F. W. Karasek R. E. Clement J. A. Sweetman Chemistry Department University of Waterloo Waterloo, Ontario N2L 3G1 Canada
Preconcentration for Trace To successfully conduct many analyses for organic compounds present at trace concentrations in gas, liquid, or solid samples, it is necessary to selectively extract these compounds and use a concentration step prior to analysis. Many of the techniques developed for preconcentration are described in specialized books and reviews (1, 2); a number appear in works on headspace analysis (3). Because of the diversity of this subject, it was felt that an exhaustive review and treatment of all these techniques would not be manageable in this REPORT. Therefore, our discussion will be confined primarily to methods we have found workable and useful for analysis of trace organic constituents in environmental samples. A complete scheme for trace analysis of organic compounds generally consists of sampling, extraction, preconcentration, prefractionation, and analysis by gas chromatography (GC), and gas chromatography/mass spectrometry (GC/MS). Determination of organics at parts-per-trillion (ppt) levels can be performed by combining sensitive and selective detection with sample preconcentration. In many instances, limits of detection can be made arbitrarily small by increasing the degree of preconcentration. The preconcentration step is often an integral part of the sampling and extraction procedure. For example, in the standard method for analysis of atmospheric airborne particulate matter, large volumes of air are drawn through a filter to obtain a sample. This represents simultaneous sampling and preconcentration. After extracting the
sorbed organics from the collected particulates, a second preconcentration step is performed before analysis. If interfering components must be removed from the sample matrix, one or more prefractionation steps may be required in the procedure. Preconcentration procedures are especially important for samples derived from the environment. Many toxic or carcinogenic compounds are distributed in the environment from a wide variety of sources. Because of their serious effects at low levels, it is necessary to achieve the greatest analytical sensitivities possible for these substances to properly assess their environmental impact. In some cases, detection limits as low as a few ppt are required. In addition to the polynuclear aromatic hydrocarbons (PAHs), much attention has been focused on many halogenated pollutants such as pesticides, polychlorinated biphenyls (PCBs), trihalomethanes (THMs), and the polychlorinated dibenzofurans (PCDFs) and dibenzo-p-dioxins (PCDDs). Such substances can be found in air, water, and solid and biological samples. Although the extraction and preconcentration procedures may vary for different sample types, problems of sample losses, contamination, interferences, and reproducibility must be considered for all preconcentration methods. Most preconcentration techniques fall into two classes: solvent extraction followed by solvent reduction, or sorbent trapping with subsequent solvent elution or thermal desorption. There are many variations of these methods, and they are frequently used in combination.
1050 A · ANALYTICAL CHEMISTRY, VOL. 53, NO. 9, AUGUST 1981
Solvent Extraction and Preconcentration Sample preconcentration by solvent reduction is most frequently performed following the extraction of solid and biological samples by liquid partition. Proper choice of the extracting solvent can often be the critical step in the sample preparation procedure. It is not always correct to assume that a solvent that efficiently removes a compound from one sample matrix will recover the same compound from a different sample type. This was recently demonstrated by a study that compared the relative efficiencies of various solvents for extraction of organic compounds from municipal incinerator fly ash (4). Methanol, which had been found to have a very high extraction efficiency for organics on airborne particulate matter (5), demonstrated very poor recoveries for organics from fly ash using Soxhlet extraction. Figure 1 illustrates the poor recoveries for the tetrachlorinated dibenzo-p- dioxins (TCDDs) from-municipal incinerator fly ash using methanol. The top trace of Figure 1 is a GC/MS selected ion monitoring (SIM) analysis of a methanol extract from fly ash using the characteristic 321.9 m/z ion of the TCDD isomers. The bottom trace shows results obtained for the same fly ash sample by following the methanol extraction with a benzene extraction. Both extractions were accomplished using a Soxhlet apparatus for 20 cycles with 200 mL of each solvent. This clearly illustrates that no preconcentration step can give adequate re0003-2700/81/A351-1050$01.00/0 © 1981 American Chemical Society
Report
Analysis of Organic Compounds suits for quantitative work unless the initial extraction technique gives high, or at least known and reproducible, recoveries of the desired compounds from the initial sample. Prior to the GC/MS analyses (shown in Figure 1) of the benzene and methanol extracts from fly ash, each extract was concentrated by a factor of 2000 by removing solvent using a rotary evaporation apparatus. Figure 2 shows one such unit, al though several other types are avail able commercially. Solvent containing the extracted compounds is rotated in a flask partially submerged in a water bath. The water bath can be main tained at temperatures lower than the normal boiling point of the solvent by reducing the internal pressure of the rotary evaporator using aspirator suc tion. Volatile components will be largely lost in this procedure. How ever, for many applications the com pounds of interest are nonvolatile compared to the extracting solvent. In these applications the rotary evapora tion technique is rapid and straight forward, although severe losses of even nonvolatiles can be experienced with out careful sample handling. Table I shows the recoveries of components of a standard mixture that was spiked into 200 mL of benzene solvent and then reduced to its original volume (100 ^L) by rotary evaporation. Losses of these magnitudes are not uncom mon when reducing sample sizes to such small volumes. A major problem exists in the physical handling of the sample. Very small volume losses to the glass walls of the recovery flask or to the disposable glass pipets com-
(a)
(b)
Time (min) Figure 1. Selected ion monitoring analysis of tetrachlorodibenzo-p-dioxins in fly ash; a and b are plotted with the same full-scale values, (a) Methanol extraction, (b) Benzene reextraction of a monly used for sample transfer may result in significant and nonreproducible component loss. Table II shows recoveries of total organic compounds after spiking 200 mL benzene with 100 μϋι of a concen trated fly ash extract and then con centrating to the original 100-μΙ_, vol ume of the spiked extract by rotary evaporation. The peaks detected by GC were divided into three categories: early-eluting compounds with GC elution temperatures less than 150 °C, middle-eluting compounds (150 to 230 °C), and late-eluting compounds with GC elution temperatures greater than 230 °C. The large losses of the early, more volatile components dem-
onstrate the difficulties of using rotary evaporation preconcentration for com pounds of high or medium volatility. To obtain the original concentrated fly ash extract, an initial rotary evapo ration step was performed, which caused losses of many of the volatile compounds prior to the additional losses reported in Table III. Many recovery studies have been reported in the literature. There is a large variation between some studies in which similar preconcentration pro cedures were employed, even though very reproducible results were ob tained for each individual study. Re coveries of PAH compounds, for ex ample, have been reported as being
ANALYTICAL CHEMISTRY, VOL. 53, NO. 9, AUGUST 1981 ·
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Table 1. Percent Recoveries of StandarcI Mixture from Rotary Evaporation Using Gas Chromatographic Peak Areas Reten tion Percent time recov (mln) eries
Compound 1.
r>-Ci2H26
2.
/7-C-)8H38
3. Diethyl phthalate 4. 5. 6. 7. 8. 9.
Figure 2. Apparatus used for solvent reduction. Left: rotary evaporator. Right: Kuderna-Danish concentrator with Snyder column
Phenanthrene 9-Fluorenone Methyl anthracene Dibutyl phthalate Fluoranthene Pyrene
10. n-C 24 H S o 11. Dioctyl phthalate 12. bis(2-ethylhexyl)phthalate 13.
C30H62
14. Benzo[a]pyrene
from 70 to 95%, When performing re covery studies, several replicates should be performed. In applications involving frequent analyses, recoveries need to be checked periodically, par ticularly when modifications to the procedure are made or if the analyses are to be performed by someone inex perienced with the procedure. New batches of solvent should always be checked for levels of trace impurities when used in procedures requiring preconcentration of the solvent ex tract before analysis. Several studies have reported the use of correction factors to allow for the organics lost during extraction and preconcentration. For such applica tions it is important that the range of recoveries for different replicates be examined, and not just the mean re covery and standard deviation. Al though the overall recovery may be ac ceptable, a single experimental value can easily vary by 30-50% from the ex pected value, especially for the deter mination of organics at ppb or lower concentrations. One method of coping with this problem is to spike the sam ple before extraction and concentra tion with an isotopically labeled stan dard that is at a concentration similar to the compound to be determined. For example, 37 Cl-labeled 2,3,7,8tetrachlorodibenzo-p- dioxin (TCDD) has been used in the determination of this compound (6). During the analy sis by GC/MS-SIM, the labeled TCDD can be determined with the nonlabeled TCDD by monitoring the m/z 321.9 ion (nonlabeled) and the 327.9 ion (labeled TCDD) in a single GC/MS analysis. The use of labeled
substances is expensive and not prac tical for analysis of a large number of compounds, but is well-suited for spe cial applications such as the analysis of TCDD.
Solvent Concentration Figure 2 also shows the apparatus that is most often used as an alterna tive to the rotary evaporation method. The Kuderna-Danish evaporative concentrator equipped with a Snyder column also operates by solvent distil lation. However, it is a more effective concentrator for compounds of higher volatility. Since the concentrator is operated at ambient pressure, the ris ing vapors must build up sufficient pressure to force their way past the stage of the Snyder column. Each stage consists of a narrow opening cov ered by a loose-fitting glass insert. Since the vapors are slow to work their
15. n-C 36 H 7 4
1.87 13.73 14.27 16.79 17.32 19.42 22.56 24.01 25.12 27.28 34.37
13 70 73 67 66 71
37.83
75 73 73 74 77 74
38.20 40.67 52.46
74 74
74
way through the different stages of the Snyder column, there is initially a large amount of condensation of these vapors, which returns to the bottom of the Kuderna-Danish apparatus. Be sides continually washing the organics from the sides of the glassware, the re turning condensate also contacts the rising vapors and helps to recondense volatile organics. Although this pro cess is slower than the rotary evapora tion method, higher recoveries of trace organics can generally be obtained. In Table III recoveries are reported of standard compounds when concen trated from an initial volume of 100 mL to 1 mL using both the rotary evaporation and Kuderna-Danish concentration methods (7). The sol vent used in this study was chloro form, and 33 μ-g of each compound in
Table II. Recovery of Total Organic Compounds in Municipal Incinerator Fly Ash Extract Concentrated by Rotary Evaporation 1 Micrograms Detected by GC-FID
Sample
Fly ash extract original solution Original solution after recovery from rotary evaporation Percent recovery 1
Early Peaks 2
Middle Peaks 2
Late Peaks 2
1600
7500
1300
640
6800
1200
40
91
92
Based on total GC-FID peak areas and estimated average response factor of 400 area counts/ng Early peaks, elution times 35 min 2
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Table III. Recoveries of Organic Compounds Using Rotary Evaporation and Kuderna-Danish Preconcentration Methods (Ref. 7) Compound
Percent recoveries Rotary Kudernaevaporation Danish
n-hexadecane
89
92
Naphthalene Phenol Acenaphthene Dibenzofuran
86
91
85 89
90 91 92
88
Table IV. Percent Recoveries Using GC/MS Analyses of Selected Compounds after Evaporation/Reconstitution of Benzene Extract of Incinerator Fly Ash Compound
Pentadecane Octadecane Heneicosane Tetracosane Dibutyl phthalate Dioctyl phthalate Biphenyl Pentachlorobenzene Anthracene Pyrene Benzopyrene Tetrachlorinated dibenzodioxins Pentachlorinated
Repli cate 1
Repli cate 2
95 95 99 98 92 86 67 95 95 91 90 91
96 78 92 90 91 89 62 97 90 88 98 88
89
86
dibenzodioxins Hexachlorinated
91
87
dibenzodioxins Heptachlorinated
97
80
90
80
dibenzodioxins Octachlorodibenzop-dioxin
Table III was spiked into the initial 100 mL of solvent. Although giving better recoveries than the rotary evap oration method, the Kuderna-Danish apparatus is slower and requires the use of more delicate and specialized equipment. Also, the recovery vessel of the Kuderna-Danish apparatus is more susceptible to violent solvent eruptions that can lead to large sam ple losses. Concentration to a final volume of less than 1 mL is seldom accomplished
in one step. Samples are usually con centrated to 5-10 mL and then trans ferred to a micro-Snyder column evaporator to further reduce the vol ume to 1.0 mL or less. Final volume adjustment or preconcentration to volumes as low as 0.1 mL can be per formed by the gas stream-water bath method. This is accomplished by pass ing a gentle gas stream over the sur face of the sample until the desired volume is reached. Gas flow must be gentle to prevent nebulization losses. High-purity air, nitrogen, and helium gases should be used to avoid the in troduction of artifacts. During the evaporation, sample cooling will be ex perienced, and the sample is normally placed in a warm water bath to speed the evaporation process. Concentration to these low volumes is possible with the micro-Snyder col umn, but care must be taken that the sample is not boiled to dryness. Large sample losses with the gas streamwater bath method are often associ ated with organic residues that are left on the glass walls of the concentrating vessel as the solvent level decreases. Once the final volume is reached, it is possible to recover most of these organics by rinsing down the glass sur face with the remaining sample using a microliter syringe. Some losses will still be experienced, but will not be critical as long as the composition of the organics remaining on the glass surface is identical to the composition of the organics in solution. Alterna tively, sample volume can be reduced to below 100 μ ι , and small amounts of fresh solvent can be used to wash the glass surface until the desired volume is reached. Measurement of final vol umes as low as 0.1 mL can be accom plished by performing the evaporation step after transfer to a graduated vial such as the reacti-vial (Pierce Chemi cal Co., Rockford 111.). Several such vials are available commercially. How ever, the graduations on the vial should be checked by calibration with clean solvent using a microliter sy ringe. Many reported preconcentration procedures require the complete evap oration of the initial solvent, usually for the purposes of changing to a sec ond solvent during sample clean-up or for sample storage. Since the largest and most irreproducible losses occur for sample concentration to volumes less than 0.5 mL, reduction below this quantity should be avoided. Even the most gentle evaporation will result in significant sample loss when allowed to continue to dryness. Table IV shows the losses obtained for different compounds when aliquots of a concen trated benzene extract of municipal incinerator fly ash were allowed to evaporate to dryness in a reacti-vial
and then were reconstituted to the original volume (8). Evaporation was accomplished using very gentle condi tions. The screw caps on the sample vials were loosely fastened, and the vials were allowed to stand in a fume hood at ambient temperatures. To re constitute the organic residues, fresh solvent was added by washing down the glass surface of the vials. Since no sample handling or transfer was per formed, except the initial transfer to the storage vials, the losses reported in Table IV can be attributed to the evaporation to dryness of the benzene extracts. It is not known whether losses are primarily due to volatilization of sample components or irreversible ad sorption on the vial walls. If the sensi tivity of the instrumental method em ployed is great enough, it would be best to preconcentrate samples to no less than 1 mL final volume.
Solvent Purity Problems In addition to the problem of sam ple losses when preconcentrating trace organics in organic solvents, severe sample contamination may be experi enced at any step in the preparation procedure. It is especially important to use the best available solvents, since impurities in the solvent will be concentrated as well as sample compo nents. A recent study compares impu rity levels in some commercially avail able solvents (9). The purity of distilled-in-glass grade solvents was found to be superior to that of pesti cide grade solvents. However, the im purity levels for even the distilled-inglass solvents were observed to vary for different batches from the same manufacturer. Figure 3 shows the im purities revealed by a GC analysis after distilled-in-glass grade cyclohexane was concentrated by a factor of 2000 (200 mL to 100 ML) using rotary evaporation. The concentrations of contaminants are significant enough to interfere in the analysis of organic compounds at the ppb level, The indi vidual impurities are not identical to those observed in other batches of this solvent. Table V shows the nature of impurities identified by GC/MS anal ysis for solvents from different sources. Benzene is one of the cleanest sol vents available. Because of its toxicity, many laboratories are unable to use benzene. In terms of extraction effi ciency, toluene is comparable to ben zene, but the batches of toluene tested in this laboratory have all contained high levels of trace organic contami nants. Preconcentrations by a factor of 2000 of distilled-in-glass grade ben zene obtained from different manu facturers have consistently produced
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ces the sorbent trap preconcentration technique offers combined sampling and preconcentration, and reduced sample handling. In this technique, a hollow tube is filled with an adsorbent material that selectively retains the desired organic compounds as sample volumes of air or water are passed through the adsorbent. Trapped organics can then be eluted from the ad sorbent by using a small amount of solvent, or removed by thermal desorption if the compounds have suffi cient volatility. Considerable effort has been directed to the study of a wide variety of adsorbent materials for application to the analysis of different compounds in air or water (11). Some of the more common adsorbent mate rials include macroreticular XAD res ins, polyurethane foams, activated carbon, and Tenax-GC.
Figure 3. Gas chromatogram of 2000-fold concentrate of distilled-in-glass grade cyclohexane showing solvent impurities acceptably low levels of impurities during several years of extractions performed in our laboratory. Solvents of initially high purity may become contaminated during storage. This is particularly true for solvents stored in bottles that are not equipped with Teflon liners. It has been demon strated that organic impurities from the glue holding foil liners to the bot tle caps will very rapidly migrate to the solvent (10).
Preconcentration with Sorbent Traps Although the solvent extractionsolvent reduction methods will con tinue to be important for trace organic analysis of solid samples, there are many applications for which these procedures are not adequate. Most of these other applications are for the analysis of trace organic compounds in air or water. For these sample matri-
The choice of the proper sorbent material is determined by the com pounds of interest, the amount and type of sample, and the desired meth od of analysis. For water samples, XAD resins have been a popular choice. The XAD series of resins is a product of the Rohm and Haas Com pany. These resins are hard insoluble spheres of 16-50 mesh available in dif fering polarities. Each bead is formed from microbeads cemented together during polymerization, giving the res ins their macroreticular, controlled pore size structure. XAD-2 and -4 are nonpolar styrene-divinylbenzene co polymers of differing pore size and surface area. XAD-7 and -8 are slight ly polar acrylate esters. XAD-2 resins have been found to give good recov-
Table V. Identities and Concentrations of Organic Compounds in Solvents by GC/MS after 200fold Concentration A Solvent
Cyclohexane
Methylene chloride
Compound C6H-12O
none
c
Β ng/mL
49
Compound
ng/mL
Compound
3.5
η-Butyl n-butyrate
1.8
Tributyl phosphate isomer 1
2.9
Tributyl phosphate isomer 2
0.9
Diethyl phthalate
0.5
Dibutyl phthalate
7.7
1,1,1-Trichloropropane and
none
—
16
1,2,3-Trichloropropane 21
Tetrachloroethane
1.9
Phthalic anhydride
0.4
Dioctyl phthalate none
29
Diethyl phthalate Dioctyl phthalate
1,1,1,2-Tetrachloropropane
Methanol
ng/mL
14
1,4-Bis(methylene)cyclohexane
1.6
2,6-Dimethylphenol
2.6
A: Caledon Laboratories (Georgetown, Ont. Canada), distilled-in-glass grade; B: Burdick and Jackson (Muskegon, Mich.), distilled-in-glass grade; C: Fisher Scientific (Fairlawn, N.J.), pesticide grade
1054 A · ANALYTICAL CHEMISTRY, VOL. 53, NO. 9, AUGUST 1981
Load Bypass
Carrier Gas
Heated Gas Switching Valve Swagelock Reducing Unions
Heated Transfer Lines
GC Inlet
GC Detector
Sorbent Trap
Desorption Oven
GC Column
Figure 4. Thermal desorption apparatus used to perform a GC analysis for organic compounds concentrated on sorbent trap
eries for a wide range of neutral compounds in the ppb range in water, including alcohols, PAH, halogen compounds, and, after acidifying the water, organic acids and phenols (72). The homogeneous surface of the macroreticular resin with its low energy binding process (van der Waals forces) allows quantitative desorption of the organics sorbed from the water sample. The organics may be readily eluted with a small amount of a watermiscible solvent, usually diethyl ether
or methanol. This property contrasts favorably with activated carbon, where physical and chemical adsorption mechanisms may occur on the heterogeneous surface, preventing quantitative desorption. Since the final eluate volume may be further concentrated, the previous discussions on solvent purity and Kuderna-Danish concentration apply here also. In addition, prior contamination of the resins must be assumed and careful conditioning is necessary.
Transfer Tube Tenax-GC Trap
Sample
Flow Meter
Glass Frit
He Gas
Figure 5. Purge and trap device used to purge and concentrate volatile organic compounds from liquid samples 1056 A · ANALYTICAL CHEMISTRY, VOL. 53, NO. 9, AUGUST 1981
For the XAD resins, sequential 8-h Soxhlet extractions with methanol, acetonitrile, and diethyl ether have been recommended to remove contaminants. After cleaning, the resins must be kept wet to avoid fracturing of the resin in which new surface contaminants are exposed. Reactions of the resin with inorganic constituents in the sample cannot be overlooked. In a study of organobromine compounds formed during the chlorination of estuarine cooling waters, some of the brominated hydrocarbons found were considered to be artifacts formed by active chlorine, bromide ion, and hydrocarbon impurities from the XAD resins, released by fracturing of the resin during sampling (13). Following sorbent trap concentration with solvent elution of the organic species and solvent reduction, only a small aliquot of the sample is needed for injection into the GC or GC/MS for analysis. Thermal desorption of a sorbent trap directly onto a GC column gives only a single sample of the total collected organics for analysis. For XAD resins used for concentration of organics from water samples, the entrained water presents a problem. Thermal desorption of XAD-2 resin has been accomplished by employing an intermediate Tenax trap (14). Tenax-GC is a porous polymer material based on 2,6-diphenyl-p-phenylene oxide. The XAD trap is thermally desorbed onto the Tenax tube. A gas flow is maintained until the water exits the hydrophobic Tenax trap, leaving the organics behind. For many applications, particularly the analysis of trace organic pollutants in air, Tenax alone is used to concentrate the organics. In fact, using this method it is possible to detect several hundred organic compounds in atmospheric air from sampling at almost any urban site. Tenax is more thermally stable than the divinylbenzene polymer XAD resins, which have been reported to outgas extraneous compounds during heating. The air sample is pumped through the trap containing Tenax that has been previously conditioned by heating with an inert gas flow. The trap is sealed and returned to the laboratory for desorption and analysis. A desorption apparatus assembled from readily available parts is shown in Figure 4. The gas switching valve allows carrier gas to flow through the GC column uninterrupted while the sorbent trap is inserted. The desorption oven is made in two sections, allowing it to be quickly clamped around the trap. The carrier gas is then diverted through the trap as it is rapidly heated. Organic compounds are thus desorbed into the inlet of the GC column held at a low temperature. A temperature-pro-
1-mL Reservoir Solvent Introduction Orifice Threaded
Viton O-ring 4-mL Reservoir Solvent Introduction Orifice Threaded
4.5 cm Figure 6. Teflon block apparatus for extraction of organic compounds from particulates collected on small dichotomous filter elements. The filter element is sealed in the block cavity in contact with the solvent reservoirs, and extraction is conducted by ultrasonic agitation. The solvent extract is then removed and concentrated by a factor of 100 or more prior to GC/MS analyses
grammed GC analysis can then be initiated. Uniformly heated transfer lines are critical features of the desorption apparatus. Cold spots will cause condensation of the less volatile compounds, resulting in incomplete transfer and loss of GC resolution. For the analysis of trihalomethanes in water, a purge and trap method followed by thermal desorption has proved to give reliable quantitation (15). The method can be used successfully for quantitation of compounds that are
