Identification and Counting of Oxygen Functionalities and Alkyl Groups

Article pubs.acs.org/EF

Identification and Counting of Oxygen Functionalities and Alkyl Groups of Aromatic Analytes in Mixtures by Positive-Mode Atmospheric Pressure Chemical Ionization Tandem Mass Spectrometry Coupled with High-Performance Liquid Chromatography Lucas M. Amundson,† Vanessa A. Gallardo,† Nelson R. Vinueza,† Benjamin C. Owen,† Jennifer N. Reece,†,‡ Steven C. Habicht,†,§ Mingkun Fu,†,∥ Ryan C. Shea,⊥ Allen B. Mossman,⊥ and Hilkka I. Kenttam ̈ aa*,† †

Department of Chemistry, Purdue University, 560 Oval Drive, West Lafayette, Indiana 47907-1393, United States British Petroleum (BP), Naperville, Illinois 60563-8460, United States

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ABSTRACT: A tandem mass spectrometric method using a commercial linear quadrupole ion trap (LQIT) mass spectrometer and another LQIT coupled with a Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometer is described for the identification and counting of different oxygen-containing functionalities and alkyl groups in unknown aromatic analytes. A total of 64 aromatic model compounds were evaporated and ionized via positive-mode atmospheric pressure chemical ionization (APCI). Ionization of the model compounds primarily results in the formation of protonated molecules, [M + H]+. In some cases, the molecular radical cation, [M]+ •, and/or a fragment ion, [M − H]+, are formed instead. Only in one case, no ions were observed near the m/z value of the molecular ion, and the ion with the greatest m/z value is a fragment ion, [M + H − H2O]+. Once ionized, the ions were subjected to multiple isolation and collision-activated dissociation (CAD) events until no more fragmentation was observed (up to MS5). In most cases, all functionalities were sequentially cleaved, one or more at a time, by the CAD events. The type of neutral molecule cleaved and the number of times that it was cleaved facilitate the identification and counting of the functionalities. The method was successfully used in concert with high-performance liquid chromatography (HPLC). The HPLC retention times offer further structural information for the analytes. This methodology benefits the chemical, pharmaceutical, and biofuels industries by facilitating the identification of previously unknown compounds directly in complex mixtures, such as crude products of chemical processes, drug metabolites, and lignin degradation products.

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INTRODUCTION The ability to rapidly identify unknown components in complex mixtures is of great importance to the chemical, pharmaceutical, and biofuels industries.1−9 For example, biofuels derived from pyrolysis of renewable plant biomass are of great interest as a possible substitute for fossil fuels.10−12 However, current biofuels are complex and unstable mixtures of numerous unknown molecules with too many oxygen atoms, which hinders their use as a transportation fuel.4−6 The ability to identify the components of these complex mixtures is critical for being able to decide how to process them further. The analysis of their conversion products that hopefully contain fewer oxygen atoms is equally challenging. The components of biofuels that originate from lignin are aromatic compounds containing various oxygen functionalities and alkyl groups. The development of suitable methodology for the analysis of these compounds in degraded biomass before and after conversion to either final fuels or valuable chemicals is the objective of this research. Mass spectrometry (MS) has evolved into an essential and powerful tool in mixture analysis.13,14 Because of its high sensitivity, specificity, and speed, MS is capable of rapidly providing useful molecular-level information for complex mixtures. The development of atmospheric pressure ionization methods, such as electrospray ionization (ESI), atmospheric pressure chemical © 2012 American Chemical Society

ionization (APCI), and atmospheric pressure photoionization (APPI), facilitated the coupling of high-performance liquid chromatography (HPLC) with MS, which has now become an invaluable tool in complex mixture analysis.2,3 However, this approach only allows for the determination of the molecular weights and elemental compositions of unknown analytes. Detailed information on the molecular structures of these analytes can be obtained using tandem mass spectrometry (MSn).15−21 MSn most often uses collision-activated dissociation (CAD) to elucidate the structures of ionized, isolated unknown compounds through their fragmentation reactions.22−24 In addition, gas-phase ion−molecule reactions have been demonstrated to facilitate the identification of various functional groups, thus aiding in the identification of unknown components in mixtures.13,21,25,26 Recently, negative-mode ESI−MS experiments were demonstrated to allow for the identification of several oxygen-containing functionalities (through MSn fragmentation reactions) in aromatic compounds.27−30 However, these methodologies are limited to highly acidic compounds, making them unsuitable for Received: December 4, 2011 Revised: March 10, 2012 Published: March 26, 2012 2975

dx.doi.org/10.1021/ef2019098 | Energy Fuels 2012, 26, 2975−2989

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Chemicals. Benzophenone and benzaldehyde were purchased from Fisher Scientific (Fair Lawn, NJ). meta-Cresol was purchased from Matheson Coleman & Bell (Cincinnati, OH; East Rutherford, NJ). Benzophenone-4,4′-dicarboxylic acid was purchased from TCI America (Portland, OR). Benzophenone-2,4,5-tricarboxylic acid was purchased from Oakwood Products, Inc. (West Columbia, SC). 9-Fluorenone-2,6-dicarboxylic acid, anthraquinone-2,6′-dicarboxylic acid, and 3,9-dicarboxybenz[c]coumarin were custom-synthesized by ChemAlong Laboratories, LLC (Lemont, IL) and provided by British Petroleum (Naperville, IL). Biphenyl-3,4-dicarboxylic acid, synthesized internally, was also provided by British Petroleum. All other chemicals were obtained from Sigma-Aldrich (St. Louis, MO). HPLC-grade H2O was purchased from Burdick & Jackson (Muskegon, MI). HPLC-grade DMSO, CH3OH, and CH3CN were purchased from Mallinckrodt Chemicals (St. Louis, MO). All chemicals were used as received without further purification.

the analysis and structural characterization of polyfunctional aromatic compounds containing no acidic functionalities, as is likely to be the case for many lignin degradation products. The utility of APCI/MSn for the identification of different oxygen-containing functionalities and alkyl groups in unknown aromatic analytes is demonstrated here. The CAD products and accompanying gas-phase ion−molecule reaction products provide valuable information on the structures of the aromatic model compounds studied. Finally, the coupling of this method with HPLC is demonstrated. The retention times provide further valuable information on the structures of the mixture components.

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EXPERIMENTAL SECTION

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Sample Preparation. Sample solutions were prepared in concentrations from 0.1 to 1 mg/mL (∼10−4−10−3 M) in 100% methanol (CH3OH), 100% acetonitrile (CH3CN), 100% dimethyl sulfoxide (DMSO), 50:50 (v/v) H2O/CH3CN, or 50:50 (v/v) H2O/ CH3OH. The product ion branching ratios given in tables are for samples dissolved in 50:50 (v/v) H2O/CH3OH. Sample solutions for HPLC analysis were prepared at a 30 μg/mL (∼10−3 M) concentration in 100% DMSO. MS. The experiments were carried out using either a Thermo Scientific LTQ linear quadrupole ion-trap (LQIT) mass spectrometer equipped with an APCI source or an identical instrument coupled to a 7 T Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometer for high-resolution measurements. Both LQITs used the Finnigan LTQ Tune Plus interface and Xcalibur 2.0 software. With helium as the buffer gas, the LQITs were maintained at a nominal pressure between 0.5 × 10−5 and 5.9 × 10−5 Torr. The sample solutions were infused at a rate of 10 μL/min using an integrated syringe drive and combined via a tee with the HPLC eluent, 50:50 (v/v) H2O/CH3CN or 50:50 (v/v) H2O/CH3OH (solvent used for the results discussed), delivered at a rate of 100 μL/min by a Finnigan Surveyor MS Pump Plus. The resulting mixture was introduced into the ion source and ionized using positive-mode APCI. Typical APCI conditions were as follows: discharge current, 5.0 μA; vaporizer temperature, 450 °C; capillary temperature, 250 °C; sheath gas (N2) flow, 50 (arbitrary units); auxiliary gas (N2) flow, 20 (arbitrary units); and sweep gas (N2) flow, 5 (arbitrary units). Voltages for the ion optics were optimized for each analyte using the tune feature of the LTQ Tune Plus interface. CAD. MSn experiments were performed using the advanced scan features of the LTQ Tune Plus interface. The analyte ion of interest was isolated using a 1−3 Th window, at a q value of 0.25, and then fragmented by applying an appropriate activation voltage (defined by the LTQ Tune Plus interface), generally 10−30% of the “normalized collision energy”, with an activation time of 30 ms.18 The fragment ions were subjected to further isolation and CAD events until no further fragmentation was observed. Xcalibur 2.0 software (Thermo) was used for both data acquisition and processing. Only product ions with a relative abundance above 2% are reported. HPLC. All HPLC experiments were performed using a Finnigan Surveyor HPLC system, consisting of an autosampler, thermostatted column compartment, quarternary pump, and photodiode array (PDA) detector. Analytes were separated on a Water Xbridge C18 reversed-phase column (150 × 2.1 mm inner diameter, 3.5 μm particle size). The mobile phases used for separation consisted of a mixture of 0.1% formic acid in water (A) and 0.1% formic acid in acetonitrile (B). A linear gradient elution of 5% B and 95% A to 70% B and 30% A over 40 min was used. The flow rate was 0.2 mL/min with an injection volume of 10 μL. The PDA detector was set to 254 nm. To minimize interference from DMSO (the solvent used for samples in the HPLC experiments) sticking onto the surfaces of the mass spectrometer, the eluent was diverted to waste instead of into the mass spectrometer during the first 5 min. Xcalibur 2.0 software (Thermo) was used for both data acquisition and processing. Only ions with a relative abundance above a 5% threshold are reported.

RESULTS AND DISCUSSION A total of 64 aromatic compounds were examined via both (+)APCI/MSn and HPLC/(+)APCI/MSn. These analytes cover a wide range of compound types because it is not known yet what types of products are obtained from lignin using different degradation and further conversion methods.31 Various solvents were used to assess the viability of (+)APCI as an effective evaporation and ionization method for the analysis of polyfunctional aromatic analytes. Most analytes primarily formed stable protonated molecules upon APCI when using 100% CH3OH, 50:50 (v/v) H2O/CH3CN, and 50:50 (v/v) H2O/ CH3OH as the solvents. Although protonated molecules were also formed upon APCI using the other test solvents (100% CH3CN and 100% DMSO), these solvents also resulted in the formation of abundant molecular radical cations as well as a large number of fragment ions. The use of 50:50 (v/v) H2O/CH3OH as the solvent produced the most stable ion currents while primarily generating only protonated analytes. Hence, only results obtained using 50:50 (v/v) H2O/CH3OH as the solvent are discussed below. The protonated molecules or other abundant ions formed upon APCI were subjected to as many as five consecutive stages of isolation followed by CAD to obtain all possible structural information. High-resolution measurements were carried out to verify the identities of the neutral molecules lost upon fragmentation. A detailed discussion on each of the different major types of analytes studied is provided below. Benzophenones. Nine different analytes containing the benzophenone backbone were studied (Table 1). Each of these molecules forms an abundant protonated molecule [M + H]+ upon ionization via positive-mode APCI, with the exception of benzhydrol, a benzophenone analogue that contains a hydroxyl instead of a carbonyl functionality. This molecule predominantly forms the [M + H − H2O]+ fragment ion. However, the hydride abstraction product, the [M − H]+ ion, was also observed. With the exception of 2-benzoyl benzoic acid and 2,4,5-tricarboxybenzophenone, all protonated benzophenones, as well as the [M − H]+ ion formed from benzhydrol, fragment upon CAD by the loss of benzene or substituted benzene. Further CAD causes these fragment ions to fragment via the loss of CO. Hence, the benzophenone backbone can be identified on the basis of the consecutive losses of the aromatic ring and CO upon CAD of the protonated analytes. The CAD spectra of protonated carboxybenzophenones (Table 1) vary based on the location of the carboxylic acid functional group. When the carboxylic acid functionality is close 2976


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Table 1. Ions Formed upon APCI as Well as the Product Ions (with Relative Abundances) Formed upon Consecutive CAD Experiments for Benzophenones

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Ion−molecule reaction with adventitious H2O.

meta and para isomers. Dissimilarities in the reactivity of the fragment ions of the protonated molecules toward H2O allowed for the unambiguous differentiation of the meta and para isomers.20 The oxygen atoms in benzophenones can be counted on the basis of the consecutive losses of oxygen-containing small

to the carbonyl group, the protonated molecule fragments via consecutive H2O and CO losses. When the carboxylic acid functionality is not close to the carbonyl group, the protonated molecule fragments predominantly via intramolecular proton transfer followed by cleavage of the carbonyl−phenyl bond, thus allowing for the distinction of the ortho isomer from the 2977


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Table 2. Ions Formed upon APCI as Well as the Product Ions (with Relative Abundances) Formed upon Consecutive CAD Experiments for Biphenyls

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Ion−molecule reaction with adventitious H2O. 2978


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Table 3. Ions Formed upon APCI as Well as the Product Ions (with Relative Abundances) Formed upon Consecutive CAD Experiments for Fluorenones

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Two methylated benzophenones (Table 1) were also studied. Upon CAD, the protonated methylated benzophenones fragment by the loss of either benzene or toluene (indicative of a single methyl moiety bound to an aromatic ring). Further CAD causes these product ions to fragment via the loss of CO, just as the other benzophenones. Therefore, the methyl moiety on a benzophenone backbone can be identified

neutral molecules (CO, CO2, and/or H2O) upon CAD (Table 1). For example, protonated 2,4,5-tricarboxybenzophenone, which contains seven oxygen atoms, first loses H2O (MS2), then CO2 (MS3), then another CO2 (MS4), and finally, two CO molecules (MS5). Interestingly, the carboxylic acid moiety is sometimes lost as two CO groups upon subsequent CAD events. 2979


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Table 4. Ions Formed upon APCI as Well as the Product Ions (with Relative Abundances) Formed upon Consecutive CAD Experiments for Anthraquinones

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Biphenyls. A total of 15 different analytes containing the biphenyl backbone were studied (Table 2). As opposed to

on the basis of the loss of toluene instead of benzene upon CAD of the protonated analyte. 2980


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Table 5. Ions Formed upon APCI as Well as the Product Ions (with Relative Abundances) Formed upon Consecutive CAD Experiments for Coumarins

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Table 6. Ions Formed upon APCI as Well as the Product Ions (with Relative Abundances) Formed upon Consecutive CAD Experiments for Diphenylmethanes

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Six biphenyls containing one or more carboxylic acid groups were also investigated (Table 2). Most of them form abundant protonated molecules [M + H]+ upon APCI, although some also show fragmentation of the protonated molecule by the loss of H2O. The protonated molecules fragment via consecutive losses of H2O and CO when subjected to CAD. This allows for the differentiation of the carboxylic acid group from the hydroxyl group that only loses H2O and from an aldehyde group that only loses CO. The loss of H2O and CO is followed by elimination of the CH3 group from the one methylated biphenyl carboxylic acid studied, thus allowing for the identification of the methyl substituent. The protonated 3,4- and 4,4′-dicarboxybiphenyl isomers display similar fragment ions upon CAD, formed via losses of H2O, CO, and CO2 from the precursor ion. However, the protonated 2,2′-isomer only dissociates via H2O loss upon

benzophenones, ionized biphenyls show only very minor losses of benzene upon CAD. Furthermore, their behavior was found to be much more diverse than that of benzophenones. For example, ionization of biphenyl (the base compound) by APCI yields an abundant molecular radical cation [M]+ • that does not produce fragment ions upon CAD, while an aldehyde group containing biphenyl forms an abundant protonated molecule, which loses CO upon CAD. On the other hand, three hydroxymethyl-containing biphenyls were found to primarily form the fragment ion [M + H − H2O]+ upon ionization. This ion can be used to identify the presence of a hydroxyl group when the molecular weight of the analyte is known from other experiments. When the [M + H − H2O]+ ions were subjected to CAD, remaining hydroxyl functionalities were lost as H2O and methylene groups were lost as CH3 (Table 2). 2981


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Table 7. Ions Formed upon APCI as Well as the Product Ions (with Relative Abundances) Formed upon Consecutive CAD Experiments for Model Compounds with Phenyl Rings Bridged by Two Methylene Groups

Table 8. Ions Formed upon APCI as Well as the Product Ions (with Relative Abundances) Formed upon Consecutive CAD Experiments for Biphenylenes

ionization via positive-mode APCI, with the exception of 1-carboxy-9-hydroxy-9H-fluorene, a fluorenone analogue that contains a hydroxyl functionality instead of a carbonyl functionality. This molecule forms the [M + H − H2O]+ fragment ion upon APCI. As mentioned above, this fragmentation is indicative of the presence of a hydroxyl group, if the molecular weight of the analyte is known. Also, an abundant [M − H]+ ion was observed. Upon CAD, protonated fluorenone and all of its derivatives lose the carbonyl group as CO after all other substituents have been cleaved off through consecutive CAD events. Carboxylic acid functionalities on fluorenones can be identified and counted on the basis of the loss of CO2 or consecutive losses of H2O and CO upon CAD of the protonated analytes, just as for benzophenones and biphenyls. The CAD spectra of the protonated carboxyfluorenones vary based on the location of the carboxylic acid functional group, similar to the results obtained for the monocarboxybenzophenones and dicarboxybiphenyls. When the carboxylic acid functionality is close to the keto group in carboxyfluorenones, the protonated molecule fragments via consecutive H2O and CO losses. When the carboxylic acid moiety is not close to the carbonyl group, the protonated molecule also fragments via H2O and CO losses but shows an additional loss of CO2 and also a simultaneous loss of

CAD. Isolation of this fragment ion followed by CAD results in a loss of CO2. Further isolation of this fragment ion followed by CAD results in a loss of CO. These results indicate that some degree of regioisomer differentiation may be achievable using this experimental approach. Furthermore, carboxylic acid functionalities can be identified and counted on the basis of the consecutive losses of CO2 or consecutive losses of H2O and CO upon CAD of the protonated analytes. Several biphenyls containing one or two CH3 groups were also investigated (Table 2). They all display an abundant protonated molecule, [M + H]+, upon positive-mode APCI, which is sometimes accompanied by a much less abundant molecular ions. The protonated molecules (as well as the molecular ion) undergo consecutive losses of one or two CH3 groups upon CAD. Thus, the number of methyl groups in these analytes can be counted on the basis of the number of CH3 group losses. It should be noted here that some of these fragmentations violate the “even-electron rule” in MS, that an even-electron ion does not fragment to give two odd-electron fragments. However, violation of this rule is common for aromatic compounds.32 Fluorenones. Six different analytes with the fluorenone backbone were studied (Table 3). Each of these molecules forms an abundant protonated molecule [M + H]+ upon 2982


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Table 9. Ions Formed upon APCI as Well as the Product Ions (with Relative Abundances) Formed upon Consecutive CAD Experiments for Fluorenones

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hydroxyl group of the carboxylic acid moiety.20 The facile loss of the carboxylic acid moiety as H2O and CO upon CAD is in agreement with this hypothesis.20 In contrast, the ions that fragment instantly after formation in the ion source by the loss of CO2 are molecules protonated at a site (possibly the aromatic ring) where a hydrogen-bond formation between the two substituents is not possible. Several examples of this type of behavior have been reported recently.20 The 3,9-dicarboxybenz[c]coumarin forms an abundant fragment ion of m/z 197 upon positive-mode APCI, which corresponds to the loss of two CO2 groups from the protonated molecule. However, the protonated molecule was not observed. Isolation of this fragment ion followed by CAD results in another loss of CO2. Hence, again, all oxygen atoms can be counted on the basis of losses of H2O, CO, and/or CO2. Diphenylmethanes. Three different analytes containing a diphenylmethane backbone were studied (Table 6). They all form a hydride abstraction product, the [M − H]+ ion, that varies in abundance. The observation of this ion is not surprising because a positively charged CH group between the two aromatic rings will enjoy substantial resonance stabilization. In addition, molecular ions and fragment ions were formed upon ionization. Upon CAD, the [M − H]+ ion fragments much like

CO2 and CO. Hence, these results indicate that this experimental approach can yield data that facilitates the differentiation of regioisomers.20 Anthraquinones. Five different analytes containing the anthraquinone backbone were studied (Table 4). Each of these molecules form an abundant protonated molecule [M + H]+ upon ionization via positive-mode APCI. Upon CAD, the protonated anthraquinone and its derivatives fragment much like the benzophenones and fluorenones. For example, carboxylic acid groups are lost as H2O and CO or CO2. All carbonyl groups are then lost as CO. The CO losses compete with CH3 losses if the molecule contains methyl groups. The one anthraquinone with a hydroxymethyl substituent loses this group as H2O or CH2O. Coumarins. Two coumarins were studied (Table 5). 3-Carboxycoumarin forms an abundant protonated molecule [M + H]+ upon ionization by positive-mode APCI. In addition, a minor CO2 loss from the protonated molecule was observed. Upon CAD, the protonated molecule dissociates via H2O loss followed by three CO losses. Thus, all four oxygen atoms are lost as either H2O or CO. The stable long-lived ions that were subjected to CAD are probably molecules protonated at the thermodynamically favored site, which is likely to be the carbonyl group of the coumarin moiety because a proton at this site can form a stabilizing internal hydrogen bond with the 2983


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Table 10. Ions Formed upon APCI as Well as the Product Ions (with Relative Abundances) Formed upon Consecutive CAD Experiments for Benzenes

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carboxylic acid functionalities can be identified and counted by the losses of CO2 or the consecutive losses of H2O and CO. Phenyl Rings Bridged by Two Methylene Groups. Three analytes containing a molecular backbone comprised of phenyl rings bridged by two methylene groups were studied (Table 7). They produced a protonated molecule [M + H]+, a

the protonated molecules discussed above, with the exception that a phenyl or a substituted phenyl group is lost as benzene or its derivative. The [M − H]+ ion formed from both diphenylmethane analytes with carboxylic acid functionalities fragments upon CAD by the loss of H2O and CO or CO2. Losses of CH3 from the methylene bridge were also observed. Hence, the 2984


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Figure 1. (a) HPLC−UV chromatogram obtained for a sixcomponent model compound mixture. The peak at 2.12 min is the start of the gradient. (b) XIC for protonated benzophenone-2,4,5tricarboxylic acid (m/z 315). (c) XIC for protonated benzophenone4,4′-dicarboxylic acid (m/z 271). (d) XIC for protonated 2- and 4benzoylbenzoic acids (m/z 227). (e) XIC for [M + H − H2O]+ pseudo-molecular ion of benzhydrol (m/z 167). (f) XIC for protonated benzophenone (m/z 183). The peak at 17.6 min is likely a decarboxylation product of protonated 2- and 4-benzoylbenzoic acids.

molecular radical cation [M]+ •, and/or a hydride abstraction product [M − H]+ upon ionization via positive-mode APCI. The 1,2-di(para-tolyl)ethane solely forms an abundant [M − H]+ ion. This is likely due to the formation of a stable benzyl cation upon dissociation of the molecular ion by hydrogen atom loss, similar to the diphenylmethane derivatives discussed above. This ion fragments by consecutive losses of two CH3 groups in competition with consecutive losses of two H2CCH2 molecules among other fragmentations. Hence, the number of carbons in the alkyl groups in this molecule can be counted. The same is true for the other two analytes that also lose two carbon atom units upon CAD of the ions formed via positive-mode APCI. 9,10-Dihydrophenanthrene forms both an abundant protonated molecule [M + H]+ and a molecular radical cation [M]+ •. The protonated molecule fragments by the loss of CH3 or ethylene. The molecular radical cation fragments either by one CH3 loss or two subsequent hydrogen atom losses, thus not providing much structural information. 9,10-Dihydroanthracene forms three ions, protonated molecule [M + H]+, molecular radical cation [M]+ •, and hydride abstraction product ion [M − H]+ (the most abundant of the three) upon ionization via positive-mode APCI. The protonated molecule fragments by the loss of CH3 or ethylene, as seen for the phenanthrene derivative. The molecular radical cation fragments by the loss of CH3, also seen for the phenanthrene derivative. The [M − H]+ ion fragments solely by the loss of a hydrogen atom. Hence, the CAD products of protonated 9,10-didehydrophenanthrene and 9,10-dihydroanthracene provide the most useful structural information. Biphenylenes. Two different analytes containing the biphenylene backbone were studied (Table 8). Biphenylene forms an abundant molecular radical cation, [M]+ •, upon ionization via positive-mode APCI that does not yield observable

Figure 2. HPLC−MSn spectra collected for protonated 2,4,5tricarboxybenzophenone directly in a mixture. The formation of the ion of m/z 153 is discussed in the literature.20

fragment ions upon CAD. A tetramethylated biphenylene forms an abundant protonated molecule [M + H]+. This ion fragments predominantly by successive losses of CH3 and/or C2H4. Again, methyl groups can be identified and counted on the basis of the consecutive losses of CH3 groups or C2H4 molecules upon CAD of the protonated analytes. Fluorenes. Six different analytes containing the fluorene molecular backbone were studied (Table 9). Each of these molecules form an abundant protonated molecule [M + H]+ upon ionization via positive-mode APCI. Upon CAD, all protonated fluorene derivatives studied, with the exception of one, lose the ring methylene group as CH3 or C2H4 (if methyl groups are also present). The total number of aliphatic carbons can be determined from the number of carbons lost as CH3 or C2H4. On the other hand, the carbonyl group of protonated fluorene-2-carbaldehyde is lost as CO upon CAD, as was observed for a biphenyl with an aldehyde functionality and most studied ketones. As discussed above for other types of analytes, the carboxylic acid functionalities can be identified on the basis of the loss of either CO2 or consecutive losses of H2O and CO. Protonated 1-carboxyfluorene fragments by consecutive losses of CO2 and CH3, while protonated 4-carboxyfluorene fragments by consecutive losses of H2O and CO or by a single loss of CO2 (this is the only analyte that shows no CH3 losses). These results indicate that some degree of regioisomer differentiation may be achievable using this experimental approach. Benzenes. A total of 13 different analytes containing a benzene backbone were studied (Table 10). All of these 2985


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Figure 3. Retention times and the observed ions for all model compounds containing only oxygen functionalities, as determined using HPLC coupled with positive-mode APCI MS.

functionalities) are also lost as CO from these ionized analytes, just like for the other analytes discussed above (Table 10). Protonated para-anisaldehyde containing a methoxy group fragments via CO loss (as expected for an aldehyde) upon the first CAD event. Upon the subsequent CAD events, losses of CH3, H2O, CO, CH2O, or CH3OH occur. The losses of CH3 and CH3OH are indicative of the presence of a methoxy functionality, but the other losses are unexpected. Three regioisomeric carboxylic acids, toluic acids, were also investigated. Upon CAD, the protonated toluic acids fragment by a single loss of CO2, which reveals the presence of a carboxylic acid moiety, and suggest that these molecules are protonated on the benzene ring instead of the carboxylic acid moiety (which should lead to the consecutive losses of H2O and CO, as observed for many other analytes studied here). However, the isomers can be distinguished on the basis of the other ions formed upon APCI and their fragmentation patterns. For example, the ortho regioisomer forms an abundant hydride abstraction product [M − H]+, in addition to the protonated molecule [M + H]+. Upon CAD, the [M − H]+ ion fragments either by the loss of H2O, CO, or CH2O. In contrast, the para regioisomer forms [M − H]+ upon CAD, as well as its fragment ion formed by the loss of H2O. The former ion fragments by the loss of CO2, as mentioned above, but the latter ion loses CO. The only isomer that only forms [M − H]+ upon APCI is

analytes, with two exceptions, produced a major protonated molecule [M + H]+ upon APCI. One of the exceptions is paraxylene, which forms an abundant molecular ion [M]+ • that fragments via the loss of one CH3 (but not two) upon CAD. This is the only compound in this group of analytes that loses a methyl group from the aromatic ring upon CAD. Hence, methyl groups bound to the benzene ring cannot be detected as easily as methyl groups bound to compounds containing more than one aromatic ring. The other exception is 4-methylbenzyl alcohol that forms a hydride abstraction product [M − H]+ upon APCI, which upon CAD fragments via the unexpected loss of CO (verified by high-resolution measurements) usually observed for carbonyl compounds. It also forms the [M + H − H2O]+ fragment ion upon APCI, and this ion fragments upon CAD by the loss of C2H2. Hence, no evidence for the presence of a benzene-bound methyl group is obtained. Three regioisomeric cresols were also investigated (Table 10). Upon CAD, all three protonated cresols fragment by the loss of H2O or CO. The ortho isomer shows different relative abundances for these fragment ions, but the meta and para isomers yield identical product distributions. On the basis of these results, both phenolic and benzylic hydroxyl groups are lost as CO from ionized substituted benzenes, which is different from what was observed for the other major analyte groups studied here. All carbonyl groups (from both aldehyde and keto 2986


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Figure 4. Retention times and the observed ions for all model compounds containing both hydrocarbon groups and oxygen functionalities, as determined using HPLC coupled with positive-mode APCI MS.

Figure 5. Retention times and the observed ions for all model compounds containing only hydrocarbon moieties, as determined using HPLC coupled with positive-mode APCI MS.

HPLC−MSn. The results obtained for the pure model compounds using positive-mode APCI were encouraging. Hence,

the meta regioisomer. Hence, regioisomer differentiation can be achieved. 2987


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this methodology was adapted to HPLC−MSn to test its applicability on the chromatographic time scale. For this experiment, an artificial mixture of six different components (2,4, 5-tricarboxybenzophenone, 4,4′-dicarboxy-benzophenone, 2-benzoylbenzoic acid, 4-benzoylbenzoic acid, benzhydrol, and benzophenone) was prepared in DMSO. DMSO is a sticky substance that remains in the instrument for a while after being introduced. The effectiveness of positive-mode APCI may be negatively influenced by the presence of DMSO because it has a proton affinity (211.4 kcal/mol) that is very close to that of the analytes; thus, it may suppress ionization of the target compounds in the positive-ion mode. To minimize this interference, the HPLC eluent was diverted to waste during the first 5 min. Further, the low mass value for the mass analysis was set to m/z 100, so that any protonated DMSO (m/z 79) formed would not dominate the mass spectra. The resulting ultraviolet (UV) and pertinent extracted ion chromatograms (XIC) are shown in Figure 1. Each of the six components was easily separated in the HPLC, including the two regioisomers studied. Five of the six components display a large peak corresponding to the protonated molecule [M + H]+ in their positive-mode APCI mass spectra, as expected. However, benzhydrol formed an abundant [M + H − H2O]+ fragment ion, which is expected for alcohols based on the results obtained for the pure compounds (Tables 1−3 and 10). The absolute abundance of each ion in the XICs varies because of the different ionization efficiencies of the corresponding neutral molecule. In addition to measuring the APCI mass spectrum for each molecule eluting from the HPLC (Figure 1), several CAD mass spectra were collected for the ions generated upon positivemode APCI. Representative spectra collected for 2,4,5tricarboxybenzophenone are shown in Figure 2. The MS scan displays an abundant ion of m/z 315 that corresponds to the protonated molecule [M + H]+. For the MS2 scan, the ion of m/z 315 was isolated and subjected to CAD. The most abundant ion formed has a m/z value of 297 (because of the loss of H2O). This ion was isolated and subjected to CAD for the MS3 scan. This experimental sequence was repeated until the isolated fragment ion did not produce further fragment ions upon CAD. The results demonstrate that as many as five stages of MS can be performed across a chromatographic peak, thus confirming that this methodology (with data-dependent scanning) is feasible for the analysis of unknown compounds on the HPLC time scale. The CAD mass spectra obtained for all six mixture components are similar to those collected during the initial model compound survey and illustrated in the tables provided. HPLC retention times were measured for all model compounds discussed above, as well as several additional compounds, to test the usefulness of retention times in the differentiation of the analytes. The varying polarities and molecular symmetries of the compounds studied resulted in different retention times (Figures 3−5). The compounds containing only oxygen functionalities had the shortest retention times as a group (Figure 3) compared to compounds containing both alkyl groups and oxygen functionalities (Figure 4) and to compounds containing no oxygen functionalities, which eluted last (Figure 5). This is consistent with the concept of reversed-phase chromatography, where less polar compounds elute at longer retention times. When comparing compounds within the first of the above three groups, the highly symmetric molecules elute at shorter retention times. This information may provide a means for the differentiation of isomers within complex mixtures. For example,

the regioisomeric compounds 4,4′-dicarboxybiphenyl, 2,2′dicarboxybiphenyl (diphenic acid), and 3,4-dicarboxybiphenyl eluted at retention times corresponding to 13.8, 14.1, and 14.9 min, respectively, with the most symmetrical molecule eluting first and the least symmetrical molecule eluting last. The same is true for the three isomeric dihydroxymethylbenzenes. Aromatic analytes in the second group, containing both aliphatic and oxygenated moieties, elute at later retention times on average (Figure 4). The more aromatic rings and alkyl groups in these analytes, the later they elute. Finally, for the third group, compounds with no oxygen functionalities, the analytes with the largest number of saturated carbon atoms elute latest.

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CONCLUSION Most of the 64 aromatic compounds studied formed stable protonated molecules [M + H]+ upon positive-mode APCI using the assortment of solvents discussed. However, 50:50 (v/v) H2O/CH3OH provided the most stable ion currents. Some compounds produced the molecular ion, M+ •, or the [M − H]+ ion, instead of [M + H]+. Only one analyte failed to yield any ions in the molecular-weight region. The protonated molecules, molecular ions, and/or fragment ions formed upon positive-mode APCI were subjected to several consecutive ion isolation and CAD events, which revealed fragmentation patterns that facilitate the identification and counting of alkyl groups (e.g., methyl and methylene substituents), as well as different oxygen-containing functionalities, such as carboxylic acid, keto, aldehyde, methoxy, hydroxymethyl, and hydroxyl groups. Multiple CAD events are needed to identify all functionalities in multi-substituted analytes. These experiments involved analytes with varying backbones and as many as four functionalities. Despite the need for multiple consecutive ion isolation and CAD events, these experiments were demonstrated to be fast enough to be applicable on the chromatographic (HPLC) time scale. The retention times provide additional information on the nature of the analytes. Hence, this analytical methodology provides useful structural information that should facilitate the identification of unknown aromatic compounds directly in complex mixtures. Application of this methodology to the characterization of unknown lignin degradation products is underway.

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AUTHOR INFORMATION

Corresponding Author

*Telephone: (765) 494-0882. Fax: (765) 494-0239. E-mail: [email protected]. Present Addresses ‡

Max Planck Institute for Coal Research, Kaiser-Wilhelm-Platz 1, D-45470 Mülheim, Germany. § Center for Naval Analysis, 4825 Mark Center Drive, Alexandria, Virginia 22311, United States. ∥ Millennium, 40 Landsdowne Street, Cambridge, Massachusetts 02139, United States. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors gratefully acknowledge British Petroleum (Vanessa A. Gallardo) and ExxonMobil (Lucas M. Amundson) for partial financial support of this work. The rest of this work was supported as part of the Center for Direct Catalytic Conversion of Biomass to Biofuels (C3Bio) and the Energy Frontier Research Center funded by the Office of Basic Energy Sciences, 2988


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Muranaka, Y.; Yasukawa, T.; Mae, K. Energy Fuels 2011, 25, 791−796. (f) Hanson, S. K.; Baker, R. T.; Gordon, J. C.; Scott, B. L.; Thorn, D. L. Inorg. Chem. 2010, 49, 5611−5618. (32) Karni, M.; Mandelbaum, A. Org. Mass Spectrom. 1980, 15, 53−64.

Office of Science, United States Department of Energy, under Award DE-SC0000997.

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Identification and Counting of Oxygen Functionalities and Alkyl Groups

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