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Elemental Dependence of Structurally Specific Isotopic Shifts in High-Field Ion Mobility Spectra Matthew A. Baird, Pratima Pathak, and Alexandre A. Shvartsburg Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.8b05801 • Publication Date (Web): 01 Feb 2019 Downloaded from http://pubs.acs.org on February 5, 2019
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Analytical Chemistry
Elemental Dependence of Structurally Specific Isotopic Shifts in High-Field Ion Mobility Spectra Matthew A. Baird, Pratima Pathak, Alexandre A. Shvartsburg* Department of Chemistry, Wichita State University, 1845 Fairmount, Wichita, Kansas 67260, United States *
[email protected] ABSTRACT: Nearly all molecules incorporate at least one element with stable isotopes, yielding ubiquitous isotopologic envelopes in mass spectra. Those envelopes split in differential ion mobility (FAIMS) spectra depending on the ion geometry, enabling a new general approach to isomer delineation as we demonstrated for chloroanilines. Here we report that analogous bromoanilines exhibit qualitatively distinct isotopic shifts under identical conditions, some changing signs depending on the gas. This dramatic elemental specificity conveys the breadth and diversity of structural isotopic effect in FAIMS, suggesting unique information-rich patterns for compounds involving various elements and feasibility of enhancing the structural elucidation by atom substitution. We also introduce the capability to make or assure structural assignments employing major isomer-specific peak broadening due to unresolved isotopomer mixtures.
Most elements in natural and biological materials, including metals (Mg, K, Ca, Ti, Fe, Cu, Zn) and non-metals (H, B, C, N, O, S, Cl, Br), have multiple stable isotopes.1 Of monoisotopic elements, mere Na and P are thought needed for life. Thus all organic and about all other compounds exist as sets of isotopologues (molecules with different isotopes for at least one atom). The associated mass distribution fingerprints stoichiometry, which can be deduced using mass spectrometry (MS) with sufficient resolving power (RMS) and measurement accuracy,2-6 but reveals nothing about the molecular geometries. Elucidating those comes to the forefront as crucial significance of isomers to biomedical functions of peptides, proteins, and metabolites gains appreciation.7-10 However, chromatography,11-13 electrophoresis,14,15 and linear ion mobility spectrometry16,17 (IMS) based on absolute ion mobility (K) at normally weak electric field (E) also discriminate non-isobaric isotopologues by mass without structural specificity. The nonlinear method of field asymmetric waveform IMS (FAIMS) captures the increment of K between two E levels (∆K).18,19 In practice, a gas flow pulls ions through a gap between two electrodes carrying the asymmetric waveform of some amplitude (dispersion voltage, DV). The ensuing periodic field deflects ions to either electrode. All would eventually be eliminated, but the compensation field EC (produced by compensation voltage, CV, superposed on the waveform) equilibrates the species with given ∆K and allows them through the gap to a detector. Scanning EC yields a spectrum of ions injected into the gap. A major inherent advantage of FAIMS over linear IMS is greater orthogonality to the MS dimension (often by 3 4×), evident from much lower r2 for linear correlation of m/z to EC than K values across many molecular classes including peptides and lipids.20-23 This facilitates resolution of similar peptide and lipid isomers22-28 and even isotopic isomers (isotopomers) labeled on alternative sites.29 We recently discovered that FAIMS broadly separated natural non-isobaric isotopologues with correlation to the ion
structure rather than mass.30,31 All three protonated monochloroanilines (MCA, monoisotopic mass of 127 Da)30 and six dichloroanilines (DCA, 161 Da)31 with variant Cl positions on the ring were delineated by EC shifts (∆EC) in He/CO2 buffers due to 13C (MCA) and 13C and/or 37Cl atoms (DCA). That work opened a new path to isomer identification and revealed a few preliminary trends:31 (a) the molecular symmetry plays no apparent role (b) heavier isotopologues can have ∆EC < 0 or ∆EC > 0, the latter (with negative EC moving toward zero) being more common (c) overall ∆EC magnitudes decrease for heavier species, in line with absolute EC (d) individual isotopic shifts are additive, with tentative discrepancies for two 13C atoms (e) the ∆EC values due to different isotopes (i.e., 13C and 37Cl) can be uncorrelated and structurally informative, enabling construction of most specific shift matrices (f) the ∆EC and EC values are generally orthogonal, thus co-eluting isomers and their mixtures are distinguishable based on ∆EC (g) isotopomers arising from isotopic substitution for inequivalent atoms have unequal EC: the consequent peak broadening varies for isomers with different nature and number of isotopomers, making a complementary structural descriptor. The next foundational question is whether the informative isotopic shifts are elementally specific, i.e., how much they vary when an atom in a molecule is replaced by one of other element. This can apply to the isotopes of both changed and retained element(s). That is, are the shifts a property of the molecular morphology or its juxtaposition with particular nuclei? The chemically closest element to Cl is Br, and they form analogous compounds. Like 35Cl and 37Cl, two stable Br isotopes are spaced by 2 Da and naturally abundant (79Br at 50.7% and 81Br at 49.3%). These aspects make halogenated species comprising Cl or Br ideal to answer the above question. Here
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we explore the MCA equivalents - monobromoanilines (MBA, 171 Da) with Br in position 2 (ortho-), 3 (meta-), or 4 (para-) on the ring (Figure 1). To compare directly, we reanalyze MCAs at same FAIMS conditions with stronger field that maximizes ∆EC and their isomer-specific differences. Experimental Methods Our FAIMS/MS platform with electrospray ionization (ESI) source comprises a FAIMS device with 1.88-mm planar gap coupled to the Thermo LTQ XL ion trap via a slit aperture/electrodynamic funnel interface.28-34 The high-definition bisinusoidal waveform has ~1.0 MHz frequency and DV of 4.0 or 5.0 kV (yielding the dispersion field, ED, of 21 or 27 kV/cm). The filtered He/CO2 carrier gas was supplied to the unit at Q = 3 L/min, which means a filtering time of ~100 ms. The maximum He fractions (v/v) avoiding electrical breakdown were 43% and 63% at DV of 5.0 and 4.0 kV, respectively.30,31 The ∆EC for DCA were larger in the {high DV; low He %} regime, which also delivered greater and more stable ion signal for more precise ∆EC measurement.31 Lower DV has enabled accessing ∆EC at higher He fractions, confirming the isomerspecific pattern over a wider gas composition range. 31 The EC scan rate was 5.3 V/(cm×min). The MCA and MBA standards (Sigma Aldrich) were dissolved to 100 μM in 99:1 MeOH/formic acid and infused at 0.3 - 1.0 μL/min. The EC scale was anchored by linear dilation utilizing a closely eluting internal calibrant: 2,4-DCA (100 μM) for MBAs and 2-MBA (1 μM) for MCAs. (Calibrating the MCAs with an MBA prevents the risk of MCA artifacts from DCA fragmentation). The calibrant concentrations were adjusted to yield ion signal comparable to the analytes and reflect the different competitive ionization efficiencies. Isomer separations were validated employing ternary mixtures. For suitable statistics, we collected ~10 - 20 spectral replicates (Figure S1). All reported error margins are 95% confidence intervals. Results and discussion Isotopic distribution and mass spectrum for MBAs All MCAs and MBAs make protonated 1+ ions in ESI, with H+ presumably on the N atom. The mass spectra for MBAs (Figure 1) match the isotopic distribution computed (Table 1) with unit resolution: disentangling isobars spaced by 104 that is beyond the capability of ion trap even in the ultrazoom mode. The mass spectra for MCAs likewise agree with the calculated distribution (Table S1).30 As with MCAs, one MBA stoichiometry dominates each nominal mass. The peaks at m/z = 173 and 175 (at +1 and +3 units above the base m/z = 172) are due to 13C at >93% level,
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Table 1. Intensities computed for H+MBA isotopologues (using the calculator on www.sisweb.com). Heavy Atoms
Accurate mass
Unit mass
M
I*
M
I*
% at unit m
None
171.976
100
172
100
100
15
N
172.973
0.361
173
6.963
5.18
13
C
172.980
6.489
93.19
D
172.982
0.112
1.61
81
Br
173.974
97.51
13
C15N
173.977
0.023
0.024
13
C2
173.983
0.175
0.179
13
CD
173.986
0.007
0.007
174
97.72
99.79
81
15
Br N
174.971
0.352
81
13
Br C
174.977
6.328
93.16
81
BrD
174.980
0.109
1.60
13
C3
174.986
0.003
81
Br13C15N
175.975
0.023
175
6.793
5.18
0.044 176
0.202
11
81
13
Br C2
175.981
0.171
85
81
13
175.984
0.007
3
Br CD
* Intensity relative to the base peak (100%) for all species with I* ≥ 0.002%. The I* values ≥ 0.02% are bolded. and that at m/z = 174 is due to 81Br at >99% (Table 1). The +4 peak (m/z = 176) with relative intensity of 0.20% is due to 81 Br13C2 at >85% level. The corresponding peak for MCAs due to 37Cl13C2 (m/z = 132) with 0.07% intensity (the 37Cl/35Cl ratio is lower than 81Br/79Br) was too tiny to reliably determine ∆EC in our initial work.30 The parallel feature for DCAs (due to 37 Cl213C2 at +6 units above the base) is yet smaller with 0.02% intensity, and ∆EC were imprecise or not measurable at all.31 With better overall signal at lower He content, we now successfully measured the pertinent ∆EC for MCAs and MBAs, permitting the evaluation of additivity for two 13C atoms. Separations of MCA and MBA isomers at DV = 5 kV The separations of MCAs at DV = 5 kV (Figure 2a) mirror those30 at 4 kV. All isomers remain type A ions19 with positive K(E) slope and thus EC < 0. As expected, the absolute EC increase upon He addition up to the 43% maximum. The peak order remains 2 > 3 > 4 by |EC|, but the maximum |EC| go up by >20% - to 300 V/cm for 2. The resolving power (defined for the full peak width at half maximum, w) also rises at higher He fractions, reaching ~130 that is near record for 1+ ions in FAIMS. In CO2, 3 and 4 still merge but 2 is resolved from them baseline. While previously30 3 and 4 were separated only at ~60% He, they are now resolved well already at 30% He: as usual, higher ED can be traded for greater He fraction.29,31,33 Separations of MBAs are similar, with same peak sequence and dependences on He content (Figure 2b). Again, 3 and 4 merge at low He % but are fully resolved by 40% He. As anticipated for heavier type A ions,21,35, the |EC| values of all MBA isomers are below those for corresponding MCAs (by ~20%).
Figure 1. Monohaloaniline isomer geometries and mass spectrum for MBA (dominant species marked per Table 1).
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Figure 2. Separations of MCA (a) and MBA (b) isomers (DV = 5 kV): EC values depending on the He fraction and spectra for mixtures and single species (scaled to fit mixtures) at 20% and 43% He. We also plot (b) the computed sum of 3 and 4.
Isotopologic shifts in FAIMS spectra for MCAs The shift patterns for MCAs (Figure 3) also track those at DV = 4 kV. The ∆EC(129 − 128) values are positive for all isomers, growing upon He addition and always dropping from 2 to 3 to 4 (Figure 3a). While slightly lower overall, the ∆EC(131 − 130) values lie close. This permits multiplexing ∆EC upon same isotopic substitution to compress error margins via the Felgett advantage (Figure 3b).30,31 Indeed, the means of these 13C shifts clearly demarcate all three isomers at any He fraction. The ∆EC(132 − 131) due to second 13C reproducibly lie under the above averages, although wide error margins due to minute signal at m/z = 132 preclude confident conclusion in most 2.5
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individual cases (Fig3.5 130 - 128 ure 3b). The system3.0 atic difference may 2 come from chemical 2.5 noise that is probabil4 2.0 istically greater for less abundant isotop1.5 ologues. Nonethe1.0 3 less, averaging all three 13C shifts im0.5 proves the isomer de0.0 lineation further (Fig0 10 20 30 40 ure 3c), as for DCAs. He fraction, % The relative shifts Figure 4. Same as Figure ∆relEC = ∆EC/|EC| rank like ∆EC 3a for 37Cl shifts. and decrease (2) or stay flat (3, 4) at higher He content (Figure 3d), as observed at lower DV. The mean 13C shifts substantially exceed those at DV = 4 kV and any He content, with the maxima (V/cm) elevating from 1.5 to 2.2 (2), 0.8 to 1.7 (3), and 0.6 to 0.8 (4). These ~30 - 110% gains uniformly exceed those of ~20 - 30% for |EC|, and ∆relEC increase from 0.2 - 0.7% to 0.3 - 1.0 %. Hence, the isotopic effect is intrinsically stronger at higher DV with less He (at DV = 4 kV, the ∆EC for 2 and 3 fall above 50% He not reached here). However, structural differentiation depends not on ∆EC itself but its inter-isomer range that widens from 1.0 V/cm at DV = 4 kV to 1.5 V/cm here (at 20% He). This happens as present conditions balance better between greater EC and lower ∆relEC at higher He content. As at lower DV, all ∆EC(130 − 128) due to 37Cl are positive and go up upon He addition (Figure 4). The values had appeared slightly greater for 2 than 3 and 4 up to 50% He. Similarly to 13 C shifts, now the maximum ∆EC for 37Cl increase by ~60% (from 1.6 - 2.0 to 2.6 - 3.2 V/cm). Their spread between 2 and {3; 4} expectedly expands, creating reliable differentiation of 2 at 0 - 20 % He with the ∆EC difference of 0.8 V/cm approaching that for 13C shifts (0.9 V/cm). At higher He fractions, the ∆EC trend for 2 bends down as previously. In summary, the 13C and 37Cl shift patterns match those at lower DV, but greater ∆EC and their inter-isomer differences (by ~60 % on average) materially simplify structural delineation. These findings copy those for DCAs.31 We now use this superior regime to study the influence of elemental substitution. EC shift, V/cm
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He fraction, %
Figure 3. Measured 13C shifts in MCA isomers (DV = 5 kV) as labeled: for first two 13C (a), third 13C (b), means from double (b) and triple (c) multiplexing, and (d) shifts from (c) in relative terms. Error bars show 95% confidence intervals.
Qualitatively different shifts for MBAs The chemical similarity and parallel separations of MCA and MBA isomers (Figure 2) tempt projecting analogous isotopic shifts. That has proved wrong. The first 13C shifts, here ∆EC(173 − 172), now rank as 3 > 4 > 2 (Figure 5a). In other words, the ∆EC for 2 moves to the bottom while the 3 > 4 order stays. The values for 2 further become negative in CO2, increasing upon He addition to cross zero at ~35% He. The ∆EC for 3 and 4 remain positive and trend flat to modestly up, with the segments at 20 - 43% He mimicking those for MCAs (Figure 3a). All MBA isomers are trivially delineated by these shifts. The next 13C shifts, ∆EC(175 − 174), virtually coincide with the first up to 40% He (Figure 5a). The systematic gap lower at 43% He may reflect some instrumental instability in the vicinity of breakdown threshold (noted previously on occa
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Analytical Chemistry (173 - 172): filled (175 - 174): empty
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The ∆EC(174 − 172) due to 81Br increase upon He addition from 0.4 - 0.5 to 1.2 - 1.6 V/cm (Figure 6). This behavior tracks that for MCAs (Figure 4), but the shifts are smaller by 2 - 3 fold. This lack of structural specificity may be due to lower ∆EC, channeling MCAs at DV = 4 kV with close ∆EC magnitudes.
(173 - 172 + 175 - 174)/2: filled (176 - 175): X
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Relative EC shift,%
0 0.036
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Figure 5. Measured 13C shifts in MBA isomers (DV = 5 kV) as labeled: for first two 13C (a), third 13C (b), mean from double multiplexing (b), and (d) multiplexed shifts from (b) in relative terms. The ratios of peak heights for m/z = 176 and 175 (at 30 - 43% He) are in (c). sion). The mean of two ∆EC confirms the isomer-specific differences even clearer (Figure 5b). The shifts due to second 13C, here ∆EC(176 − 175), match those averages for 4 and are close to them (a bit more positive at low He content) for 2 (Figure 5b). The same ∆EC for 3 lie well below the averages for 3, actually matching those for 2 and 4. That violates the shift additivity found heretofore for all natural or artificially labeled compounds. To test if a hidden isobaric interference might be responsible, we compared the actual and theoretical ratios of peak heights at m/z = 176 and 175. The measurements for 2, 3, and 4 essentially match each other and the ideal level, proving no significant contamination (Figure 5c). Therefore, the shifts for two 13C in 3 appear truly non-additive. The reason for and extent of this manifestation remain to be grasped. Anyhow, we left the last 13C shift alone and kept the mean of first two as the structural descriptor. The maximum 13C shifts for 3 and 4 (1.2 and 0.6 V/cm at 43% He) are ~2/3 of those for respective MCAs (1.8 and 0.9 V/cm, Figure 3b), while the values at lower He fractions are similar. The moderately lower ∆EC for MBAs mainly reflect smaller absolute EC. The ∆relEC (Figure 5d) are close to those for MCAs (Figure 3d): 0.1 - 0.3 % (MBA) ver1.6 (174 - 172) 4 sus 0.2 - 0.3 % (MCA) 1.4 for 3 and 0.7 - 1.0 % (MBA) versus 0.6 - 0.7 1.2 % (MCA) for 4. Both 2 1.0 ∆EC and ∆relEC values 0.8 for 2 are obviously 3 lower than those for 0.6 MCA. The widest inter0.4 isomer ∆EC range for MBAs (1.4 V/cm at 0% 0.2 0 10 20 30 40 He) almost equals that He fraction, % for MCAs, and the relaFigure 6. Same as Figure 4 tive range is actually wider for 81Br shifts in MBAs. at 1.3% versus 0.8%.
Shifts for MBAs at higher He fractions To explore the trends at higher He fractions and directly compare with same for MCAs,30 we probed the shifts for MBAs up to 63% He using DV = 4 kV (Figure 7). These confirm and extend the key trends for 13C shifts above: (i) equal 1st and 2nd shifts with 3 > 4 > 2 order at any He %, (ii) positive ∆EC for 3 and 4, near-flat for 3 and rising upon He addition for 4, (iii) ∆EC for 2 negative in CO2, but increasing and crossing zero upon He addition (at ~50% He here: once more, higher He fraction substitutes for lower DV).29,31,33 These results show constant shift patterns over fair ranges of DV and He fractions. As with MCAs,30 the 13C shifts begin dropping for the isomer with highest ∆EC (here 3) and converging for all isomers above 50% He, so the best isomer delineation is at ~30 - 50% He. The 81Br shifts also mirror those at DV = 5 kV: the ∆EC are positive and close for all isomers, growing at higher He fractions. The fall-over of 81Br shift for 2 (but not 3 or 4) at ~60 63% He follows that for MCAs.30 The maximum ∆EC are 0.8 V/cm (13C) and 0.9 V/cm (81Br), with the widest inter-isomer range of 0.9 V/cm (for 13C). These values amount to ~60% of those at DV = 5 kV, similarly to MCAs and DCAs. Again, the widest inter-isomer range is only slightly narrower than that for MCAs in same regime (1.0 V/cm).30 1.0 0.8
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Figure 7. Same as Figures 5a and 6 at DV = 4 kV, up to 63% He. Structurally informative peak broadening for MCAs and MBAs While we have mapped all haloaniline isomers by ∆EC for non-isobaric isotopologues, separations between isotopomers also depend on the ion geometry. We have not yet resolved isotopomers for natural compounds, but the MCA peaks with one 13C (comprising isotopomers for all isomers, Figure S2) were slightly broader than those with just 12C and 35Cl or 37Cl (with none for any isomer). A convolution over unresolved isotopomers is the sole plausible rationale. Although a larger number of isotopomers does not automatically translate into a wider peak (more isotopomers for one structure may cluster tighter in EC than fewer for other structure), a positive statistical correlation is intuitive. Indeed, the broadening (∆w) for 2 and 3 with six distinct 13C sites exceeded that for symmetric 4 with four sites. However, the subtle effect at DV = 4 kV (relative ∆w = 2 ± 1.5% for 4 and 7 ± 2% for 2 and 3) required ~100 replicates to distinguish 4 from 2 or 3 and failed to distinguish 2
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Figure 8. Widths of base peaks for MCA (a) and MBA (b) isomers (DV = 5 kV). from 3. As the ∆EC for isotopologues at DV = 5 kV here consistently exceed those at 4 kV, one may anticipate the same between isotopomers and thus more pronounced and isomer-selective peak broadening. That turned out to be true. We started by assessing the evolution of widths for base MCA and MBA peaks (Figure 8). First, those for each isomer narrow upon He addition, in concord with FAIMS fundamentals (the ion mobility increases at higher He fractions, while peak widths scale approximately as K−1/2)37,38 and past observa tions.24,28,33 Second, the peak widths at given He content rank as 2 < 3 < 4 for same reason of greater collision cross section (Ω) and thus lower K for less compact geometries. Again, all peaks due to 13C are broader than the base. (We averaged ∆w over non-zero He fractions for tighter statistics.) For MCAs (Figure 9a), the ∆w quantity at m/z = 129 and 131 with one 13C (at 20 - 43% He) is ~10 - 15% for 4, ~25 - 30% for 3, and ~35 - 45% for 2. The values exceed those at DV = 4 kV by about 5×, revealing far stronger isotopomer separations. The ∆w values for double-13C peak (m/z = 132) are same to slightly greater, in line with increased number of isotopomers (15 versus 6 for 2 or 3 and 9 versus 4 for 4, Figure S3). With much more explicit peak broadening, the 2 and 3 become distinguishable. For each isomer, ∆w is less at m/z = 130 (with 37Cl) than any 13C peak, validating the effect. The origin of ~10% broadening is unclear. The peaks ought to be wider for heavier isotopologues that are less mobile because of the reduced mass factor µ in Mason-Schamp equation.16,37,38 However, that effect upon incrementing from 128 to 130 Da is 3 > 4 (MCAs) to 3 > 4 > 2 (MBAs). Hence the informative shifts for analogous isomers qualitatively depend on the elements involved, even with a single atom in play. This conveys the specificity of isotopic shifts in high-field ion mobility spectra, wherein one may affirm or detail structural conclusions via chemical substitution. The shifts and their isomer-specific differences maximized at stronger field with lower He fractions (~20%). This optimum, also noted for DCAs and peptide isotopomers,29,31 differs from that typical for structural isomer separations26,41,42 and may reflect the inevitably weaker isotopic effect on ion mobilities in lighter gases with smaller µ and collisional recoil.16 Raising the dispersion voltage from 4 to 5 kV has magnified ∆EC by >50%, leading to more robust isomer delineation. All MCA and MBA isomers are immediately distinguishable by 13C shifts. In particular, that for 2-MBA uniquely transitions from ∆EC < 0 to ∆EC > 0 upon He addition. While ∆EC can have either sign,31,43 this first report of ∆EC for a natural isotopologue switching sign depending on the gas composition illustrates the importance of gas choice as an extra dimension in shift matrices. The absolute 13C shifts are overall smaller in MBAs than MCAs, but relative ∆EC/EC are not - despite greater mass of MBAs. The inter-isomer ∆EC diapason only marginally narrows
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Figure 10. The FAIMS spectra for 2-MCA isotopologues at m/z = 128, 130, and 132 (DV = 5 kV, He 43%). The broadening of 37Cl13C2 peak is apparent. from MCAs to MBAs. Hence the isomer differentiation capability does not necessarily diminish for heavier ions and may extend to yet larger species. The 37Cl and 81Br shifts lie close across isomers, although greater ∆EC now elicited real differences for 2-MCA. Both absolute and relative shifts are substantially smaller for 81Br than 37 Cl, possibly reflecting a lower relative atomic mass increment (2.5% for Br versus 5.7% for Cl). Present ∆EC in 2-MCA are the largest encountered so far in natural or artificial isotopologues for both single and multiple heavy atoms: 3.2 V/cm for 37Cl and 7.3 V/cm for 37Cl13C2, allowing first baseline resolution of natural isotopologues (Figure 10). This indicates trying yet higher DV at lower He fractions, now possible with the upgraded FAIMS system.42 We had asserted the additivity of EC shifts for natural and artificial isotopologues.29-31,43 Deviations for two 13C in haloanilines were conjectured31 for DCAs and now convincingly observed for MBAs. The causes for and prevalence of this situation remain to be understood. Such non-additivity compromises the multiplexing of ∆EC from successive instances of same isotope, but establishes new potentially informative shifts. The EC spreads between isotopomers expand more drastically. While the features for individual species (mostly with distinct 13C sites) are still unresolved, the peak broadening for their ensembles (∆w) increased by over 5-fold, to the maximum of >40% (for MCAs). Major ∆w differences between MCA or MBA isomers also delineate them and verify the assignments extracted from EC shifts. These results promise the molecular geometry fingerprints made of resolved isotopomers 42 upon attainable improvement of FAIMS resolving power. The order of ∆w does not change from MCAs to MBAs (Figure 9). Indeed, the separations of isotopomers and non-isobaric isotopologues should differ across isomers in unrelated ways, revealing complementary structural facets. Accordingly, the ∆w values make multidimensional matrices homologous to but independent of those for ∆EC. These arrays (depending on the gas composition) jointly provide exceptionally specific structural descriptors. Analogy to NMR We have commented that the compound identification residing on FAIMS isotopic shifts operationally resembles NMR,
where chemical shifts for active nuclei contain structural information.30 Then the shift matrices for different elements are parallel to those in multidimensional (heteronuclear correlation) NMR.31.45 Continuing that storyline, the striking dependence of isotopic shifts in FAIMS on elemental identities compares to that of NMR chemical shifts on the constituent nuclei, e.g., those shifts in proton NMR of a molecule and 19F NMR of its fluorinated derivative (with F replacing H) totally differ.46 The additivity of isotopic shifts in FAIMS (with putative deviations for two 13C atoms in some cases) mirrors that known (also with minor deviations) for isotope effects in NMR.47-50 For example, the measured D isotope effect on 13C shifts in CH3D, CHD3, and CD4 are −0.202, −0.601, and −0.795 ppm.50 The fact that MCA, DCA, and MBA isomers differ mostly or only in the 13C (not 37Cl or 81Br) shifts in FAIMS, whereas the geometries differ in Cl or Br positions, seems surprising. However, the primary isotope effect (upon substitution of active nuclei) in NMR is commonly smaller than the secondary effect (upon substitution of neighboring atoms, as for CH4 above). For example, representative perylenequinones cercosporine, isocercosporine, noranhydrocercosporin, and phleichrome exhibit primary D effects of 0.31 - 0.50 ppm, but secondary effects of 0.68 - 0.89 ppm on the 13C adjacent to O - H/D bond.51 The difference can expand for heavier detected nuclei, e.g., the secondary effect in 31PH3 is >0.8 ppm per D atom versus the primary D effect of ~0.1 ppm.50 Whether this trend is relevant to FAIMS remains to be seen.
ACKNOWLEDGMENT We thank Julia L. Kaszycki (WSU), Gordon A. Anderson (GAACE), Keqi Tang (Ningbo University), and Will Flannery (GenTech) for help with FAIMS and MS hardware and data processing. This research was supported by NSF CAREER Award (CHE-1552640).
Supporting Information Available Calculated MCA isotopologue abundances, examples of replicate FAIMS spectra for MBAs, and depictions of 13C and 13C2 isotopomers for each isomer.
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