Research Article Received: 29 August 2014
Revised: 28 October 2014
Accepted: 31 October 2014
Published online in Wiley Online Library
Rapid Commun. Mass Spectrom. 2015, 29, 91–99 (wileyonlinelibrary.com) DOI: 10.1002/rcm.7091
Gas chromatography with parallel hard and soft ionization mass spectrometry† Leila Hejazi1,2*, Michael Guilhaus2, D. Brynn Hibbert1* and Diako Ebrahimi3 1
School of Chemistry, UNSW Australia, Sydney 2052, Australia Bioanalytical Mass Spectrometry Facility, UNSW Australia, Sydney 2052, Australia 3 Centre for Vascular Research, UNSW Australia, Sydney 2052, Australia 2
RATIONALE: Mass spectrometric identification of compounds in chromatography can be obtained from molecular masses from soft ionization mass spectrometry techniques such as field ionization (FI) and fragmentation patterns from hard ionization techniques such as electron ionization (EI). Simultaneous detection by EI and FI mass spectrometry allows alignment of the different information from each method. METHODS: We report the construction and characteristics of a combined instrument consisting of a gas chromatograph and two parallel mass spectrometry ionization sources, EI and FI. When considering both ion yield and signal-to-noise it was postulated that good-quality EI and FI mass spectra could be obtained simultaneously using a post-column splitter with a split fraction of 1:10 for EI/FI. This has been realised and we report its application for the analysis of several complex mixtures. RESULTS: The differences between the full width at half maximum (FWHM) of the EI and FI chromatograms were statistically insignificant, and the retention times of the chromatograms were highly correlated (r2 =0.9999) with no detectable bias. The applicability and significance of this combined instrument and the attendant methodology are illustrated by the analysis of standard samples of 13 compounds with diverse structures, and the analysis of mixtures of fatty acids, fish oil, hydrocarbons and yeast metabolites. CONCLUSIONS: This combined dual-source instrument saves time and resources, and more importantly generates equivalent chromatograms aligned in time, in EI and FI (i.e. peaks with similar shapes and identical positions). The identical FWHMs and retention times of the EI and FI chromatograms in this combined instrument enable the accurate assignment of fragment ions from EI to their corresponding molecular ions in FI. Copyright © 2014 John Wiley & Sons, Ltd.
The development of various mass spectrometry ionization methods in combination with separation techniques has resulted in many advanced analytical tools. There have also been many reports on the development and use of multiple ionization sources in order to take advantage of their different characteristics. The purpose of the design and use of multiple source mass spectrometers can be classified into two categories: • Increasing the range of ionized compounds: There is not a single ionization method in mass spectrometry that is equally effective for all types of molecules. Therefore, analysis using two or more ionization sources can lead to the detection of a greater number of compounds than when using single-source instruments. In 1976 a combined field ionization (FI)/electron ionization (EI) source was
* Correspondence to: L. Hejazi and D. B. Hibbert, School of Chemistry, UNSW Australia, Sydney 2052, Australia. E-mail: [email protected]; [email protected] †
Rapid Commun. Mass Spectrom. 2015, 29, 91–99
Copyright © 2014 John Wiley & Sons, Ltd.
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This paper is dedicated to the memory of our colleague Professor Michael Guilhaus, who passed away in 2009.
invented by Brunnee for the analysis of high molecular weight compounds with low volatility.[1] Other examples include the EI/chemical ionization (CI)/fast atom bombardment (FAB) combined ion source of a JEOL JMS700 magnetic sector instrument and the FI/field desorption (FD)/EI combined ion source of a modified AEI MS9 magnetic sector mass spectrometer. These combined sources are of compact design to simplify switching from EI to CI/FAB or to FI/FD for the analysis of different groups of samples.[1,2] A dual source comprising laser ionization and photoemission EI was developed in a time-of-flight (TOF) instrument by Colby and Reilly.[3] The two methods were described as being complementary with laser ionization providing good temporal and spatial resolution, controlled fragmentation and high spectrometric selectivity, and EI providing highly reproducible spectra. Siegel et al. modified an atmospheric pressure ionization (API) source to develop a mass spectrometer that utilizes, simultaneously, an electrospray ionization (ESI) probe and an atmospheric pressure chemical ionization (APCI) corona discharge needle. Rapid switching between the two ionization modes resulted in spectra being recorded alternatingly across the peak. The approach was developed to enable the analysis of
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compounds having a wide range of polarities. The shortcoming of ESI in ionizing less polar compounds was compensated for by the incorporation of an APCI source.[4] In a different study, atmospheric pressure photoionization (APPI) was combined separately with APCI and ESI to produce two dual sources: APCI/APPI and ESI/APPI. Simultaneous use of the two sources as well as switching between the sources during a single chromatography run was applied to extend the range of compounds that could be simultaneously analyzed. Better ionization efficiency of APPI than of APCI and ESI for non-polar compounds such as naphthalene was demonstrated.[5] A dual-source linear ion trap mass spectrometer design for the Mars Organic Molecular Analyser (MOMA) takes advantage of laser desorption ionization and GC/EI techniques to analyse molecules from both powdered and liquid samples.[6] • Structural analysis: A second application of multiple ionization sources is in structural analysis. In this context, hard and soft ionization techniques are used together to obtain both molecular ions and fragment ions. For example, it is known that EI, a hard ionization technique, has a high ion yield and sensitivity, and generates highly reproducible spectra for which there is a large database of spectra of known compounds. The downside of EI is in some cases the lack of a molecular ion. Alternative ionization methods, which produce spectra with less fragmentation (i.e. soft ionization techniques) and display the molecular ions (or peaks from which the molecular mass can be inferred), are often used. FI is an example of these ionization methods and with few exceptions it produces an abundant molecular ion. In contrast to EI, FI provides little fragmentation and structural information. The two methods are complementary and have a long history of being employed together. In the following some examples of multiple hard/soft ionization instruments are provided. In 1967, Wanless and Glock reported the application of a dual source in organic geochemistry that could provide EI and FI spectra successively. The importance of using dual sources was indicated by the authors, but the focus of the study was on FI which produced valuable primary metastable ions. The instrument was used for the analysis of a wide range of aliphatic compounds.[7] In a study by Kuhlman et al. mixed EI/CI (chemical ionization) spectra were obtained by adding perfluorokerosene and adjusting the pressure in the dual source. The importance of this design for the analysis of medicinal agents was highlighted.[8] Ryhage modified a GC/EI-MS instrument and designed a new source that allowed operation in either EI or CI mode.[9] Hogg and Payzant used a combined FI/FD (field desorption)/EI source for the analysis of hydrocarbon mixtures. Quick and easy switching between FI/FD and EI (or CI) was reported and the ion source could be rapidly interchanged with existing EI or CI sources.[2] Byrdwell et al. developed an instrument consisting of two parallel mass spectrometers, providing ESI and APCI spectra, connected to one LC system (LC/APCI/ESI). This instrument was used for the analysis of sphingolipid, glycerophospholipid and plasmalogen species.[10] In that work ESI-MS and APCI-MS were used as complementary techniques with ESI providing only protonated molecules
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and APCI producing diagnostic fragment ions as well as small amounts of protonated molecules. Later the same technique was used in combination with tandem mass spectrometry (MS/MS) for the analysis of triacylglycerols and their oxidation products.[11] In 2009 a combined EI/FI ion source was demonstrated with a partially modified GC/orthogonal acceleration time-offlight (oaTOF) mass spectrometer (JEOL AccuTOFGCv). This system is equipped with a single EI/FI/FD shared ion source enabling facile switching between the ionization modes without changing the ion sources and breaking the ion source vacuum, and it has been used to perform accurate qualitative analysis of different alkanes.[12] The above examples demonstrate the advantages of using multiple ionization systems in terms of increasing the range of ionized compounds as well as enabling structural analysis in a single run, therefore saving time and resources. However, it is important to note from all the above studies, that several ’separate’ analyses, one by each single ionization source, provide the same amount of information as a single analysis using multiple sources; the difference is that the latter is quicker and more economical. In this paper we present the design, construction and application of a new instrument with a dual EI/FI source and a single gas chromatography stream (GC/parallel EI/FI-MS). Similar to the previously reported dual-source instruments, the new design saves time and resources but, more importantly, it produces data with a significant and unique structure, that is, "equivalent EI and FI chromatograms". This allows accurate matching of hard and soft ionization data thus providing structural information about individual compounds and the composition of complex mixtures for which single-source instruments fall short. The combined dual-source instrument has another advantage over other dual-source designs by taking benefit of the maximum duty cycle of each mass spectrometer involved. In this instrument two analysers and two detectors are involved for separation, identification and data acquisition; therefore, the full duty cycle of each mass spectrometer gives more scans and better sensitivity for any type of analysis. To demonstrate the broad application of this instrument, simultaneous EI and FI mass spectra and chromatograms from several samples including yeast metabolites, hydrocarbons and fatty acids, as well as standard compounds with diverse structures were acquired, each in a single run.
EXPERIMENTAL Chemicals Thirteen structurally diverse compounds were used in this study: methyl stearate (99%), hexachlorobenzene (99%), 1-octadecanol (99%), octafluoronaphthalene (96%), 1,8-ethylenenaphthalene (99%), benzoin methyl ether (99%) and tert-butyl bromoacetate (98%) from Aldrich (Castle Hill, Australia), anthraquinone (≥99%), benzophenone (≥99%) and 1-bromodecane (≥97%) from Fluka (Castle Hill, Australia), hexadecane (≥99%) and β-1,2,3,4,5,6-hexachlorocyclohexane
Copyright © 2014 John Wiley & Sons, Ltd.
Rapid Commun. Mass Spectrom. 2015, 29, 91–99
GC/MS with parallel EI and FI detection (97%) from Sigma (Sydney, Australia), and p-chloroanaline (99%) from Unilab (Sydney, Australia). A standard mixture of 37 FAMEs (fatty acid methyl esters; Supelco, 18919, total mass concentration 10 mg mL 1 in methylene chloride) was purchased from Sigma-Aldrich (Castle Hill, Australia). A bottle of fish oil capsules (Blackmores Ltd) was purchased from a local pharmacy in Sydney. A sample of a hydrocarbon mixture was obtained from the School of Biological, Earth and Environmental Sciences, UNSW, Sydney, Australia. A sample of yeast (Saccharomyces cerevisiae) metabolites was obtained from the School of Biotechnology and Biomolecular Sciences, UNSW. Chloroform (99.8%), diethyl ether (99%), acetone (99.5%), methanol (99.5%), and xylene (98.5%) solvents were from Univar (Sydney, Australia). Hexane (99%) and methanol (99.7%) were purchased from Unichrom (Ajax Finechem Ltd, Thermo Fisher Scientific, Auckland, New Zealand) and sulfuric acid (99.999%) from Aldrich. Toluene (100%), diethyl ether (98%) and sodium hydrogen carbonate (99.7%) were purchased from Univar and sodium sulfate anhydrates (≥99%) from Fluka. Butylated hydroxytoluene was obtained from MP Biomedicals., Inc. (Aurora, OH, USA). A 1.0 mL reference mixture for calibration of the mass scale in FI mode was prepared to contain 20% by volume of perfluoro-n-buthylamine (heptacosa), 2% hexafluorobenzene, 2% pentafluorobenzene, 2% chloropentafluorobenzene (≥99% purity from Aldrich); 2% acetone, 2% xylene (99.5% and 98.5% purity, respectively, from Univar); and 70% perfluorotrimethylcyclohexane (90% purity from Fluorochem, Novachem, Collingwood, Australia). Tris(trifluoromethyl) triazine (99.9% pure, Aldrich) was used to generate a lock mass molecular ion at m/z 284.9949 to improve mass accuracy.
Metabolites A sample of yeast (Saccharomyces cerevisiae) was cultured in defined medium for 24 h. Cells were then removed by centrifugation and filtration. Of the spent medium, 5 mL was added to a standard GC vial with excess NaCl. After liquid/liquid extraction using 2 mL diethyl ether, the ether layer was collected and 1 μL of the solution was injected into the instrument. Gas chromatography (GC)
Fish oil
GC analysis was performed by a HP-6890 series GC system (Hewlett Packard, Agilent Technologies, Wilmington, DE, USA). Different capillary columns were used as follows: a HP-5ms column (5% phenylmethylpolysiloxane, 25 m ×250 μm i.d., 0.25 μm film thickness; Agilent J&W, Burwood Highway, Forest Hills, Victoria, Australia) was used for the analysis of 13 structurally diverse compounds and the hydrocarbon sample; a BPX70 column (60 m ×0.25 mm ×0.25 μm; SGE Altech, Baulkham Hills, NSW, Australia) was used for the analysis of the 37 FAMEs mixture and the fish oil sample; and a BP20 column (60 m ×0.25 mm ×0.25 μm; SGE Altech) was used for the analysis of the metabolite sample. The injector was operated with manual injections of 1 μL of solutions in splitless mode at 240 °C for all compounds. The carrier gas was helium with a constant flow rate of 1.1 mL min 1 for the 30 m column and 0.6 mL min 1 for the 60 m column. To split the column eluent flow between the two instruments used for collecting EI and FI data, a vitreous silica outlet splitter (VSOS, 1:10 split ratio, SGE International Pty, Ringwood, Australia) was connected to the analytical column. The outlet connections from the splitter were arranged to give flow rates of 1.0 and 0.1 mL min 1 to the FI and EI sources, respectively, when the 30 m column was used and 0.54 and 0.06 mL min 1, respectively, for the 60 m column. Different temperature programs were applied depending on the analytical sample. The temperature was held at 60 °C for 3 min then increased at 24 °C min 1 to 240 °C for the 13 structurally diverse compounds. The temperature of the transfer lines was set to 280 °C. To analyse the 37 FAMEs mixture and the fish oil sample the temperature ramp program was: 60 °C for 1 min, then increased at 5 °C min 1 to 80 °C, and at 2 °C min 1 to 230 °C. The temperature of the transfer lines was set to 280 °C. For the hydrocarbon sample, the initial temperature was 70 °C for 1 min before increasing it at 12 °C min 1 to 300 °C. The temperature of the transfer lines was set to 310 °C. The temperature program for the metabolite sample was as follows: 50 °C for 2 min then increased by 10 °C min 1 to 220 °C and remaining at 220 °C for 21 min. The temperature of the transfer lines was set to 260 °C.
The fish oil sample was esterified using the method described previously[13] and stored at 20 °C until use.
Mass spectrometry
Sample preparation Thirteen structurally diverse compounds With each compound, a test solution containing an accurately weighed mass of the compound was prepared to make a 10 mL stock solution of 1μg μL 1 from which 100 μL was diluted to 1.0 mL in GC/MS vials. The main dilution solvent was hexane and the concentrations of samples were in the range 10–100 ng μL 1. The relative standard uncertainty due to the gravimetric and volumetric steps was estimated to be 1.7%.[13] Standard 37 fatty acid methyl esters (FAMEs) mixture 1.0 μL of a diluted solution (1.0 mg mL 1) of a 37 FAMEs standard mixture with original concentration of 10.0 mg mL 1 was used.
FI-MS Hydrocarbons
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The mass spectrometer used for FI measurements was a Waters Micromass GCT (Manchester, UK) orthogonal acceleration time-of-flight (oa-TOF) instrument, which can provide exact mass measurement and elemental composition
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The hydrocarbon mixture sample was a membrane fouled with oil (80 mL) extracted three times in hexane: 1.0 μL of this solution was injected into the instrument.
L. Hejazi et al. determination. At high mass resolution (5000–7000 FWHM), a mass accuracy of about 5 ppm is achievable. The GCT mass spectrometer has different ionization source options, such as electron ionization (EI), chemical ionization (CI) and field ionization (FI), and the FI source was used for our parallel EI/FI design. Positive molecular ions are produced based on quantum tunneling of a valence electron at the tip of an FI emitter. The mass spectrometer was operated at a resolution of 7000 FWHM. The push-out pulse frequencies were as recommended by the manufacturer at 30 kHz (mass range m/z 10–800). The 3.6 GHz time-to-digital converter (TDC) was set up with a 350 mV trigger threshold and a 42 mV signal threshold. FI was performed with a 12 kV extraction voltage. The data was acquired using Masslynx software (version 4, Waters Micromass).
and the higher signal-to-noise ratio in FI than in EI suggested that a constant split of 10:1 should give good-quality FI and EI spectra for most compounds.[13] The transfer column to the FI source was heated by the HP-6890 transfer line, and the heat for the transfer column going to the EI source was provided by the detector cables of a HP-5890 gas chromatograph. The transfer lines were thoroughly wrapped in fiberglass to ensure that the system was thermally isolated. The same temperature program was used in both the EI and the FI instruments where the data acquisition was started simultaneously upon the injection of sample into the analytical GC column in the HP-6890. GCT FI data and 5971A MSD EI data were recorded and analyzed by Masslynx and Chem Station software, respectively. The schematic of the GC/parallel EI/FI-MS system is shown in Fig. 1.
EI-MS The mass spectrometer used for EI measurements was a HP 5971A mass-selective detector (MSD) instrument with a quadrupole mass analyzer from Agilent Technologies (Forest Hill, Victoria, Australia). In our experiments the m/z range 40–400 was used for all samples except for the hydrocarbon sample for which m/z 40–600 was employed. Scan rates of 1 and 1.2 scans/s were used for m/z 40–400 and 40–600, respectively. New design, GC/parallel EI/FI-MS The new instrument consists of two mass spectrometers (GCT running in FI and HP 5971A running in EI) connected in parallel to a single HP-6890 gas chromatograph, The exit of the GC analytical capillary column was connected to a vitreous silica splitter whose outlet was connected to two transfer columns (~5 m of 0.22 mm i.d. deactivated fusedsilica tubing) with different lengths to provide a flow ratio of 1:10 for the EI/FI sources. As has been described in our previous study,[13] to find the best post-column split ratio for acquiring good-quality spectra in both FI and EI in the parallel source instrument, a figure of merit – defined as the geometrical mean of the ratios of ion yield and signal-to-noise ratio – was investigated. The greater ion yield in EI than in FI
RESULTS AND DISCUSSION The use of FI alongside EI has two main advantages. First, in cases where the molecular ion is absent or appears with low intensity in EI, being able to obtain or confirm a molecular mass is important for the characterization of unknown compounds. It is particularly essential when the EI spectrum does not yield a conclusive match with a database record.[13] Secondly, complex samples such as metabolites and petroleum oils can contain hundreds to thousands of compounds, many of which have considerable chromatographic overlap in a typical GC/MS analysis. In those cases, FI with much cleaner mass spectra (only a few m/z peaks), than EI, is highly useful. This unique feature of FI facilitates the determination of the number of chromatographically overlapped compounds, in particular when accurate mass data are available. This is not usually possible using EI which produces many fragment ions for which discrimination from molecular ions (and attribution to their corresponding molecular ion, if there is any) is challenging. The fragment ions which are common among the overlapping compounds and the similar mass spectra for some compounds with different structures (e.g. trienes and higher unsaturated fatty acids, see Fig. 2) add to the difficulty of identification of overlapping peaks using EI analysis. As described below simultaneous recording of EI and FI data so that similar chromatograms are obtained for EI and FI is essential to achieve the abovementioned advantages. Alignment of total ion chromatograms
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Figure 1. Schematic of the GC/parallel EI/FI-MS system. GC1: HP 6890; GC2: HP 5890; GCT: orthogonal acceleration time-of-flight mass spectrometer, 5971A; MSD: Quadrupole mass-selective detector, 1: injector, 2 and 3: interface heater wiring from detector A and B, 4: fixed outlet splitter.
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A standard mixture of 13 structurally diverse compounds (described above) gave peaks in EI and FI modes that essentially overlapped (see Supplementary Fig. S1, Supporting Information, and an example in Fig. 3). In all cases good-quality data (sharp and well-shaped peaks) are obtained simultaneously for EI and FI. These results confirm the optimum split ratio 1:10 EI/FI that we proposed in our previous feasibility study.[13] Total ion chromatograms of samples of FAMEs, fish oil, hydrocarbons and yeast metabolites using the new GC/parallel EI/FI-MS instrument are shown in Figs. 4–7. What is noticeable in these figures is that the ’positions’ of the chromatographic peaks in EI and FI are identical. This is because a single chromatography step is performed
Copyright © 2014 John Wiley & Sons, Ltd.
Rapid Commun. Mass Spectrom. 2015, 29, 91–99
GC/MS with parallel EI and FI detection
Figure 2. EI and FI spectra of two polyunsaturated fatty acid methyl esters, C22:6n3 and C20:5n3. The EI spectra of these compounds are almost indistinguishable and more importantly they lack molecular ion peaks. The FI spectra show sharp molecular ion peaks and unique patterns by which the compounds can be distinguished.
Figure 3. EI and FI chromatograms of 1-octadecanol (C18H38O, 100 ng μL 1) and tert-butyl bromoacetate (C6H11BrO2, 100 ng μL 1) obtained using GC/parallel EI/ FI-MS.
Figure 4. Total ion chromatograms of 37 FAMEs mixture obtained using GC/parallel EI/FI-MS.
Rapid Commun. Mass Spectrom. 2015, 29, 91–99
reason the shapes of the peaks from EI and FI are very similar, although not necessarily identical. For example, both sets of peaks show the same pattern in terms of tailing
Copyright © 2014 John Wiley & Sons, Ltd.
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which is then followed by a detection step using two ionization sources. Therefore, the chromatographic conditions under which the compounds elute remain identical for the two ionization sources: EI and FI. For the same
Figure 5. Total ion chromatograms of a fish oil sample obtained using GC/parallel EI/FI-MS.
L. Hejazi et al. Retention time
Figure 6. Total ion chromatograms of a hydrocarbon sample obtained using GC/parallel EI/FI-MS.
Figure 7. Total ion chromatograms of a yeast metabolite sample obtained using GC/parallel EI/FI-MS.
To investigate whether there is a significant difference in the retention times of the EI and FI chromatograms in our parallel EI/FI data acquisition approach, a regression analysis was used. Linear correlation between the EI and FI data over a retention time range of 10.7 to 76.5 min returned a coefficient of determination (r2) 0.9999, following the relationship RTFI = (0.001 ± 0.015) + RTEI × (1.0000 ± 0.0003) for retention time in units of minutes. The production of EI and FI chromatographic peaks with identical position and similar shapes is an important feature of GC/parallel EI/FI-MS in that it facilitates the matching of fragment and molecular ions, thus enabling accurate association of fragment ions from EI with their corresponding molecular ion from FI. This is essential for the identification of compounds, particularly in complex mixtures. To illustrate this strength, Fig. 8 shows a small region of the chromatogram of Fig. 6. The FI data revealed that there are (at least) two isomers with a nominal molecular mass of 196 and a third compound (at least one isomer) with a nominal molecular mass of 184, which elutes between the two isomers. It should be noted that not all ions observed in a FI spectrum are necessarily molecular ions; we have shown before that alcohols, as an example, can undergo loss of water from the molecular ion to yield [M–H2O]+• as the dominant ion in the FI spectrum.[13] The molecular ion chromatograms from EI and FI and those of one fragment ion from each compound (obtained from EI) are also presented in Fig. 8. The combined EI mass spectrum of the region showed a weak molecular ion for the two isomers but none for the third compound (see
but can exhibit different intensities (see the EI and FI chromatograms of C18H38O and C16H11BrO2 in Fig. 3). The latter is because the EI and FI peaks are generated using different ionization mechanisms, thus having different ionization efficiencies and sensitivities. Another example of different efficiencies is seen in the latter peaks of the fish oil analysis (Fig. 5). The peak at 72 min, yielding the ion at m/z 316.2382, is C21H32O2 (C20:5 methyl eicosapentaenoate) and the peak at 78.6 min, with the ion at m/z 342.2543, is C23H34O2 (C22:6 methyl docosahexaenoate). Highly unsaturated fatty acids have low ionization efficiency in FI, an opposite characteristic to EI. To investigate the degree of the equivalence between the EI and FI chromatograms, two figures of merit were considered using 31 resolved peaks from a standard mixture of FAMEs: FWHM and retention time.
FWHM
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To investigate whether there is a significant difference between the shapes of the EI and FI chromatograms, a twotailed t-test of the difference between the FWHM of the EI and FI peaks returned a P value of 0.38. A difference that is significant at the 95% level would have P
Revised: 28 October 2014
Accepted: 31 October 2014
Published online in Wiley Online Library
Rapid Commun. Mass Spectrom. 2015, 29, 91–99 (wileyonlinelibrary.com) DOI: 10.1002/rcm.7091
Gas chromatography with parallel hard and soft ionization mass spectrometry† Leila Hejazi1,2*, Michael Guilhaus2, D. Brynn Hibbert1* and Diako Ebrahimi3 1
School of Chemistry, UNSW Australia, Sydney 2052, Australia Bioanalytical Mass Spectrometry Facility, UNSW Australia, Sydney 2052, Australia 3 Centre for Vascular Research, UNSW Australia, Sydney 2052, Australia 2
RATIONALE: Mass spectrometric identification of compounds in chromatography can be obtained from molecular masses from soft ionization mass spectrometry techniques such as field ionization (FI) and fragmentation patterns from hard ionization techniques such as electron ionization (EI). Simultaneous detection by EI and FI mass spectrometry allows alignment of the different information from each method. METHODS: We report the construction and characteristics of a combined instrument consisting of a gas chromatograph and two parallel mass spectrometry ionization sources, EI and FI. When considering both ion yield and signal-to-noise it was postulated that good-quality EI and FI mass spectra could be obtained simultaneously using a post-column splitter with a split fraction of 1:10 for EI/FI. This has been realised and we report its application for the analysis of several complex mixtures. RESULTS: The differences between the full width at half maximum (FWHM) of the EI and FI chromatograms were statistically insignificant, and the retention times of the chromatograms were highly correlated (r2 =0.9999) with no detectable bias. The applicability and significance of this combined instrument and the attendant methodology are illustrated by the analysis of standard samples of 13 compounds with diverse structures, and the analysis of mixtures of fatty acids, fish oil, hydrocarbons and yeast metabolites. CONCLUSIONS: This combined dual-source instrument saves time and resources, and more importantly generates equivalent chromatograms aligned in time, in EI and FI (i.e. peaks with similar shapes and identical positions). The identical FWHMs and retention times of the EI and FI chromatograms in this combined instrument enable the accurate assignment of fragment ions from EI to their corresponding molecular ions in FI. Copyright © 2014 John Wiley & Sons, Ltd.
The development of various mass spectrometry ionization methods in combination with separation techniques has resulted in many advanced analytical tools. There have also been many reports on the development and use of multiple ionization sources in order to take advantage of their different characteristics. The purpose of the design and use of multiple source mass spectrometers can be classified into two categories: • Increasing the range of ionized compounds: There is not a single ionization method in mass spectrometry that is equally effective for all types of molecules. Therefore, analysis using two or more ionization sources can lead to the detection of a greater number of compounds than when using single-source instruments. In 1976 a combined field ionization (FI)/electron ionization (EI) source was
* Correspondence to: L. Hejazi and D. B. Hibbert, School of Chemistry, UNSW Australia, Sydney 2052, Australia. E-mail: [email protected]; [email protected] †
Rapid Commun. Mass Spectrom. 2015, 29, 91–99
Copyright © 2014 John Wiley & Sons, Ltd.
91
This paper is dedicated to the memory of our colleague Professor Michael Guilhaus, who passed away in 2009.
invented by Brunnee for the analysis of high molecular weight compounds with low volatility.[1] Other examples include the EI/chemical ionization (CI)/fast atom bombardment (FAB) combined ion source of a JEOL JMS700 magnetic sector instrument and the FI/field desorption (FD)/EI combined ion source of a modified AEI MS9 magnetic sector mass spectrometer. These combined sources are of compact design to simplify switching from EI to CI/FAB or to FI/FD for the analysis of different groups of samples.[1,2] A dual source comprising laser ionization and photoemission EI was developed in a time-of-flight (TOF) instrument by Colby and Reilly.[3] The two methods were described as being complementary with laser ionization providing good temporal and spatial resolution, controlled fragmentation and high spectrometric selectivity, and EI providing highly reproducible spectra. Siegel et al. modified an atmospheric pressure ionization (API) source to develop a mass spectrometer that utilizes, simultaneously, an electrospray ionization (ESI) probe and an atmospheric pressure chemical ionization (APCI) corona discharge needle. Rapid switching between the two ionization modes resulted in spectra being recorded alternatingly across the peak. The approach was developed to enable the analysis of
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compounds having a wide range of polarities. The shortcoming of ESI in ionizing less polar compounds was compensated for by the incorporation of an APCI source.[4] In a different study, atmospheric pressure photoionization (APPI) was combined separately with APCI and ESI to produce two dual sources: APCI/APPI and ESI/APPI. Simultaneous use of the two sources as well as switching between the sources during a single chromatography run was applied to extend the range of compounds that could be simultaneously analyzed. Better ionization efficiency of APPI than of APCI and ESI for non-polar compounds such as naphthalene was demonstrated.[5] A dual-source linear ion trap mass spectrometer design for the Mars Organic Molecular Analyser (MOMA) takes advantage of laser desorption ionization and GC/EI techniques to analyse molecules from both powdered and liquid samples.[6] • Structural analysis: A second application of multiple ionization sources is in structural analysis. In this context, hard and soft ionization techniques are used together to obtain both molecular ions and fragment ions. For example, it is known that EI, a hard ionization technique, has a high ion yield and sensitivity, and generates highly reproducible spectra for which there is a large database of spectra of known compounds. The downside of EI is in some cases the lack of a molecular ion. Alternative ionization methods, which produce spectra with less fragmentation (i.e. soft ionization techniques) and display the molecular ions (or peaks from which the molecular mass can be inferred), are often used. FI is an example of these ionization methods and with few exceptions it produces an abundant molecular ion. In contrast to EI, FI provides little fragmentation and structural information. The two methods are complementary and have a long history of being employed together. In the following some examples of multiple hard/soft ionization instruments are provided. In 1967, Wanless and Glock reported the application of a dual source in organic geochemistry that could provide EI and FI spectra successively. The importance of using dual sources was indicated by the authors, but the focus of the study was on FI which produced valuable primary metastable ions. The instrument was used for the analysis of a wide range of aliphatic compounds.[7] In a study by Kuhlman et al. mixed EI/CI (chemical ionization) spectra were obtained by adding perfluorokerosene and adjusting the pressure in the dual source. The importance of this design for the analysis of medicinal agents was highlighted.[8] Ryhage modified a GC/EI-MS instrument and designed a new source that allowed operation in either EI or CI mode.[9] Hogg and Payzant used a combined FI/FD (field desorption)/EI source for the analysis of hydrocarbon mixtures. Quick and easy switching between FI/FD and EI (or CI) was reported and the ion source could be rapidly interchanged with existing EI or CI sources.[2] Byrdwell et al. developed an instrument consisting of two parallel mass spectrometers, providing ESI and APCI spectra, connected to one LC system (LC/APCI/ESI). This instrument was used for the analysis of sphingolipid, glycerophospholipid and plasmalogen species.[10] In that work ESI-MS and APCI-MS were used as complementary techniques with ESI providing only protonated molecules
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and APCI producing diagnostic fragment ions as well as small amounts of protonated molecules. Later the same technique was used in combination with tandem mass spectrometry (MS/MS) for the analysis of triacylglycerols and their oxidation products.[11] In 2009 a combined EI/FI ion source was demonstrated with a partially modified GC/orthogonal acceleration time-offlight (oaTOF) mass spectrometer (JEOL AccuTOFGCv). This system is equipped with a single EI/FI/FD shared ion source enabling facile switching between the ionization modes without changing the ion sources and breaking the ion source vacuum, and it has been used to perform accurate qualitative analysis of different alkanes.[12] The above examples demonstrate the advantages of using multiple ionization systems in terms of increasing the range of ionized compounds as well as enabling structural analysis in a single run, therefore saving time and resources. However, it is important to note from all the above studies, that several ’separate’ analyses, one by each single ionization source, provide the same amount of information as a single analysis using multiple sources; the difference is that the latter is quicker and more economical. In this paper we present the design, construction and application of a new instrument with a dual EI/FI source and a single gas chromatography stream (GC/parallel EI/FI-MS). Similar to the previously reported dual-source instruments, the new design saves time and resources but, more importantly, it produces data with a significant and unique structure, that is, "equivalent EI and FI chromatograms". This allows accurate matching of hard and soft ionization data thus providing structural information about individual compounds and the composition of complex mixtures for which single-source instruments fall short. The combined dual-source instrument has another advantage over other dual-source designs by taking benefit of the maximum duty cycle of each mass spectrometer involved. In this instrument two analysers and two detectors are involved for separation, identification and data acquisition; therefore, the full duty cycle of each mass spectrometer gives more scans and better sensitivity for any type of analysis. To demonstrate the broad application of this instrument, simultaneous EI and FI mass spectra and chromatograms from several samples including yeast metabolites, hydrocarbons and fatty acids, as well as standard compounds with diverse structures were acquired, each in a single run.
EXPERIMENTAL Chemicals Thirteen structurally diverse compounds were used in this study: methyl stearate (99%), hexachlorobenzene (99%), 1-octadecanol (99%), octafluoronaphthalene (96%), 1,8-ethylenenaphthalene (99%), benzoin methyl ether (99%) and tert-butyl bromoacetate (98%) from Aldrich (Castle Hill, Australia), anthraquinone (≥99%), benzophenone (≥99%) and 1-bromodecane (≥97%) from Fluka (Castle Hill, Australia), hexadecane (≥99%) and β-1,2,3,4,5,6-hexachlorocyclohexane
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GC/MS with parallel EI and FI detection (97%) from Sigma (Sydney, Australia), and p-chloroanaline (99%) from Unilab (Sydney, Australia). A standard mixture of 37 FAMEs (fatty acid methyl esters; Supelco, 18919, total mass concentration 10 mg mL 1 in methylene chloride) was purchased from Sigma-Aldrich (Castle Hill, Australia). A bottle of fish oil capsules (Blackmores Ltd) was purchased from a local pharmacy in Sydney. A sample of a hydrocarbon mixture was obtained from the School of Biological, Earth and Environmental Sciences, UNSW, Sydney, Australia. A sample of yeast (Saccharomyces cerevisiae) metabolites was obtained from the School of Biotechnology and Biomolecular Sciences, UNSW. Chloroform (99.8%), diethyl ether (99%), acetone (99.5%), methanol (99.5%), and xylene (98.5%) solvents were from Univar (Sydney, Australia). Hexane (99%) and methanol (99.7%) were purchased from Unichrom (Ajax Finechem Ltd, Thermo Fisher Scientific, Auckland, New Zealand) and sulfuric acid (99.999%) from Aldrich. Toluene (100%), diethyl ether (98%) and sodium hydrogen carbonate (99.7%) were purchased from Univar and sodium sulfate anhydrates (≥99%) from Fluka. Butylated hydroxytoluene was obtained from MP Biomedicals., Inc. (Aurora, OH, USA). A 1.0 mL reference mixture for calibration of the mass scale in FI mode was prepared to contain 20% by volume of perfluoro-n-buthylamine (heptacosa), 2% hexafluorobenzene, 2% pentafluorobenzene, 2% chloropentafluorobenzene (≥99% purity from Aldrich); 2% acetone, 2% xylene (99.5% and 98.5% purity, respectively, from Univar); and 70% perfluorotrimethylcyclohexane (90% purity from Fluorochem, Novachem, Collingwood, Australia). Tris(trifluoromethyl) triazine (99.9% pure, Aldrich) was used to generate a lock mass molecular ion at m/z 284.9949 to improve mass accuracy.
Metabolites A sample of yeast (Saccharomyces cerevisiae) was cultured in defined medium for 24 h. Cells were then removed by centrifugation and filtration. Of the spent medium, 5 mL was added to a standard GC vial with excess NaCl. After liquid/liquid extraction using 2 mL diethyl ether, the ether layer was collected and 1 μL of the solution was injected into the instrument. Gas chromatography (GC)
Fish oil
GC analysis was performed by a HP-6890 series GC system (Hewlett Packard, Agilent Technologies, Wilmington, DE, USA). Different capillary columns were used as follows: a HP-5ms column (5% phenylmethylpolysiloxane, 25 m ×250 μm i.d., 0.25 μm film thickness; Agilent J&W, Burwood Highway, Forest Hills, Victoria, Australia) was used for the analysis of 13 structurally diverse compounds and the hydrocarbon sample; a BPX70 column (60 m ×0.25 mm ×0.25 μm; SGE Altech, Baulkham Hills, NSW, Australia) was used for the analysis of the 37 FAMEs mixture and the fish oil sample; and a BP20 column (60 m ×0.25 mm ×0.25 μm; SGE Altech) was used for the analysis of the metabolite sample. The injector was operated with manual injections of 1 μL of solutions in splitless mode at 240 °C for all compounds. The carrier gas was helium with a constant flow rate of 1.1 mL min 1 for the 30 m column and 0.6 mL min 1 for the 60 m column. To split the column eluent flow between the two instruments used for collecting EI and FI data, a vitreous silica outlet splitter (VSOS, 1:10 split ratio, SGE International Pty, Ringwood, Australia) was connected to the analytical column. The outlet connections from the splitter were arranged to give flow rates of 1.0 and 0.1 mL min 1 to the FI and EI sources, respectively, when the 30 m column was used and 0.54 and 0.06 mL min 1, respectively, for the 60 m column. Different temperature programs were applied depending on the analytical sample. The temperature was held at 60 °C for 3 min then increased at 24 °C min 1 to 240 °C for the 13 structurally diverse compounds. The temperature of the transfer lines was set to 280 °C. To analyse the 37 FAMEs mixture and the fish oil sample the temperature ramp program was: 60 °C for 1 min, then increased at 5 °C min 1 to 80 °C, and at 2 °C min 1 to 230 °C. The temperature of the transfer lines was set to 280 °C. For the hydrocarbon sample, the initial temperature was 70 °C for 1 min before increasing it at 12 °C min 1 to 300 °C. The temperature of the transfer lines was set to 310 °C. The temperature program for the metabolite sample was as follows: 50 °C for 2 min then increased by 10 °C min 1 to 220 °C and remaining at 220 °C for 21 min. The temperature of the transfer lines was set to 260 °C.
The fish oil sample was esterified using the method described previously[13] and stored at 20 °C until use.
Mass spectrometry
Sample preparation Thirteen structurally diverse compounds With each compound, a test solution containing an accurately weighed mass of the compound was prepared to make a 10 mL stock solution of 1μg μL 1 from which 100 μL was diluted to 1.0 mL in GC/MS vials. The main dilution solvent was hexane and the concentrations of samples were in the range 10–100 ng μL 1. The relative standard uncertainty due to the gravimetric and volumetric steps was estimated to be 1.7%.[13] Standard 37 fatty acid methyl esters (FAMEs) mixture 1.0 μL of a diluted solution (1.0 mg mL 1) of a 37 FAMEs standard mixture with original concentration of 10.0 mg mL 1 was used.
FI-MS Hydrocarbons
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The mass spectrometer used for FI measurements was a Waters Micromass GCT (Manchester, UK) orthogonal acceleration time-of-flight (oa-TOF) instrument, which can provide exact mass measurement and elemental composition
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The hydrocarbon mixture sample was a membrane fouled with oil (80 mL) extracted three times in hexane: 1.0 μL of this solution was injected into the instrument.
L. Hejazi et al. determination. At high mass resolution (5000–7000 FWHM), a mass accuracy of about 5 ppm is achievable. The GCT mass spectrometer has different ionization source options, such as electron ionization (EI), chemical ionization (CI) and field ionization (FI), and the FI source was used for our parallel EI/FI design. Positive molecular ions are produced based on quantum tunneling of a valence electron at the tip of an FI emitter. The mass spectrometer was operated at a resolution of 7000 FWHM. The push-out pulse frequencies were as recommended by the manufacturer at 30 kHz (mass range m/z 10–800). The 3.6 GHz time-to-digital converter (TDC) was set up with a 350 mV trigger threshold and a 42 mV signal threshold. FI was performed with a 12 kV extraction voltage. The data was acquired using Masslynx software (version 4, Waters Micromass).
and the higher signal-to-noise ratio in FI than in EI suggested that a constant split of 10:1 should give good-quality FI and EI spectra for most compounds.[13] The transfer column to the FI source was heated by the HP-6890 transfer line, and the heat for the transfer column going to the EI source was provided by the detector cables of a HP-5890 gas chromatograph. The transfer lines were thoroughly wrapped in fiberglass to ensure that the system was thermally isolated. The same temperature program was used in both the EI and the FI instruments where the data acquisition was started simultaneously upon the injection of sample into the analytical GC column in the HP-6890. GCT FI data and 5971A MSD EI data were recorded and analyzed by Masslynx and Chem Station software, respectively. The schematic of the GC/parallel EI/FI-MS system is shown in Fig. 1.
EI-MS The mass spectrometer used for EI measurements was a HP 5971A mass-selective detector (MSD) instrument with a quadrupole mass analyzer from Agilent Technologies (Forest Hill, Victoria, Australia). In our experiments the m/z range 40–400 was used for all samples except for the hydrocarbon sample for which m/z 40–600 was employed. Scan rates of 1 and 1.2 scans/s were used for m/z 40–400 and 40–600, respectively. New design, GC/parallel EI/FI-MS The new instrument consists of two mass spectrometers (GCT running in FI and HP 5971A running in EI) connected in parallel to a single HP-6890 gas chromatograph, The exit of the GC analytical capillary column was connected to a vitreous silica splitter whose outlet was connected to two transfer columns (~5 m of 0.22 mm i.d. deactivated fusedsilica tubing) with different lengths to provide a flow ratio of 1:10 for the EI/FI sources. As has been described in our previous study,[13] to find the best post-column split ratio for acquiring good-quality spectra in both FI and EI in the parallel source instrument, a figure of merit – defined as the geometrical mean of the ratios of ion yield and signal-to-noise ratio – was investigated. The greater ion yield in EI than in FI
RESULTS AND DISCUSSION The use of FI alongside EI has two main advantages. First, in cases where the molecular ion is absent or appears with low intensity in EI, being able to obtain or confirm a molecular mass is important for the characterization of unknown compounds. It is particularly essential when the EI spectrum does not yield a conclusive match with a database record.[13] Secondly, complex samples such as metabolites and petroleum oils can contain hundreds to thousands of compounds, many of which have considerable chromatographic overlap in a typical GC/MS analysis. In those cases, FI with much cleaner mass spectra (only a few m/z peaks), than EI, is highly useful. This unique feature of FI facilitates the determination of the number of chromatographically overlapped compounds, in particular when accurate mass data are available. This is not usually possible using EI which produces many fragment ions for which discrimination from molecular ions (and attribution to their corresponding molecular ion, if there is any) is challenging. The fragment ions which are common among the overlapping compounds and the similar mass spectra for some compounds with different structures (e.g. trienes and higher unsaturated fatty acids, see Fig. 2) add to the difficulty of identification of overlapping peaks using EI analysis. As described below simultaneous recording of EI and FI data so that similar chromatograms are obtained for EI and FI is essential to achieve the abovementioned advantages. Alignment of total ion chromatograms
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Figure 1. Schematic of the GC/parallel EI/FI-MS system. GC1: HP 6890; GC2: HP 5890; GCT: orthogonal acceleration time-of-flight mass spectrometer, 5971A; MSD: Quadrupole mass-selective detector, 1: injector, 2 and 3: interface heater wiring from detector A and B, 4: fixed outlet splitter.
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A standard mixture of 13 structurally diverse compounds (described above) gave peaks in EI and FI modes that essentially overlapped (see Supplementary Fig. S1, Supporting Information, and an example in Fig. 3). In all cases good-quality data (sharp and well-shaped peaks) are obtained simultaneously for EI and FI. These results confirm the optimum split ratio 1:10 EI/FI that we proposed in our previous feasibility study.[13] Total ion chromatograms of samples of FAMEs, fish oil, hydrocarbons and yeast metabolites using the new GC/parallel EI/FI-MS instrument are shown in Figs. 4–7. What is noticeable in these figures is that the ’positions’ of the chromatographic peaks in EI and FI are identical. This is because a single chromatography step is performed
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GC/MS with parallel EI and FI detection
Figure 2. EI and FI spectra of two polyunsaturated fatty acid methyl esters, C22:6n3 and C20:5n3. The EI spectra of these compounds are almost indistinguishable and more importantly they lack molecular ion peaks. The FI spectra show sharp molecular ion peaks and unique patterns by which the compounds can be distinguished.
Figure 3. EI and FI chromatograms of 1-octadecanol (C18H38O, 100 ng μL 1) and tert-butyl bromoacetate (C6H11BrO2, 100 ng μL 1) obtained using GC/parallel EI/ FI-MS.
Figure 4. Total ion chromatograms of 37 FAMEs mixture obtained using GC/parallel EI/FI-MS.
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reason the shapes of the peaks from EI and FI are very similar, although not necessarily identical. For example, both sets of peaks show the same pattern in terms of tailing
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which is then followed by a detection step using two ionization sources. Therefore, the chromatographic conditions under which the compounds elute remain identical for the two ionization sources: EI and FI. For the same
Figure 5. Total ion chromatograms of a fish oil sample obtained using GC/parallel EI/FI-MS.
L. Hejazi et al. Retention time
Figure 6. Total ion chromatograms of a hydrocarbon sample obtained using GC/parallel EI/FI-MS.
Figure 7. Total ion chromatograms of a yeast metabolite sample obtained using GC/parallel EI/FI-MS.
To investigate whether there is a significant difference in the retention times of the EI and FI chromatograms in our parallel EI/FI data acquisition approach, a regression analysis was used. Linear correlation between the EI and FI data over a retention time range of 10.7 to 76.5 min returned a coefficient of determination (r2) 0.9999, following the relationship RTFI = (0.001 ± 0.015) + RTEI × (1.0000 ± 0.0003) for retention time in units of minutes. The production of EI and FI chromatographic peaks with identical position and similar shapes is an important feature of GC/parallel EI/FI-MS in that it facilitates the matching of fragment and molecular ions, thus enabling accurate association of fragment ions from EI with their corresponding molecular ion from FI. This is essential for the identification of compounds, particularly in complex mixtures. To illustrate this strength, Fig. 8 shows a small region of the chromatogram of Fig. 6. The FI data revealed that there are (at least) two isomers with a nominal molecular mass of 196 and a third compound (at least one isomer) with a nominal molecular mass of 184, which elutes between the two isomers. It should be noted that not all ions observed in a FI spectrum are necessarily molecular ions; we have shown before that alcohols, as an example, can undergo loss of water from the molecular ion to yield [M–H2O]+• as the dominant ion in the FI spectrum.[13] The molecular ion chromatograms from EI and FI and those of one fragment ion from each compound (obtained from EI) are also presented in Fig. 8. The combined EI mass spectrum of the region showed a weak molecular ion for the two isomers but none for the third compound (see
but can exhibit different intensities (see the EI and FI chromatograms of C18H38O and C16H11BrO2 in Fig. 3). The latter is because the EI and FI peaks are generated using different ionization mechanisms, thus having different ionization efficiencies and sensitivities. Another example of different efficiencies is seen in the latter peaks of the fish oil analysis (Fig. 5). The peak at 72 min, yielding the ion at m/z 316.2382, is C21H32O2 (C20:5 methyl eicosapentaenoate) and the peak at 78.6 min, with the ion at m/z 342.2543, is C23H34O2 (C22:6 methyl docosahexaenoate). Highly unsaturated fatty acids have low ionization efficiency in FI, an opposite characteristic to EI. To investigate the degree of the equivalence between the EI and FI chromatograms, two figures of merit were considered using 31 resolved peaks from a standard mixture of FAMEs: FWHM and retention time.
FWHM
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To investigate whether there is a significant difference between the shapes of the EI and FI chromatograms, a twotailed t-test of the difference between the FWHM of the EI and FI peaks returned a P value of 0.38. A difference that is significant at the 95% level would have P
