Patent Publication Number: US-2023152277-A1

Title: De-clustering ion guide

Description:
CROSS-REFERENCE TO RELATED APPLICATION 
     This application claims priority from and the benefit of United Kingdom patent application No. 2004961.5 filed on 3 Apr. 2020. The entire contents of this application are incorporated herein by reference. 
     FIELD OF THE INVENTION 
     The present invention relates generally to a declustering device that removes adducted species from analyte ions, and to a mass or mobility spectrometer containing such a device. 
     BACKGROUND 
     There is increasing focus on the study of relatively large molecules such as biomolecules in mass spectrometry, e.g. from areas including the biopharmaceutical interest in intact antibodies or the structural biology interest in protein complexes and membrane proteins. Analytes such as these are ionised by an ion source prior to being analysed in a mass spectrometer. However, the resulting analyte ions tend to be clustered with other species when leaving the ion source, such as when leaving an electrospray ion source. These other species are known as adducts and may comprise species such as salts or solvent molecules, for example. As the cluster of an analyte ion with an adduct species will have different physicochemical properties to the analyte ion alone (e.g. a different mass to charge ratio and/or ion mobility), it may be desired to remove the adduct species from the analyte ions prior to analysis or selection of the analyte ions. For example, it may be desired to remove the adduct molecules prior to mass filtering the analyte ions, e.g. because it may be desired to transmit analyte ions having a specific mass to charge ratio, whereas the cluster will have a different, possibly unknown, mass to charge ratio. 
     Declustering techniques are known for removing adduct species from analyte ions. For example, the ions generated by the ion source may be accelerated by the potential differences that drive the ions downstream through the instrument and such that the analyte-adduct clusters collide with background gas molecules and shed the adduct species. It is also known to radially confine the ionic clusters within an RF ion guide and to urge the clusters radially outwards towards the RF electrodes of the ion guide so as to cause RF heating of the clusters such that the analyte ions and adduct species separate. 
     However, it is desired to provide an alternative technique for declustering the adduct species from the analyte ions. 
     SUMMARY 
     The present invention provides a method of mass and/or ion mobility spectrometry comprising: providing an ion guide comprising a plurality of electrodes and having a background gas therein; applying an RF voltage to electrodes of the ion guide for radially confining ions therein; transmitting clusters of analyte ions and adduct species into the ion guide; applying, in a first mode, one or more AC voltage to the ion guide so as to oscillate the clusters such that they collide with molecules of the background gas and cause adduct species in the clusters to detach from the analyte ions, wherein the one or more AC voltage has a different amplitude and/or frequency to that of said RF voltage; and (i) varying the speed with which the clusters are urged along the ion guide during the first mode; and/or (ii) varying the amplitude and/or frequency of the one or more AC voltage as the clusters travel along the ion guide. 
     The speed at which the clusters are urged axially along the ion guide in the first mode may be varied so as to vary the transit time of the clusters through the declustering ion guide and hence vary the time that the clusters are subjected to oscillation by the AC voltage. This varies the amount of declustering performed in the first mode. Similarly, amplitude and/or frequency of the one or more AC voltage may be varied so as to vary the amount of declustering performed (on an ion of a given mass to charge ratio), and/or so as to vary (e.g. optimise) the amount of declustering for ions of different mass to charge ratios at different times. 
     Embodiments of the invention vary the conditions in which the clusters are subjected to the one or more AC voltage so as to vary or control the amount of declustering. This may be advantageous, for example, if the presence of some adduct species is relevant for analysis of the analyte. For example, it may be important to remove solvent adducts but preserve non-covalently bound drug molecules. 
     Embodiments of the invention vary the conditions in which the clusters are subjected to the one or more AC voltage so as to vary or control the amount of declustering or amount of conformation changes to the (e.g. protein) analyte ions. For example, it may be desired to provide a relatively low amount of conformational rearrangements or unfolding of the analyte ions. 
     Embodiments decouple the requirements of the RF voltage for radially confining ions from those of the AC voltage used for declustering, thereby allowing more degrees of freedom for optimisation of the radial confinement and/or declustering. 
     The amplitude and/or frequency of the one or more AC voltage may be varied with time. 
     The method may comprise transmitting ions from the ion guide into a mass filter and mass filtering the ions in the mass filter. The mass to charge ratio, or range of mass to charge ratios, that is selectively transmitted by the mass filter may be varied with time in synchronism with the variation of the amplitude and/or frequency of the one or more AC voltage with time, optionally so as to substantially only transmit ions that have been declustered in the ion guide. 
     Alternatively, the method may comprise transmitting ions from the ion guide into a mobility filter and mobility filtering the ions in the mobility filter; wherein the mobility, or range of mobilities, that is selectively transmitted by the mobility filter is varied with time in synchronism with the variation of the amplitude and/or frequency of the one or more AC voltage with time, optionally so as to substantially only transmit ions that have been declustered in the ion guide. 
     The method may comprise separating the clusters by mass to charge ratio or ion mobility prior to transmitting the clusters into the ion guide, and varying the amplitude and/or frequency of the one or more AC voltage with time based on the mass to charge ratio or ion mobility of the clusters being transmitted into the ion guide. 
     For example, the clusters may be separated by scanning/stepping a mass filter (e.g. quadrupole mass filter), mass selective ion trap or other separator device so as to transmit ions having different mass to charge ratios to the ion guide at different times. The separator may be scanned/stepped in this manner over a time period and the variation of the one or more AC voltage may be synchronised with this time period such that clusters having different mass to charge ratios experience different AC amplitudes and/or frequencies in the ion guide. Similarly, the clusters may be separated by an ion mobility separator so as to transmit ions having different mobilities to the ion guide at different times. The separator may separate the ions over a time period and the variation of the one or more AC voltage may be synchronised with this time period such that clusters having different mobilities experience different AC amplitudes and/or frequencies in the ion guide. 
     The method may comprise mass analysing ions from the ion guide in a mass analyser; wherein operation of the mass analyser is varied with time so as to vary the mass to charge ratio, or range of mass to charge ratios, that the mass analyser is capable of analysing or is optimised to analyse; and wherein this mass to charge ratio, or range of mass to charge ratios, is varied with time in synchronism with the variation of the amplitude and/or frequency of the one or more AC voltage, optionally so as to substantially only mass analyse ions that have been declustered in the ion guide 
     For example, the mass analyser may be a quadrupole mass analyser comprising a quadrupole mass filter that is scanned or stepped with time (in synchronism with the one or more AC voltage) so as to transmit different mass to charge ratios at different times to a detector. 
     Alternatively, the mass analyser may be a Time of Flight mass analyser that comprises a pusher and/or puller electrode that is repeatedly and intermittently activated so as to pulse the ions into a time of flight region to a detector. The operation of the Time of Flight mass analyser may be varied with time for mass analysing different ranges of mass to charge ratios at different times, wherein the range is varied with time in synchronism with the variation of the amplitude and/or frequency of the one or more AC voltage. 
     The Time of Flight mass analyser may be operated in an Enhanced Duty Cycle (EDC) mode. In this mode ions are pulsed towards the Time of Flight mass analyser and to the pusher and/or puller electrode, and the time at which the pusher and/or puller electrode is activated is synchronised with the time that the ions are pulsed towards the Time of Flight mass analyser such that ions having one or more predetermined mass to charge ratio are pulsed towards the detector by the pusher and/or puller electrode. The time delay between pulsing ions towards the Time of Flight mass analyser and activating the pusher and/or puller electrode may be varied for different pulses towards the Time of Flight mass analyser, e.g. so as to optimise the mass analysis of ions having different mass to charge ratios in different ones of these pulses. This time delay may be varied with time in synchronism with the variation of the amplitude and/or frequency of the one or more AC voltage. 
     It is alternatively contemplated that the mass analyser may record mass spectral data as a function time (i.e. as a function of the frequency and/or amplitude of the one or more AC voltage). The recorded data may then be post-processed to obtain mass spectral data at the desired AC voltage properties. For example, the mass spectral data may be filtered so as to only retain data that is for declustered ions. 
     The one or more AC voltage may be a plurality of different AC voltages having different amplitudes and/or frequencies, and the different AC voltages may be applied at different axial locations along the length of the ion guide. 
     The AC voltages may be applied at progressively more downstream axial locations of the ion guide have progressively lower amplitudes. 
     This is beneficial since as clusters move along the ion guide and shed adduct ions their mobility tends to increase, and so reducing the amplitude of the AC voltages along the ion guide helps prevent these ions oscillating with large amplitudes that will cause them to be lost to electrodes of the ion guide. The different AC voltages may (alternatively or additionally) have different frequencies for the same purpose. 
     The electrodes of the ion guide may define a conduit through which the clusters are guided, wherein the RF voltage applied to the electrodes radially confines the ions and urges them towards a central axis through the conduit, and wherein the AC voltage causes the clusters to oscillate about the central axis, in the first mode. 
     This enables the clusters to be oscillated with a relatively large amplitude and relatively low risk of them striking the electrodes of the ion guide and being lost to the system. This is in contrast to some conventional techniques in which ions are driven radially towards the RF electrodes so as to oscillate and heat them. 
     The AC voltage may cause the ions to oscillate about the central axis such that the clusters have substantially the same average amplitude of oscillation either side of the axis. 
     There may be substantially no gas flow through the ion guide, at least in the first mode. Additionally, or alternatively, a gas flow may be provided through the ion guide in either the downstream or upstream direction and voltages may be applied to the ion guide to oppose the force on the ions due to the gas flow, wherein the gas flow and/or voltages are selected or varied so as to slow or vary the transit time of the clusters through the ion guide. 
     The step of varying the speed with which clusters are urged along the ion guide may comprise repeatedly travelling a transient DC voltage along the ion guide so as to urge the clusters along the ion guide; and the amplitude of the transient DC voltage, and/or the speed and/or frequency with which the transient DC voltage moves along the ion guide, may be varied with time so as to vary the speed with which the clusters are urged along the ion guide in the first mode. 
     The ion guide may comprise a plurality of electrodes spaced along its longitudinal axis and each time the transient DC voltage is travelled along the ion guide, the transient DC voltage may be successively applied to different electrodes, or successively applied to different groups of multiple electrodes, along the ion guide so that the transient DC voltage moves along the ion guide. 
     The step of varying the speed with which clusters are urged along the ion guide may comprise generating an axial electric field along the ion guide by simultaneously applying different DC voltages to different electrodes of the ion guide, and varying the different voltages so as to vary the magnitude of the electric field and hence vary the speed with which the clusters are urged along the ion guide in the first mode. 
     Alternatively, or additionally, the step of varying the speed with which clusters are urged along the ion guide may comprise providing a gas flow through the ion guide so as to urge clusters along the ion guide and varying the speed of the gas flow. 
     During the first mode, the background gas may be maintained at a pressure between 0.01 and 10 millibar. 
     The method may comprise varying the pressure and/or composition of the background gas over time, e.g. during the first mode. 
     The background gas may be maintained at a first pressure, or be varied within a first range of pressures, during the first mode, whereas the background gas may be maintained at a pressure that is lower than the first pressure, or first range of pressures, during a second mode in which the AC voltage is not applied to the ion guide. 
     The method may comprise operating the ion guide in a second mode in which said one or more AC voltage is not applied to the ion guide. 
     The RF voltage desirably does not cause the clusters to oscillate to the extent that the analyte ions detach from the adduct species in the clusters. 
     In this second mode, the clusters may substantially not be declustered. In this mode the speed with which the clusters are urged along the ion guide may, or may not, be varied. 
     In the second mode the ion guide may operate in a higher transmission mode than the first mode, i.e. a greater proportion of the analyte ions may be transmitted in the second mode. 
     The method may comprise switching between the first and second modes whilst said clusters are passing through the ion guide. 
     The method may comprises repeatedly switching between the first and second modes whilst said clusters are passing through the ion guide. 
     The method may comprise ionising an analyte solution so as to produce said clusters, wherein the analyte solution comprises a membrane protein dissolved in a solvent using a detergent, and wherein the analyte ion in the cluster is a membrane protein ion and the adduct species in the cluster is a detergent molecule. 
     The method subsequently comprises said step of transmitting clusters of analyte ions and adduct species into the ion guide. 
     The method may comprise a step of forming the analyte solution by dissolving the membrane protein in the solvent using the detergent. 
     By way of example only, the membrane protein and/or solvent and/or detergent may any of those used in Lengqvist et al. JBC, Vol. 279, No. 14, Issue of April 2, pp. 13311-13316, 2004. For example, the membrane protein may be Microsomal glutathione transferase-1 (MGST1). The detergent may be Triton X-100. The solvent may be an aqueous solution of ammonium acetate. 
     It is alternatively contemplated that the membrane protein and/or solvent and/or detergent may any of those used in Barrera et al. Science, 321, 243, 2008. For example, the membrane protein may be Heteromeric (ATP)—binding cassette transporter complex (BtuC 2 D 2 ) and/or the detergent may be n-dodecyl-β-D-maltoside (DDM). 
     It is alternatively contemplated that the membrane protein and/or solvent and/or detergent may be any of those used in US 2015/0346214. For example, the membrane protein may be one of: ammonium channel C-terminally fused to green fluorescent protein (AmtB-GFP), aquaporin Z membrane protein complex (AQPZ), ammonium channel membrane protein complex (AmtB), mechanosensitive channel of large conductance (MscL), acriflavine resistance protein B (AcrB), G protein-coupled receptor (GPCR), multidrug transporter protein (EmrE), integral membrane protein (LmrP), multidrug resistance protein (MexB), inner membrane protein (MacB), transmembrane P-glycoprotein 1 (P-gp), lipid A export ATP-binding/permease protein (MsbA), probable multidrug resistance protein (NorM), or inward rectifier potassium channel (Kirbac3.1). The detergent may be n-decyl-β-D-maltoside (DM), n-undecyl-β-D-maltoside (UDM), n-dodecyl-β-D-thiomaltopyranoside (DDTM), Cymal-5, Cymal-6, octyl glucose neopentyl glycol (OGNG), n-octyl-β-D-glucopyranoside (OG), tetraethylene glycol monooctyl ether (C8E4), pentaethylene glycol monooctyl ether (C8E5), octaethylene glycol monododecyl ether (C12E8), or anapoe-58 (Brij-58, C16E20). The solvent system may be an aqueous solution of: ammonium acetate; ammonium bicarbonate; sodium chloride, TRIS and beta-mercaptoethanol; sodium chloride, glycerol, TRIS, and beta-mercaptoethanol; sodium chloride, imidazole, TRIS, and beta-mercaptoethanol; or sodium chloride, imidazole, glycerol, TRIS, and beta-mercaptoethanol. 
     Although the clusters have been described as being clusters of membrane protein ions and detergent, it is contemplated that the analyte ions and/or adduct species may be other types of species. For example, the adduct species in the clusters may be salts or molecules of the solvent. 
     The method may comprise mass analysing and/or ion mobility analysing the analyte ions and any remaining clusters downstream of the ion guide so as to obtain mass and/or mobility peaks, respectively, of the analyte ions and remaining clusters. 
     The method may comprise determining the width and/or signal-to-noise ratio of one or more of the peaks and varying the frequency and/or amplitude of the one or more AC voltage during the first mode so as to alter the width and/or signal-to-noise ratio of peaks for subsequently analysed analyte ions and clusters. 
     The width of the peak may be the FWHM width. 
     The method may be performed on a mass or mobility spectrometer comprising: a first vacuum chamber having an inlet aperture; a second vacuum chamber adjacent the first vacuum chamber; and a differential pumping aperture separating the first and second vacuum chambers; wherein said ion guide is arranged in the first vacuum chamber. 
     The first vacuum chamber may comprise an ion guiding device having a first portion that guides ions along a first axial path, a second portion that guides ions along a second different axial path, and a transition portion that urges ions from the first axial path onto the second axial path. 
     The first axial path may be substantially parallel to, but radially displaced from, the second axial path. This ion guiding device may be arranged so as to guide ions from the inlet aperture to and through the differential pumping aperture. 
     Said ion guide may be part of, or downstream of, said ion guiding device. 
     For example, the ion guide may form part, or all, of the second portion of the ion guiding device. 
     The first vacuum chamber comprises a gas pumping port for evacuating the first vacuum chamber of gas, and at least part of the second portion of the ion guide may be shielded from the gas pumping port by a barrier. Alternatively, or additionally, the ion guiding device may be arranged such that a central axis of the first axial path is coaxial with a central axis said gas pumping port. 
     A mass and/or ion mobility analyser may be arranged in the second vacuum chamber or in a further vacuum chamber downstream of the second vacuum chamber. 
     The mass analyser may be a Time of Flight mass analyser. 
     The present invention also provides a mass or mobility spectrometer configured to perform any of the methods described herein. 
     Accordingly, the present invention provides a mass or mobility spectrometer comprising: an ion guide comprising a plurality of electrodes and a background gas therein; an RF voltage supply for applying an RF voltage to electrodes of the ion guide for radially confining ions therein; one or more AC voltage supply for applying, in a first mode, one or more AC voltage to the ion guide so as to oscillate clusters of analyte ions and adduct species such that they collide with molecules of the background gas and cause adduct species in the clusters to detach from the analyte ions, wherein the AC voltage has a different amplitude and/or frequency to that of said RF voltage; and control circuitry configured to control the spectrometer so as to: (i) vary the speed with which the clusters are urged along the ion guide during the first mode; and/or (ii) vary the amplitude and/or frequency of the one or more AC voltage as the clusters travel along the ion guide. 
     The spectrometer may comprise a DC voltage supply connected to electrodes of the ion guide, wherein the control circuitry controls the DC voltage supply to successively apply a DC voltage to different ones of the electrodes so as to repeatedly travel a transient DC voltage along the ion guide; wherein the amplitude of the transient DC voltage, and/or the speed and/or frequency with which the transient DC voltage moves along the ion guide, is varied with time for varying the speed with which the clusters are urged along the ion guide. 
     The spectrometer may comprise a DC voltage supply connected to electrodes of the ion guide, wherein the control circuitry controls the DC voltage supply to simultaneously apply different DC voltages to different ones of the electrodes for generating an axial electric field along the ion guide, and vary the different voltages with time so as to vary the magnitude of the electric field for varying the speed with which the clusters are urged along the ion guide. 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
       Various embodiments will now be described, by way of example only, and with reference to the accompanying drawings in which: 
         FIGS.  1 A and  1 B  show schematics of declustering devices according to embodiments of the present invention; 
         FIG.  2    schematically illustrates the upstream end of a mass spectrometer, including the ion source; 
         FIGS.  3 A- 3 C  show views of an ion guide that may be used in embodiments of the present invention; 
         FIG.  4 A  shows a schematic side view of the of the ion guide shown in  FIGS.  3 A- 3 C , and  FIG.  4 B  shows a schematic of a declustering device arranged within the ion guide, according to an embodiment; 
         FIGS.  5 A- 5 C  show mass spectral data according to both a known declustering technique and a declustering technique according to an embodiment of the present invention; 
         FIGS.  6 A- 6 C  also show mass spectral data according to both a known declustering technique and a declustering technique according to an embodiment of the present invention; 
         FIGS.  7 A- 7 D  show mass spectral data from a declustering technique according to an embodiment of the present invention, wherein the declustering AC voltage has different frequencies; 
         FIGS.  7 A- 7 D  show mass spectral data from a declustering technique according to an embodiment of the present invention, wherein the declustering AC voltage has different frequencies; 
         FIGS.  8 A- 8 E  show mass spectral data from a declustering technique according to an embodiment of the present invention, wherein the declustering AC voltage has a first frequency and different amplitudes; 
         FIGS.  9 A- 9 E  show mass spectral data from a declustering technique according to an embodiment of the present invention, wherein the declustering AC voltage has a second frequency and different amplitudes; 
         FIG.  10    shows plots of how the FWHM of a m/z peak varies as a function of the declustering AC voltage amplitude, for declustering AC voltages having four different frequencies; 
         FIG.  11    shows plots of how the peak area of a certain mass range varies as a function of the declustering AC voltage amplitude, for declustering AC voltages having different frequencies; and 
         FIGS.  12 A- 12 D  show mass spectral data according to an embodiment in which a mass filter is scanned in synchronism with the amplitude of the declustering AC voltage. 
     
    
    
     DETAILED DESCRIPTION 
     Embodiments of the present invention provide sustained activation of ion species, such as relatively large analyte ion species, in order to remove adduct species prior to selection and/or analysis of the analyte ions, e.g. by mass filtering and/or mass analysing the analyte ions. 
       FIGS.  1 A and  1 B  show schematics of declustering devices according to embodiments of the present invention. 
     Referring to  FIG.  1 A , the declustering device comprises two planar upper and lower electrodes  1  that are spaced apart from each other in a y-dimension and have major faces that face each other. In use, a voltage supply  9  applies DC voltages to these electrodes so as to generate an electric field that confines ions between the electrodes  1 . Alternatively, the voltage supply  9  may supply RF voltages to these electrodes  1  so as to confine the ions therebetween. The planar electrodes  1  are shown as being parallel to each other, although it is contemplated that they may be angled to each other, e.g. such that when DC and/or AC voltages are applied to these electrodes ions are driven through the device between the planar electrodes  1 . The declustering device also comprises side electrodes  3  that are spaced apart from each other in an x-dimension and are arranged such that the side electrodes  3 , together with the upper and lower electrodes  1 , define a conduit therebetween through which ions  5  can be guided. In the embodiment depicted in  FIG.  1 A , each side wall of the declustering device comprises multiple electrodes  3  that are each elongated in the z-dimension, i.e. in the direction that ions  5  travel through the device, and which are spaced apart along the direction between the upper and lower electrodes  1  (i.e. in the x-dimension). These electrodes  3  may be planar and may have major faces that face towards the upper and lower electrodes  1 . These electrodes  3  may be arranged parallel to each other. In use, a voltage supply  11  may apply RF voltages to these side electrodes  3  so as to generate an AC electric field and pseudo-potential barrier that radially confines ions between the electrodes  3  (in the x-dimension). For example, opposing phases of an RF voltage may be applied to electrodes  3  that are adjacent each other within each side wall. Side electrodes  3  that are adjacent and oppose each other in the x-dimension may be maintained at the same RF phase. 
     Although each side wall has been described as comprising multiple RF electrodes  3 , it is contemplated that it may instead comprise only a single RF electrode. Less preferably, rather than applying RF voltages to the electrode(s)  3  in each side wall it is contemplated that a DC voltage may be applied to the electrode(s)  3  in each side wall so as to confine ions in the x-dimension. 
     As described above, in use, the RF and/or DC voltages applied to the upper, lower and side electrodes are such that ionic clusters  5  entering the ion transmission conduit along an axis arranged in the z-dimension are radially confined relative to that axis (i.e. 
     radially confined in the x- and y-dimensions). 
     In a first, declustering mode, one or both of voltage supplies  9 , 11  apply an AC voltage to one or more of the electrodes  1 , 3  so as to cause the clusters of analyte ions and adduct species to oscillate in the radial direction. For example, the AC voltage may be applied between the upper and lower electrodes  1  so as to cause ionic clusters to oscillate in the y-dimension. A background gas is present in the ion transmission conduit and as such, the oscillation of the ionic clusters causes the clusters to collide with the molecules of the background gas, causing the clusters to increase their internal energy and decluster, i.e. the adduct species detach from the analyte ions. It is contemplated that the background gas pressure may be maintained at sub-atmospheric pressure or atmospheric pressure during this process. The energy input to the cluster is related to its size, the strength of the electric field generated by the AC voltage and the gas pressure. 
     The amplitude and frequency of the AC voltage are selected such that at least some of the analyte ions remain radially confined by the electrodes  1 , 3  and desirably such that the analyte ions are not fragmented by collisionally induced dissociation (CID). For example, the AC voltage may be applied around the DC offset potential of the RF voltage used to radially confine the ions. The ensures that the ion excursions due to the declustering AC voltage are symmetrical about the central axis, rather than extending relatively far from the axis and striking an electrode  1 , 3 . The frequency of the declustering AC voltage may also be selected such that the excursion of the ions of interest from the central axis is such that the ions are not lost to the electrodes  1 , 3 . The most efficient form of the declustering AC voltage is a square wave, since ions experience the de-clustering field essentially all of the time. After declustering, the analyte ions may then be onwardly transmitted in a downstream direction through the conduit for further analysis. 
     The clusters can be driven through the declustering device in order to control and vary the amount of declustering. This may be achieved by urging the clusters through the declustering device and varying the force that is applied to the clusters to achieve this so as to vary the speed of the clusters through the device. For example, if the clusters are urged through the declustering device relatively slowly then, in the first declustering mode of operation, they will be subjected to the oscillations caused by the declustering AC voltage for a relatively long period of time, thus causing a relatively large amount of declustering (e.g. a relatively large proportion of the analyte will be declustered and/or a relatively large number of adducts will be declustered from each cluster). In contrast, if the clusters are urged through the declustering device more quickly then, in the first declustering mode of operation, they will be subjected to the oscillations for a shorter period of time, thus causing a smaller amount of declustering (e.g. a smaller proportion of the analyte will be declustered and/or a smaller number of adducts will be declustered from each cluster). 
     The clusters may be urged through the declustering device by one or more of several means, including one or more of the following. A transient DC voltage may be repeatedly travelled along the declustering device so as to urge the clusters therethrough. The transient DC voltage is successively applied to electrodes that are axially spaced along the declustering device so as to generate a DC potential barrier that moves along the declustering device, thereby causing the ions that are radially confined within the declustering device to be urged through it. These axially spaced electrodes are not shown in  FIG.  1 A , but electrodes  3  in  FIG.  1 B  may be used for this purpose. The step of travelling the transient DC voltage along the declustering device may comprise controlling a voltage supply  13  to successively applying the transient DC voltage to each and every one of the axially spaced electrodes  3 . Alternatively, the transient DC voltage may be successively applied only to every nth electrode that is downstream of the electrode that the transient DC voltage was last applied to, where n is an integer greater than 1 (e.g. only applied to alternate axially spaced electrodes). It is also contemplated that the transient DC voltage may be simultaneously applied to a group of multiple electrodes at any given time, and the transient DC voltage may be sequentially applied to different groups of multiple electrodes at different respective times such that the transient DC voltage moves along the declustering device. Each group of electrodes may consist of axially consecutive electrodes of the declustering device. The properties of the transient DC voltage, such as amplitude and/or speed and/or frequency of travel may be varied so as to vary the speed with which the clusters are urged through the declustering device. 
     As an alternative to the transient DC voltages, or in addition thereto, a static electric field may be generated by the voltage supply  13  applying a potential difference across the axial length of the declustering device so as to urge the clusters through it. The magnitude of the electric field may be varied with time so as to vary the speed with which the clusters are urged through the declustering device. 
     In addition, or as an alternative, to the other techniques described herein for varying the amount of declustering, a gas flow may be used to urge the clusters through the declustering device and the gas flow speed may be varied with time so as to vary the speed with which the clusters are urged through the declustering device. 
     In addition, or as an alternative, to the other techniques described herein for varying the amount of declustering, it is contemplated that the clusters may be axially trapped within the declustering device, e.g. by applying voltages to the upstream and downstream portions of the declustering device, and the amount of declustering may be varied by varying the time that the clusters are trapped for before being released from the declustering device. 
     In addition, or as an alternative, to the other techniques described herein for varying the amount of declustering, the composition or pressure of the background gas within the declustering device may be varied so as to vary the amount of declustering. 
     The declustering device may be operated in a second, different mode in which the declustering AC voltage is not applied such that the clusters are not de-clustered. For example, in this mode only DC voltages (and no AC/RF voltages) may be applied to the upper and lower electrodes  1  so as to confine the ions in the y-dimension. The DC voltages (i.e. potentials) applied to upper and lower electrodes  1  may be higher than the DC bias of the side electrodes  3  (about which the RF voltages are applied to confine ions in the x-direction). However, it is contemplated that in less preferred embodiments RF voltages may be applied to upper and lower electrodes  1  in addition to, or instead of, the DC voltages so as to confine ions in the y-dimension (but not so as to cause declustering). The clusters may be radially confined by the electrodes  1 , 3  and guided through the conduit. In the second mode, ions  5  enter the declustering device in the z-direction. 
     The declustering device may be switched between the two modes whilst clusters are flowing through the conduit by switching the declustering AC voltage on or off. 
       FIG.  1 B  shows another embodiment of the declustering device that is the same as that described above in relation to  FIG.  1 A , except that the arrangements of the side electrodes  3  differ. In the embodiment depicted in  FIG.  1 B , each side wall of the declustering device comprises multiple electrodes  3  that are each elongated in the y-dimension, i.e. in the direction between the upper and lower electrodes  1 . These electrodes  3  may be planar and may have major faces that are substantially orthogonal to the major faces of the upper and lower electrodes  1 . The electrodes  3  in each side wall may be spaced apart from each other in a direction along the z-dimension. The electrodes  3  in each side wall may be arranged parallel to each other. In use, RF voltages are applied to these side electrodes  3  so as to generate an AC electric field and pseudo-potential barrier that radially confines ions between the side walls (in the x-dimension). For example, opposing phases of an RF voltage may be applied to electrodes that are adjacent each other within each side wall. Side electrodes  3  that are adjacent and oppose each other in the x-dimension may be maintained at the same RF phase. Less preferably, rather than applying RF voltages to the electrodes in each side wall it is contemplated that a DC voltage may be applied to the electrodes  3  in each side wall so as to radially confine ions in the x-dimension. 
     As has been described above, various techniques may be used for varying the amount of declustering, such as varying properties of a transient DC voltage that is repeatedly travelled along the declustering device or varying the magnitude of a static electric field across the axial length of the declustering device. It will be appreciated that the transient DC voltage may be travelled along the declustering device by applying the transient DC voltage to different ones of the electrodes  3  in  FIG.  1 B  at different times. Similarly, the static electric field may be arranged by simultaneously applying different DC voltages to different ones of the electrodes  3 . 
     Although the upper and lower electrodes  1  have been described as being parallel to each other in the above embodiments, it is contemplated that they may be angled to each other, e.g. such that when DC and/or AC voltages are applied to these electrodes ions are driven through the device between the planar electrodes. Alternatively, or additionally, although each of the upper and lower electrodes  1  have been described as being single electrodes, it is contemplated that one or each of these may be replaced with an array of multiple RF electrodes (or DC electrodes), e.g. so as to form the upper and/or lower wall from multiple electrodes in a corresponding manner to that in which one of the side walls  3  is formed. 
     For the avoidance of doubt, although electrodes  1  have been described as upper and lower electrodes, the y-dimension may be arranged in any orientation and not necessarily vertically. 
     In mass spectrometry, analyte ions are often generated by relatively high pressure ion sources, e.g. by atmospheric pressure ion sources. It is then necessary to transmit these ions into a vacuum region of the mass spectrometer, since the processing or analysis of the ions is required to be performed at relatively low vacuum pressures. 
       FIG.  2    schematically illustrates a known arrangement comprising an electrospray ionisation (ESI) probe  2  arranged in an atmospheric pressure region  4 , a low pressure vacuum chamber  6  of a mass spectrometer, and an intermediate pressure chamber  8  arranged between the atmospheric pressure region  4  and the vacuum chamber  6  of the mass spectrometer. A cone  10  is arranged between the atmospheric pressure region  4  and the intermediate pressure chamber  8  so that the intermediate pressure chamber  8  is able to be maintained at a lower pressure than the atmospheric pressure region  4 , and a differential pumping aperture  12  is arranged between the vacuum chamber  6  and the intermediate pressure chamber  8  so that the vacuum chamber is able to be maintained at a lower pressure than the intermediate pressure chamber. An ion guide  14 , such as an ion tunnel or multipole ion guide, is arranged in the intermediate pressure chamber  8  for guiding ions received through the cone  10  towards and through the differential pumping aperture  12 . 
     In operation, the intermediate pressure chamber  8  is pumped to a lower pressure than the atmospheric pressure region  4 , and the vacuum chamber  6  is pumped to a lower pressure than the intermediate pressure chamber  8 . Analyte solution is then delivered to the capillary  16  of the ESI probe  2  and is sprayed from the tip thereof so as to produce analyte ions  18  in the atmospheric pressure region  4 . The analyte ions  18  then pass through the cone  10  and into the ion guide  14  in the intermediate pressure chamber  8 . The ion guide  14  guides the ions through the intermediate pressure chamber and through the differential pumping aperture  12  into the vacuum chamber  6 . The ions may then be fragmented in the vacuum chamber  6 , or in a further downstream vacuum chamber of the spectrometer which may be pumped to an even lower pressure. In conventional declustering techniques, the voltage applied to the cone  10  may be selected so as to accelerate the clusters through the background gas in atmospheric pressure region  4  at a speed such that they decluster. 
     According to an embodiment of the present invention, the declustering device may be arranged downstream of the ESI probe  2  so as to receive the clusters. For example, the declustering device may replace the ion guide  14 . Alternatively, the declustering device may be arranged within, upstream or downstream of the ion guide  14  so as to decluster the analyte ions before they pass through differential pumping aperture  12  into the vacuum chamber  6 . 
       FIG.  3 A  shows part of another embodiment of the present invention that is similar to that shown in  FIG.  2   , except that the ion guide  14  has been replaced by another type of ion guide that guides ions along a first axial path and then onto and along a second axial path that is displaced from the first axial path. Voltages may be applied to the ion guide so as to urge ions along the ion guide in the downstream direction. 
     In the embodiment of  FIG.  3 A , the cone  20  separates the relatively high pressure region  22  (such as an atmospheric pressure region) from the first vacuum chamber  24 . An electrospray ionisation (ESI) probe, or other source of ions, may be arranged in high pressure region  22 . A differential pumping aperture  26  is arranged between the first vacuum chamber  24  and a second vacuum chamber  28  so that the second vacuum chamber  28  is able to be maintained at a lower pressure than the first vacuum chamber  24 . The ion guide is arranged in the first vacuum chamber  24  for guiding ions received through the cone  20  towards and through the differential pumping aperture  26 , as will be described in more detail below. A mass analyser  29 , such as an orthogonal acceleration Time of Flight mass analyser, may be arranged in the second vacuum chamber  28  for analysing ions transmitted through the differential pumping aperture  26 . 
     The ion guide comprises a first portion  30  for guiding ions along a first axial path, a second portion  32  for guiding ions along a second axial path (which may be parallel to and displaced the first axial path), and a transition portion  33  for transferring ions from the first axial path to the second axial path. In the depicted embodiment, each of the first and second ion guide portions  20 , 32  comprises a plurality of axially separated apertured electrodes (e.g. ring electrodes) for radially confining the ions along their respective axial paths. RF voltages are applied to these electrodes so as to radially confine the ions. For example, different (e.g. opposite) phases of an RF voltage supply may be applied to adjacent apertured electrodes in the known manner so as to radially confine the ions. 
       FIG.  3 B  shows three cross-sectional views of the electrode arrangement in the ion guide at different axial points along the ion guide. View  30  shows the electrode arrangement proximate the cone  20 , where the ions are confined in the first portion  30  of the ion guide to the first axial path by the apertured electrodes  34 . View  32  shows the electrode arrangement proximate the differential pumping aperture  26 , where the ions are confined in the second portion of the ion guide  32  to the second axial path by the apertured electrodes  35 . View  33  shows the electrode arrangement in the transition region  33  of the ion guide, in which the ions are transferred from the first axial path of the first ion guide portion  30  to the second axial path of the second ion guide portion  32 . This transfer may be achieved by: providing one or more electrodes  36  in the transition region, each of which only partially encircles the first axial path and has a radial opening in its side that is directed towards the second axial path (e.g. an arc-shaped electrode); providing one or more electrodes  37  in the transition region, each of which only partially encircles the second axial path and has a radial opening in its side that is directed towards the first axial path (e.g. an arc-shaped electrode); and urging ion from the first axial path, through the radial openings in the electrodes, and onto the second axial path. This urging of the ions may be performed by providing an electrical potential difference, e.g. by applying voltages to the electrodes in the transition region so as to provide a potential difference in the radial direction. 
       FIG.  3 C  shows a perspective view of the electrode arrangement in the transition region. 
     Referring back to  FIG.  3 A , the first ion guide portion  30  may be arranged in the first vacuum chamber  24  such that the aperture of the cone  20  is aligned (e.g. coaxial) with the first axial path defined by the first ion guide portion  30 . The second ion guide portion  32  may be arranged in the first vacuum chamber  24  such that the aperture in the differential pumping aperture  26  is aligned (e.g. coaxial) with the second axial path defined by the second ion guide portion  32 . 
     A vacuum pump is provided for evacuating the first vacuum chamber  24  through a gas pumping port  38 . The opening of the gas pumping port  38  may be aligned (e.g. coaxial) with the first axial path of the first ion guide portion  30 . The end of the ion guide formed by the second portion  32  may be physically shielded from the gas pumping port  38  by a barrier  40 . 
     In operation, ions are generated in high pressure region  22 . The pressure differential between the high pressure region  22  and the first vacuum chamber  24  causes gas and ions to pass through the cone  20  and into the first vacuum chamber  24 , whereby the gas and ions tend to expand into the lower pressure region. The ions enter into the first portion  30  of the ion guide and are radially confined thereby, but may be relatively diffuse, as shown by ion cloud  42 . The ions are driven axially along the first portion  32  of the ion guide, at least partially by the gas flow towards the gas pumping port  38 . When ions reach the transition portion  33  of the ion guide, they are urged in the radial direction and onto the second axial path defined by the second portion  32  of the ion guide, as shown by ion trajectories  43 . As described above, this may be caused by applying a potential difference in the radial direction. As a result, ions are caused to migrate from the first ion guide portion  30  to the second ion guide portion  32 . In contrast, the majority of the gas flow continues substantially along the axis defined by the first ion guide portion  30  towards and through the gas pumping port  38 , as shown by arrow  44 . Ions are therefore radially confined in the second ion guide portion  32  and travel along the second axial path towards the differential pumping aperture  26 , whereas the majority of the gas is routed in a different direction towards the gas pumping port  38 . At least part of the second portion  32  of the ion guide may be shielded from the pumping port by a barrier  40 , so that the gas flow towards the pumping port  38  is directed away from the second axial path of the second ion guide portion  32 . 
     The second ion guide portion  32  may have a smaller radial cross-section than the first portion  30  so that the ions are radially compressed in the second portion as compared to the first portion, as shown by ion beam  46 . Ions are then guided by the second ion guide portion  32  through the differential pumping aperture  26  and into the second vacuum chamber  28 . 
     The clusters and other ions may be urged along the ion guide by a static DC electric field. For example, a DC voltage gradient may be arranged between a point in the first vacuum chamber  24  towards the cone  20  and a point towards the differential pumping aperture  26 , e.g. by applying different DC voltages to electrodes of the ion guide. The DC voltage gradient may be arranged along the first and/or second axis of the ion guide (and/or the transition region  33 ), e.g. by applying different voltages to electrodes of the ion guide at different axial locations. Alternatively, or additionally, clusters and other ions may be urged along the ion guide by repeatedly travelling one or more DC potential barrier along the first and/or second ion guide portions  30 , 32 . This may be performed by successively applying one or more transient DC voltage to electrodes along the ion guide. The one or more DC potential barrier may be repeatedly travelled along the ion guide. 
     As will be described below, the declustering device may be located upstream, downstream or within the ion guide. It has been recognised that the ion guide in the above-described arrangement is able to handle relatively high gas loads (e.g. since the ion guide initially conveys the ions with the gas flow towards the pumping port and then moves the ions out of the gas flow), and that the ion guide therefore enables the first vacuum chamber  24  to be operated at relatively high pressures. This is advantageous as the relatively high pressure provides a suitable background gas in which the declustering device can oscillate the clusters so as to cause them to decluster. 
     As described above, the declustering device may be located upstream, downstream or within the ion guide.  FIGS.  4 A and  4 B  schematically illustrate the position of the declustering device within the ion guide in one embodiment. 
       FIG.  4 A  shows a schematic side view of the of the ion guide shown and described above in relation to  FIGS.  3 A- 3 C . The declustering device may be located within or downstream of the ion guide. For example, the declustering device may be located downstream of transition region  33  of the ion guide, in portion  32  of the ion guide (shown in  FIG.  3 A ). This location is illustrated by the circled portion in  FIG.  4 A . 
       FIG.  4 B  shows a schematic, perspective view of the circled portion of  FIG.  4 A . This shows the declustering device arranged within portion  32  the ion guide. The declustering device shown is the same as that of the embodiment in  FIG.  1 B , although other embodiments of the declustering device may be used. Printed circuit boards  35  may be arranged on opposing sides of the ion guide and declustering device and connected to the electrodes  1 , 3 , 34 - 37  of these components for applying the various voltages to the electrodes. 
       FIGS.  5 A- 5 C  show mass spectral data for the analysis of alcohol dehydrogenase protein complex dissolved to 10 uM in 200 mM ammonium acetate and infused at 5 μL/min via a standard flow electrospray ionisation source on a Q-ToF mass spectrometer. No desalting procedure was used prior to the infusion. 
       FIG.  5 A  shows mass spectral data obtained using a Synapt G2-Si mass spectrometer, in which a relatively low voltage of 50V was applied to the sampling cone. This caused the ions to be accelerated through the device at a relatively low rate such that relatively low energy collisions occurred between the clusters and background gas. As can be seen from  FIG.  5 A , the observed multiply charged peaks are relatively broad due to the ion clusters.  FIG.  5 B  shows mass spectral data obtained using a Synapt G2-Si mass spectrometer, in which a relatively high voltage of 200V was applied to the sampling cone. This caused the ions to be accelerated through the device at a relatively high rate such that relatively high energy collisions occurred between the clusters and background gas, causing declustering of the analyte ions and adduct species. As can be seen from  FIG.  5 B , the observed mass spectral peaks are relatively narrow and the signal-to-noise ratio has improved, as compared to  FIG.  5 A . 
       FIG.  5 C  shows mass spectral data obtained according to an embodiment of the present invention, in which the relatively low cone voltage of  FIG.  5 A  was used, but a declustering device as described herein was provided. The declustering AC voltage applied to the device had a square waveform, a frequency of 45 kHz and a peak-to-peak voltage of 280V. As can be seen from  FIG.  5 C , even using a relatively low cone voltage, but with the declustering AC voltage applied, the mass spectral peaks observed have a relatively high definition and signal-to-noise ratio, e.g. as compared to  FIG.  5 B . 
     Further experimental data was obtained by mass analysing alcohol dehydrogenase protein complex dissolved to 10 uM in 200 mM ammonium acetate and infused at 5 μL/min via a standard flow electrospray ionisation source. No desalting procedure was used prior to the infusion. 
       FIGS.  6 A- 6 C  show plots of mass spectral data obtained using the known cone voltage declustering technique and obtained using an embodiment of the invention.  FIG.  6 A  shows mass spectral data obtained when a relatively low voltage of 50V was applied to the sampling cone. This caused the ions to be accelerated through the device at a relatively low rate such that relatively low energy collisions occurred between the clusters and background gas. As can be seen from  FIG.  6 A , the m/z peaks are relatively broad, e.g. the peak closest to m/z=5466 has a FWHM of 35 m/z.  FIG.  6 B  shows mass spectral data obtained when a relatively high voltage of 200V was applied to the sampling cone. This caused the ions to be accelerated through the device at a relatively high rate such that relatively high energy collisions occurred between the clusters and background gas, causing declustering of the analyte ions and adduct species. As can be seen from  FIG.  6 B , the observed mass spectral peaks are narrower (e.g. the peak closest to m/z=5466 has a FWHM of 18 m/z) and the signal-to-noise ratio has improved.  FIG.  6 C  shows mass spectral data obtained according to an embodiment of the present invention, in which the relatively low cone voltage of 50V was used, but a declustering device as described herein was provided. The declustering AC voltage applied to the device had a substantially sinusoidal waveform, a frequency of 60 kHz and a peak-to-peak voltage of 280V. As can be seen from  FIG.  6 C , even using a relatively low cone voltage, but with the declustering AC voltage applied, the m/z peaks observed have a high definition (e.g. the peak closest to m/z=5466 has a FWHM of 16 m/z) and signal-to-noise ratio. 
       FIGS.  7 A- 7 D  show plots of mass spectral data corresponding to that obtained as described in relation to the embodiment in  FIG.  6 C , except wherein  FIG.  7 A  shows spectral data obtained using a declustering AC voltage having a frequency of 55 kHz,  FIG.  7 B  shows spectral data obtained using a declustering AC voltage having a frequency of 60 kHz,  FIG.  7 C  shows spectral data obtained using a declustering AC voltage having a frequency of 65 kHz, and  FIG.  7 D  shows spectral data obtained using a declustering AC voltage having a frequency of 70 kHz. It can be seen from  FIGS.  7 A- 7 D  that the width of the peaks observed become smaller as the frequency of the declustering AC voltage is reduced. For example, the peak closest to m/z=5466 has a FWHM of 15 m/z in  FIG.  7 A , 16 m/z in  FIG.  7 B , 18 m/z in  FIG.  7 C , and 23 m/z in  FIG.  7 D . 
       FIGS.  8 A- 8 E  show plots of mass spectral data obtained as described in relation to the embodiment in  FIG.  6 C , except wherein  FIGS.  8 A- 8 E  show spectral data obtained using a declustering AC voltage having a frequency of 55 kHz, and wherein the peak-to-peak amplitude of the declustering AC voltage is different for each of the different plots of  FIGS.  8 A- 8 E .  FIG.  8 A  shows a plot obtained when the declustering AC voltage had a peak-to-peak amplitude of 280V,  FIG.  8 B  shows a plot obtained when the declustering AC voltage had a peak-to-peak amplitude of 260V,  FIG.  8 C  shows a plot obtained when the declustering AC voltage had a peak-to-peak amplitude of 175V,  FIG.  8 D  shows a plot obtained when the declustering AC voltage had a peak-to-peak amplitude of 90V, and  FIG.  8 E  shows a plot obtained when the declustering AC voltage had a peak-to-peak amplitude of 0V. 
       FIGS.  9 A- 9 E  show plots of mass spectral data obtained as described in relation to the embodiment in  FIG.  6 C , except wherein  FIGS.  9 A- 9 E  show spectral data obtained using a declustering AC voltage having a frequency of 70 kHz, and wherein the peak-to-peak amplitude of the declustering AC voltage is different for each of the different plots of  FIGS.  9 A- 9 E .  FIG.  9 A  shows a plot obtained when the declustering AC voltage had a peak-to-peak amplitude of 280V,  FIG.  9 B  shows a plot obtained when the declustering AC voltage had a peak-to-peak amplitude of 260V,  FIG.  9 C  shows a plot obtained when the declustering AC voltage had a peak-to-peak amplitude of 175V,  FIG.  9 D  shows a plot obtained when the declustering AC voltage had a peak-to-peak amplitude of 90V, and  FIG.  9 E  shows a plot obtained when the declustering AC voltage had a peak-to-peak amplitude of 0V. 
     As can be seen from  FIGS.  8 A- 8 E , once the amplitude of the declustering AC voltage has been increased above a certain level the signal to noise ratio for the ions of interest (e.g. the peak nearest to m/z=5466) significantly increases, and the peak width decreases as the amplitude is further increased. The same trends can be observed in  FIGS.  9 A- 9 E . 
       FIG.  10    shows four plots of how the width (FWHM) of the peak closest to m/z=5466 varies as a function of declustering AC voltage amplitude, for four declustering AC voltages having four different frequencies. Plot A is for a declustering AC voltage having a frequency of 70 kHz, plot B is for a declustering AC voltage having a frequency of 65 kHz, plot C is for a declustering AC voltage having a frequency of 60 kHz, and plot D is for a declustering AC voltage having a frequency of 55 kHz.  FIG.  11    shows four plots of how the peak area between m/z=5440 and 5540 varies as a function of declustering AC voltage amplitude, for four declustering AC voltages having four different frequencies. Plot A is for a declustering AC voltage having a frequency of 70 kHz, plot B is for a declustering AC voltage having a frequency of 65 kHz, plot C is for a declustering AC voltage having a frequency of 60 kHz, and plot D is for a declustering AC voltage having a frequency of 55 kHz. 
     The frequency and amplitude of the declustering AC voltage determine the position of the low-m/z cut off. For maximum “declustering” the low-m/z cut off can be set just above the m/z of ion of interest. This however results in some ion losses. There is no enhancement in transmission, however spectrum quality is improved (lower FWHM). 
     It can be seen from the above-described Figures that increasing the amplitude of the declustering AC voltage generally increases the signal-to-noise-ratio and reduces the FWHM and area of the peaks. Also, the width of the peaks observed generally becomes smaller as the frequency of the declustering AC voltage is reduced. This is because the declustering AC voltage causes the device to operate as a high-pass filter to some extent. In general, ions having relatively low mass to charge ratio tend to have a relatively high mobility, and so these ions tend to be oscillated by the declustering AC voltage with a greater amplitude than ions having a higher mass to charge ratio. Accordingly, when the declustering AC voltage is set at a frequency and amplitude necessary to decluster ions having a relatively high mass to charge ratio, then this will generally cause ions having lower mass to charge ratios to oscillate with a relatively amplitude and be lost to the electrodes of the declustering device (e.g. by hitting the electrodes to which the declustering AC voltage is applied). This effect can be seen in  FIGS.  8  and  9   . For example, the group of ions having a relatively low mass to charge ratio around m/z=1000 disappear as the amplitude of the declustering AC voltage is increased. These ions hit upper and lower electrodes  1  of the declustering device and are not transmitted through the device. As the declustering AC voltage is increased further (e.g. from  FIG.  8 C to  8 B ), the group of ions having a mass to charge ratio around m/z=3500 disappear, leaving only ions of interest having a mass to charge ratio around m/z=5500. However, if the amplitude of the declustering AC voltage is increased too far then some ions of interest may be lost, such as in  FIG.  8 A  in which ions having a mass to charge ratio around m/z=5500 are lost. Similarly, varying the frequency of the declustering AC voltage alters the ions that are transmitted by the declustering device. 
     Therefore, it may be desired to vary the amplitude and/or frequency of the declustering AC voltage in order to optimise the declustering and/or transmission of ions of interest. 
     For example, the amplitude and/or frequency of the declustering AC voltage may be varied with time. In these embodiments, the clusters may be separated by mass to charge ratio or ion mobility prior to transmitting the clusters into the declustering device, and the amplitude and/or frequency of the declustering AC voltage may be varied with time based on the mass to charge ratio or ion mobility of the clusters being transmitted into the declustering device. For example, the clusters may be separated by scanning/stepping a mass filter (e.g. quadrupole mass filter), mass selective ion trap or other separator device so as to transmit ions having different mass to charge ratios to the declustering device at different times. The separator may be scanned/stepped in this manner over a time period and the variation of the declustering AC voltage may be synchronised with this time period such that clusters having different mass to charge ratios experience different AC amplitudes and/or frequencies in the declustering device. Similarly, the clusters may be separated by an ion mobility separator so as to transmit ions having different mobilities to the declustering device at different times. The separator may separate the ions over a time period and the variation of the declustering AC voltage may be synchronised with this time period such that clusters having different mobilities experience different AC amplitudes and/or frequencies in the declustering device. 
     Alternatively, or additionally, a plurality of different declustering AC voltages having different amplitudes and/or frequencies may be applied at different axial locations along the length of the declustering device. The declustering AC voltages applied at progressively more downstream axial locations of the declustering device may have progressively lower amplitudes. This is beneficial since as clusters move along the declustering device and shed adduct ions their mobility tends to increase, and so reducing the amplitude of the declustering AC voltages along the declustering device helps prevent these ions oscillating with large amplitudes that will cause them to be lost to electrodes of the declustering device. The different declustering AC voltages may (alternatively or additionally) have different frequencies for the same purpose. 
     A mass filter, such as a resolving quadrupole mass filter, may be provided downstream of the declustering device. The mass filter has a mass to charge ratio transmission window and ions having mass to charge ratios within this window are transmitted, whereas ions having mass to charge ratios outside of this window are not transmitted and are filtered out by the mass filter. The window may be scanned with time such that the mass filter is (only) capable of transmitting different mass to charge ratios at different times. The amplitude and/or frequency of the declustering AC voltage that is applied to the declustering device may be varied with time, together with the mass to charge ratio transmission window of the mass filter. For example, the amplitude and/or frequency of the declustering AC voltage may be varied in synchronism with the mass to charge ratio transmission window of the mass filter, e.g. in order to only transmit ions that have been declustered by a relatively high amount. An example of this will be described in relation to  FIGS.  12 A- 12 D . 
       FIGS.  12 A- 12 C  show schematic plots of how a mass spectrum may change as the amplitude of the declustering AC voltage is increased.  FIG.  12 A  shows a mass spectrum obtained using a relatively low amplitude declustering AC voltage on the declustering device. Ions represented by the peak of the lowest mass to charge ratio have been relatively well declustered, which is shown by the peak being relatively narrow. However, the two higher m/z peaks are relatively wide, due to relatively poor declustering of those ions.  FIG.  12 B  shows a mass spectrum obtained using a higher amplitude declustering AC voltage on the declustering device. The intensity of the lowest m/z peak has been significantly reduced, due to the increased amplitude of the declustering AC voltage having resulted in the low-mass cut-off of the declustering device being increased, due to the reasons described above (i.e. the lower m/z ions are oscillated to higher amplitudes by the increased amplitude of the declustering AC voltage and so are lost to the electrodes). The ions represented by the two higher m/z peaks have been declustered to a greater extent, which is why these two peaks are narrower than in  FIG.  12 A . It can be seen that the middle m/z peak in  FIG.  12 B  is relatively well declustered and narrow, whereas the highest m/z peak is still relatively broad and not well declustered.  FIG.  12 C  shows a mass spectrum obtained using a higher amplitude declustering AC voltage than in  FIG.  12 B . The lowest m/z peak has disappeared and the intensity of the middle m/z peak has been significantly reduced, due to the increased amplitude of the declustering AC voltage having resulted in the low-mass cut-off of the declustering device being increased. The ions represented by the highest m/z peak have been declustered to a relatively high extent, which is why this peak is narrower than in  FIGS.  12 A and  12 B . Alternatively, or additionally, the frequency of the declustering AC voltage may be varied with time so as to achieve this. 
     As described above, a mass filter may be provided downstream of the declustering device having a mass to charge ratio transmission window that is scanned in synchronism with the amplitude (and/or frequency) of the declustering AC voltage that is applied to the declustering device. The mass filter may be scanned in synchronism with the declustering AC voltage so that substantially only declustered ions are transmitted downstream by the mass filter. The upper and lower limits of the mass to charge ratio transmission window are shown as vertical dashed lines in  FIGS.  12 A- 12 C . As can be seen from  FIG.  12 A , at a first time when the declustering AC voltage has a first (low) amplitude, the mass to charge ratio transmission window of the mass filter is set so as to transmit ions having a first (low) range of mass to charge ratios. As such, only the highly declustered ions represented by the lowest m/z peak in  FIG.  12 A  are transmitted. As can be seen from  FIG.  12 B , at a second time when the declustering AC voltage has a higher (medium) amplitude, the mass to charge ratio transmission window of the mass filter is set so as to transmit ions having a higher (medium) range of mass to charge ratios. As such, only the highly declustered ions represented by the middle m/z peak in  FIG.  12 B  are transmitted. As can be seen from  FIG.  12 C , at a third time when the declustering AC voltage has a higher (highest) amplitude, the mass to charge ratio transmission window of the mass filter is set so as to transmit ions having a higher (highest) range of mass to charge ratios. As such, only the highly declustered ions represented by the highest m/z peak in  FIG.  12 C  are transmitted. 
       FIG.  12 D  shows the combined mass spectrum obtained by scanning the mass filter in this manner. It can be seen by comparing  FIG.  12 D  with  FIG.  12 A  that this significantly reduces the widths of the m/z peaks and provides higher resolution mass spectral data. 
     Removing adduct ions removes low m/z ions and so reduces the amount of charge entering the mass analyser, which reduces potential detrimental space-charge effects in the instrument. 
     Although the present invention has been described with reference to preferred embodiments, it will be understood by those skilled in the art that various changes in form and detail may be made without departing from the scope of the invention as set forth in the accompanying claims. 
     For example, although the declustering AC voltage has been described above as having a substantially square or sinusoidal waveform, it is contemplated that it may have a waveform of any other shape, such as triangular. Desirably, the peak positive and peak negative amplitudes of the waveform are the same, i.e. the waveform is symmetric, although it is contemplated that these peak amplitudes could be different and the waveform may be asymmetric. 
     The declustering device described herein may be provided in an atmospheric pressure region or vacuum chamber and utilise the background gas therein for providing the collisions during the declustering mode. Alternatively, the declustering device may be provided in a gas cell (e.g. its own dedicated gas cell) and the gas pressure and/or gas composition in that gas cell may be controlled, e.g. so as to be optimised for the declustering and/or non-declustering modes. The gas cell may be maintained with different gas pressures and/or gas compositions in the two different modes. 
     Although the declustering device has been described as being part of or immediately downstream of the ion source, it is contemplated that it may be arranged at other downstream locations in the spectrometer.