WO2011017409A1

WO2011017409A1 - Apparatus for determining masses at high pressure

Info

Publication number: WO2011017409A1
Application number: PCT/US2010/044367
Authority: WO
Inventors: David E. Clemmer; Stephen J. Valentine
Original assignee: Indiana University Research And Technology Corporation
Priority date: 2009-08-05
Filing date: 2010-08-04
Publication date: 2011-02-10

Abstract

Apparatus, systems, and methods for determining the mass of one or more ions at high pressure are disclosed. The subject devices may operate by and the subject methods may include receiving one or more drift time signals indicative of the time required by a first ion or group of ions to traverse a drift tube operating at or near atmospheric pressure, receiving one or more drift time signals indicative of the time required by a second ion or group of ions to traverse the drift tube, wherein the second ion or group of ions have similar collision cross-sections but different ion masses from the first ion or group of ions, and calculating a mass measurement for at least one of the first ion or group of ions or the second ion or groups of ions which is substantially independent of the collision cross-sections.

Description

APPARATUS FOR DETERMINING MASSES AT HIGH PRESSURE

CROSS REFERENCE TO RELATED CASE

[0001] This application claims priority to U.S. Provisional Patent Application No.

61/231,336 filed August 9, 2009, the entire disclosure of which is hereby incorporated by reference.

GOVERNMENT RIGHTS

[0002] This invention was funded in whole or in part by grants from the National

Institutes of Health (NIH Grants P41 -RROl 8942 and AG-024547-04); the United States Government may have rights in this invention.

FIELD OF THE INVENTION

[0003] The present invention relates generally to ion separation instruments, and more specifically to instruments that operate to separate ions in time as a function of ion mobility.

BACKGROUND

[0004] Despite interest in ion populations under ambient conditions over the last century, no direct measurements of ion masses have been made at high pressures. This is because mass spectrometry measurements are based on determination of the motion of ions in a vacuum (usually < 10^"4 Torr). As ions are extracted from a high-pressure, or ambient, environment into the low pressure regions of mass analyzers, the population evolves due to shifts in equilibrium as pressures, temperatures, and species concentrations change. A direct method for measuring the fundamental property of ion mass at high pressures would find applications in a range of problems, from understanding and monitoring ions in the environment to delineating details of ion formation in a range of new sources that operate in ambient conditions.

[0005] These and other background considerations are described in the following references: B.H. Clowers et al., 80 Analytical Chemistry 612-623 (2008); S.L. Koeniger et al., 110 J. Physical Chemistry B 7017-7021 (2006); SJ. Valentine et al., 5 J. Proteome Research 2977-2984 (2006); G.A. Eiceman et al., Ion Mobility Spectrometry (2005); K. Tang et al., 77 Analytical Chemistry 3330-3339 (2005); Z. Takats et al., 306 Science 471 (2004); V.V. Laiko et al., 72 Analytical Chemistry 652-647 (2000); CA. Srebalus et al., 71 - ? -

Analytical Chemistry 3918-3927 (1999); A. A. Shvartsburg et al., 108 J. Chem. Physics 2416- 2423 (1998); C. Wu et al., 70 Analytical Chemistry 4929-4938 (1998); D.E. Clemmer et al., 32 J. Mass Spectrometry 577-592 (1997); P. Dougard et al., 1 19 Rev. Sci. Instruments 2240 (1997); T. Wyttenbach et al., 8 J. Amer. Soc'y Mass Spectrometry 275-282 (1997); M.F. Mesleh et al., 100 J. Physical Chemistry 16082-16086 (1996); A.A. Shvartsburg et al., 261 Chem. Physics Letters 86-91 (1996); W.F. Siems et al., 66 Analytical Chemistry 4195 (1994); P.R. Kemper et al., 95 J. Physical Chemistry 5134-5146 (1991); J.B. Fenn et al., 246 Science 64 (1989); M. Karas et al., 60 Analytical Chemistry 2299 (1988); E.A. Mason et al., Transport Properties of Ions in Gases (1988); E. E. Ferguson, Kinetics of Ion-Molecule Reactions (P. Ausloos ed., 1979); A.P. Krueger et al., 193 Science 2309-1213 (1976); D.I. Carroll et al., 47 Analytical Chemistry 2369-2973 (1975); H.E. Revercomb et al., 47

Analytical Chemistry 970-983 (1975); S.N. Lin et al., 60 J. Chem. Physics 4994 (1974); N. Robinson et al., 4 Int'l J. Biometeorology 101-110 (1963); P. Langevin, 21 ACP 483-530 (1903); JJ. Thomson, 44 Phil. Mag. 293 (1897) ; J. Elster et al., 4 Terrestrial Magnetism & Atmospheric Electricity 213 (1894). Each of the above listed references is hereby expressly incorporated by reference in its entirety. This listing is not intended as a representation that a complete search of all relevant prior art has been conducted or that no better reference than those listed above exist; nor should any such representation be inferred.

SUMMARY

[0006] The present application discloses one or more of the features recited in the appended claims and/or the following features which, alone or in any combination, may comprise patentable subject matter.

[0007] According to one an aspect, apparatus for determining the mass of one or more ions at high pressure may include a drift tube that operates at or near atmospheric pressure, a detector configured to produce a drift time signal indicative of the time required by a particular ion to traverse the drift tube, and a processor. The processor may be configured to (i) receive drift time signals from the detector representative of at least two ions or groups of ions with similar collision cross-sections but different ion masses and (ii) calculate a mass measurement of at least one of the two ions or groups of ions which is substantially independent of the collision cross-sections. The two ions or groups of ions may be related as isotopic species. [0008] In some embodiments, the processor may calculate the mass measurement (w_/) using a formula which relates the mass measurement to the difference in ion mass (X) between the at least two ions or groups of ions with similar collision cross-sections, the mass of a buffer gas (/«_#) in the drift tube, and the drift times of the at least two ions or groups of ions (toi and tp₂, respectively). The drift times may be determined based upon the drift time signals. The drift tube of the apparatus for determining the mass of one or more ions at high pressure may contain a buffer gas at or near atmospheric pressure. In some embodiments, the buffer gas may be selected from the group consisting of He, N₂, CO₂, Xe, SF₆, Ar, and mixtures thereof. In other embodiments, the buffer gas may be Xe. In still other

embodiments, the buffer gas may have an average molecular mass of at least 100 u.

[0009] According to another aspect, a method of determining the mass of one or more ions at high pressure may include receiving one or more drift time signals indicative of the time required by a first ion or group of ions to traverse a drift tube operating at or near atmospheric pressure and also receiving one or more drift time signals indicative of the time required by a second ion or group of ions to traverse the drift tube, wherein the second ion or group of ions have similar collision cross-sections to but different ion masses from the first ion or group of ions. The method may further include calculating a mass measurement for at least one of the first ion or group of ions or the second ion or groups of ions which is substantially independent of the collision cross-sections. The first ion or group of ions and the second ion or groups of ions may be related as isotopic species.

[0010] In some embodiments, calculating the mass measurement (mj) may include solving a formula which relates the mass measurement to the difference in ion mass (X) between the first ion or group of ions and the second ion or groups of ions, the mass of a buffer gas (m«) in the drift tube, a first drift time (t_Df) of the first ion or group of ions, and a second drift time (foi) of the second ion or group of ions. The first and second drift times may be determined based upon the drift time signals. The method may further comprising plotting a representation of drift time and mass-to-charge ratio (mlz) distribution patterns using at least the drift time signals indicative of the time required by the first and second ions or group of ions to traverse the drift tube.

[0011 ] In some embodiments, the method of determining the mass of one or more ions at high pressure may further include at least partially filling the drift tube with a buffer gas at or near atmospheric pressure. In some embodiments, the buffer gas may be selected from the group consisting of He, N₂, CO₂, Xe, SF₆, Ar, and mixtures thereof. In other embodiments, the buffer gas may be Xe. In still other embodiments, the buffer gas may have an average molecular mass of at least 100 u.

[0012] According to yet another aspect, a computer readable medium may embody a program of instructions executable by a processor to perform process steps for determining the mass of one or more ions at high pressure. The process steps may include receiving one or more drift time signals indicative of the time required by a first ion or group of ions to traverse a drift tube operating at or near atmospheric pressure and also receiving one or more drift time signals indicative of the time required by a second ion or group of ions to traverse the drift tube, wherein the second ion or group of ions have similar collision cross-sections to but different ion masses from the first ion or group of ions. The process steps may further include calculating a mass measurement for at least one of the first ion or group of ions or the second ion or groups of ions which is substantially independent of the collision cross- sections.

[0013] In some embodiments, the process step of calculating the mass measurement

(mj) may include solving a formula which relates the mass measurement to the difference in ion mass (X) between the first ion or group of ions and the second ion or groups of ions, the mass of a buffer gas (m_B) in the drift tube, a first drift time (t_D1) of the first ion or group of ions, and a second drift time (t_D2) of the second ion or group of ions. The first and second drift times may be determined based upon the drift time signals. In some embodiments, X may be an integer. The program of instructions executable by the processor further may further include the process step of plotting a representation of drift time and mass-to-charge ratio (mlz) distribution patterns using at least the drift time signals indicative of the time required by the first and second ions or group of ions to traverse the drift tube.

[0014] Additional features, which alone or in combination with any other feature(s), including those listed above and those listed in the claims, may comprise patentable subject matter and will become apparent to those skilled in the art upon consideration of the following detailed description of illustrative embodiments exemplifying the best mode of carrying out the invention as presently perceived.

BRIEF DESCRIPTION OF DRAWINGS

[0015] The detailed description particularly refers to the accompanying figures in which: [0016] FIG. 1 is a simplified diagram of an illustrative embodiment of an ion mobility spectrometer;

[0017] FIG. 2 is a graph of ion mobility distributions for two isomers (Ii and I₂) of a hypothetical ion which might be achieved with an ion mobility spectrometer having various resolving powers;

[0018] FIG 3. is a graph of ion mobility distributions for the two isomers of FIG. 2 which might be achieved using an ion mobility spectrometer having sufficient resolving power and employing either He or Xe as a buffer gas;

[0019] FIG. 4 is a graph predicting the resolving power required for an ion mobility spectrometer using various illustrative buffer gases to determine molecular masses;

[0020] FIG. 5 is a graph of ion mobility distributions for two isotopic peaks (M and

M+l) of a hypothetical ion;

[0021] FIG. 6 is a graph predicting the accuracy of ion mobility spectrometer mass determinations as a function of resolving power;

[0022] FIG. 7 is a flowchart demonstrating an illustrative method of using or programming an ion mobility spectrometer according to the present disclosure;

[0023] FIG. 8 is a graph predicting the resolving power required for an ion mobility spectrometer to separate various ion species of butane and isobutane;

[0024] FIG. 9 is a two-dimensional graph of drift time and mass-to-charge ratio for a hypothetical distribution of various ion species of butane, along with ion mobility

distributions for different isotopic species exhibiting different numbers of ¹³C atoms; and

[0025] FIG. 10 is a two-dimensional graph of drift time and mass-to-charge ratio for a hypothetical distribution of various ion species of isobutane, along with ion mobility distributions for different isotopic species exhibiting different numbers of ¹³C atoms.

DESCRIPTON OF THE ILLUSTRATIVE EMBODIMENTS

[0026] Although direct measurements of ion mass in ambient environments are not feasible with conventional mass analyzers or mass spectrometers, it is possible to record ion mobilities at high pressures. As used in this disclosure, "high pressure" refers to pressures at or near the ambient atmospheric pressure, as opposed to the low pressure, or vacuum, environments required by conventional mass spectrometers. The mobility of an ion through a buffer gas (K = v/E) is a measure of the ion's velocity (v) under the influence of an applied electric field (E). Thus, K is a fundamental property of the ion in a defined buffer gas. It is generally understood that an ion's mobility is inversely related to its collision cross-section (Ω) with the buffer gas as shown by the following equation:

1 - 2

1 (18;r)"² ze 1 1 760 T 1

K = - +• (I)-

Ω 16 (k_bT)^{] 2} m, P 273.2 N

In this equation, the parameters of temperature (T), pressure (P), and average buffer gas mass (me) define properties of the buffer gas; the variables z and m_/, define the ion's charge state and ion mass, respectively. The terms N, k_/» and e are constants corresponding to the neutral number density (at standard temperature and pressure, or STP), Boltzmann's constant, and electron charge, respectively.

[0027] A measurement of ion mobility may be made using an ion mobility

spectrometer to determine the relative drift times of various ions generated from a sample. Such instruments allow detection and identification of very low concentrations of chemicals based upon the differential migration of gas phase ions through an electric field. These gas phase ions may be produced from a variety of sources including, but not limited to, complex biological samples. Illustrative examples of ion mobility spectrometers include those described in U.S. Patent No. 7,077,944, issued July 18, 2006, U.S. Patent Application

Publication No. 2007/0114382, published May 24, 2007, International Publication No. WO 2008/028159, published March 6, 2008, and International Publication No.

PCT/US2009/031263, filed Jan. 16, 2009, the disclosures of which are each expressly incorporated herein by reference. The present disclosure contemplates the use and/or modification of these instruments, as well as other known ion mobility separation

instruments, to practice the apparatus, systems, and methods described herein. For ease of reference, however, an illustrative ion mobility spectrometer will now be described.

[0028] Referring to FIG. 1, a simplified diagram is shown of one illustrative embodiment of an ion mobility spectrometer instrument 100. In the illustrated embodiment, the ion mobility spectrometer instrument 100 includes an ion source 102 having an ion outlet that is coupled to an ion inlet of a drift tube 104. An ion outlet of the drift tube 104 is coupled to an ion detector 106 having a signal output that is electrically connected to an input of a control circuit 108. The control circuit 108 illustratively includes a conventional processor 120, conventional memory unit 122, a conventional clock circuit 124 that may be controlled by the processor 120 to produce periodic signals of desired frequency, and may include additional conventional components, such as a communications interface 126 configured for wired or wireless communications with one or more other electronic devices.

[0029] The ion source 102 may be any conventional ion source that is configured to controllably produce ions from one or more samples. Ion source 102 is illustratively shown as including an electrospray ionizer (ESI) 110 which is operable to provide ions in the form of an ionized spray or mist in a known manner. Examples of additional conventional ion sources include, but are not limited to, ion sources using radiation source to desorb ions from a sample, e.g., matrix-assisted laser desorption ion sources (MALDI), ion sources that collect generated ions in an ion trap for subsequent release, and the like. Illustratively, the ion source 102 may be configured to continuously produce ions, or may alternatively be configured to produce discrete packets of ions. The control circuit 108 is electrically connected to an input of the ion source 102 to provide control signals. Alternatively or additionally, the ion source 102 may be or include one or more conventional ion separation instruments configured to separate ions in time as a function of one or more characteristics. Examples include, but are not limited to, a conventional liquid or gas chromatograph, a conventional mass spectrometer, a conventional ion mobility spectrometer, a capillary electrophoresis instrument, or the like.

[0030] The drift tube 104 of the ion mobility spectrometer instrument 100 may be constructed using a number of electrically conductive rings each separated by electrically insulating rings, connected together in a linear, cascaded fashion to produce the drift tube 104. In some embodiments, the drift tube 104 may further include one or more ion funnels. Further details relating to the construction of the drift tube 104 are provided in co-pending U.S. Patent Application Publication No. 2007/0114382, discussed above. It will be understood, however, that this disclosure contemplates other embodiments in which the drift tube 104 is provided in other conventional forms which may be linear or non-linear. For example, the drift tube 104 may alternatively be provided in the form of a circular drift tube. Details relating to exemplary circular drift tube arrangements are provided in co-pending International Publication No. WO 2008/028159, discussed above.

[0031] During operation, the interior of the drift tube 104 is at least partially filled with a buffer gas, which may include He, N₂, CO₂, Xe, or mixtures thereof, by way of illustrative example. Those of skill in the art will recognize that other gases or mixtures of gases which are amenable to ion mobility separations, such as SF₆ or Ar, may be used as a buffer gas. The buffer gas may be supplied to the drift tube 104 via a buffer gas inlet using a fluid pump (not shown) in a conventional manner. The control circuit 108 applies a constant or variable voltage to the electrically conductive rings of the drift tube 104 to produce an electric field within the drift tube 104. Ions which are introduced by the ion source 102 at the ion inlet of the drift tube 104 travel across the drift region, through the buffer gas, under the influence of the electric field until reaching the ion outlet and the ion detector 106. The time a particular ion or group of ions require to traverse the length of the drift tube 104, also known as the "drift time," is related to the characteristic ion mobility of that ion or group of ions.

[0032] Illustratively, the drift tube 104 also includes a so-called ion inlet "gate" 1 14 in the form of a grid or plate positioned at the end that is closest to the ion source 102, and a so-called ion outlet "gate" 116 in the form of a grid or plate positioned at the opposite end, i.e., closest to the ion outlet of the drift tube 104. It will be understood, however, that this disclosure also contemplates embodiments that do not include any such grids or plates between the ion outlet of the ion source 102 and the ion inlet of the drift tube 104 or adjacent to the ion outlet of the drift tube 104. In such embodiments, ions generated by the ion source 102 enter and travel through the drift tube 104 and through the ion outlet solely under the influence of electric fields applied.

[0033] The ion detector 106 is conventional and is configured to produce an ion intensity signal that is proportional to the number of ions that reach, and are detected by, the ion detector 106 at a particular instant in time. The ion intensity signal is supplied to the control circuit 108, which then processes the ion intensity signal using processor 120 to calculate drift times and produce ion mobility spectral information. The memory unit 122 includes one or more computer-readable media having instructions stored therein that are executable by the processor 120 of the control circuit 108 to control the operation of the various components of the ion mobility spectrometer instrument 100 discussed above. It will be understood, however, that this disclosure also contemplates embodiments in which the operations of the ion mobility spectrometer instrument 100 are controlled by and the ion intensity signal is processed by a processor of an external electronic device (not shown), alone or in conjunction with control circuit 108. Such external electronic devices include, but are not limited to, a conventional personal computer, laptop or notebook computer, hand-held computer device (e.g., personal data/digital assistant, PDA), application specific computer, signal analyzer, or the like. [0034] Returning to Equation 1, there are two fundamental properties that are not known in the measurement of ion mobility (K) for a particular ion, namely Ω and mi. Ω, the collision cross-section, depends on the ion's shape, the nature of the buffer gas (polarizability and dipole moment) and to a lesser extent, the masses of the colliding pair (the ion and the buffer gas molecule). If Ω was defined (e.g., from theoretical calculations), then, tn_/ could be determined directly from a measurement of ion mobility, because Equation 1 can be rewritten as:

m, =

(2).

[0035] The inventors of the present application have determined that the separation of isotopes of the same ion will allow for the derivation of a mass scale based solely on mobility measurements at high pressure. Neglecting small variations in scattering (discussed in more detail below), different isotopes of the same ion will have identical collision cross-sections with the buffer gas; however, the mobilities of isotopes must differ because of differences in mj (Equation 1). Thus, where the resolving power of the ion mobility measurement is sufficient to distinguish these isotopes, one need not know the cross-section a priori to determine m_/. Rather, mi could be directly determined from the separation of the isotopes. Apparatus, systems, and methods applying this principle may be used to rigorously determine ion masses under ambient conditions.

[0036] Referring now to FIG. 2, distributions of drift times (fø) are shown which were simulated for two isomers (Ii and I₂) of a hypothetical ion having a charge-to-mass ratio (mlz) of 100. The plots of FIG. 2 represent the ion mobility spectral information which might be generated by ion mobility spectrometers having resolving powers of 25, 75, 225 and 675, respectively. Resolving power {R_IMS) quantifies the ability of a particular ion mobility spectrometer to differentiate between two ions having similar, but distinct, drift times and is defined as tlΔt (where Δt is the full width at half maximum, FWHM, of the peak). In this simulation, two isomers that differ in collision cross-section by 2.5% were assumed— the ions and conditions were defined such that peaks in the drift time distribution would be centered at 50 and 51.25 milliseconds. As can be seen in FIG. 2, drift times for Ii and I₂ are unresolved at R_IMS ⁼ 25, but almost fully resolved at R_IMS ~ 75. At RIM_S ⁼ 225 and RI_MS ~ 675, the two isomers were baseline resolved. The insets of FIG. 2 show an enlarged view of the drift time region containing the two features to emphasize the change in drift time distributions as resolving power increases.

[0037] At this point in the development of ion mobility spectrometer instrumentation, a value ofR/M_S ⁼ 25 is attainable by almost any reasonable ion mobility instrument and is considered a relatively low resolving power. A value of R_/Ms = 75 is attainable in many laboratories for many types of ions and is considered fairly typical. A value of R_IMS = 225 is currently attainable in several laboratories and is considered a relatively high resolving power. Such higher-resolution measurements are quickly becoming routine in the art.

Although R_IMS ⁼ 675 has not been achieved, the inventors anticipate that this resolving power will be realized in the next several years using high-pressure trapping and focusing techniques, circular instruments, and new approaches, such as the use of overtone mobility techniques. Several of these techniques and approaches are described in more detail in the references discussed above.

[0038] Referring now to FIG. 3, distributions of drift times (to) are shown which were simulated for two different isotopic peaks (M and M+2) representing populations of ions with ³⁵Cl and ³⁷Cl. In FIG. 3, the dashed lines correspond to the distributions for ions with a molecular weight of 100 u, and the solid lines correspond to the distributions for ions with a molecular weight of 102 u. Such a 2.0 u shift is typical of a chlorinated molecule, and this example uses the relative isotopic composition for that of ³⁵C1:³⁷C1 (i.e., 3:1). The lower spectra represents the results which would be achieved if He were used as a buffer gas, while the upper spectra represents the results which would be achieved if Xe were used as a buffer gas.

[0039] When He is used as the buffer gas, only slight shifts in the positions of peak centers occur: 50.00 and 50.02 ms for the M and M+2 isotopes, respectively (for isomer 1); and, 51.25 and 51.27 ms for the M and M+2 peaks, respectively (for isomer 2), as shown in FIG. 3. While the centers of the M and M+2 pairs of peaks shift slightly for each isomer in He, the resolving power of R_IMS ⁼ 675 is insufficient to resolve these isotopes. Instead, peaks in the measured drift time distributions in He will appear broadened, compared with what is expected for a single isotope (or, a single peak that is calculated using an isotopically averaged mass). An evaluation of Equation 1 indicates that He is a poor choice for the buffer gas because m_\\t = 4.0 u is a value that dominates the reduced mass term. The inventors of the present application have determined, however, that isotope separation can be favored by use of a heavy collision gas (e.g., Xe with m = 131.29 u). As shown in FIG. 3, when Xe is used as the collision gas, the transport equation predicts that the M and M+2 isotopic peaks will be well resolved at R_/MS ⁼ 675 for each of the isomers.

[0040] The above considerations may be extended to develop a general mass scale based solely on mobility measurements at high pressure. By way of example, pairs of M and M+l isotopes, i.e. ions that are separated by 1.0 u, as in the case of the ¹²C:^{| J>}C isotopic pairs, might also be resolved. Again, because the collision cross-sections of the isotopic peaks are equal and because the relationship of the masses is known, the ratio of the drift times for the isotopes (i.e., tpj for M ion, and toi for the M+l ion) is:

1 1

— +—

W m, m_t

(3).

^■ D\ 1 1

m, + 1 m_B

Solving Equation 3 for m_f yields:

[0041] Thus, the difference in the drift times of the ion pairs are all that is required to determine m_\. This quadratic form of Equation 4 yields two solutions. However, because the denominator is negative, the only meaningful solution is the one for which the numerator is negative as well. For example, if values of toi— 75.00 ms and tp₂ =75.15 ms were obtained for mi and m_f + 1 , then m; = 146.4 u. In another part of the same spectrum, a pair of peaks might have tpi = 50.00 ms and tp₂ =50.20 ms such that m_/ = 77.7 u. In this manner, the drift times become a mass spectrum.

[0042] As noted above with reference to FIGS. 2 and 3, the ability of an ion mobility spectrometer to differentiate the drift times of ions having similar masses is a function both of resolving power (Rms) and of the mass of the buffer gas (m_#). Equation 2 may be rewritten such that K is expressed in terms of E, to, and L (the drift tube length). This provides an expression that relates the mass resolving power {R_maSs ⁼ mlΔm, where Δm is the peak at FWHM) to R_IMS- To obtain this expression, we find the rate of change of the mass with respect to the rate of change of the drift time, i.e., (3w_/)/(δ/_/)) as:

[0043] The mass resolving power of an ion mobility spectrometer may be obtained by combining the revised expression for Equation 2 (in terms of for E, to, and L) with Equation 5. Additionally, Am_/ and Δto for the respective peak widths at FWHM have been substituted for dm_/ and dtp to generate a relationship between R_maSτ and R_/MS as given by:

From this, it can be seen that the ability to resolve different masses is directly proportional to

RIMS-

[0044] Additionally, Equation 6 makes it possible to estimate values of R_/MS that would be required to determine the masses of different ions. For example, if it is desirable to resolve isotopic peaks differing by 1.0 u, then R_mass must be equivalent to the numerical value of the mass of the ion. To illustrate this, FIG. 4 shows the minimum required R_/MS as a function of the m_/ results in resolving the M and M+l isotope pairs, based on differences in ion mobilities. These minimum criteria are predicted for different illustrative buffer gasses: regression 402 represents using He as a buffer gas, regression 404 represents using N₂ as a buffer gas, regression 406 represents using CO₂ as a buffer gas, and regression 408 represents using Xe as a buffer gas. As shown in FIG. 4, a value of R_/MS ⁼ 1000 would limit the maximum mass that could be resolved with its M+l isotope to ~40, ~100,— 110, and ~200 u, for separations in He, N₂, CO₂, and Xe, respectively. While these values show that analyses are limited to relatively low molecular weights (compared with mass analyzers), they also suggest that many new experiments for directly assessing masses of ions at high pressues may be feasible. The applicable mass range would be extended for isotopic pairs separated by more than one atomic mass unit.

[0045] The mass accuracy associated with the measurements that are described above may also be predicted. For ease of calculation, a resolving power of R_/MS ^~ 10⁴ is assumed. Referring now to FIG. 5, distributions of drift times (to) are shown which were simulated for two different isotopic peaks at 100 u and 101 u, having drift times of 50.00 and 50.14 ms, respectively. In FIG. 5, the dashed line corresponds to the isotopic peak of mass 100 u, and the solid line corresponds to the isotopic peak of mass 101 u. A measurement in which R_IMS = 10 would result in a peak capacity of 56 for the range between the two features. Assuming that each peak is defined by approximately 10 data points and that the center can be measured to ± 0.5 increments then the difference in drift bins would be ± 1 increment of 1 120 (or ± 1.25 x 10^" ms). To determine the error associated with the w_/, it is necessary to propagate this error in drift times (to/ and tp?) from Equation 4 via

[4 _D2)f (7),

where ε{mi), ε(toι), and ε(fø?) correspond to the errors in the mass of the ion and the measured drift times of the isotopic peaks, respectively. Using Equation 7 and the uncertainty in the drift times, the mass accuracy associated with the calculated error in w_/ is approximately 0.9

PPt-

[0046] This approach can also be used to predict the dependence of mass accuracy on

R_IMs and w/ for values of m/ equal to 50, 100, and 200 u, as shown in FIG. 6. Using R_/Ms = 225 will result in mass accuracy uncertainties of 9, 40, and 201 for ions of mass 50, 100, and 200 u, respectively. Increasing the mass from 50 to 100 u would result in the mass accuracy decreasing by a factor of about 4.5 for all values of R_IMS- Increasing the mass from 100 to 200 u would result in a similar decrease in the mass accuracy by a factor of about 5.0 for all values of R_IMS- Thus, for a defined value of R_IM_S, the accuracy of mass determinations with this approach will be mass dependent. The results of these considerations suggest that current ion mobility spectrometers may be capable of resolving some isotopes for light ions at high pressures; moreover, as significant improvements in mobility resolving power are made, the present considerations indicate that isotopic structure could become a routine feature of ion mobility data sets, analogous to the patterns observed from mass spectrometry

measurements.

[0047] Referring now to FIG. 7, an illustrative embodiment of a method of

determining the mass of one or more ions at high pressure is illustrated as a simplified flow diagram. An ion mobility spectrometry (IMS) process 700 may include manually or automatically operating an ion mobility spectrometer, similar to those discussed above. By way of example, the control circuit 108, including processor 120, shown in FIG. 1 may operate the ion mobility spectrometer instrument 100 in accordance with IMS process 700. Additionally or alternatively, IMS process 700 may be implemented as a program of instructions on a computer readable medium, such as the memory 122 of the control circuit 108. The program of instructions may be executable by the processor 120 to perform the steps of IMS process 700 to determine the mass of one or more ions at high pressure.

Implementation of the IMS process 700 as program code will be readily apparent to those of skill in the art. As further described below, IMS process 700 includes a number of process steps 702-710, as shown in FIG. 7.

[0048] The IMS process 700 may optionally begin with process step 702, in which a drift tube of an ion mobility spectrometer is at least partially filled with a buffer gas. As noted above, isotope separation can be favored by use of a relatively heavy buffer gas (e.g., Xe with m = 131.29 u). The buffer gas may include, but is not limited to, He, N₂, CO₂, Xe, SF₆, Ar, and mixtures thereof. In other embodiments, however, a buffer gas need not be pumped into the drift tube. In such embodiments, ambient atmospheric air may provide the "buffer gas" through which the ions will pass in the drift tube.

[0049] After optional process step 702, if used, the IMS process 700 proceeds to process step 704, in which ions having various masses but similar collision cross-sections (Ω) are introduced to the drift tube of the ion spectrometer from a conventional ion source. These ions may be produced from one or more samples, including complex biological samples. A time (to) at which the ions are introduced to the drift tube is recorded by a control circuit for later use. The control circuit also applies an electric field to the drift tube at, or prior to, the introduction of the ions. As described above, the ions migrate through the drift tube under the influence of the electric field with speeds proportional to their respective ion mobilities.

[0050] After process step 704, the IMS process 700 proceeds to process step 706, in which a first ion or group of ions reaches the end of the drift tube and is sensed by a detector of the ion mobility spectrometer. The detector is configured to produce a drift time signal indicative of the time required by a particular ion to traverse the drift tube. This drift time signal may be the elapsed time from to until the first ion or group of ions reaches the detector. The processor of the control circuit may receive the drift time signal and determine a first drift time (t_D/) for the first ion or group of ions based upon the drift time signal.

[0051] After process step 706, the IMS process 700 proceeds to process step 708, in which a second ion or group of ions reaches the end of the drift tube and is sensed by the detector of the ion mobility spectrometer, in a similar fashion to that described in process step 706. The detector will produce a drift time signal which may be the elapsed time from to until the second ion or group of ions reaches the detector. The processor of the control circuit may receive the drift time signal and determine a second drift time (to?) for the second ion or group of ions based upon the drift time signal. As described above, the detection of the second ion or group of ions may occur within milliseconds, microseconds, or nanoseconds of detection of the first ion or group of ions.

[0052] As noted above, the second ion or group of ions have similar collision cross- sections (Ω) to, but different ion masses from, the first ion or group of ions. In some embodiments, the first ion or group of ions have an ion mass which may be represented as M, while the second ion or group of ion have an ion mass which may be represented as M+X, where X is rounded to an integer value. In such embodiments, the second ion (or ions in the second group) includes a heavier isotope of an element or elements contained in the first ion (or ions in the first group), and X represents the difference in isotope mass.

[0053] After process step 708, the IMS process 700 concludes with process step 710, in which the processor calculates a mass measurement for at least one of the first ion or group of ions or the second ion or groups of ions which is independent of the collision cross- sections. This calculation is based primarily on the differential in the first and second drift times {toi and toi)- In some embodiments, the processor may calculate the mass

measurement (mi), at least in part, by solving a formula which relates the mass measurement (mi) to the difference in ion mass (X) between the first ion or group of ions and the second ion or groups of ions, the mass of the buffer gas (m_B) in the drift tube, the first drift time (to_/) of the first ion or group of ions, and the second drift time (fo_?) of the second ion or group of ions. This formula may be based on Equation 3, discussed above, and may have the general form:

— 1 ₊— 1

m, m_B (8). tn.

Tn₁ + X m_B

The processor of the ion mobility spectrometer may solve this Equation for a value of mi, the mass of the first ion or group of ions to reach the detector, using any number of known methods. For instance, where X equals 1 , the processor may utilize the quadratic form shown in Equation 4. Additional methods of calculating the mass measurement (mi) using the Equation 8 with known and/or measured values of X, me, tpi, and t^2 will be readily apparent to those of skill in the art.

[0054] As discussed above, the ability to determine accurate ion masses based on mobility measurement is dependent upon the resolving power of the IMS instrument. Extending the above methodology and assuming sufficient resolving power, other ion properties and information may be obtained from high-resolution mobility measurements. Specifically, such high-resolution measurements may provide information regarding the structures of ions with respect to the covalent bonds between different constituent atoms. Referring now to FIG. 8, a graph is shown predicting the resolving power required for an ion mobility spectrometer to separate various ion species of butane and isobutane. As discussed above, a relatively low resolving power of R_/MS ~ 100 will be sufficient to resolve isomeric species having approximately the same mass, but differing collision cross-sections (for example, butane versus isobutane, as illustrated in the bottom trace of FIG. 8). In the remainder of FIG. 8, theoretical distributions of various ion species of butane are shown. For instance, a slightly higher IMS resolving power (for example, R_/MS ~ 1000) will allow the separation of specific isotopes of a given molecule. As already explained in this disclosure, this separation forms the basis for ion mass determinations from mobility measurements.

[0055] Such determinations of ion mass from mobility measurements generally assume that the related isotopic species have the same or similar collision cross-sections and neglect any small variations in scattering. In practice, the relatively large shift in drift time between isotopes having similar collision cross-sections will be due to differences in ion mass. However, there will also be minute differences in shape arising from the fact that different isotopic species have different zero-point energies. This property difference gives rise to characteristic bond lengths. For example the bond length of a carbon-hydrogen (C-H) single bond is 1.11 A while the bond length for the equivalent carbon-deuterium (C-D) is 1.09 A. Thus, some relatively small contribution to differences in drift time among isotopic species will result from these differences in shape. Adapting the methodology of this disclosure, it should be theoretically possible to resolve isotopic species consisting of the same constituent atoms but differing in arrangement (e.g., the location of a single deuterium). Preliminary molecular dynamics simulations have shown that, for a molecule such as butane, an ion mobility spectrometer resolving power of at least R_/MS ~ 10,000 will be required to resolve the location of a single deuterium, as illustrated in FIG. 8. Expanding upon this idea, the resolving power required for the resolution of molecular ions that differ by the position of other atomic isotopes (e.g., the position of a single ¹³C atom in butane) may be estimated. Because the ¹²C/¹³C percentage increase in mass is approximately an order of magnitude smaller than the Η/²H percentage increase in mass, a ten-fold increase in R_IMS would be required. The upper trace of FIG. 8 depicts a separation based upon the position of a single ^ljC atom on the molecule butane at R_IMS - 100,000.

[0056] Having described the resolution of molecular ions with different positions of specific atomic isotopes, it is also possible to consider the types of drift time and mass-to- charge ratio (ml z) distribution patterns that would be observed for different molecular species. For discussion purposes, we will consider the two isomers butane (FIG. 9) and isobutane (FIG. 10). A hypothetical, two-dimensional plot of drift time and mlz for a population of butane ions is illustrated in FIG. 9. The level of ¹³C incorporation among the various butane ion species is denoted in FIG. 9 with a number system in which the number 1 represents no incorporation of ¹³C (all ' C atoms) and the number 5 represents complete incorporation of ¹³C (no ¹²C atoms). Hypothetical drift time distributions, along with corresponding molecular structures, for each level of ¹³C incorporation (numbers 1-5) are shown along the right edge of FIG. 9. As can be seen in the figure, the monoisotopic ¹²C peak (1) at mass 59 is singular in the drift time distribution. The incorporation of a single ¹³C atom results in a doublet (2), as the C isotope can be positioned in exactly two unique locations in the molecule. In this hypothetical distribution, there is an equal probability that the ¹³C atom will be located in either position, so the peak heights are equal. The incorporation of two ¹³C isotopes results in a quartet (3) in the drift time distribution. The smaller outside peaks of the quartet (3) result from the single possibilities of locating a ¹³C atom on either end of the molecule (1st and 4th positions) or locating the two ¹³C atoms adjacent to one another at the 2d and 3d positions. The larger middle peaks result from doubly degenerate molecules: for example, locating the two ¹³C atoms at the first and third positions would be the same as locating them at the second and fourth positions. Placing three ¹³C atoms on the molecule would result in a doublet (4) considering the fact that there are only two unique locations for the lone ¹²C atom. Finally, the molecule exhibiting all ¹³C atoms would produce a single peak (5) in the mobility distribution.

[0057] The hypothetical, two-dimensional pattern described above for population of butane ions will be unique. For example, we can contrast this pattern with a hypothetical, two-dimensional plot of drift time and mlz for a population of isobutane ions, as illustrated in FIG. 10. Here again, a population of ions contains species with differing numbers of incorporated ¹³C atoms, and the same numbering scheme (1-5) as described for FIG. 9 is also used in FIG. 10. Once again, the species containing all ¹²C atoms exhibits a single peak (1) in the drift time distribution. Incorporation of a single ¹³C isotope, however, results in a doublet (2) with a 3:1 peak height ratio. This ratio results from the fact that, in isobutane, the ¹³C atom can be placed on three separate exterior positions compared to the single interior position. Furthermore, incorporation of two ¹³C atoms results in a doublet (3), as opposed to a quartet for butane, because there are only two different arrangements for incorporation of two ¹³C atoms in isobutane. Because there is an equal level of redundancy for both arrangements, the two peaks of the doublet (3) have the same height. Incorporation of three ¹³C isotopes results in a doublet (4) in the drift time distribution with a 1 :3 peak height ratio. This is explained again by the concept of three-fold degeneracy for location of the single ¹²C atom at an exterior position while only a single location is available for the interior position. As before, complete incorporation of ¹³C would result in a single drift time distribution peak.

[0058] Thus, the two-dimensional (m/z and drift time) plot will be unique for a given ion type, and the drift time profiles for all related isotopes may provide further information to determine molecular structure. Briefly, the process flow for such a determination might proceed as follows. First, by zooming in to a particular region on a two-dimensional plot, the position of single molecular species might be determined. Second, the drift spacing between isotopes could be used to determine the mass, as disclosed above. Finally, the drift spacing for resolved species of given isotopes could then be used to piece together the isotopic constituents of the molecular ions. By combining the information from all observed isotopes, it may be possible to not only determine the three-dimensional structure of the molecule, but also the position of specific isotopes. One illustrative application of such capabilities would be the determination of hydrogen/deuterium (HfD) exchange positions. In summary, high resolution ion mobility separation may open a whole new field of molecular characterization techniques.

[0059] While the disclosure has been illustrated and described in detail in the drawings and foregoing description, such an illustration and description is to be considered as exemplary and not restrictive in character, it being understood that only illustrative embodiments have been shown and described and that all changes and modifications that come within the spirit of the disclosure are desired to be protected. For instance, while the examples described above have made reference to ion mobility spectrometers, the

methodology of this disclosure may be used with different types of mobility-based separation devices, provided that the separation is carried out in the low-field limit where ion drift velocities are defined as the product KE. Additionally, the methodology of this disclosure may be used with any isotopically related ions from any type of ion source. [0060] There are a plurality of advantages of the present disclosure arising from the various features of the apparatus, systems, and methods described herein. It will be noted that alternative embodiments of the apparatus, systems, and methods of the present disclosure may not include all of the features described yet still benefit from at least some of the advantages of such features. Those of ordinary skill in the art may readily devise their own implementations of the apparatus, systems, and methods that incorporate one or more of the features of the present invention and fall within the spirit and scope of the present disclosure as defined by the appended claims.

Claims

1. Apparatus for determining the mass of one or more ions at high pressure, the apparatus comprising:

a drift tube that operates at or near atmospheric pressure;

a detector configured to produce a drift time signal indicative of the time required by a particular ion to traverse the drift tube; and

a processor configured to (i) receive drift time signals from the detector representative of at least two ions or groups of ions with similar collision cross-sections but different ion masses and (ii) calculate a mass measurement of at least one of the two ions or groups of ions which is substantially independent of the collision cross-sections.

2. The apparatus of claim 1 wherein the two ions or groups of ions are related as isotopic species.

3. The apparatus of claim 1 wherein the processor calculates the mass measurement (mi) using the formula:

1 1

¹Dl ^mi ^mB

¹ DX

In₁ + X m_c

where X is the difference in ion mass between the at least two ions or groups of ions with similar collision cross-sections, TH_B is the mass of a buffer gas in the drift tube, and tpi and to₂ are drift times of the at least two ions or groups of ions, respectively, the drift times being determined based upon the drift time signals.

4. The apparatus of claim 1 wherein the drift tube contains a buffer gas at or near atmospheric pressure.

5. The apparatus of claim 4 wherein the buffer gas is selected from the group consisting of He, N₂, CO₂, Xe, SF₆, Ar, and mixtures thereof.

6. The apparatus of claim 4 wherein the buffer gas is Xe.

7. The apparatus of claim 4 wherein the buffer gas has an average molecular mass of at least 100 u.

8. A method of determining the mass of one or more ions at high pressure, the method comprising:

receiving one or more drift time signals indicative of the time required by a first ion or group of ions to traverse a drift tube operating at or near atmospheric pressure,

receiving one or more drift time signals indicative of the time required by a second ion or group of ions to traverse the drift tube, wherein the second ion or group of ions have similar collision cross-sections to but different ion masses from the first ion or group of ions; and

calculating a mass measurement for at least one of the first ion or group of ions or the second ion or groups of ions which is substantially independent of the collision cross- sections.

9. The method of claim 8 wherein the first ion or groups of ions and the second ion or group of ions are related as isotopic species.

10. The method of claim 8 wherein calculating the mass measurement (mi) comprises solving the formula:

1 1

^■ + - m, + X m_g

where X is the difference in ion mass between the first ion or group of ions and the second ion or groups of ions, TH_B is the mass of a buffer gas in the drift tube, toi is a first drift time of the first ion or group of ions, and tø? is a second drift time of the second ion or group of ions, the first and second drift times being determined based upon the drift time signals.

1 1. The method of claim 8 further comprising plotting a representation of drift time and mass-to-charge ratio (m/z) distribution patterns using at least the one or more drift time signals indicative of the time required by the first ion or group of ions to traverse the drift tube and the one or more drift time signals indicative of the time required by a second ion or group of ions to traverse the drift tube.

12. The method of claim 8 further comprising at least partially filling the drift tube with a buffer gas at or near atmospheric pressure.

13. The method of claim 12 wherein the buffer gas is selected from the group consisting of He, N₂, CO₂, Xe, SF₆, Ar, and mixtures thereof.

14. The method of claim 12 wherein the buffer gas is Xe.

15. The method of claim 12 wherein the buffer gas has an average molecular mass of at least 100 u.

16. A computer readable medium embodying a program of instructions executable by a processor to perform process steps for determining the mass of one or more ions at high pressure, said process steps comprising:

17. The computer readable medium of claim 16 wherein the process step of calculating a mass measurement (mi) comprises solving the formula:

1 I

2 +

m,

2 1 1

+ - where X is the difference in ion mass between the first ion or group of ions and the second ion or groups of ions, m_B is the mass of a buffer gas in the drift tube, t_D! is a first drift time of the first ion or group of ions, and to₂ is a second drift time of the second ion or group of ions, the first and second drift times being determined based upon the drift time signals.

18. The computer readable medium of claim 17 wherein X is rounded to an integer value.

19. The computer readable medium of claim 16 wherein the program of instructions executable by the processor further comprises the process step of plotting a representation of drift time and mass-to-charge ratio (m/z) distribution patterns using at least the one or more drift time signals indicative of the time required by the first ion or group of ions to traverse the drift tube and the one or more drift time signals indicative of the time required by a second ion or group of ions to traverse the drift tube.