, and the extracts are combined for the analytical step. This technique has the benefit of shortened extraction times, but it sacrifices performance relative to the Soxhlet technique. The solvent receives only minor heating of a few degrees above room temperature and, thus, cannot provide as thorough extraction of difficult matrices such as aged soil samples. Supercritical fluid extraction (EPA Methods 3560, 3561, and 3562): These three methods use supercritical carbon dioxide or carbon dioxide with a modifier to extract total recoverable hydrocarbons, polycyclic aromatic hydrocarbons (PAHs), polychlorinated biphenyls (PCBs), and organochlorine pesticides. Supercritical carbon dioxide or carbon dioxide–organic modifier extracts the sample, which is held in an extraction vessel within a closed system. Supercritical fluids such as carbon dioxide have properties of both liquids and gases, which make them desirable for extraction. When its temperature and pressure are controlled, carbon dioxide has the penetrating characteristics of gases and the solvating properties of liquids. An organic modifier such as methanol, acetonitrile, or isopropanol can be used to assist the extraction of polar analytes. The primary operating parameters are the carbon dioxide or carbon dioxide–modifier flow rates, temperature, pressure, and dynamic or static mode of extraction. Figure 4 shows a schematic of a typical supercritical fluid extraction (SFE) system. In the static mode, the extraction cell fills the extraction vessel with the supercritical fluid and holds it in the vessel for a specified period of time. In the dynamic mode, the supercritical fluid passes through the extraction vessel continuously. The depressurized carbon dioxide or carbon dioxide– modifier exits the system, and the target compounds are collected in a vessel that contains a suitable solvent or sorbent material. For more information about SFE, consult reference 5. Comparison of solid sample extraction techniques: Table I compares the EPA sample preparation methods for solid samples. It compares the above techniques with regard to solvent use, extraction time, acquisition costs, and operating costs. Clearly, the modern techniques provide more rapid extraction with a minimal amount of organic solvent required. However, some of them are expensive compared with the classic methods. Aqueous samples: Several solvent extraction techniques for the analysis of non- and semivolatile organic compounds in a liquid state are available under SW846. Separatory funnel liquid–liquid extraction, continuous liquid–liquid extraction, and solid-phase extraction (SPE) techniques most often are used for liquid matrices. The solvents used for liquid–liquid extraction techniques are insoluble in the aqueous sample. The techniques are applicable for the extraction of water-insoluble and slightly water-soluble organic compounds. Separatory funnel liquid–liquid extraction (EPA Method 3510C): This technique is a classic approach to extraction for liquid samples for a spectrum of non- and semi- Temperature probe Cryogenic zone 4 Cryogenic zone 3 Restrictor Seal cover Liner Chamber with sample Cryogenic zone 2 Cryogenic zone 1 Impinged surface or region Preheat Microwave energy Carbon dioxide pump Sample and solvent Liquid carbon dioxide Vessel support module Extraction Figure 3: Sample and solvent in a GreenChem extraction vessel. Contents are rapidly heated to elevated temperatures and pressures using microwave energy. (Courtesy of CEM Corp., Matthews, North Carolina.) Expansion Collection Reconstitution Figure 4: Schematic diagram of a generic SFE system showing four cryogenic zones. (Courtesy of Agilent Technologies, Wilmington, Delaware.) NOVEMBER 2001 LCGC VOLUME 19 NUMBER 11 www.chromatographyonline.com 1125 Table I: Comparison of 3500 series extraction techniques for solid samples* EPA Method Number Extraction Technique 3540B 3541 3545A 3546 3550C 3560, 3561, and 3562† Soxhlet Automated Soxhlet Pressurized-fluid extraction Microwave-accelerated extraction Ultrasonic nebulization SFE Average Solvent Use (mL/sample) Average Extraction Time (min/sample) 300 50 10–30 25–40 300 10 960–1440 120 10–15 10–20 30 20–50 Acquisition Cost Operating Cost per Sample Very low Very high Moderate Low to moderate High Low Moderate Low Low High Moderate to high Moderate to high * Examples of solid samples include soils, sediments, fly ashes, sludges, and solid wastes that are amenable to extraction with conventional solvents. † SFE is limited to the analysis of total recoverable hydrocarbons, PAHs, organochlorine pesticides, and PCBs. volatile organic compounds. An aqueous sample is mixed in a separatory funnel with an immiscible organic solvent that is denser than water. After standing, the mixture will separate into two phases with the analytes partitioning toward the organic phase. The solvent is drawn off and saved, and the extraction step is repeated multiple times. The solvent extracts are combined for the analytical step. For a basic discussion of liquid–liquid extraction, please see reference 6. Continuous liquid–liquid extraction (EPA Method 3520C): This technique is an automated version of the separatory funnel technique for a spectrum of non- and semivolatile organic compounds. Figure 5 illustrates the construction principles of two types of continuous liquid–liquid extraction systems. The solvent is added to the top of a liquid–liquid extractor, which contains the aqueous sample. The solvent extracts the analytes as it passes through the sample. The extract is collected in a boiling flask and distilled, and fresh solvent is sent to the top of the extractor to create a continuous process. This process runs for 12–24 h, and it is used in situations in which large sample sizes with low analyte concentrations are needed. The extract contained in the boiling flask is used for the analytical step. Circle 16 Solid-phase extraction (EPA Method 3535C): This extraction technique for aqueous samples is the latest to be added to the SW-846 manual, and it involves the most recent advances in technology. SPE isolates analytes using the same principles as those used in liquid chromatography, though much less efficiently. As Figure 6 depicts, in SPE, compounds are retained and eluted as a mobile phase transports them over a stationary phase (sorbent) that has been conditioned with an organic solvent to activate it. In the most common use of SPE, the mobile phase is the aqueous sample to retain the analytes onto the sorbent. This step is followed with a solu- 1126 LCGC VOLUME 19 NUMBER 11 NOVEMBER 2001 bilizing solvent as the mobile phase to elute the analytes, which are collected for analysis. The SPE packing is housed in a cartridge or disk. The cartridge is a disposable syringe with a frit on each end of the packing, and the disk is a membrane filter. The smaller length-to-diameter ratio of a disk allows greater flow and extraction rates relative to the cartridges. www.chromatographyonline.com This technique’s benefits are that it significantly reduces extraction times and solvent consumption and has a concentration step. With automated filtering systems, multiple samples can be processed simultaneously. The downside of SPE is the cost of the cartridges and disks. For a series of reviews of various aspects of SPE, please see reference 7. Postextraction Handling and Cleanup (a) Condenser Condensed solvent Concentrated solutes Solute solution Heating mantle (b) Condenser Condensed solvent After the extraction step, chemists rarely perform analysis directly without further sample handling. Postextraction handling includes steps as simple as sample–extract separation, water removal, and solvent exchange or a more involved, multiplestep cleanup. The cleanup methods are designed to remove interferences that cause poor analytical results and increased analytical instrument downtime. Postextraction handling steps are dependent upon the matrix, the analytes of interest, and the solvent. Sample–extract separation: The objective is to separate the original matrix from the extract. Two approaches are available: filtration and centrifugation. Filtration: The sample–extract mixture is passed through a filter to remove the solid sample from the solvent. Fresh solvent washes the solid sample on the filter to ensure all the analyte goes into the collected solvent. Two or three wash steps can be used with minimal solvent to prevent further dilution. Centrifugation: The sample–extract mixture is centrifuged, and the extract is decanted and removed. The residual sample is washed two or three times with minimal solvent to prevent further dilution. Water removal: Water is extremely polar and will adversely affect most column packing materials, especially GC stationary phases and some normal-phase HPLC packings. Therefore, analysts should remove water from the extract before injecting it into the analyzer. A common technique to remove any water from the sample or extract is to pass it over anhydrous sodium sulfate. The sodium sulfate is a water scavenger, and it will dry the sample solvent without absorbing any of the analyte of interest. Sodium sulfate water removal usually is performed in conjunction with a filtration step. The sodium sulfate is added to the filter before filtering the sample–extract mixture. Another approach is to mix and swirl the sodium sulfate with the mixture solution before filtration. Solvent concentration: This technique concentrates the analyte of interest, so the analytical signal intensity is increased. This task is performed by evaporating the solvent to a 1–2 mL volume and then making it up to a 5-mL volume in a volumetric flask or GC–HPLC vials. Automatic solvent concentration systems are commercially available. Solvent exchange: This technique separates the extracted molecules by their polarity to eliminate extraneous peaks in subsequent analysis or to move the analytes to a different solvent that is more compatible with the subsequent analytical technique. Solvent exchange is performed as a liquid–liquid extraction in a separatory funnel. This step is one most analysts would prefer to avoid. However, they may need an aggressive solvent to extract the analytes from the matrix and remove extraneous analytes. Sometimes, a polar solvent Solute solution (a) Conditioning solvent Concentrated solutes A A A I I A I IA I A I AI I I IA I IAA IA I AI A I A A AA (c) Collection reservoir (d) Sample Washing solvent Analytes SPE cartridge Heating mantle (b) A A AA A A AA A A A AAA A A AA I II I II I I II I I I I I I II II I I I I Interference Figure 5: Schematics of a continuous liquid– liquid extraction system in which the extraction solvent is (a) less dense and (b) more dense than the solution from which the solute is being extracted. Eluting solvent A A A AA A A AAAA A A Analytes Figure 6: Steps in an SPE experiment: (a) sorbent conditioning; (b) sample loading; (c) washing, in which the analytes are retained and the interferences are washed into the collection reservoir; and (d) elution, in which the analytes are eluted with a strong solvent. 1128 LCGC VOLUME 19 NUMBER 11 NOVEMBER 2001 www.chromatographyonline.com is necessary to remove the analytes of interest that can not be used in the chromatographic analysis. The cleanup methods are covered by the 3600 series methods of SW-846 (see Table II). They include adsorption chromatography to separate compounds based on differences in polarity, gel-permeation chromatography to remove interferences with high molecular weights or high boiling points, acid–base partitioning to separate acidic or basic organic compounds from neutral ones, and oxidation of interfering components with acids, alkalis, and oxidizing agents. Adsorption chromatography: This technique is used to separate analytes of a relatively narrow polarity range from interfering peaks of different polarity. Adsorption chromatography is used primarily for the cleanup of nonpolar compounds such as organochlorine pesticides and PAHs. In addition to removing interferences, adsorption chromatography can be used to fractionate complex mixtures of analytes. Gel-permeation chromatography: This technique is used to remove high molecular weight or high-boiling-point interferences from the target compounds. High molecular weight compounds can contami- Table II: 3600 series cleanup method summary EPA Method Method Name Number (Technique) Objective Procedure Comments 3610B Alumina cleanup (adsorption chromatography) To separate analytes from interfering compounds of different polarity Elute sample through basic- to neutral-pH alumina with suitable solvents to leave interfering compounds on the column Suitable for extracts that contain nitrosamines and phthalate esters 3611B Alumina column cleanup and separation of petroleum wastes (adsorption chromatography) To separate petroleum waste extracts into base– neutral aliphatic, aromatic, and polar fractions Elute sample through neutral-pH alumina with suitable solvents to leave interfering compounds on the column Not recommended for extracting petroleum wastes with predominantly polar solvents; perform acid–base partition cleanup on extract before alumina cleanup 3620C Florisil cleanup* (adsorption chromatography) To separate analytes from interfering compounds of different polarity or fractionate groups of target compounds Elute extract through Florisil to leave interfering compounds on the column or cartridge or to fractionate target compounds Suitable for extracts that contain aniline and its derivatives, chlorinated hydrocarbons, haloethers, nitroaromatics, nitrosamines, organochlorine and organophosphorus pesticides, organophosphates, PCBs, and phthalate esters 3630C Silica-gel cleanup† (adsorption chromatography) To separate analytes from interfering compounds of different polarity Elute extract through silica gel to leave interfering compounds on the column or cartridge Primary use is for extracts that contain PAHs, derivatized phenolic compounds, organochlorine pesticides, and PCBs 3640A Size separation (sizeexclusion chromatography) To remove high molecular weight, high-boiling-point materials from target analytes Elute extract through column packed with hydrophobic gels of varying pore sizes to separate its components by molecular weight Universal technique for semivolatile organic compounds and pesticides 3650B Acid–base partition cleanup (liquid–liquid partitioning) To separate acid analytes from base to neutral analytes in petroleum waste extracts Mix extract with methylene chloride and water at pH 12–13 in separatory funnel; separate aqueous (acidic) and organic (base to neutral) fractions Useful for separating neutral PAHs from acidic phenols; base–neutral fraction may require an alumina column cleanup before analysis 3660B Sulfur cleanup (oxidation and reduction) To eliminate sulfur from an extract and prevent the masking of organochlorine pesticides and organophosphorus pesticides in GC analysis Mix sample with either copper or tetrabutylammonium sulfite, shake, and separate the sample from the sulfur cleanup reagent Sulfur has solubility characteristics similar to organochlorine and organophosphorus pesticides; typically used for sediment, marine algae, and industrial waste samples 3665A Sulfuric acid– permanganate cleanup (oxidation and reduction) To decompose organic compounds that cause baseline elevation or complex chromatograms and prevent the accurate quantitation of PCBs Exchange extracting solvent with hexane, sequentially treat with 98% sulfuric acid and, if necessary, 5% potassium permanganate Decomposes most other organic chemicals, so it is not applicable for other target analytes * Florisil is magnesium silicate with basic properties. † Sulfuric acid with sodium silicate. 1130 LCGC VOLUME 19 NUMBER 11 NOVEMBER 2001 www.chromatographyonline.com nate HPLC columns and be difficult to remove by washing. High-boiling-point compounds can contaminate GC injection ports and column heads, thus requiring more instrument maintenance. Gelpermeation chromatography, also known as size-exclusion chromatography, is the most universal cleanup method for semivolatile organic compounds and pesticides. Acid–base partitioning: This technique is used to separate neutral PAHs from the acidic PAHs that can appear in petroleum waste samples. Acid–base partitioning also can be used to fractionate base–neutral compounds. Oxidation of interfering components: Copper or tetrabutylammonium sulfite is used to eliminate the sulfur contamination that can mask pesticide peaks in certain GC detectors. Sulfuric acid and potassium permanganate are used to oxidize organic compounds that cause interferences for PCB analysis. Oxidation is a very rigorous but nonspecific technique. Summary Sample preparation is a critical step in the overall process of obtaining reliable and accurate data, especially in the environmental analysis of nonvolatile and semivolatile organic compounds. Extraction techniques are devised to remove a spectrum of compounds. This removal requires subsequent handling and cleanup of the extract before analytical measurement. In this “Sample Prep Perspectives” column, I attempted to review the extraction and cleanup techniques available to analysts according to SW-846 requirements for nonvolatile and semivolatile organic compounds. I have observed a noticeable improvement in sample preparation capabilities with SW-846’s inclusion of extraction techniques such as SPE for aqueous samples and pressurized-fluid extraction, SFE, and microwave extraction for solid samples. These newer methods reduce extraction times and solvent consumption. References (1) S. Arment, Current Trends and Developments in Sample Preparation, LCGC 17(6S), S38–S42 (1999). (2) R.E. Majors, Current Trends and Developments in Sample Preparation, LCGC 17(6S), S8–S13 (1999). (3) B.E. Richter, Current Trends and Developments in Sample Preparation, LCGC 17(6S), S22–S28 (1999). (4) G. LeBlanc, Current Trends and Developments in Sample Preparation, LCGC 17(6S), S30–S37 (1999). (5) J.M. Levy, Current Trends and Developments in Sample Preparation, LCGC 17(6S), S14–S21 (1999). (6) R.E. Majors, LCGC 14(11), 936–943 (1996). (7) R.E. Majors, Current Trends and Developments in Sample Preparation, LCGC May 1998, S8–S15 (1998). Greg LeBlanc is the new business development manager at CEM Corp., P.O. Box 200, Matthews, NC 28106-0200, e-mail greg.leblanc@cem.com. Ronald E. Majors “Sample Prep Perspectives” editor Ronald E. Majors is business development manager, consumables and accessories business unit, Agilent Technologies, Wilmington, Delaware, and is a member of LCGC’s editorial advisory board. Direct correspondence about this column to “Sample Prep Perspectives,” LCGC, 859 Willamette Street, Eugene, OR 97401, e-mail lcgcedit@ lcgcmag.com. Circle 19
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