Organic analyte procedures are characterized

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1 1120 LCGC VOLUME 19 NUMBER 11 NOVEMBER Sample Prep Perspectives Review of EP Sample Preparation Techniques for Organic Compound nalysis of Liquid and Solid Samples Guest uthor 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 Environmental 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 gency (EP) 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 EP 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 ct. Furthermore, SW-846 outlines test procedures used to characterize solid waste in accordance with 40 Code of Federal Regulations (CFR) Part 261, dentification and Listing of Hazardous Waste. The sample preparation and analytical procedures or determinative steps are categorized by the analyte, either inorganic or organic. norganic 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). norganic analysts are concerned with approximately 30 analytes or elements for environmental analysis. Organic analyte procedures are characterized by 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) ( 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. s 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. n this report, 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. ll samples analyzed for nonvolatile or semivolatile organic compounds require a extraction step, with the exception of nonaqueous soluble samples. The -soluble samples use a simple dilution step, a so-called dilute-and-shoot method. Because both solid and liquid samples are injected as an extracted liquid, first will discuss sample preparation techniques for solid samples and later those for aqueous samples.

2 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 vanti automated extraction system. Shown are (a) the solubilization of extractable matter from sample immersed in boiling, (b) rinsing of extracted (similar to conventional Soxhlet extraction), and (c) concentration of the extracted sample by evaporation and collection of distilled for reuse or disposal. During evaporation, is blocked from returning to the extraction cup and flows into a collection tank. (Courtesy of Foss North merica, Eden Prairie, Minnesota.) Load sample into cell Fill cell with Heat and pressurize cell Hold sample at pressure and temperature Pump clean into sample cell Purge from cell with nitrogen gas Extract ready for analysis Solvent Pump Nitrogen method used to enhance the action of the for the extraction. They range from classic Soxhlet extraction to modern microwave extraction. For this discussion, will define a solid sample as clay, soil, sludge, sediment, or waste. Soxhlet extraction (EP Method 3540C): nalytical 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. t 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. Thus, the extraction is performed at temperatures below the s boiling point. n 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 h at 4 6 cycles/h. utomated Soxhlet extraction (EP 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. Then, the thimble is moved above the to mimic the rinse-extraction step of Soxhlet extraction. Finally, a concentration step using modern automated equipment reduces the final vol- Oven Collection vial Extraction cell Figure 2: Schematic diagram of a pressurized-fluid extraction system. (Courtesy of Dionex Corp., Sunnyvale, California.) Vent NOVEMBER 2001 LCGC VOLUME 19 NUMBER 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 at the s boiling point. t also reduces the consumption of. For more details about automated Soxhlet extraction, please see rment s review (1). Pressurized-fluid extraction (EP Method 3545): Pressurized-fluid extraction is one of the latest technologies to be approved for solid-sample extraction. The method performs extractions at elevated temperatures and pressures to achieve performance comparable to the Soxhlet technique with a significant reduction in time and consumption. The instrumentation to perform pressurized-fluid extraction, more commonly known by its trade name of accelerated extraction, is semiautomated (see Figure 2). fter 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 ; the extracting 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. n pressurized-fluid extraction, the sample is diluted by the volume of extraction and must be concentrated before analysis. For more details about the pressurized-fluid extraction technique, please see the review by Richter (3). Microwave extraction (EP 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- mixtures in a sealed (closed) vessel with microwave energy under temperature-controlled conditions. lthough 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

3 1124 LCGC VOLUME 19 NUMBER 11 NOVEMBER the, 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 min per batch. Solvent consumption is only ml per sample. fter 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 (EP 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 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 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 (EP 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 (PHs), 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. n 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. n 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. n 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 or sorbent material. For more information about SFE, consult reference 5. Comparison of solid sample extraction techniques: Table compares the EP sample preparation methods for solid samples. t compares the above techniques with regard to use, extraction time, acquisition costs, and operating costs. Clearly, the modern techniques provide more rapid extraction with a minimal amount of organic required. However, some of them are expensive compared with the classic methods. queous samples: Several extraction techniques for the analysis of non- and semivolatile organic compounds in a liquid state are available under SW Separatory funnel liquid liquid extraction, continuous liquid liquid extraction, and solid-phase extraction (SPE) techniques most often are used for liquid matrices. The s 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 (EP 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 Microwave energy Vessel support module Seal cover Liner Sample and Figure 3: Sample and in a GreenChem extraction vessel. Contents are rapidly heated to elevated temperatures and pressures using microwave energy. (Courtesy of CEM Corp., Matthews, North Carolina.) Liquid carbon dioxide Cryogenic zone 1 Extraction Chamber with sample Carbon dioxide pump Restrictor Cryogenic zone 2 Preheat Expansion mpinged surface or region Collection Reconstitution Figure 4: Schematic diagram of a generic SFE system showing four cryogenic zones. (Courtesy of gilent Technologies, Wilmington, Delaware.)

4 NOVEMBER 2001 LCGC VOLUME 19 NUMBER Table : Comparison of 3500 series extraction techniques for solid samples* verage Solvent Use verage Extraction Operating Cost EP Method Number Extraction Technique (ml/sample) Time (min/sample) cquisition Cost per Sample 3540B Soxhlet Very low Very high 3541 utomated Soxhlet Moderate Low to moderate 3545 Pressurized-fluid extraction High Low 3546 Microwave-accelerated extraction Moderate Low 3550C Ultrasonic nebulization Low High 3560, 3561, and 3562 SFE 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 s. SFE is limited to the analysis of total recoverable hydrocarbons, PHs, organochlorine pesticides, and PCBs. volatile organic compounds. n aqueous sample is mixed in a separatory funnel with an immiscible organic that is denser than water. fter standing, the mixture will separate into two phases with the analytes partitioning toward the organic phase. The is drawn off and saved, and the extraction step is repeated multiple times. The extracts are combined for the analytical step. For a basic discussion of liquid liquid extraction, please see reference 6. Continuous liquid liquid extraction (EP 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 is added to the top of a liquid liquid extractor, which contains the aqueous sample. The extracts the analytes as it passes through the sample. The extract is collected in a boiling flask and distilled, and fresh is sent to the top of the extractor to create a continuous process. This process runs for 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. Solid-phase extraction (EP 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. s 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 to activate it. n 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- Circle 16

5 1126 LCGC VOLUME 19 NUMBER 11 NOVEMBER bilizing 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. (a) (b) Solute solution Condenser Condenser Condensed Concentrated solutes Heating mantle Condensed This technique s benefits are that it significantly reduces extraction times and 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 fter 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 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. 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. Fresh washes the solid sample on the filter to ensure all the analyte goes into the collected. Two or three wash steps can be used with minimal 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 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. 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 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. nother 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 to a 1 2 ml volume and then making it up to a 5-mL volume in a volumetric flask or GC HPLC vials. utomatic 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 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 to extract the analytes from the matrix and remove extraneous analytes. Sometimes, a polar Solute solution (a) (b) (c) (d) Concentrated solutes SPE cartridge Conditioning Sample nalytes Washing Eluting Heating mantle Figure 5: Schematics of a continuous liquid liquid extraction system in which the extraction is (a) less dense and (b) more dense than the solution from which the solute is being extracted. Collection reservoir 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. nterference nalytes

6 1128 LCGC VOLUME 19 NUMBER 11 NOVEMBER 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 ). 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. dsorption chromatography: This technique is used to separate analytes of a relatively narrow polarity range from interfering peaks of different polarity. dsorption chromatography is used primarily for the cleanup of nonpolar compounds such as organochlorine pesticides and PHs. n 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 : 3600 series cleanup method summary EP Method Method Name Number (Technique) Objective Procedure Comments 3610B lumina cleanup To separate analytes from Elute sample through basic- to Suitable for extracts that contain (adsorption interfering compounds of neutral-ph alumina with nitrosamines and phthalate chromatography) different polarity suitable s to leave esters interfering compounds on the column 3611B lumina column To separate petroleum Elute sample through neutral-ph Not recommended for extracting cleanup and separation waste extracts into base alumina with suitable s petroleum wastes with of petroleum wastes neutral aliphatic, to leave interfering compounds predominantly polar s; (adsorption aromatic, and polar on the column perform acid base partition chromatography) fractions cleanup on extract before alumina cleanup 3620C Florisil cleanup* To separate analytes from Elute extract through Florisil to Suitable for extracts that contain (adsorption interfering compounds of leave interfering compounds on aniline and its derivatives, chromatography) different polarity or the column or cartridge or to chlorinated hydrocarbons, fractionate groups of fractionate target compounds haloethers, nitroaromatics, target compounds nitrosamines, organochlorine and organophosphorus pesticides, organophosphates, PCBs, and phthalate esters 3630C Silica-gel cleanup To separate analytes from Elute extract through silica gel to Primary use is for extracts that (adsorption interfering compounds of leave interfering compounds on contain PHs, derivatized chromatography) different polarity the column or cartridge phenolic compounds, organochlorine pesticides, and PCBs 3640 Size separation (size- To remove high molecular Elute extract through column Universal technique for exclusion weight, high-boiling-point packed with hydrophobic gels of semivolatile organic compounds chromatography) materials from target varying pore sizes to separate its and pesticides analytes components by molecular weight 3650B cid base partition To separate acid analytes Mix extract with methylene Useful for separating neutral PHs cleanup (liquid liquid from base to neutral chloride and water at ph from acidic phenols; base neutral partitioning) analytes in petroleum in separatory funnel; separate fraction may require an alumina waste extracts aqueous (acidic) and organic column cleanup before analysis (base to neutral) fractions 3660B Sulfur cleanup To eliminate sulfur from an Mix sample with either copper or Sulfur has solubility characteristics (oxidation and extract and prevent the tetrabutylammonium sulfite, similar to organochlorine and reduction) masking of organochlorine shake, and separate the sample organophosphorus pesticides; pesticides and organo- from the sulfur cleanup reagent typically used for sediment, phosphorus pesticides in marine algae, and industrial GC analysis waste samples 3665 Sulfuric acid To decompose organic Exchange extracting with Decomposes most other organic permanganate cleanup compounds that cause hexane, sequentially treat with chemicals, so it is not applicable (oxidation and baseline elevation or 98% sulfuric acid and, if for other target analytes reduction) complex chromatograms necessary, 5% potassium and prevent the accurate permanganate quantitation of PCBs * Florisil is magnesium silicate with basic properties. Sulfuric acid with sodium silicate.

7 1130 LCGC VOLUME 19 NUMBER 11 NOVEMBER 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. cid base partitioning: This technique is used to separate neutral PHs from the acidic PHs that can appear in petroleum waste samples. cid 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. n this Sample Prep Perspectives column, attempted to review the extraction and cleanup techniques available to analysts according to SW-846 requirements for nonvolatile and semivolatile organic compounds. 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 consumption. References (1) S. rment, Current Trends and Developments in Sample Preparation, LCGC 17(6S), S38 S42 (2) R.E. Majors, Current Trends and Developments in Sample Preparation, LCGC 17(6S), S8 S13 (3) B.E. Richter, Current Trends and Developments in Sample Preparation, LCGC 17(6S), S22 S28 (4) G. LeBlanc, Current Trends and Developments in Sample Preparation, LCGC 17(6S), S30 S37 (5) J.M. Levy, Current Trends and Developments in Sample Preparation, LCGC 17(6S), S14 S21 (6) R.E. Majors, LCGC 14(11), (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 , greg.leblanc@cem.com. Circle 19 Ronald E. Majors Sample Prep Perspectives editor Ronald E. Majors is business development manager, consumables and accessories business unit, gilent 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, lcgcedit@ lcgcmag.com.