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Identification and Quantification of Aerosol Polar Oxygenated Compounds Bearing Carboxylic or Hydroxyl Groups. 1. Method Development M. Jaoui,*,† T. E. Kleindienst,‡ M. Lewandowski,‡ and E. O. Edney‡
ManTech Environmental Technology, Inc., P.O. Box 12313, Research Triangle Park, North Carolina 27709, and National Exposure Research Laboratory, U.S. Environmental Protection Agency, Research Triangle Park, North Carolina 27711
In this study, a new analytical technique was developed for the identification and quantification of multifunctional compounds containing simultaneously at least one hydroxyl or one carboxylic group, or both. This technique is based on derivatizing first the carboxylic group(s) of the multifunctional compound using an alcohol (e.g., methanol, 1-butanol) in the presence of a relatively strong Lewis acid (BF3) as a catalyst. This esterification reaction quickly and quantitatively converts carboxylic acids to their ester forms. The second step is based on silylation of the ester compounds using bis(trimethylsilyl) trifluoroacetamide (BSTFA) as the derivatizing agent. For compounds bearing ketone groups in addition to carboxylic and hydroxyl groups, a third step was used based on PFBHA derivatization of the carbonyls. Different parameters including temperature, reaction time, and effect due to artifacts were optimized. A GC/MS in EI and in methane-CI mode was used for the analysis of these compounds. The new approach was tested on a number of multifunctional compounds. The interpretation of their EI (70 eV) and CI mass spectra shows that critical information is gained leading to unambiguous identification of unknown compounds. For example, when derivatized only with BF3methanol, their mass spectra comprise primary ions at m/z M•+ + 1, M•+ + 29, and M•+ - 31 for compounds bearing only carboxylic groups and M•+ + 1, M•+ + 29, M•+ - 31, and M+. - 17 for those bearing hydroxyl and carboxylic groups. However, when a second derivatization (BSTFA) was used, compounds bearing hydroxyl and carboxylic groups simultaneously show, in addition to the ions observed before, ions at m/z M•+ + 73, M•+ - 15, M•+ - 59, M•+ - 75, M•+ - 89, and 73. To the best of our knowledge, this technique describes systematically for the first time a method for identifying multifunctional oxygenated compounds containing simultaneously one or more hydroxyl and carboxylic acid groups. Atmospheric aerosols, which include biogenic and anthropogenic aerosols, play an important role in global climate and * To whom correspondence should be addressed. Phone: (919) 541-7728. Fax: 919-541 3566. E-mail:
[email protected]. † ManTech Environmental Technology, Inc. ‡ U.S. Environmental Protection Agency. 10.1021/ac049919h CCC: $27.50 Published on Web 07/14/2004
© 2004 American Chemical Society
atmospheric chemistry. Their direct environmental impact involves increasing the scattering and absorption of solar radiation, which leads to visibility degradation1,2 and alters the amount of solar radiation that reaches the Earth’s surface.3 Aerosols indirectly affect cloud properties and the hydrological cycle of the climate by acting as cloud condensation nuclei.3,4 Recently, concerns have increased about the apparent relationship between exposure to PM2.5 (particulate matter with an aerodynamic diameter less than 2.5 µm) and adverse health effects observed in many places around the world.5,6 As the understanding of the toxicology associated with these particles develops, more accurate compositional data will be required. Oxygenated organic compounds are ubiquitous organic aerosol constituents throughout the troposphere, and numerous recent studies establish that a significant portion of these compounds result from the photooxidation of biogenic and anthropogenic hydrocarbons.7,8 In addition, these compounds are emitted directly into the atmosphere from vegetation,9 combustion engines,10 or biomass burning.11 In fact, a number of products identified in the particle phase from field samples and smog chamber experiments are polar oxygenated compounds (POCs) containing one, two, three, or more oxygenated functional groups (e.g., hydroxyl, carboxylic, ketone, aldehyde), and an understanding of their molecular composition still remains limited.7,8 The high polarity and low levels of these compounds preclude direct gas chromatography/mass spectrometry (GC/MS) analysis, and their identification and quantification are somewhat challenging. Recently, several on-line techniques were developed for the identification or quantification of organic products from the (1) Watson, J. G. J. Air Waste Manage. Assoc. 2002, 52, 628-713. (2) Charlson, R. J.; Schwartz, S. E.; Hales, J. M.; Cess, R. D.; Coakley, J. A.; Hansen, J. E., Hoffmann, D. J. Science 1992, 255, 423-430. (3) Haywood, J.; Boucher, O. Rev. Geophys. 2000, 38, 513-543. (4) Ramanathan, V.; Crutzen, P. J.; Kiehl, J. T.; Rosenfeld, D. Science 2001, 294, 2119-2124. (5) Samet, J. M.; Dominici, F.; Curriero, F. C.; Coursac, I.; Zeger, S. L. N. Eng. J. Med. 2000, 343, 1742-1749. (6) Gamble, J. F. Environ. Health Perspect. 1998, 106, 535-549. (7) Kubatova, A.; Vermeylen, R.; Claeys, M.; Cafmeyer, J. Atmos. Environ. 2000, 34, 5037-5051. (8) Edney, E. O.; Kleindienst, T. E.; Conver, T. S.; McIver, C. D.; Weathers, W. S. Atmos. Environ. 2003, 37 (28), 3947-3965. (9) Kesselmeier, J.; Staudt, M. J. Atmos. Chem. 1999, 33, 28-88. (10) Westerholm, R.; Karl-Eric, E. Environ. Health Perspect. 1994, 102, 13-23. (11) Graham, B., Mayol-Bracero, O. L.; Guyon, P.; Roberts, G. C. J. Geophys. Res. 2002, 107, D20, 8047.
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oxidation of biogenic compounds12,13 or from field samples.14 Examples of these techniques are atmospheric pressure chemical ionization source coupled with an ion trap mass spectrometer;12 atmospheric sampling glow discharge ionization coupled with a quadrupole ion trap mass spectrometer;13 proton-transfer reaction coupled with a gas chromatograph, which is itself coupled with an ion trap mass spectrometer;14 and in situ long-path Fourier transform infrared spectroscopy. The first three techniques are in development, and isomers are difficult to resolve using these techniques. In addition, the interpretation of mass spectra is complicated by the fragmentation of product ions and the formation of cluster ions. This is further complicated when complex mixtures need to be analyzed, such as those from field or chamber experiments, because of the lack of separation prior to detection. Current procedures for analyzing POCs are based on singlestep or multistep derivatization techniques. One of the most common derivatization techniques used in atmospheric chemistry is based on derivatizing carbonyl groups using O-(2,3,4,5,6pentafluorobenzyl)hydroxylamine (PFBHA) or methoxyamine along with a silylation agent (e.g., bis(trimethylsilyl) trifluoroacetamide, BSTFA) to trimethylsilylate carboxylic and hydroxyl groups simultaneously.8,15 Only the desired derivatives are expected from these techniques; however, a number of artifacts are formed.16 When one or more hydroxyl groups coexist in the same molecule with a carboxylic group, this method leads to ambiguity between these two different groups. Also, a number of artifacts are reported from the silylation reaction that can affect product identification,16 which was also observed in this study. Most of the artifacts noted in our laboratory are reported by Little16 and are only briefly discussed here. We refer the reader to this interesting paper for more information. Scientists should be wary when using silylation reactions in atmospheric chemistry as a tool to identify organic compounds bearing OH groups. In fact, current analytical methods do not allow the complete identification of the organic compounds comprising PM2.5. Recently, Edney et al.8 identified a number of POCs in field samples using the PFBHA + BSTFA double-derivatization technique. However, it was difficult for these authors to assign an unambiguous structure to compounds bearing more than one hydroxyl or one carboxylic group simultaneously. The primary motivation of the present study is (1) to investigate the possibility of identifying compounds bearing hydroxyl, ketone, and carboxylic groups simultaneously that current derivatization methods are unable to unambiguously differentiate and (2) to extend the capability of current analytical techniques used for aerosol analysis. In fact, a new analytical method was developed for the characterization of aerosol oxygenated organic compounds bearing one or more of the following groups: hydroxyl (-OH), carboxylic (>COOH), and ketone or aldehyde (>CO). This approach is based on derivatizing carboxylic groups using methanol or 1-butanol as derivatizing agents in the presence of a (12) Warscheid, B.; Ku ¨ckelmann, U.; Hoffmann, T. Anal. Chem. 2003, 75, 14101417. (13) Dalton, C.; Jaoui, M.; Kamens, R. M.; Glish, G. Submitted to Anal. Chem. (14) Warneke, C.; De Gouw, J.; Kuster, W. C.; Goldan, P. D.; Fall, R. Environ. Sci. Technol. 2003, 37, 2494-2501. (15) Yu, J.; Flagan, R. C.; Seinfeld, J. H. Environ. Sci. Technol. 1998, 32, 23572370. (16) Little, J. M. J. Chromatogr., A 1999, 844, 1-22.
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relatively strong acid (BF3 or BCl3), followed by silylation using BSTFA to derivatize OH groups or PFBHA to derivatize ketone and aldehyde groups. Special emphasis was given to POCs bearing both hydroxyl and carboxylic groups. The new method was successfully tested on 52 model compounds, on samples from chamber experiments (secondary organic aerosol (SOA) from photooxidation of biogenic and aromatic compounds such as R-pinene, β-pinene, and toluene), and on field samples (PM2.5). Due to lack of space, results from smog chamber experiments and field samples are reported in a separate paper.17 Several parameters of the esterification were investigated and optimized, including derivatizing reagent (BF3-methanol, BCl3-methanol, and BF3-1-butanol), temperatures, and reaction times. The new method (BF3-methanol + BSTFA/PFBHA) was compared to current derivatization methods (BSTFA, PFBHA, and PFBHA + BSTFA). Also, several combinations of the three derivative agents (BF3-methanol, BSTFA, PFBHA) were used, and artifacts are reported. Note that Kubatova et al.7 used diazomethane to methylate field samples followed by silylation, and the derivatives were analyzed only in electron impact ionization (EI) mode. However, to our knowledge, this is the first time that a derivatization based on a combination of BF3-methanol (or BF3-1butanol), PFBHA, or BSTFA coupled with EI and chemical ionization (CI) mass spectrometry for the identification and quantification of POC compounds is reported. Tandem mass spectrometry (MS/MS) spectra were also obtained for the molecular or fragment ions of each compound, which were used for further identification of the reaction products and to elucidate dissociation pathways. EXPERIMENTAL METHOD Chemicals and Solvents. All chemicals of analytical reagent grade were purchased from Aldrich Chemical Co. (Milwaukee, WI) except dimethyl esters, which were purchased from Acros Organics (Geel, Belgium). All chemicals were purchased at the highest purity available and used without further purification. All solvents were from Burdick and Jackson (Muskegon, MI) and specified as GC2 quality. Reagent agents used for the different derivatizations (BF3-methanol, BCl3-methanol, BF3-1-butanol, BSTFA 1% trimethylchlorosilane, TMCS, and PFBHA) were obtained from Aldrich Chemical Co. Pinonaldehyde was synthesized in our laboratory.18 Glassware was washed with soap, rinsed with hot water, and dried overnight at 200 °C. Just before use, the glassware was rinsed three times with acetone and three times with methylene chloride and dried at 120 °C. Model Compounds. A total of 52 standard compounds (see Tables 1 and S-1 (Supporting Information)) that exist commercially or were synthesized in our laboratory were selected to optimize the analytical method developed in this study. Five groups of compounds shown in Table 1 were used: mono- and multicarboxylic acid compounds (1-20), hydroxycarboxylic acids (compounds 21-27), carboxylic acid with carbonyl groups (28-31), hydroxy/oxo aldehydes or ketones (32-34), and hydroxy compounds (35-41). These 41 analytes were derivatized using the procedures described below and analyzed in EI and CI modes. Also, MS/MS spectra were recorded for most of these standards (17) Jaoui M.; Corse, E. W.; Kleindienst, T. E.; Lewandowski, M.; Offenberg, J.; Edney, E. O. Submitted to Environ. Sci. Technol. (18) Jaoui, M.; Kamens, R. M. Atmos. Environ. 2003, 37, 1835-1851.
Table 1. Model Compounds
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Table 1. (Continued)
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Table 1. (Continued)
to help depict dissociation pathways for the known (authentic existing standards) and unknown compounds. The remaining 11 esters (Table S-1) were used to estimate the efficiency of the derivatization technique based on esterification (see below). These standards were chosen for their relevance to atmospheric systems, for structural variety, and to cover a range of polarity. The primary standards consisted of four different mixtures of 6-20 components prepared gravimetrically with an estimated overall precision of 0.1%. In addition to molecular weight (MW), systematic nomenclature, and structure, each compound was numbered (column 1) for easy reference in the text, figures, and tables. Derivatization Procedures. In addition to the BF3-methanol + BSTFA double derivatization method discussed in detail in this study, we investigated a number of multistep derivatization techniques as summarized in Table S-2 (Supporting Information). The objective of testing these different derivatization techniques
is to confirm and validate the key information that the developed method was able to provide for the identification of POCs. Preparation of Methyl Esters and n-Butyl Esters. We chose esterification of organic acids using BF3-methanol, because it is simple and environmentally safe esters are formed. Esterification also provides a convenient alcohol-catalyst system that, when used in excess with heating, quickly and quantitatively converts carboxylic acids to their methyl or n-butyl esters. The method described here was based on techniques reported by Morrison and Smith19 and by Metcalfe and Schmitz.20 Standards (10 µg/mL of each compound in dichloromethane) were placed in a 15-mL round-bottom tube and placed into an N-Evap evaporation device water bath (Organomation Associates, Inc., Berlin, MA) used at room temperature. The solution was con(19) Morrison, W. R.; Smith, L. M. J. Lipid Res. 1964, 5, 6000-608. (20) Metcalfe, L. D.; Schmitz, A. A. Anal. Chem. 1961, 33, 363-364.
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Scheme 1
centrated to dryness by a blowdown using a stream of ultrapure dry nitrogen passed through an oxy-trap and gas-drying tube and then treated with 0.5 mL of 14% BF3-methanol. The tube was capped tightly, sealed with Parafilm, and heated for 20 min at 65 °C. After the mixture was allowed to cool to room temperature, 1 mL of water saturated with sodium chloride was added to neutralize the excess of BF3 and the solution was allowed to stand for 2 min. To extract the sample for analysis, 2 mL of an appropriate solvent (see below) was added to the sample tube, and then the tube was capped tightly and shaken vigorously for 3 min. The organic layer was transferred into a 15-mL tube containing ∼1 g of anhydrous sodium sulfate to dry the extracts. The extraction procedure was repeated two more times. After the solution was allowed to dry for 30 min, the extraction solution was decanted into a 15-mL test tube to separate the extract from the sodium sulfate. The test tube containing the sodium sulfate was rinsed with 2 mL of the extraction solvent, and the extract was added to the test tube containing the sample extract. The extract was filtered (Millipore 0.2-µm PTFE hydrophobic) to remove fine particles from the extract and concentrated to ∼0.3 mL by a blowdown using a stream of ultrapure nitrogen passed through an oxy-trap and gas-drying tube. The sample was directly analyzed by GC/MS, without further purification or cleanup in EI or CI mode. A 1- or 2-µL aliquot was injected in each mode. The same procedure was used with 14% BCl3-methanol and 14% BF3-1-butanol as derivatizing reagents. BSTFA Derivatization. The trimethylsilyl derivatives of the underivatized samples or the remaining solutions of the methyl, n-butyl esters, PFBHA oximes, etc., (see Table S-2) were obtained using BSTFA containing 10% TMCS as reagent. This procedure is similar to that reported in detail elsewhere8,21 and will be described here only briefly. After an aliquot of the underivatized standards solution or the derivatized solution (methylation, nbutylation, PFBHA, etc.) was transferred to a 15-mL round-bottom tube and dried completely under a gentle stream of ultrapure nitrogen, 250 µL of BSTFA/TMCS (90:10, v/v) and 100 µL of pyridine were added to the solution. The tube was sealed with a Teflon-coated cap and Parafilm and allowed to react at 70 °C for 2 h. After cooling to room temperature, the solution was filtered, after which the derivatives were analyzed by GC/ITMS in EI and methane-CI modes. (21) Kleindienst, T. E.; Conver, T. S.; McIver, C. D.; Edney, E. O. J. Atmos. Chem. 2004, 47, 79-100.
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PFBHA Derivatization. This procedure was used to identify oxygenated compounds bearing carbonyl groups (ketone or aldehyde) using PFBHA. This procedure is similar to that described earlier by our group,8,21 and only a brief summary is given here. Standard mixtures were evaporated to dryness using the N-Evap analytical evaporator. After samples were removed from the evaporator, 1 mL of distilled, deionized water was added to the tube containing the sample followed by 250 µL of PFBHA reagent (50 µg/µL in water). The solution was capped and allowed to stand for 24 h at room temperature. After the reaction was complete, three drops of concentrated HCl was added. The sample was capped and shaken vigorously for 2 min and then was extracted with n-hexane-dichloromethane (2:1 v/v) using the same procedure described in BF3-methanol derivatization. After drying the extract under 1 g of sodium sulfate for ∼30 min and placing it in a new tube, the extract was filtered and concentrated to ∼0.3 mL using the N-Evap evaporator. The sample was directly analyzed by GC/MS, without further purification or cleanup, in EI or methane-CI mode. Multistep Derivatization. A number of oxygenated organic compounds found in PM2.5 contain multiple functional groups, and a single derivatization may not be sufficient to elucidate complete structural information.8 In fact, multistep derivatization techniques combined with GC/ITMS analysis in EI and CI modes, with the possibility of using MS/MS, make possible more accurate identification and elucidation of dissociation pathways. Table S-2 in Supporting Information presents the single-step and multistep derivatization procedures investigated in this study. Our main focus in this paper is the identification of compounds containing OH and COOH groups. Therefore, most of our discussion is directed toward derivatization in which methylation and silylation take place. As an example, Scheme 1 shows derivatization reactions occurring with citramalic acid. This compound contains both a carboxylic and a hydroxyl group, which were derivatized using BF3-methanol, followed by BSTFA-TMCS. The first derivatization converts the carboxylic acid groups to their ester form, and the second converts the methylated compounds or compounds bearing only hydroxyl groups to their trimethylsilyl derivatives. However, care must be taken, especially with silylation, when analyzing mass spectra because some compounds form additional derivatives or artifacts. In fact, for compounds containing CO, COOH, and OH groups simultaneously, derivatization D-13 (Table S-2) was used. The PFBHA
needed to be used before BSTFA derivatization to prevent the enolation of the ketone group (see BSTFA artifacts below). Multistep derivatization techniques have been reported in the literature to detect and identify multifunctional groups. For example, a number of two-step derivatizations such as PFBHA + BSTFA and PFBHA + BF3-methanol have been used by Le Lacheur et al.22 to analyze reaction products from the photooxidation of isoprene. Yu et al. used PFBHA + BSTFA to measure reaction products from the ozonolysis of monoterpene.23 Recently, Kubatova et al.7 used diazomethane to methylate atmospheric samples followed by silylation using BSTFA, or methoxyamine to derivatize carbonyl groups, and the derivatives were analyzed only in EI mode. In our study, a more systematic analysis was undertaken and the derivatives were analyzed in EI, methane-CI, and MS/MS modes. GC/MS Analysis. Analyses were performed on a ThermoQuest (Austin, TX) GC coupled with an ion trap mass spectrometer (ITMS). The injector, operated in splitless mode, was heated to 260 °C and aliquots of 1 or 2 µL of the derivatized samples (standard mixture) were injected. Separation was carried out on an RTx-5MS of 60-m length and 0.25-mm internal diameter, with a 0.25-µm film thickness (Restek, Inc., Bellefonte, PA). Proper compound resolution was obtained from an initial hold of 84 °C for 1 min followed by a temperature program of 8 °C min-1 to 200 °C, held for 2 min, then 10 °C min-1 to 300 °C, and held for 15 min. All samples were analyzed in electron impact positive (EI+) mode and in positive chemical ionization (CI+) mode with methane as the reagent gas over a m/z range between 25 and 1000 amu. The ion source, ion trap, and interface temperatures were 200, 200, and 275 °C, respectively. Tandem Mass Spectrometry. Tandem mass spectrometry is a method that can assist in identification and structure elucidation of organic compounds. Therefore, collisionally activated dissociation (CID) of a series of compounds was carried out. Fragmentation of the precursor ion (M•+) or of the abundant ion was enhanced by CID using helium gas in the collision cell of the mass spectrometer. RESULTS AND DISCUSSION Optimization of the Esterification Method. One of the aims of this study was qualitative and quantitative analysis of organic compounds bearing at least one carboxylic, one hydroxyl group, or both. Initially, the methylation parameters, including derivatization reagent, extraction solvent, catalyst, temperature, and reaction time, were evaluated to achieve the most efficient method. Derivatization Reagent. In this set of experiments, three derivatization reagents were investigated: BF3-methanol, BCl3methanol, and BF3-1-butanol. To cover a wide range of polarity and for structural variety, a mixture of 41 standard compounds (Table 1) was used for this optimization. EI and methane-CI mass spectra of esters from the esterification of 1-41 using BF3methanol and BCl3-methanol, respectively, were recorded. Analysis of these mass spectra shows that both BF3 and BCl3 in the presence of methanol lead to similar spectra, as expected. The recoveries of these compounds in both techniques were also (22) Le Lacheur, R. M.; Sonnenberg, L. B.; Singer, P. C.; Chrisman, R. F.; Charles, M. J. Environ. Sci. Technol. 1993, 27, 2745-2753. (23) Yu, J.; Cocker, D.; Griffin, R.; Flagan, R. C.; Seinfeld, J. J. Atmos. Chem. 1999, 34, 207-258.
similar. Therefore, only BF3-methanol was used for real environmental samples. Methyl esters with low molecular weight (e.g., glycolic acid (21) and pyruvic acid (30)) were not observed because of their high volatility and were lost during the evaporation process (see Preparation of Methyl Esters and n-Butyl Esters). The problem can be alleviated by using the n-butyl derivatization technique. Methyl esters or n-butyl esters bearing two or more hydroxyl groups (e.g., esters from L-tartaric acid (26) and 3,4,5trihydroxybenzoic acid (27)) were not observed in the chromatogram because of their high polarity and were probably lost in the injector or in the column during analysis. However, their presence was confirmed when double derivatization (BF3-methanol followed by BSTFA) was used. Therefore, special attention is required when analyzing a complex mixture that contains compounds bearing carboxylic and hydroxyl compounds. Solvents. Four different solvents were evaluated. A mixture of ester compounds (Table S-1) prepared in methylene chloride (500 ppbv of each analyte) was extracted with methylene chloride, n-hexane, petroleum ether, and methylene chloride/n-hexane (2:1 v/v) using the same procedure discussed previously for the BF3methanol derivatization technique. When methylene chloride and n-hexane were used as the extraction solvent, recoveries were between 80 and 89% for most compounds. The best results were obtained when extracting with petroleum ether or methylene chloride/n-hexane, with recoveries higher than 96% for most compounds. The number of extractions was optimized, and three extractions were enough to extract the analytes studied here with the recoveries reported above. The temperatures at which the highest efficiency for methylation was observed were between 60 and 70 °C when the mixture was allowed to react between 15 and 30 min. Note that only temperatures between room temperature (25 °C) and 75 °C were evaluated. It is important to note that the same conditions were used for BF3-1-butanol derivatization. Artifacts. Trimethylsilyl Derivatization. In analytical chemistry24 and particularly in silylation reactions, artifacts are reported for a wide variety of compounds. Little16 has reviewed and reported silylation artifacts observed in a variety of laboratories. In our study, a number of artifacts were noted during analysis of silylation derivatives from model compounds (Table 1) and from smog chamber experiments samples or field studies.17 Most of the artifacts noted in our laboratory are reported by Little and are only briefly discussed here. For qualitative and quantitative analysis, silylation used alone or in combination with other derivatization can lead to confusion in identifying ketones and aldehydes with R-hydrogen (aldehyde and ketone artifacts). In fact, BSTFA can react with the enolic form of these compounds, leading to their misidentification. As an example, Figure 1 shows the PFBHA, PFBHA + BSTFA, and BSTFA (enolic form) spectra of 3-hydroxy-2-butanone. This compound was identified in the field study, and its identification was confirmed by an authentic standard. Only the enolic form of 3-hydroxy-2-butanone was observed, and no trimethylsilyl derivative of the ketone form was observed. The same artifact was observed for 3-acetylpentanedioic acid and other R-hydrogen-containing compounds as exemplified for 2-oxopentanedioic acid, known also as 2-ketoglutaric acid (see Figure 2). (24) Pierce, A. E. Silylation of Organic Compounds; Pierce Chemical Co.:, Rockford, IL, 1968; pp 33-39.
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Figure 1. PFBHA, PFBHA + BSTFA, and the BSTFA (enolic form) mass spectra of 3-hydroxy-2-butanone.
One interesting observation was found in our laboratory when a mixture of R-pinene/NOx was irradiated in the presence and absence of sulfur dioxide in a smog chamber.25 When SO2 was present, no trimethylsilyl derivatives of the aerosol phase were observed; however, a large number of these derivatives were 4772
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present when SO2 was absent.25 The only difference between the two experiments is the presence of SO2, hence sulfuric acid. A close analysis of the mass spectra from aerosol samples collected when SO2 was present shows the presence of one peak with a molecular weight of 242 amu, tentatively identified as SO2[OSi-
Figure 2. Mass spectrum of the enolic form of 2-oxopentanedioic acid observed in summer field sample.
(CH3)3]2 formed from the silylation of H2SO4. Note that this peak was not observed when silylated field samples were analyzed. BF3-Methanol. In the methane-CI mode, dimers of some esters used in this study were observed, as shown in Figure 3 for methyl octanoate and methyl decanoate. Note that no dimers could be identified when smog chamber or field samples were analyzed.17 The mass spectra of these dimers show ions at m/z [(2M•+) + 1], M•+ + 1, and weak 59, M•+ - 31, and no fragments at m/z M•+ - 59 were observed. BF3-1-Butanol. Kawamura reported the formation of acetals and ketals when 1-butanol was allowed to react with aldehydes and ketones at 100 °C for 30 min in the presence of BF3.26 In our study, none of these derivatizations took place when compounds containing ketones and aldehydes (e.g., pinonaldehyde) were derivatized with BF3-1-butanol using the same procedure as BF3methanol (T ) 65 °C). This technique is under investigation in our laboratory, and the results of this derivatization are reported briefly. BF3 is a relatively strong Lewis acid and may cause some compounds to undergo reactions or rearrangements under acidic conditions. However, no such artifacts were observed in this study. Reference Spectra and Mass Spectral Analyses. Understanding the ion formation processes occurring in the ion source (EI or CI), particularly when analyzing oxygenated organic substances, requires detailed studies of the fragmentation patterns in EI and methane-CI modes for the chemical class of interest. After optimization of the analytical method using the 41 standard compounds commercially available or synthesized in our laboratory, their EI and methane-CI spectra were recorded for the different derivatizations discussed above. These standards were (25) Kleindienst, T. E.; Lewandowski, M.; Edney, E. O.; Jaoui M.; Corse, E. W. SOA formation from the irradiation of R-pinene-NOX in the absence and presence of sulfur dioxide. Presented at the AAAR conference, Anaheim, CA, October 2003. (26) Kawamura, K. Anal. Chem. 1993, 65, 3505-3511.
chosen for their relevance to atmospheric systems and for structural variety. First, mass spectra recorded for each compound were closely analyzed to aid in understanding fragmentation patterns in the ion trap using the two ionization modes (EI and CI). The interpretation of PFBHA, BSTFA, and PFBHA + BSTFA mass spectra has been reported in a number of papers and therefore is not discussed here.8,21,27 EI and CI Mass Spectral Analysis of BF3-Methanol of Model Compounds. The EI (70 eV) and methane-CI mass spectra of the methylated model compounds were evaluated to determine whether molecular weight information of BF3methanol along with BSTFA or PFBHA derivatives of unknown oxygenated compounds could be obtained by interpreting their mass spectra. Shown in Figure 4 are GC chromatograms (TIC) of the CI methyl esters of carboxylic acid, which include 1-20 (top), and silylated 21-34, which include hydroxy carboxylic acids and carboxylic acids with carbonyl groups (bottom). In the CI mode, the most abundant and common ion fragments or adduct for all these methyl esters are M•+ - 59, M•+ - 31, and M•+ + 1, in addition to weak adducts M•+ + 15, M•+ + 29, and M•+ + 41. The base peaks are mostly M•+ + 1 or M•+ - 31 for monoesters, M•+ - 31 or M•+ - 59 for alkene esters or branched esters, and M•+ - 31 or M•+ + 1 for the other methyl esters. The methaneCI mass spectra of the methyl derivatives give immediate information on the presence of one, two, or more carboxylic groups on the molecule, as can be seen from the mass spectra shown in Figure 5 for propanedioic acid, p-toluic acid, and norpinic acid. EI mass spectra were also obtained for the model compounds containing at least one COOH group. These mass spectra are characterized by ions at m/z 41, 43, 55, M•+ - 43, M•+ - 29, and a weak M•+ for methyl esters, and at m/z 41, 43, 55, M•+ - 31, (27) Yu, J.; Jeffries, H. E.; Le Lacheur, R. M. Environ. Sci. Technol. 1995, 29, 1923-1932.
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Figure 3. Methane-CI mass spectra of methyl octanoate and methyl decanoate showing the formation of dimers.
and M•+ - 59 for the dimethyl or trimethyl esters. The base peak in the EI mass spectra is either m/z 43 or 55. The presence of ions M•+ - 29 and M•+ - 31 at 10-55% relative intensity provides an immediate indication that one carboxylic group is in the original molecule. Also, the presence of ions M•+ - 31 and M•+ - 59 provides an immediate indication that two or more carboxylic groups are in the original molecule. CI and EI fragment charac4774
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teristics are presented in Table 2 for the ester form of the 41 compounds shown in Table 1. EI and CI Mass Spectral Analysis of the Silylated and Methylated Model Compounds. The methylated model compounds were allowed to react with BSTFA according to the above protocol. As can be expected, only compounds containing OH groups were derivatized (21-27, 33-41). In the CI mode, all
Figure 4. Chromatograms of methylated model compounds 1-20 (top) and 21-34 (bottom).
compounds containing COOH and OH groups share several common ion fragments and adducts at m/z 73, M•+ - 75, M•+ 59, M•+ - 15, M•+ + 1, M•+ - 29, M•+ + 41, and M•+ + 73. The base peak is M•+ - 15 for most compounds. In EI mode, each derivative of the tested compounds gives rise to weak fragment ions at 73, M•+ - 15, M•+ - 43, M•+ - 73, M•+ - 89, etc. The base peak is usually m/z 43 or 55. Therefore, these fragments
provide important and immediate information that can be used to differentiate between compounds containing OH, carboxylic groups, and others. Figure 6 shows mass spectra of citramalic acid and L-tartaric acid. The interpretation of the data has shown that the mass spectra from the derivatization methods described above contain important structural information about the compound. The methane-CI mass Analytical Chemistry, Vol. 76, No. 16, August 15, 2004
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Figure 5. Methane chemical ionization mass spectra of the methyl derivatives of propanedioic acid, p-toluic acid, and norpinic acid.
spectra provide unambiguous molecular weight in addition to the presence and distinction between OH and COOH groups; however, the sensitivity was found to be lower than in the EI mode. Quantitative Measurements. The derivatization techniques discussed here give important structural information in addition 4776 Analytical Chemistry, Vol. 76, No. 16, August 15, 2004
to molecular weight. A complementary development to these techniques involves combining them with quantitative analysis using recovery, internal standards, or both. For quantitative purposes, only two derivatization procedures, namely, methylation (D-6) and silylation (D-11) were investigated because of their high
Figure 6. Mass spectra of citramalic acid and L-tartaric acid derivatized by BF3-methanol followed by derivatization with BSTFA.
efficiencies. Double-derivatization techniques were not tested. However, before making quantitative measurements, the methylation efficiency was investigated. Calibration curves were made for 11 esters for which corresponding acid forms exist commercially (see Table S-1) by plotting relative amount (mass) injected on column versus relative area. Relative amount (area) is defined as mass (area) of analyte divided by mass (area) of internal standard. Three internal or recovery standards were used including bornyl acetate, ketopinic acid, and p-menth-6-ene-2,8diol. Bornyl acetate was chosen for compounds that do not
undergo reaction with the derivative reagent, and ketopinic acid and p-menth-6-ene-2,8-diol were chosen for compounds that methylate or silylate in the presence of BF3-methanol or BSTFA. Ten carboxylic acids commercially available of the corresponding esters (see Table S-1) were derivatized using the methylation method described above combined with the internal/recovery standards reported above. When the calculated concentration of the derivatized esters (using calibration curves obtained for their authentic standards) was compared to the original concentration of the corresponding underivatized carboxylic acid compounds, Analytical Chemistry, Vol. 76, No. 16, August 15, 2004
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Table 2. Methane-CI and EI Spectra Patterns of Methyl Esters for Model Compounds Bearing One or More Carboxylic Groups (% Relative Intensity)a methane-CI/m/z at cpds
59
2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20
70 20 5 7 75 1 2 1 1 2 2 1 3 4 2 4
22 24 26 27 29 31
3 4 5 20
EI (70 eV)/m/z at
M•+ -
M•+ -
M•+ -
M•+ +
M•+ +
M•+ +
M•+ +
63
59
31
1
15
29
41
68
3 4 8 8 6 18 15 84 13 4 27
2 100 100 100 82 100 89 70 45 43 100 100 45 38 100 55 39 100 100
100 83 7 8 8 20 72 35 7 20 18 15 60 35 27 100 100 15 3
100 58 3 100 35 9
14 4 40 22 38 100
55
6 100 95 100 35 6 37 45 3
57 30 100 7 32
3 3 4 1 4 2 1 1 2 2 4 4 2 2 3 3 2
M•+ 100%
39
55
63
Mono- and Multicarboxylic Acids 17 7 M•+ + 1 2 3 M•+ - 31 5 2 1 M•+ - 31 8 2 1 M•+ - 31 25 15 1 M•+ - 59 38 15 4 M•+ - 31 62 17 4 M•+ - 63 42 25 5 111 51 6 2 M•+ - 63 9 8 139 65 24 5 M•+ - 31 50 22 4 M•+ - 31 40 25 1 139 41 6 6 M•+ - 31 35 21 3 M•+ - 31 36 14 7 M•+ + 1 7 5 1 100 10 7 2 M•+ - 31 8 16 3 M•+ - 31 15
6 3 10 50 100 80 100 8 100 65 70 73 65 80 5 15 9 6
45 2 3 5 100 70 63 25 15 10 5 4 100 2 0 2 95
Hydroxycarboxylic Acids and Carboxylic Acids with Carbonyl Groups 16 2 1 M•+ M•+ 10 4 2 40 10 2 121 40 45 1 •+ 100 3 5 2 M +1 40 10 2 17 2 2 2 M•+ - 59 20 5 85 2 21 5 181 30 10 30 2 6 4 M•+ - 31 25 10
M•+ 59
M•+ 31
M•+
100%
1 73 35 72 3 10 1 15 5 10 12 1 8 10 93 38 15 20
30 100 100 25 21 29 22 5 7 12 13 5 10 25 100 100 100 100
2 1
