STRUCTURAL ANALYSIS OF IONISED MOLECULES

Abstract

An ion mobility spectrometry method is described comprising: providing a sample; generating molecular ions from the sample; separating the molecular ions according to their mobility characteristics; fragmenting at least some of the separated molecular ions to form sub-molecular fragment ions in a fragmentation zone; separating at least some of the fragment ions according to their mobility characteristics; wherein the separation and fragmentation steps are performed at a pressure of at least 50 mbar; detecting at least some of the separated fragment ions; and identifying at least one molecular ion based on its mobility characteristics and/or the mobility characteristics of at least one detected fragment ion.

Claims

1. A method of ion mobility spectrometry comprising: providing a sample; generating molecular ions from the sample; separating the molecular ions according to their mobility characteristics; thermally fragmenting at least some of the separated molecular ions to form sub-molecular fragment ions in a fragmentation zone, wherein the fragmentation zone comprises a heated gas at a temperature above 300° C., wherein a residence time of the molecular ions in the fragmentation zone is in the range of 0.1-5 milliseconds; separating at least some of the fragment ions according to their mobility characteristics; wherein each step of separating the molecular ions, fragmenting at least some of the separated molecular ions and separating at least some of the fragment ions is performed at a pressure of at least 50 mbar; detecting at least some of the separated fragment ions; and identifying at least one molecular ion based on its mobility characteristics and/or the mobility characteristics of at least one detected fragment ion.

2. (canceled)

3. A method of ion mobility spectrometry according to claim 1, wherein thermally fragmenting the molecular ions comprises transporting the molecular ions through the fragmentation zone by an electric and/or gas flow field.

4. A method of ion mobility spectrometry according to claim 1, wherein the heated gas is at a temperature of at least 400° C.

5. A method of ion mobility spectrometry according to claim 1, wherein T*ln(1/τ) is above 3200, or above 4000, r above 5000, where T is the temperature of the heated gas in Kelvin and τ is the residence time of the molecular ions in the fragmentation zone in seconds.

6. A method of ion mobility spectrometry according to claim 1, wherein thermally fragmenting the molecular ions is carried out in the absence of any additional charged species or electromagnetic radiation in the fragmentation zone.

7. A method of ion mobility spectrometry according to claim 1, further comprising generating a fragment ion mobility spectrum from detecting two or more fragments from a molecular ion and comparing the spectrum with a library of fragment ion mobility spectra to identify the molecular ion.

8. A method of ion mobility spectrometry according to claim 1, wherein a residence time of the molecular ions in the fragmentation zone is in the range of 0.1-1 millisecond.

9. A method of ion mobility spectrometry according to claim 1, wherein each step of separating the molecular ions, fragmenting at least some of the separated molecular ions and separating at least some of the fragment ions is performed at atmospheric pressure.

10. A method of ion mobility spectrometry according to claim 1, wherein separating the molecular ions and/or separating at least some of the fragment ions is caused by a combination of crossed electric and gas flow fields.

11. (canceled)

12. A method of ion mobility spectrometry according to claim 1, wherein a gas circulating in a closed loop is used for both separating the molecular ions according to their mobility characteristics and separating at least some of the fragment ions according to their mobility characteristics.

13. (canceled)

14. (canceled)

15. A method of ion mobility spectrometry according to claim 1, wherein the molecular ions are separated and fragmented in parallel and the fragmentation zone comprises an array of fragmentation channels.

16. A method of ion mobility spectrometry according to claim 1, wherein the molecular ions are separated and sequentially scanned into a single fragmentation channel.

17. A method of ion mobility spectrometry according to claim 16, wherein the molecular ions are sequentially scanned into a single fragmentation channel using an ion mobility separator that is a differential mobility analyser (DMA), or other ion mobility separator that separates a continuous beam of molecular ions in space based on their ion mobilities, and scanning or stepping an electric field thereof.

18. A method of ion mobility spectrometry according to claim 1, wherein more than one fragment ion from a given molecular ion is detected sequentially or in parallel.

19. A method of ion mobility spectrometry according to claim 18, wherein more than one fragment ion from a given molecular ion is detected in parallel and the detector comprises an array detector comprising a plurality of spatially separated individual detectors.

20. A method of ion mobility spectrometry according to claim 18, wherein the fragment ions are separated and sequentially scanned into a single detector channel.

21. A method of ion mobility spectrometry according to claim 20, wherein the fragment ions are sequentially scanned into a single detector channel using an ion mobility separator that is a differential mobility analyser (DMA), or other ion mobility separator that separates a continuous beam of fragment ions in space based on their ion mobilities, and scanning or stepping an electric field thereof.

22. A method of ion mobility spectrometry according to claim 1, wherein more than one molecular ion is separated in space along a first direction of separation (x) and more than one fragment ion is separated in space along a second direction of separation (y), wherein the first and second directions are substantially orthogonal to each other.

23. A method of ion mobility spectrometry according to claim 1, wherein the molecular ions are separated and fragmented in parallel along the first direction of separation (x) and more than one fragment ion from each molecular ion is separated and detected in parallel along the second direction of separation (y), wherein the detector comprises a two-dimensional array detector.

24. A method of ion mobility spectrometry according to claim 1, wherein for a period the molecular ions are not fragmented but are separated and detected as molecular ions, wherein the molecular ions either bypass the fragmentation zone or are transmitted through the fragmentation zone wherein the conditions are adjusted for the period so that they do not permit fragmentation.

25. A method of ion mobility spectrometry according to claim 1, further comprising detecting the fragment ions as a function of a gas temperature in the fragmentation zone.

26. (canceled)

27. An ion mobility spectrometer comprising: an ion source for receiving a sample and generating molecular ions from the sample; a first ion mobility separator for separating the molecular ions according to their mobility characteristics; a fragmentation zone for fragmenting at least some of the separated molecular ions to form sub-molecular fragment ions, wherein the fragmentation zone comprises a heated gas at a temperature above 300° C., wherein the molecular ions are transported through the fragmentation zone by an electric field and/or gas flow such that a residence time of the molecular ions in the fragmentation zone is in the range of 0.1-5 milliseconds; a second ion mobility separator for separating at least some of the fragment ions according to their mobility characteristics; and a detector for detecting at least some of the separated fragment ions; wherein the first ion mobility separator, fragmentation zone and second ion mobility separator are adapted to be held at a pressure of at least 50 mbar in use.

28. An ion mobility spectrometer as claimed in claim 27, wherein the first ion mobility separator, fragmentation zone and second ion mobility separator are adapted to be held at atmospheric pressure in use.

29. An ion mobility spectrometer as claimed in claim 27, further comprising a data processing system for receiving data from the detector representative of the ion mobility of detected fragment ions and processing the data to provide an ion mobility spectrum of the fragment ions.

30. An ion mobility spectrometer as claimed in claim 27, wherein the first ion mobility separator and/or second ion mobility separator comprise crossed electric and gas flow fields.

31. (canceled)

32. An ion mobility spectrometer as claimed in claim 27, further comprising a closed gas circulation loop for continuously circulating gas between the first and second ion mobility separators.

33. An ion mobility spectrometer as claimed in claim 27, wherein the fragmentation zone comprises: an open jet of heated gas, a flame, or a heated channel, tube or capillary.

34. An ion mobility spectrometer as claimed in claim 27, wherein the fragmentation zone comprises a single fragmentation channel and the first ion mobility separator comprises an ion mobility separator that separates a continuous beam of molecular ions in space based on their ion mobilities having an electric field that can be scanned for sequentially scanning molecular ions into the single fragmentation channel.

35. An ion mobility spectrometer as claimed in claim 27, wherein the first ion mobility separator comprises an ion mobility separator that separates a continuous beam of molecular ions in space based on their ion mobilities and the fragmentation zone comprises an array of fragmentation channels to receive the separated molecular ions in parallel.

36. An ion mobility spectrometer as claimed in claim 27, wherein the detector comprises an array detector comprising a plurality of spatially separated individual detectors and the second ion mobility separator comprises an ion mobility separator that separates a continuous beam of fragment ions in space based on their ion mobilities such that two or more fragment ions are detected in parallel by the array detector.

37. An ion mobility spectrometer as claimed in claim 27, wherein the detector comprises a single detector and the second ion mobility separator comprises an ion mobility separator that separates a continuous beam of fragment ions in space based on their ion mobilities having an electric field that can be scanned for sequentially scanning fragment ions to the single detector.

38. An ion mobility spectrometer as claimed in claim 27, wherein the first ion mobility separator and the second ion mobility separator each comprise crossed electric and gas flow fields, wherein more than one molecular ion is separated in space along a first direction of separation (x) and more than one fragment ion is separated in space along a second direction of separation (y), wherein the first and second directions are substantially orthogonal to each other.

39.-47. (canceled)

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0117] FIG. 1 shows schematically an embodiment using dual stage DMA.

[0118] FIG. 2 shows schematically an embodiment utilising thermal atmospheric pressure fragmentation using an open gas jet.

[0119] FIG. 3 shows schematically an embodiment utilising thermal atmospheric pressure fragmentation in a heated channel.

[0120] FIG. 4 shows schematically a heating device for producing a heated gas jet.

[0121] FIG. 5 shows experimental data obtained on fragmenting peptide ions (neurotensin) by adding products of negative corona discharge.

[0122] FIG. 6 shows experimental results of thermal fragmentation of neurotensin.

[0123] FIG. 7 shows a thermal fragmentation mass spectrum of neurotensin.

[0124] FIG. 8 shows a mass spectrum of the thermal fragmentation of the peptide Leucine-Enkephalin (Leu-Enk).

[0125] FIG. 9 shows the dynamics of fragment formation with temperature of the peptide Leu-Enk.

[0126] FIG. 10 shows plots of the total fragment intensity and the parent intensity normalized to their sum for thermal fragmentation of the peptide Leu-Enk.

[0127] FIG. 11 shows schematically an atmospheric pressure embodiment of dual IMS comprising closed-loop recycling of gas between a first IMS and second IMS.

[0128] FIG. 12 shows schematically a reduced pressure embodiment of dual IMS comprising closed-loop recycling of gas between a first IMS and second IMS.

[0129] FIG. 13 shows schematically an embodiment of an ion mobility spectrometer using a pulsed ion source, first IMS drift tube with ion gating, thermal fragmentation and second IMS drift tube.

DETAILED DESCRIPTION

[0130] In order to enable a more detailed understanding of the invention, various embodiments and examples will now be described.

[0131] Referring to FIG. 1, there is shown schematically one preferred embodiment of the present invention. As will become apparent, in this embodiment, there is provided a parallel analysis of molecular ions and their fragments ions, in which the ions are separated by a continuous two stage differential mobility analyser (DMA) and detected by a two-dimensional (2D) detector array.

[0132] A sample (not shown) is introduced into an ion source 2 of an ion mobility spectrometer 1, in this embodiment a multi-stage ion mobility analyzer. The sample contains one or more components in the form of molecules of one or more different chemical structure. Molecular ions are formed in the ion source from the molecules. In some embodiments, the sample has been subjected to liquid or gas chromatographic separation prior to introduction into the ion source.

[0133] As the molecular ions are formed by the ion source 2 (e.g. formed as an ion spray 3 by an electrospray or atmospheric-pressure chemical ionization (APCI), or other ion source, as known in the art), they traverse the ion source chamber 5 and are pushed towards an entrance or sampling aperture 4 of the first ion mobility analyzer 10, for example by a voltage and/or gas flow. The entrance aperture 4 is located in a shield 8, such as a plate. This may be a voltage on the ion source (such as a voltage on the sprayer of an ESI, APCI source). It is preferable to improve desolvation of the molecular ions by using a heated (e.g. 200-500° C.) desolvation gas (e.g. in a curtain or orthogonal flow as shown by gas flow 6). A typically desolvation gas is nitrogen (preferably dry nitrogen) or dry air, with a typical flow rate of 10 L/min. The gas flow may move the ions towards the sampling aperture 4.

[0134] The ions preferably enter the sampling aperture 4 at an angle to the plane of the aperture, i.e. to the plane of the shield 8. The angle is generally less than 90 degrees, for example 60 degrees or less, more preferably 45 degrees or less, e.g. 30 degrees or less. It is preferred to sample the ions through the aperture using primarily an electric field rather than a gas flow field.

[0135] The first ion mobility analyzer 10 is a differential mobility analyser (DMA). Once molecular ions from the ion source 2 enter the first ion mobility analyzer 10 via the aperture 4 for separation they are picked up by a gas flow field V.sub.1, which has a direction shown by arrows 12 (which is along axis x of the three dimensional axes x, y, z). In this embodiment, the gas flow field V.sub.1 is at atmospheric pressure and is transverse to the direction in which the ions enter the DMA. The gas V.sub.1 is typically not heated, at least not heated enough to cause any fragmentation of the molecular ions, e.g. less than 200° C. Perpendicular to the gas flow field V.sub.1 is provided an electric field E.sub.1, the direction of which is shown by arrow 14 (which is along axis z of the three dimensional axes x, y, z). The molecular ions are thereby spatially separated in the drift space of the DMA according to their ion mobility in the crossed electric E.sub.1 and gas flow V.sub.1 fields. Ions species of different mobilities thus arrive at different parts of the array of fragmentation channels 16, each molecular ion species of a particular ion mobility arriving at its own channel. In total there are n fragmentation channels (16.sup.1, 16.sup.2, . . . 16.sup.n) in the array 16. At the entrance to the array of fragmentation channels 16 is a multi-apertured plate 18 having n apertures denoted 17.sup.1, 17.sup.2, . . . 17.sup.n. Thus, each n.sup.th fragmentation channel of the array of fragmentation channels 16 has its own respective entrance aperture. The field E.sub.1 is created between shield 8 and the array 16 (e.g. with voltages applied to shield 8 and to multi-apertured plate 18 below it). In some embodiments, to confine the electric field, printed-circuit boards on each side of each gap (drift space) could be used. In some embodiments, the gas flow field could be made more uniform using, for example, grids as known in the art.

[0136] The different channels thus receive different molecular ions based on their ion mobility in the first stage of ion mobility separation 10. The resolution of the mobility selection is determined by the size of the apertures 17 and the amount of diffusion in the DMA 10. The fragmentation channels 16 form a fragmentation zone 20. The individual channels 16.sup.1, 16.sup.2, . . . 16.sup.n are provided between walls 22 that separate adjacent channels. In the shown embodiment, the walls 22 are planar. The channels are thereby also planar. The walls may be made, for example, from material, such as resistive glass or ceramics or SiC, that can be heated and/or the walls can comprise resistive coatings and inks used in resistor technology, etc. The walls can be heated by a heater (not shown). The walls may be heated by a resistive heater, ceramic heater or cartridge heater for example. The heater may be placed in contact with the walls for this purpose. In some alternative embodiments, the multiple fragmentation channels could be provided as respective tubes or capillaries for example, which may have a circular or rectangular cross-sectional profile. These may also be made, for example, from material, such as resistive glass or ceramics or SiC, that can be heated and/or the walls can comprise resistive coatings and inks used in resistor technology, etc.

[0137] Each of the selected molecular ion species then transverse the fragmentation zone 20 through their respective fragmentation channel under the force of an electric field E.sub.f, the direction of which is shown by arrow 26 (which is, like E.sub.1, along axis z), while they are subjected to heating by a flow of heated gas at atmospheric pressure and a temperature in the range of 400-700° C. In this embodiment, the electric field E.sub.f is applied by a voltage difference between the multi-apertured plate 18 at the fragmentation zone entrance and the bottom of the fragmentation channels 16, the channels 16 being made from resistive material to sustain the field. In pushing the ions through the fragmentation channels by the electric field formed using the resistive channels, it can be possible to keep the ion residence time fixed per molecular species (depending on the ion mobility). This arrangement of electric driving field is preferred to an alternative use of a gas flow Vz in z direction for ion transfer through channels, as Pouseille profiles of Vz(xy) could spread the residence time widely, and if boundary layers do not merge and profiles are not formed, then the flow core may not be sufficiently heated by walls. Another preferred alternative embodiment comprises using a gas flow Vz in the z direction, especially using capillaries as fragmentation channels, using pre-heating of the gas (as described further below).

[0138] The heated gas flow in the embodiment shown in FIG. 1 is represented by gas flow field V.sub.f, which has a flow direction shown by arrows 24 (which is along axis y). In the fragmentation zone 20, the gas flow field V.sub.f is perpendicular (or crossed) with respect to the electric field E.sub.f. Furthermore, the gas flow field V.sub.f in the fragmentation zone is perpendicular to the gas flow field V.sub.1 in the first DMA 10. Thus, although the gas flow field V.sub.1 and the gas flow field V.sub.f are both in the x-y plane, V.sub.1 is in the x direction while V.sub.f is in the y direction. The heated gas may be provided by heating the walls of the fragmentation channels, as described above, to heat the gas flow field V.sub.f inside the channels. As an alternative to heating the walls of the channels, the channels may be provided with gas flow field V.sub.f that has been already heated by a separate heater before it enters the channels. The heated gas flow V.sub.f may be provided as one or more open or free jets of heated gas, such as multiple open jets (e.g. one for each channel). Examples of heated open gas jets are described in other embodiments below. The ion transfer through heated channels or tubes is conveniently compatible with the shown IMS-IMS scheme.

[0139] In other embodiments, a lower temperature may be usable for the heated gas flow, for example at least 200° C. (e.g. such as 200-400° C. or 200-300° C.). or at least (preferably above) 300° C. (e.g. such as 300-400° C.). Preferably though the temperatures is at least 400° C., or at least 450° C., or at least 500° C. The temperature may be up to 1200° C., or up to 1100° C., or up to 1000° C., or up to 900° C., or up to 800° C., or up to 700° C. More preferred for fragmentation is a gas temperature of at least (preferably above) 300° C. and better still at least (preferably above) 400° C. or 450° C. or 500° C. The temperature may preferably lie in the range 300-900° C., or more preferably 400-700° C., especially 400-600° C. or 500-700° C. As described below with reference to FIG. 6, the temperature choice can depend on the class of analyzed compounds and on the heating residence time.

[0140] In various embodiments of the invention, the gas temperature may be measured directly or indirectly using a temperature measuring device, e.g. a thermocouple located in or adjacent to the fragmentation zone. In some embodiments, a controller connected to a power supply can adjust the power provided by the supply to the gas heating means based on a temperature provided to it by the temperature measuring device. In this way, the gas temperature may be controlled by the controller, e.g. to maintain a target gas temperature that is preferably optimised for fragmentation of the molecular ions. The controller may comprise a computer and/or electronics for this purpose. The target gas temperature can be optimised for fragmentation of the particular sample being analysed (i.e. the molecular ions thereof). The target gas temperature may be predetermined, e.g. according to software or firmware operating the controller, or may be input by a user, such as via a user interface of the controller.

[0141] The transit time through the fragmentation zone (i.e. the ion residence time in the zone) can be defined, for example, by the length of the zone, the speed of the heated gas and/or the electric field E.sub.f transporting the ions. Preferably, E.sub.f>E.sub.1 for improving capture of ions into a channel. The transit time through the channel (equivalent to residence time in the heated gas) is arranged to lie in the range of 0.1-5 millisecond (ms). More preferably is arranged to lie in the range of 0.5 ms-5 ms or 1 ms-5 ms. Residence time preferably should be at least 0.1-1 ms (at least 0.1 ms, at least 0.5 ms, or at least 1 ms), for ions of m/z in a range 400-700. On average, fragmentation rate roughly doubles per 15 C per each individual compound. Especially preferred is a residence time in the heated gas of at least 1 ms.

[0142] As the molecular ions travel through the heated gas flow in the fragmentation zone 20, in their respective fragmentation channel, at least some of the molecular ions fragment to produce sub-molecular fragment ions that are related to the structure of the molecular ion (i.e. a subunit thereof). As fragment ions are formed, they, along with any unfragmented molecular ions, reach the second ion mobility analyzer 30, which, like the first ion mobility analyser 10, is a differential mobility analyser (DMA). The gas used for the heated gas flow field V.sub.f in the fragmentation zone may be same or different to the gas used in the gas flow fields V.sub.1 and V.sub.2 in the first and second ion mobility separators 10 and 30. Preferably, it is the same gas for V.sub.f, V.sub.1 and V.sub.2. The gas or gases for flows V.sub.f, V.sub.1 and V.sub.2 may be selected from inert gases such as nitrogen or argon or helium. Nitrogen is a preferred gas. The gas is preferably dried and optionally purified.

[0143] The second ion mobility analyzer 30 separates the fragment ions and any unfragmented molecular ions in space based on their ion mobility and they are detected by an array of individual detectors 36 for each fragmentation channel. The ions enter the second ion mobility analyzer 30 after exiting the fragmentation channels of the fragmentation zone. As mentioned, the second ion mobility analyzer 30 is a DMA, wherein the ions are picked up by a gas flow field V.sub.2, which has a direction shown by arrows 42 (which is, like gas flow field V.sub.f in the fragmentation zone, directed along axis y). In this embodiment, the gas flow field V.sub.2 is again at atmospheric pressure. The gas V.sub.2 is typically not heated, at least not heat enough to cause any further fragmentation of the ions, e.g. less than 100° C. Perpendicular to the gas flow field V.sub.2 is provided an electric field E.sub.2, the direction of which is shown by arrow 44 (which is, like electric fields E.sub.1 and E.sub.f, along axis z) to move the ions in the z direction through the second DMA 30 the towards a detector 36. Preferably, E.sub.2>E.sub.f for improving transfer of the ions into the second stage DMA 30. The molecular and fragment ions are thereby spatially separated in the drift space of the second DMA according to their ion mobility in the crossed electric E.sub.2 and gas flow V.sub.2 fields in the DMA 30. Particular parameters of field strengths E.sub.1, E.sub.f and E.sub.2, and gas flow speeds for V.sub.1, V.sub.f and V.sub.2 etc. will depend on the required resolving power as known in the art. As an example, a typical length of each separation channel is in the range 10-100 mm or 20-50 mm, gas speed is in the range 10-100 m/s, and field strengths E.sub.1 and E.sub.2 are in the range 2×10.sup.4-1×10.sup.5 V/m.

[0144] Furthermore, the gas flow field V.sub.2 in the second DMA 30 is perpendicular to the gas flow field V.sub.1 in the first DMA 10. Thus, although the gas flow field V.sub.1 and the gas flow field V.sub.2 are both in the x-y plane, V.sub.1 is in the x direction while V.sub.2 is in the y direction. The gas flow fields V.sub.f and V.sub.2 may be the same gas flow field, i.e. a single gas flow field, thus comprising the same gas flowing in the same direction. The single gas flow field for may be heated specifically within the fragmentation zone by heated walls of the fragmentation channels.

[0145] As shown, for example, in the second DMA 30 the fragment ions from the eighth fragmentation channel 16.sup.8 become separated from each other (and from any molecular ions) in the y direction and are detected by a one-dimensional array of individual detectors 32.sup.1-32.sup.m (i.e. m detectors positioned in a row 32 shown in the y direction). Similarly, in the second DMA 30 the fragment ions from the twelfth fragmentation channel 16.sup.12 become separated from each other (and from any molecular ions) in the y direction and are detected by a one-dimensional array of detectors (i.e. in a row 34 shown in the y direction). There are n rows (n 1D arrays) of detectors corresponding to the n fragmentation channels. Thus, a 2D array 36 of n×m detectors is provided. In some embodiments, the individual detectors may be arranged at regularly spaced locations in the 2D array (in x and/or y). In some other embodiments, the individual detectors may be arranged in a 2D array not at regular spaced locations but at specific locations only, e.g. the individual detectors may be provided in locations so as to detect only a limited number of molecular ions and, for each detected molecular ion, one or more fragment ions. The latter may be a more dedicated (molecule-specific) detector than a universal (broad range) detector.

[0146] In one embodiment, the detector array comprises a set of ion collectors connected to one or more electrometers. The collectors can be arranged to accumulate charge for sequential reading by a single electrometer. To enhance detector sensitivity, the ions can be field accelerated in front of the detector. The detector or detectors may comprise MCPs or electron multipliers, e.g. an array thereof. The detectors may comprise photodetectors, such as an array photomultiplier tube (PMT) or diode array. In one embodiment, the ions can be field accelerated to sharp tips to produce a photo signal, which is read by a photodetector such as an array photomultiplier tube (PMT) or diode array. The latter may be more practical at fore-vacuum gas pressures produced by a mechanical pump (e.g. rotary or roots pump) in case of single channel detection, or when operating IMS at sub-atmospheric pressures. Even at reduced efficiencies of producing photons, ion counting with a PMT may be much more sensitive than the collector current measurements.

[0147] The detector array 36 is connected to a data processing device (not shown) for producing a spectrum of the fragments from data provided by the detector. The data processing device also comprises an instrument interface to operate the spectrometer 1.

[0148] Although the embodiment in FIG. 1 has been described as having each of the ion source chamber 5, first ion mobility separator 10, fragmentation zone 20 and second ion mobility separator 30 at atmospheric pressure, in some other embodiments the first or sampling aperture 4 may separate an atmospheric pressure ion source (such as ESI or APCI) and a fore-vacuum stage e.g. at 0.1-100 mbar, or 1-100 mbar, or 0.1-10 mbar, or 1-10 mbar. The fore-vacuum pressure may be produced by a mechanical pump (e.g. rotary or roots pump). In such embodiments, the first ion mobility separator 10, fragmentation zone 20 and second ion mobility separator 30 may be provided at the fore-vacuum pressure. In some embodiments, the operation at fore-vacuum gas pressures may have some advantages such as: lower gas consumption and lower power of gas compressors; induction of gas flows by pumps; non compromised mobility resolution, where lower pressure P is compensated by a linearly scaled dimension of mobility separation L at the same voltage U (defining mobility resolution) in turn, being limited by L/P product; and possibly easier to manufacture devices of larger size.

[0149] Although, parallel detection of multiple fragment ions for multiple precursor (molecular) ions is described with reference to FIG. 1 and requires a 2D detector, a simpler design would involve just one selection (fragmentation) channel with different ion mobility molecular ions being sequentially directed to it by the first DMA by changing electric field E.sub.1 and/or gas flow field V.sub.1 (preferably by changing electric field E.sub.1 as it is easier to change the electric field in a controlled way). For each molecular ion being fragmented in the single fragmentation channel in sequence the fragment ions may be separated from each other and detected by a one-dimensional (1D) array of individual detectors. In another, similar embodiment, just one selection (fragmentation) channel may be provided with different ion mobility molecular ions being sequentially directed to it by the first DMA and a single detector provided with different ion mobility fragment ions being sequentially directed to it (for each molecular ion) by the second DMA by changing electric field E.sub.2 and/or gas flow field V.sub.2 (preferably by changing electric field E.sub.2 as it is easier to change the electric field in a controlled way). In a further, similar embodiment, multiple (fragmentation) channels may be provided with different ion mobility molecular ions being directed to it in parallel by the first DMA as shown in FIG. 1 and a single detector provided for each fragmentation channel (i.e. a 1D array of detectors spaced in the same direction as the fragmentation) channels, wherein different ion mobility fragment ions for each molecular ion/fragmentation channel are sequentially directed to a detector for that molecular ion/channel by the second DMA. Thus, individual detection channels, rather than the detection array of FIG. 1, may be used for target analyses. Such target analyzers may have some advantages such as lower gas consumptions due to using fewer separation channels; lower operation cost at fewer detection channels; and selecting fragmentation temperatures for efficiency, since it can be difficult in some cases to fragment all species with the highest efficiency using a single temperature setting.

[0150] The fragmentation zone may comprise: an open jet of heated gas, a flame, or a heated channel, tube or capillary.

[0151] In some embodiments, the fragmentation zone may be provided, for example, in the form of an open or free jet, e.g. a region containing one or more jets of pre-heated gas (thus passing the sample molecular ions through one or more jets (beams) of heated gas). The term free gas jet or open gas jet herein refers to a stream of gas that is projected into the fragmentation zone, generally from a nozzle or aperture. The free gas jet typically has a higher momentum compared to the surrounding gas. In a gas jet fragmentation, at atmospheric pressures, the gas flow velocity in the jet may be, for example, 0.5-100 m/s, or 0.5-50 m/s, or 0.5-10 m/s, preferably 1-10 m/s, e.g. 2, 3, 4, 5, 6, 7, 8 or 9 m/s. At sub-atmospheric pressures, vacuum pumping may produce gas jets at nearly sonic velocities, e.g. up to 300 m/s for nitrogen, or even supersonic velocities. The gas jet may also be constrained in a channel. In such cases, the channel itself preferably needs to be heated to enable the required gas temperature and therefore fragmentation in the channel.

[0152] In other embodiments, the fragmentation zone may be provided, for example, in the form of a heated channel, heated tube or heated capillary. It may be provided, for example, in the form of enclosed channels (e.g. tube-like or flute-like enclosed channels with sampling apertures) through which the sample ions and gas flows, or capillaries through which the ions and gas pass (including a direct current heated capillary), or flames through which the ions pass. The heated channel, tube or capillary may be heated, e.g. from the outside, thereby to heat the gas flowing inside. The heated channel, tube or capillary may comprise a heater inside, e.g. a wire or filament heater, thereby to heat the gas flowing inside. The channel, tube or capillary may receive a gas that has been pre-heated before entry into the channel, tube or capillary.

[0153] An example of an embodiment utilising thermal atmospheric pressure fragmentation in open jet is shown schematically in FIG. 2. An atmospheric pressure ion spray source 102 at 3 kV sprays a cloud of positive molecular ions 108 towards a shield 110 held at 0-1 kV. A gas flow 106 such as nitrogen at 2 L/min assists with transporting and/or desolvating the ions. The molecular ions are sampled through an aperture 104 in the shield. The molecular ions then travel through a fragmentation zone provided by an atmospheric pressure free jet 120 of heated nitrogen gas emitted from a nozzle 130. The free jet 120 has a higher momentum compared to the surrounding gas 124. The free jet is directed substantially transverse to the direction of ion travel. Temperature measuring devices in the form of thermocouples TC1 and TC2 measure the gas temperature. The gas jet temperature is approximately 565° C. where the thermocouple readings differ, an average may be taken as the gas temperature. Typically, a spread or range of gas temperatures in the fragmentation zone is less than 30° C. or less than 20° C., for example achieved by ion sampling primarily with electric field and by reducing the sampling of the relatively colder gas of flow 106 through the aperture 104. The gas jet 120 is generated by a flow of nitrogen at 2 L/min through a heated tube 132 wherein the gas temperature is approximately 700° C. before it leaves the tube. The estimated heating time is about 1-2 ms, based on a 3 m/s average jet velocity. The capillary 132 is a resistively heated quartz tube. The molecular ions fragment into sub-molecular fragments in the heated free gas jet and the fragment ions then enter an RF only transfer quadrupole 160 of a mass analyser at 2 torr through a nozzle 150, which is held at a lower potential (50V). The ions are analysed by their mass-to-charge ratio (M/z) in the mass analyser. The use of open or free jets has been found to provide very reproducible fragmentation of ions. Furthermore, good transmission of ions can be obtained by fragmenting using open jets.

[0154] An embodiment utilising thermal atmospheric pressure fragmentation in a heated channel is shown schematically in FIG. 3. The embodiment of FIG. 3 shares numerous similar components to the embodiment of FIG. 2 so like components are given like reference numerals. An atmospheric pressure ion spray source 102 at 3 kV sprays a cloud of positive molecular ions 108 towards a tube or flute (denoting tube with sampling aperture) 210 held at 100-1000 V. A gas flow 106 such as nitrogen at 2 L/min assists with transporting and/or desolvating the ions as they flow towards an entrance or sampling aperture 104 in the tube 210. The molecular ions are sampled through the aperture 104 in the tube. The molecular ions then travel through the tube in an atmospheric pressure flow 220 of heated nitrogen gas (0.5-10 L/min) emitted from a heated capillary 230, which forms a fragmentation zone. The capillary 230 is a resistively heated quartz tube. The gas flow is directed transverse to the direction of ion travel as the ions enter the tube. Temperature measuring devices in the form of thermocouples TC1 and TC2 measure the gas temperature. The heating time of the ions (i.e. the residence time of the ions in the gas flow) can be controlled by the gas flow rate in the tube. The molecular ions fragment into sub-molecular fragments in the heated gas flow and the fragment ions then enter an RF only quadrupole 160 of a mass analyser at 2 torr through a nozzle 150 located in an exit aperture of the tube. The ions are analysed by their mass-to-charge ratio (M/z) in the mass analyser.

[0155] Variants of the embodiments of FIG. 2 or FIG. 3 preferably could be implemented with a stage of ion mobility separation, such as a first DMA as shown in FIG. 1, between the ion source and the entrance to the fragmentation zone. In such embodiments, different ion mobility molecular ions could be sequentially directed to it by the first DMA, i.e. by changing an electric field and/or gas flow field (preferably by changing an electric field) in the DMA. For each molecular ion then fragmented in sequence, the fragment ions could be analysed by their mass-to-charge ratio (M/z) in the mass analyser.

[0156] The invention can therefore be implemented using a number of different designs for providing a heated fragmentation zone: free jet, tube or flute-type enclosed channels, heated capillaries, including direct current heated capillaries, examples including tantalum (Ta) or tungsten (W) capillaries or tubes, Kanthal™, Nicrothal™ (FeCrAl, NiCr alloys) or SiC-like semiconductor tubes. Tantalum, Kanthal™, Nicrothal™ are preferred because of their tendency for resistance to oxidation at higher temperatures. Another embodiment may comprise a rolled Ta foil that is directly heated.

[0157] Surfaces adjacent the fragmentation zone and/or exposed to the heated gas can be made from, e.g., oxidation-resistant refractory metal, such as tantalum (Ta), or carbides (SiC, WC), or, depending on thermal wall conductance, of stainless steel or tungsten (W). The heating of the gas may be provided by a gas heating means, such as one or more resistive heaters, wire heaters, ceramic heaters, silicon carbide (SiC) heaters, or cartridge heaters, or other heaters, preferably resistant to oxidation and specified for at least 300° C. temperatures by wire insulation). The one or more heaters may comprise one or more heaters located external to a region, channel or tube through which the ions and gas flow, such as to heat the gas through one or more walls adjacent the region, channel or tube. Alternatively, or additionally, the one or more heaters may comprise one or more heaters located internal to a region, channel or tube through which the ions and gas flow, such as a heated wire or filament in the region, channel or tube. An example of one preferred heating arrangement is a resistive heater located around a tube or channel (such as a quartz tube), the gas being passed through tube when the tube is heated by the heater. These may be easily used for gas temperatures up to 700° C. In another embodiment, the gas heating means heats a flow of gas so as to provide a heated gas jet that is directed into the fragmentation zone.

[0158] A heating device for producing a heated gas jet is shown schematically in FIG. 4. A quartz tube 330 is provided with a nichrome coil heater 320 wound around its external surface to heat the tube (connections to the heater are shown at 322 and 324 that supply a 30V, 10 A current). Two layers of 0.1 mm stainless steel shield 342, 344 are provided around the tube and heater. Gas, such as nitrogen, is supplied to an inlet of the quartz tube via a Swagelok™ connection 350 to a gas source (not shown). A typical gas flow rate is 3 L/min. A gas flow is heated by the hot tube 330 and emitted from a 4 mm diameter nozzle 360 to form a hot gas jet.

[0159] As described in the Background, there are multiple known ways to fragment molecules at atmospheric pressure. For example, FIG. 5 shows experimental data obtained on fragmenting peptide ions (neurotensin with charge states .sub.+1, .sub.+2 and .sub.+3) by adding products of negative corona discharge (n-CD) into a gas flow to fragment the peptide ions, arranged without removing electrospray solvent. In the plot shown, the 3.sub.+, 1.sub.+ designate charge states of molecular ions; X7 is the X7 fragment of neurotensin, M0 3.sub.+″ is the oxide ion of M3H.sup.3+ and M0 2.sub.+″ is the oxide ion of M2H.sup.2+. The scales are logarithmic. The fragment X7 was observed at 1×10.sup.−3 relative intensity of the M3.sub.+ molecular ion peak. The trends illustrated in FIG. 5 include the drop of absolute signal intensities versus drop of the overall signal intensity, induced by larger n-CD currents or faster delivery of n-CD products. Overall, the primarily observed effects of n-CD, as illustrated in FIG. 5, are: (a) ionization of air impurities, thus, forming additional chemical background, (b) charge reduction of peptide ions with increasing share of lower charged ions; (c) significant drop of the overall intensity; (d) formation of oxide ions, most probably produced by ozone, produced in the n-CD; (e) formation of fragments at low intensity.

[0160] In contrast to n-CD, rather than employing ionizing fragmentation methods, the present invention utilizes a novel method of thermal ion fragmentation at atmospheric pressure or higher pressure vacuum, i.e. fragmentation caused by interacting the sample molecules with a heated gas so as to transfer thermal energy. The thermal ion fragmentation method provided by the invention can: (a) produce an abundant and reproducible ion fragmentation depending solely on the structure of fragmented molecule, gas temperature and optionally the residence time of the molecule in the heated gas; (b) not introduce additional chemical background (ionizing fragmentation methods, for example, may produce a high background of newly formed ions from impurities and background gases, thereby complicating the interpretation of the sample fragment spectra; and (c) not affect the overall signal intensity (e.g. by charge reduction). FIGS. 6 and 7 illustrate this for the same sample, neurotensin. FIG. 6 shows the absolute signal intensity of all precursor ions (P) with 3.sub.+, 2.sub.+ and 1.sub.+ charge states and absolute signal intensity of all fragments (Fr) vs temperature of heated gas flow using the hot gas jet set-up. All scales are linear. Substantial fragmentation is observed at 500-600° C. The optimum fragmentation temperature can depend on the compound type to be fragmented and optionally on the ions' residence time (longer residence times may allow a lower temperature). In our experiments, doubling of residence time does drop the characteristic fragmentation temperature (for 50% degree of fragmentation) by 15-20° C. for about 50 tested compounds of different chemical classes. The effect is known from IR PD studies, where fragmentation occurred at about 200-300° C. at residence times on the scale of minutes. FIG. 7 shows a mass spectrum of neurotensin subjected to thermal fragmentation, which is similar to spectra produced by collisional induced dissociation (CID) in vacuum. The spectrum is composed of y-, b-, x- and z-ions, mainly b- and y-, containing structural information, suitable for library identification of the peptide. FIGS. 8, 9 and 10 show the results of the thermal fragmentation of another peptide sample, Leucine-Enkephalin (Leu-Enk). FIG. 8 shows the mass spectrum of the fragmentation and FIG. 9 shows the dynamics of the fragment formation with temperature. The decrease of the molecular ion MH.sup.+ particularly above 400° C. is accompanied by the increase in the fragment intensities. The intensity scale is logarithmic. The degree of molecular ion fragmentation may be used as a calibrant or molecular thermometer. As seen in FIG. 9, a fragment ion ratio varies with temperature much slower than the degree of fragmentation. Indeed, the curves for fragment ions stay nearly parallel over a wide temperature range, compared to the curve for fragmentation of the molecular ion. This means that the degree of (molecular ion) fragmentation can serve as a temperature calibrant or thermometer. This can allow adjustment of the fragmentation temperature (and/or residence time) to optimize the sensitivity of the method for target compounds, especially where fast adjustments of the reactor temperature can be achieved, for example by mixing hot and cold gas jets. A ratio of one fragment per parent may be used in the method. One way of measuring the degree of fragmentation is to measure at least one fragment ion and the molecular ion. Another method is to introduce heated gas as a pulse, i.e. detecting molecular ions with and without fragmentation. FIG. 10 plots the total fragments intensity and the parent intensity normalized to their sum. Early thermal fragmentation starts appearing at gas temperatures above 250 C. Notable fragmentation occurs above 350° C. and above 400° C. In some embodiments, the fragmentation temperature can be changed, for example stepped, during the analysis to adjust the fragmentation temperature to be optimal for the target compound(s). This may not be required, for example, if it is a single channel analysis. Such variation of the fragmentation temperature, for example, can be achieved by mixing hot and cold gas at specific calibrated ratios. Stepping the fragmentation zone temperature or residence time is a method of improving selectivity, since a curve of fragmentation degree versus temperature, as shown FIG. 10, depends on the compound.

[0161] The thermal fragmentation method generates intense fragments (unlike ECD or ETD). A hot gas allows substantial fragmentation, e.g. a degree of fragmentation (total fragments intensity per total signal) of 90% in some cases occurs above 500° C. Furthermore, the total ion current only slightly drops at higher temperatures, thus, heating does not cause ion discharging. The hot gas method does not produce any new background ions (contrary to ionizing fragmentation methods). Thus, thermal fragmentation can be a useful fragmenting method at atmospheric pressure for the tandem identification of compounds, e.g. in set-ups such as the IMS-IMS systems described herein.

[0162] Other known fragmentation methods such as irradiation by photons (e.g. photons of any of the following: vacuum UV, UV, IR or visible) or electrons (e.g. from glow or corona discharge, or from a vacuum tube) or metastable atoms and molecules could be used, e.g. in addition, but it is preferable to use a non-ionizing fragmentation method, i.e. thermal energy only.

[0163] In further embodiments, to preferably reduce consumptions of power and purified gas (or at least dried gas), gas from a first stage of ion mobility separation (e.g. DMA) is re-used for a second stage of ion mobility separation (e.g. DMA), as well as preferably in the fragmentation zone, and then re-cycled back into the first stage by a close-loop compressor. Such an embodiment is shown in FIGS. 11 and 12. FIGS. 11 and 12 are similar but whereas the FIG. 11 shows an atmospheric pressure (1 atm=1 bar) embodiment, FIG. 12 shows a vacuum embodiment (10 mbar). Similar features are given the same reference numerals in each of FIGS. 11 and 12. An LC separation provides sample molecules to an ion source 402. Molecular ions 404 are sprayed under the influence of a voltage on the ion source towards an entrance or sampling aperture 406 of a first stage of ion mobility separation. The ions are desolvated using a curtain gas (N.sub.2) as they travel to the aperture 406. The first stage of ion mobility separation is a first differential mobility analyser DMA (DMA1). The DMA1 contains a flowing gas field (N.sub.2) in the direction indicated by arrow 408. Perpendicular to the gas flow field 408 is provided an electric field in the direction indicated by arrows 409. The ions separate in DMA1 according to their differential mobility in the crossed gas and electric fields and selected molecular ions enter an entrance aperture 432 of a thermal fragmentation channel in the form of a heated capillary 430 (e.g. a capillary made of tantalum, tungsten or other oxidation resistant material, or steel (preferably coated by tantalum, tungsten or other oxidation resistant material)). Molecular ions of different ion mobility may be scanned into the aperture 432 by changing either the electric field 409 or gas velocity 408. The aperture 432 samples the ions by means of gas flow from the DMA1 into the heated capillary 430 in the direction of arrow 435. The capillary is heated for example to heat the gas therein to 400-600° C. as described above. Therein the molecular ions fragment into sub-molecular fragment ions, which are carried by heated gas flow together with any unfragmented molecular ions into a second stage ion mobility analyser in the form of second DMA (DMA2).

[0164] The drift space 410 of DMA1 is in fluid communication with a first gas conduit 420 such that the gas field 408 flows gas though the drift space 410 of DMA1 and into the first gas conduit 420. The gas flow direction is indicated by the arrows in gas conduit 420. The gas then flows into drift space 440 of DMA1 in the direction indicated by arrow 448 to provide a gas flow field in DMA2. It is noted that gas flow field 448 of DMA2 is in the opposite direction to the gas flow field 408 in DMA1. Perpendicular to the gas flow field 448 is provided an electric field in the direction indicated by arrows 449. The ions separate in DMA2 according to their differential mobility in the crossed gas and electric fields and selected molecular ions enter an entrance aperture 442 of a detector 470, which can be a simple ion detector or a mass spectrometer. In DMA 2, fragment ions of different ion mobility may be scanned into the aperture 442 of the ion detector by changing the electric field 449.

[0165] The drift space 440 of DMA2 is in fluid communication with a second gas conduit 422 such that the gas field 448 flows gas though the drift space 440 of DMA2 and into the second gas conduit 422. The second gas conduit 422 is in fluid communication with drift space 410 of DMA1 such that the gas is thereby re-circulated into DMA1 again. A sealed blower or compressor 450 in second gas conduit 422 drives the gas back into the DMA1 in the circulation loop.

[0166] Preferably, metal fan blades (squirrel wheel type or radial rotary type, similar to ones used for industrial hot gas processing, see for example www.chuanfan.com/showrooml.html) of the compressor 450 are remote from a motor to avoid contaminating fumes. Preferably, a mesh and/or dust filter (such as porous metal, also serving as heating means) 452 are used for gas flow laminarisation. Though the IMS spectrometer of FIG. 11 should be more compact at 1 atm pressure, a larger size of gas blower might make the device as bulky, and similar cost as the 10 mbar IMS spectrometer of FIG. 12, where operation at 10 mbar is expected to provide benefits of easier construction and of higher parameters with easily achieved laminar gas flow.

[0167] The ion transfer through heated channels or tubes 430 is conveniently compatible with the shown IMS-IMS scheme based on a cycled DMA analyzer. The gas flow scheme may be modified for a gas pressure drop between the IMS stages (which may occur in the cycled gas scheme proposed in FIGS. 11 and 12), e.g. wherein the second IMS stage is lower pressure than the first IMS stage, so that a gas flow is achieved through the fragmentation device that connects the IMS stages, whereby the gas flow samples the molecular ion precursors into the fragmentation device 430 and then into the second stage IMS (DMA2).

[0168] The overall specificity of molecular identification is proportional to the multiplication of the ion mobility resolutions of the first stage IMS (resolution R1) and second stage IMS (R2), i.e. R1×R2. Further separation stages could be incorporated in the design for further improvements in this regard. Selection of multiple fragment ions (at least 2 but, preferably, 3-6 characteristic fragments) improves both specificity and confidence of the identification.

[0169] In some embodiments, the relative intensity, i.e. abundance, of fragment ions to each other and/or to their parent molecular ion can be used for additional confidence or confirmation of molecular identification, e.g. as known in triple quadrupole mass spectrometry (multiple reaction monitoring method, MRM) or high resolution mass spectrometry (parallel reaction monitoring method, PRM). Such additional confidence is best enabled by reference/comparison of the acquired fragment IMS spectrum to a library of fragments (fragment IMS spectra or MS spectra) that has been created for each of a plurality of analytes of interest. A sufficient match of fragments acquired from the sample to fragments in the library can be used to identify the molecule(s). The library is preferably a library of fragments or fragment spectra that has been acquired using the same type of thermal fragmentation, and preferably IMS separation, as described herein. The library is preferably a library of fragments or fragment spectra that has been acquired using the same type of thermal fragmentation, and preferably IMS separation, as used to acquire the fragment IMS spectrum for the sample. The library preferably also contains fragments (fragment IMS spectra or MS spectra) of one or more calibrants. In this way, samples of interest can be analysed using the invention along with at least one calibrant. The calibrant could be external (i.e. run in another experiment than the sample of interest) or internal (i.e. part of the same mixture as the sample). The main function of calibration would be to use the calibrant(s) as a so-called molecular thermometer to establish optimum effective temperature (and optionally other conditions) of fragmentation, preferably to provide corresponding fragmentation to the calibrant(s) in the library). Thus, K1 and K2n (i.e. the ion mobilities of the parent molecular ions (K1) and each of their n fragment ions (K2n), n is 1, 2 . . . n) can be measured, while the matrix may vary. The method preferably selects those fragments which exhibit the correct intensity ratio, even in the presence of the matrix. A high reproducibility of temperature calibration (e.g. using chemical thermometers as described) of the thermal fragmentation method enables a high reproducibility of fragment ratios and therefore confidence in comparison with a library of fragment ion IMS spectra.

[0170] In some embodiments, internal calibrants can also be used for quantitation in targeted analysis, in particular, if they are provided in the form of isotopically labelled variants of analytes of interest. Comparing to mass spectrometry, labelling with .sup.2H (Deuterium) or .sup.13O, for example, needs to be more extensive to enable a larger mass difference and therefore larger mobility difference in order to accommodate the generally lower levels of ion mobility resolution (30-200 for each of stages) comparing to even nominal-mass mass spectrometry resolution (200-2000). Preferably, at least 6-15 Da mass shift is to be provided in internal calibrants, or a chemically attached tag is provided for sufficient mobility shift. In case of GC or LC separation, this might result in a significant shift in retention times that needs to be taken into account during quantitation.

[0171] In FIG. 13 another embodiment of an ion mobility spectrometer 500 is schematically shown, wherein a pulsed ion source is used. For a pulsed ion source, a drift tube, preferably a linear drift tube, is desirable. A drift tube is more suitable as it allows selection of one or more species of molecular ions by gating one or more packets of molecular ions of interest, e.g. gating the packet(s) of ions after an appropriate delay from the pulse (i.e. after a first stage of IMS). A pulsed ion gate is thus provided for this purpose. If desired, to improve duty cycle, multiple packets could be selected with an appropriate delay between gating pulses. Temporal broadening of the packet in the fragmentation zone could be reduced by eluting ions out of this zone at lower electric field and then step-wise applying a stronger, spatially inhomogeneous electric field in order to reduce the duration of the peak at the expense of its size.

[0172] In detail, FIG. 13 shows a pulsed laser source 502, e.g. for implementing a MALDI source. The laser is arranged to irradiate a sample held on a sample plate 504 at atmospheric pressure and produce a pulse of molecular ions. The pulse of produced molecular ions then enters a first buffer gas-filled ion mobility drift tube 506 wherein the molecular ions 505 of the ion pulse separate based on their ion mobility in an axial DC potential provided by a series of ring electrodes 508 axially spaced apart along the length of the drift tube, as known in the art. The buffer gas is arranged flowing in the opposite direction to the direction of ion travel but that is not necessary. The ions reach the exit of the drift tube at different times dependent on their ion mobility. At the exit of the first drift tube 506 is a Buckbee-Mears ion gate 518 for gating (i.e. selection) of molecular ions. The ion gate is optional. After (optional) gating, the molecular ions enter a fragmentation zone 520 provided inside a fragmentation tube 522. An entrance aperture 524 is provided in the tube for sampling the molecular ions in this way. A hot gas e.g. at 400-700° C. is arranged to flow through the tube in the direction shown by the arrow 530. The tube may be heated for this purpose. The gas flows from an entrance 523 to an exit 525 of the tube. The molecular ions are subject to thermal fragmentation in the fragmentation zone and the produced fragment ions travel in the hot gas flow 530 along the fragmentation tube 522. Ion extraction optics 538 downstream of the fragmentation zone, which may be pulsed, extract the ions from the fragmentation zone via exit aperture 528. The fragment ions, and optionally any unfragmented molecular ions, then enter a second buffer gas-filled ion mobility drift tube 546, wherein the ions separate based on their ion mobility in an axial DC potential provided by a series of ring electrodes 548 axially spaced apart along the length of the drift tube. The buffer gas is arranged flowing in the opposite direction to the direction of ion travel but, again, that is not necessary. The separated ions are finally detected by an ion detector 550, which is connected to a data processing device 560 for producing a spectrum of the fragments. The data processing device also provides control of the spectrometer 500.

[0173] Although the FIG. 13 embodiment has been described as an atmospheric pressure system, as described above, the ion mobility stages and fragmentation zone could be maintained at a vacuum, e.g. 1 mbar or above, preferably 50 mbar or above, in that case, preferably, using radiofrequency (RF) fields for radial ion confinement.

[0174] In a variation of the embodiment shown in FIG. 13, a travelling DC wave can be applied to the series of ring electrodes 508 of the first ion mobility drift tube to select molecular ions of certain mobility, for example as disclosed in U.S. Pat. No. 5,789,745. Optionally, a travelling DC wave could be applied to the series of ring electrodes 548 of the second ion mobility drift tube to select fragment ions of certain mobility.

[0175] In some embodiments, wherein the sample is delivered in a continuous mode, the fragmentation conditions (e.g. gas temperature or power density) could be changed over time in order to construct fragmentation curves (e.g. degree of fragmentation versus temperature), which can be indicative of the analyte structure, i.e. the identity of the molecular ion. When interfaced to a mass spectrometer, such a 1-dimensional (1D) scan could be complemented by a 2.sup.nd dimension of scanning fragmentation spectra inside mass spectrometer (e.g. collision energy in collision-induced dissociation, or exposure in infrared or ultra-violet photodissociation, interaction time in electron-transfer dissociation, etc.).

[0176] In some embodiments, wherein the sample is delivered in a time-dependent manner (e.g. from a liquid or gas separation, such as LC or GC) and both precursor (molecular ion) and fragment spectra need to be collected, a pulsed operation of the thermal fragmentation can be arranged by using pulsed valves to mix cold and hot gas streams for rapid temperature variations, thereby to effectively switch the fragmentation between on and off, or alternatively using ion optics to electrically steer ions to by-pass the fragmentation zone.

[0177] In any of the foregoing embodiments, the ions can be produced by any of the following ion sources: ESI, APCI, APPI, APGC with glow discharge, AP-MALDI, LD, inlet ionization, DESI, LAESI, ICP, LA-ICP, etc. these can be interfaced to any of the following separations: LC, IC, GC, CZE, GC×GC, LC-LC, etc. Multiple ion sources or ionization sprayers or channels could be used in parallel and gated either mechanically or electronically as known in the art. Any type of ion mobility separation can be used as described herein. Any combinations of these units can be used to create analytical instruments with any combination or number of stages of analysis.

[0178] A number of preferred embodiments of analytical methods can be implemented according to the invention: [0179] a. Single compound monitoring at nearly unity transmission and at very high speed analysis of a few ms (1-3 ms may be required for monitoring ultra-fast processes, e.g., engine control, or selected reaction monitoring). Such embodiments preferably comprise fixing the fragmentation temperature T, and passing ions at fixed first and second mobilities K1 and K2, to the detector, which we abbreviate as K1, T, K2. The occurrence and intensity of individual target compounds are thereby detected, e.g. as function of an upfront chromatographic separation time (retention time), or the occurrence and intensity of the target compounds in air monitoring with a mobile laboratory can be performed, or similarly in the monitoring of a technological process. [0180] b. Multiple reaction monitoring (MRM) with preselected channels for ultra-trace and/or ultra-fast analysis by switching K1, T and/or K2 for each particular reaction. Preferably, the fragmentation temperature is adjusted between multiple channels of MRM. [0181] c. Increased sensitivity by sampling ions using a mechanical pump, accelerating to a scintillator tip, and then detecting individual ions. Selectivity of dual IMS can be comparable to a single MS (e.g. resolution of 50×50=2500). Selectivity or specificity of detection using multiple channel MRM can be much higher. [0182] d. Parallel 2D analysis of ions, i.e. comprising separating molecular ions in one dimension and their fragments in another, orthogonal direction (for example as illustrated by the embodiment shown in FIG. 1). [0183] e. 2D analysis of ions using thermal scans, where parent ions are separated by their mobility K1 in the first IMS1 stage; the fragmentation temperature T varies in time at slower time scales compared to IMS1 time scale; and the overall intensity of (all or majority of) fragment ions is detected as the function of K1 (mobility) and T (fragmentation temperature) only, thus avoiding slow scanning of three parameters—K1, T and K2. To detect overall fragment intensity, the second mobility filter is time-linked to the first one. In one method, IMS2 passes to the detector only those ions whose K2 is less than K1 (the method is useful for small molecules producing 1+ ions predominantly). In another method, IMS2 forms a notch, to pass all ions with K2 being not equal to K1. This method is more appropriate for large peptides and proteins, where multiply charged fragments may have K2>K1. [0184] f. 3D analysis: IMS1-Th.Scan-IMS2 [0185] g. IMS-Th.Frag-MS. This can be implemented, for example, in embodiments where the detector 470 of the system shown in FIG. 11 is a mass spectrometer. [0186] h. IMS-Th.Scan-MS. This too can be implemented, for example, in embodiments where the detector 470 of the system shown in FIG. 11 is a mass spectrometer. [0187] (above Th.Frag means thermal fragmentation and Th.Scan means thermal fragmentation with scanned temperature, i.e. detection of fragments as a function of temperature in the fragmentation device. Generally, fragmentation temperature variations are made on a slower time scale compared to IMS1 separation time) [0188] i. Tracking M-dM patterns for fragments in the case of MS analysis (thus providing a “3D analysis”), becoming 4D with thermal profiles, wherein M refers to the fragment mass and dM is the difference of the mass from the integer mass, i.e. the so-called mass defect. For example, for a homologous series (e.g. polymer series), all compounds of the same series will fall on one line.

[0189] Further preferred embodiments include: [0190] j. LC or GC followed by sequential dual stages of DMA for MRM monitoring. In these embodiments, multiple fragment ions can be detected for each molecular ion. It is most preferably implemented using a single channel detector, wherein the ion mobilities of the parent, molecular ions (K1) are mapped over the LC/GC retention time, and ion mobilities of the fragments (K2) are scanned by stepping field strength, preferably stepping the electric field strength of a second DMA. [0191] k. LC-IMS-Thermal Fragmentation-MS, which is particularly useful for compound identification via fragment libraries.

[0192] The use of any and all examples, or exemplary language (“for instance”, “such as”, “for example” and like language) provided herein, is intended merely to better illustrate the invention and does not indicate a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.

[0193] As used herein, including in the claims, unless the context indicates otherwise, singular forms of the terms herein are to be construed as including the plural form and vice versa. For instance, unless the context indicates otherwise, a singular reference herein including in the claims, such as “a” or “an” means “one or more”.

[0194] Throughout the description and claims of this specification, the words “comprise”, “including”, “having” and “contain” and variations of the words, for example “comprising” and “comprises” etc, mean “including but not limited to”, and are not intended to (and do not) exclude other components.

[0195] The present invention also covers the exact terms, features, values and ranges etc. in case these terms, features, values and ranges etc. are used in conjunction with terms such as about, around, generally, substantially, essentially, at least etc. (e.g., “about 3” shall also cover exactly 3, or “substantially constant” shall also cover exactly constant).

[0196] The term “at least one” should be understood as meaning “one or more”, and therefore includes both embodiments that include one or multiple components. Furthermore, dependent claims that refer to independent claims that describe features with “at least one” have the same meaning, both when the feature is referred to as “the” and “the at least one”.

[0197] Any steps described in this specification may be performed in any order or simultaneously unless stated or the context requires otherwise.

[0198] All of the features disclosed in this specification may be combined in any combination, except combinations where at least some of such features and/or steps are mutually exclusive. In particular, the preferred features of the invention are applicable to all aspects of the invention and may be used in any combination. Likewise, features described in non-essential combinations may be used separately (not in combination).