Tandem time of flight mass spectrometer and method of use

Field of invention The present invention relates to the field of mass spectrometry, particularly to high throughput comprehensive tandem mass spectrometry in a device including two time-of-flight mass spectrometers.

Background of the invention A mass spectrometer is a device that vaporizes and ionizes a sample and then uses a static or dynamic electric field to measure the mass-to-charge ratio of the formed ions. Tandem mass spectrometry is the structural analysis and identification of compounds in complex mixtures.<u style="single">Use in</u>Used. In virtually all applications, the MS-MS procedure involves the following sequence of operations: mass-to-charge ratio (m / z) mass selection of parent ions, fragmentation of those ions, Follow the order of fragment mass spectrometry. There is a great variety of tandem MS-MS equipment, with their own strengths and weaknesses, but they all have one common feature: they all use one parent ion at a time. is there. The rest of the ion species is removed from the primary ion beam and lost.

Triple quadrupole equipment is the most common MS-MS equipment. For example, a continuous ion source such as an electrospray (ESI) introduces ions into a first quadrupole mass filter, which is tuned so that only the ions of interest pass through the mass filter. The rest of the primary beam components are rejected and lost. The selected ion is<u style="single">, M</u>It is filled with gas at a pressure of Torr (1 Torr corresponds to 0.00133 bar and so on) and is transmitted to a so-called "collision-induced dissociation" (CID) cell with a radio frequency (RF) quadrupole guide. .. The kinetic energy of the injected ions is controlled by the electrostatic bias of the mass filter.<u style="single">that is</u>It is regulated to induce ion fragmentation through gas collisions. Fragment ions are impact attenuated within the CID cell and then introduced into the second quadrupole for mass spectrometry. Since scanning the mass at the second quadrupole is time consuming and causes an additional ion loss of about 1000 times, triple quadrupole instruments are known for parent and fragment ion mass. Mainly used for the detection of known species.

The introduction of a quadrupole time-of-flight tandem mass spectrometer (Q-TOF) has greatly improved the throughput of MS-MS equipment (Maurice et al.)<u style="single">Rap. Comm</u>. Mass Spectrom. V.10, pp.889-896, 1996). The triple quadrupole was modified so that the second quadrupole mass filter was replaced by an orthogonal TOF MS (oa-TOF MS). This substitution provides the benefits of parallel analysis of all fragment ions at once, thus providing the benefits of high sensitivity and fast acquisition in the second MS, and the resolution and mass accuracy of the second MS. Improved. However, quadrupoles are still used for parent ion selection with rejection of all but one ion species. The idea of parallel analysis has not been extended to the parent ion.

Another common MS-MS instrument is the Paul Ion Trap Mass Spectrometry (ITMS), March, RE, Hughes RJ Quatrupole Storage Mass Spectrometry, Willey-Interscience, Well explained in New York 1989. Ions generated by the ion source are periodically injected into the IMTS and captured within the IMTS by a radio frequency (RF) field. The "undesirable" species are removed, for example, by applying a broadband resonant AC signal, leaving only the ions of interest in the trap. The selected parent ion is then excited by another AC field that resonates with the perennial motion of the precursor. The parent ion obtains kinetic energy and becomes fragmented by the collision of energy with the buffer gas. Fragments are mass spectrometrically analyzed using resonant emission techniques. The amplitude of the RF field is tilted so that the ions in turn exit the trap according to their m / z value.

It is known to combine 3-D pole traps with TOF analyzers for more accurate mass spectrometry of fragment ions. See Quin and D. Lubman, Rap. Comm. Mass. Spectrom. 10, 1079, 1996 and WO 099/39368 by Shimadzu. US Pat. No. 5,847,386 by D. Douglas,<u style="single">BA Thomson and LL Joliffe</u>U.S. Pat. No. 6,111,250,<u style="single">T. Dresch et al.</u>U.S. Pat. No. 6,020,586,<u style="single">B. Reinhold and A. Verentchikov</u>In WO 01/15201 by, a linear ion trap (LIT) is coupled to the TOF analyzer. All ion trap tandems are primarily aimed at multi-stage MS-MS analysis. The parent ion is selected by losing the components of other ions.

The recently introduced tandem time-of-flight mass spectrometer (TOF-TOF) is the closest basic form to the invention described below due to the similar hardware used. Examples of TOF-TOF are US Pat. No. 5,032,722 by Schlag et al., US Pat. No. 5,464,985 by TJ Kornich et al., US Pat. No. 5,854,485 by T. Bergmann, US Pat. No. WO 99/40610 by ML Vestal,<u style="single">US Pat. No. 6,300,627 by C. Koster et al.,</u>And WO 99/01889 by C. Hop. In all TOF-TOF tandems, the pulsed ion beam is time separated by a first high energy TOF, filtered by a timed ion selector, and only the ions of interest pass through the CID cell. The CID cell is filled with a gas of low gas pressure (usually lower than 1 mtorr) and introduces a single high energy collision with the buffer gas, sufficient for ion fragmentation, but still ion packets.<u style="single">To maintain</u>Keep a short duration. The pulsed beam of fragment ions is analyzed in a second high energy TOF. To deal with the large energy diffusion of fragmented ions, the second TOF uses a secondary field potential or additional pulse acceleration.

In WO 00/77823 by A. Verentchikov, variants of the TOF-TOF tandem use slow injection of parent ions into the CID cell using fragment collision attenuation, followed by injection into the orthogonal TOF. This device is the closest basic form of the invention, given the components used. Collision damping in fragmented cells is the second TOF<u style="single">Upstream</u>Improves the characteristics of the ion beam in, enabling high resolution and accurate measurement of the mass of fragment ions. The first TOF operates with an energy of 1 kV and a short time scale. The time gate in front of the CID cell contains only the mass of one parent ion at a time.

In all the tandems described, the first mass spectrometer (quadrupole, ion trap or TOF) is one parent at a time.<u style="single">ion</u>Select the mass of and reject all other components. For some applications, such as drug metabolism studies, it is acceptable to track one compound of interest. For complex mixtures (such as protein characterization from gels), however, it is necessary to analyze multiple parent ions. Continuous MS-MS analysis of multiple precursors using known techniques is redundant and insensitive.

Recently introduced tandem IMS-CID-TO<u style="single">F is</u>Uses the principle of time-nested acquisition, which can be achieved without ion loss. WO 00/7033 by D. Clemmer<u style="single">5</u>Please refer. Separation on ion mobility analyzer (IMS) is in milliseconds<u style="single">Time scale</u>TOF mass spectrometry is performed in microseconds<u style="single">Scale</u>Therefore, it is possible to obtain a fragment spectrum for the mobile debris of each ion.<u style="single">Tona</u>To. The drawback of this technique is the poor isolation of IMS with a resolution of mobility below R = 50, which is<u style="single">About 10</u>Corresponds to mass resolution. This invention because the IMS-TOF tandem uses the principles of comprehensive tandem mass spectrometry with time-nested acquisitions.<u style="single">Base of</u>Book<u style="single">With shape</u>Is selected.

The idea of MS-MS spectrometry without loss of parent ion is WO 01/15201 by B. Reinhold and A. Verentchikov.<u style="single">Also disclosed in</u>.. Ions are selected by resonant excitation and are moved between ion traps without rejecting other ion components. This procedure is verbose<u style="single">long</u>, Ions from the ion source are lost. So-called parallel ion treatment is used in multiple ion traps in WO92 / 14259 by Kirchner, where the beam is split by multiple traps. Sensitivity<u style="single">sacrifice</u>This saves time.

There is still a need for equipment that provides rapid and sensitive MS-MS analysis for multiple parent ions in parallel without rejecting ions coming from the ion source. Such instruments further improve the throughput of MS-MS analysis and are desirable for the analysis of complex mixtures.

<p> Outline of the invention<u style="single">Invention of the present application</u>Nested using two time-of-flight (TOF) mass spectrometers, ie, slow TOF1 for parent ion separation and fast TFO2 for fragment mass spectrometry. Hara<u style="single">Li</u>Can be realized<u style="single">Is evaluating</u>.. Therefore, the tandem mass spectrometry of the present invention uses two time-of-flight separations, and for the same mass-to-charge ratio, the flight time in the first separation step is greater than the flight time in the second separation step. Much longer, multiple parent ions are separated, fragmented and mass spectrometrically processed per injection of ions from the ion source.</p><p> The tandem mass spectrometer of the present invention is a pulse ion source, a time-of-flight mass spectrometer (TOF1) for time separation of parent ions, a fragmented cell, and a second time-of-flight mass spectrometer for mass analysis of fragment ions. Includes mass spectrometer (TOF2) and data acquisition system.<u style="single">Uninflected word</u>For the TOF-TOF system, the flight time at TOF1 is the transit time through the fragmented cell and the flight time at TOF2.<u style="single">Both</u>Substantially than<u style="single">Long</u>I<u style="single">.. Nori</u>Typewise in the millisecond range<u style="single">T</u>Extended separation in OF1 is typically<u style="single">about</u>This can be achieved by operating a longer TOF1 with a much lower kinetic energy around 1 to 100 eV, while using a shorter TOF2 with an energy of 3 to 10 keV. The time between arrivals of adjacent parent ion species<u style="single">, Decline</u>Sufficient for mass spectrometry of disintegration and fragments. Therefore, the present invention enables rapid MS-MS analysis of a plurality of parent ions in real time without rejecting the parent ions. The MS-MS acquisition cycle lasts several milliseconds and can be repeated multiple times to improve sensitivity and signal quality.</p><p> To avoid ion loss, the ion source operates in pulse mode at a repetition rate of approximately 100 Hz, which is compatible with the millisecond time of the MS-MS cycle. The matrix-assisted laser desorption / ionization (MALDI) ion source is an example of a available pulse ion source. The invention is also compatible with a variety of continuous ion sources such as ESI, MALDI with gas cooling, and photoionized ion sources filled with chemical ionization and gas. The ion flow is continuously stored in the stored radio frequency (RF) device and periodically pulsed to TOF1. The storage device can be a pole trap or a storage multi-pole, preferably a quadrupole.</p><p><u style="single">As far as the author knows</u>The new time-nested TOF-TOF method cannot be achieved with existing TOF-TOF equipment without seriously sacrificing performance.<u style="single">I.</u>This invention<u style="single">, Low</u>Operates with low ion energy (1 to 100 eV) to extend the separation time,<u style="single">Five</u>Disclosure of the new TOF1 separator.</p><p> Two of these new TOF1 analyzers use a combination of radio frequency (RF) field confinement and direct current secondary fields to provide a temporary ion beam with relatively large energy diffusion. Provide focusing. These analyzers can operate at particularly low ion energies of 1 to 10 eV. In a preferred embodiment, the novel TOF1 analyzer comprises a linear multipole ion guide, preferably a quadrupole, surrounded by a DC mirror. The DC mirrors at both ends are turned on and off to allow injection of ions from one end of the TOF1, reflection of multiple ions, and release of subsequent ions from the other end. In another preferred embodiment, the novel TOF1 analyzer comprises two outer rows of DC electrodes and two inner rows of RF-only rods oriented across the TOF1 axis. This structure forms a two-dimensional RF-tunnel combined with a second-order potential distribution along the axis of the TOF. Ions are injected into TOF1 at a small angle to the axis, causing multiple reflections along the axis, moving slowly across the axis, leaving TOF1 after several reflections.</p><p> Another three new analyzers are 10<u style="single">0e</u>It is an electrostatic device that operates with medium energy around V. One of them, the "spiratoron", contains a pair of coaxial cylindrical electrodes to which a DC voltage is applied. Ions are injected between the electrodes at a small angle with respect to their axes. Medium energy (100 eV) ions drift slowly along the axis, turning around the central electrode. After a few turns, the ions exit TOF1 through a cutoff region formed by a double-sided printed circuit board to avoid DC field disturbances. The other two electrostatic separators are planar and cylindrical multipath analyzers that use gridless mirrors and act like lenses at the same time. The effective flight path is extended by the use of multipath mode, achieving a 10ms timescale (compared to RF-assisted TOF1), despite high energy.</p><p> The present invention is based on gas collisions, surface collisions and light.<u style="single">including</u>Compatible with various fragmentation methods. The fragmentation cell design is tuned to reduce transmission time and time spread. The CID cell is short (around 1 cm), filled with a relatively high pressure gas (over about 0.1 mBar), and supplemented by an axial DC field to accelerate transmission and modulate the ion beam in synchronization with TOF2. .. Surface-induced dissociation (SID) cells use a pulse lens to provide spatial focusing as well as temporary focusing (bunching). Ions are ejected from the SID cell by a pulse probe potential synchronized with the bunching lens and TOF2 pulse (through time-shifting).</p><p> The choice of a second time-of-flight analyzer is not critical, but in most tandem examples, TOF with orthogonal ion implantation (o-tof) is more preferred. In order to improve the efficiency of orthogonal injection (so-called operating cycle), it is preferable to eject ions from the fragmented cell in synchronization with and shortly before the orthogonal injection pulse.</p><p><u style="single">Of this invention</u>The TOF-TOF tandem is expected to separate parent ions with medium resolution, for example, limited by the speed of 10us, primarily the second TOF MS. The estimated resolution of TOF1 on the order of 300 (see detailed description) is still sufficient to isolate the group of parent ion isotopes.<u style="single">Prior art</u>Much higher than the resolution of parental separation on an ion mobility analyzer. Higher resolution of separation can be achieved within longer TOF1 or by regular selection of ions by the time gate in front of the CID cell.</p><p> The present invention enables multiple strategies for data acquisition. In the simplest and most robust method, MS-MS data is acquired continuously and the MS-MS spectra of multiple parent ions are later reconstructed. However, it is advisable to perform MS-MS analysis in two stages. In the first, MS-only stage, the parent ion is continuously placed in TOF2 for mass spectrometry of the parent ion. Information about the mass of the parent ion is used in the second MS-MS stage. The time gate opens only upon arrival of multiple parents of interest in order to improve the resolution of parent ion separation and avoid signals from chemical backgrounds. The TOF2 signal is acquired only for the time window selected to deny the flow of meaningless data. Similar information about the parent ion can be obtained using any online detector somewhere after TOF1.<u style="single">To.</u></p><p> In addition to sensitive and rapid MS-MS analysis, the present invention provides multiple types of MS-only analysis. TOF1 alone can be used in MS-only analysis to diffuse peaks over time, avoid detector saturation, and use inexpensive, slow, temporary recorders. A better quality spectrum of the parent ion can be obtained with TOF2 while using TOF1 in pass mode. The so-called "parental scan", i.e., the spectrum of parent ions with a particular set of fragments, can be reconstructed from MS-MS data averaged by injection of multiple sources. Data can ultimately be accumulated only for the mass of the parent.</p><p> Since the MS / MS spectrum is obtained for all precursor ions of interest with a single ion implantation, the invention presents an exceptionally fast MS estimated to be 10 to 30 full cycles per second. / Provides MS analysis. The speed of MS-MS analysis is compatible with the time scale of chromatographic separation, and real-time LC-MS-MS analysis is previously used in ion traps and Q-TOF, such as "data-dependent acquisition". It is possible without any restrictions. The high acquisition speed and sensitivity of the MS-MS tandem of the present invention also opens up opportunities for the latest trends in nested LC-LC analysis.</p><p> The invention is described in detail in the claims. The above-mentioned advantages and yet another advantages of the present invention may be better understood by reference to the following description along with the accompanying drawings.</p>

Detailed description of the invention<u style="single">This</u>The method of tandem mass spectrometry of the present invention includes the step of generating an ion pulse with an ion source containing a mixture of different sample ions, and<u style="single">Operates with low energy</u>A step of separating sample ions according to flight time in a first time-of-flight mass spectrometer to generate a sequence of ion packets in the order of their masses, and sample ions without mixing the separated ion packets. Fragmented ions in the second time-of-flight mass spectrometer on a time scale much shorter than the time scale of the sequential fragmentation step and the first separation step.<u style="single">Quality</u>A single ion pulse from a quantitative source and a fragment mass spectrum for the mass-to-charge ratio of multiple sample ions, optionally for each of the sample ions across multiple source pulses. Includes a step of summing the fragment spectra.

Of this way<u style="single">Basic</u>The point is that the separation time at the first TOF is arranged much longer than the fragmentation time and the time of fragment mass spectrometry for the same mass-to-charge ratio. Substantial differences on the time scale are used to separate, fragment and mass spectrometric fragments for multiple parent ions per single ion implantation from an ion source. Substantial differences on the time scale are achieved by selecting longer flight paths and / or lower ionic energies in the first TOF.

With reference to FIG. 1, this method is illustrated by a block diagram of the main tandem MS-MS components. A typical TOF-TOF device using a time-nested acquisition 11 is a pulsed ion source 12 that communicates in sequence, a first time-of-flight mass spectrometer-TOF113, a fragmented cell-CID / SID14, and a second. Includes a time-of-flight mass spectrometer TOF2 15 and a data system 16 for time-nested acquisition. The pulse ion source is biased by a voltage source 17 with a small potential difference compared to the TOF1 analyzer, and the TOF1 is biased by a voltage source 18 with a voltage source 18 compared to a CID cell. Any timed gate 19 may be inserted between the TOF 1 13 and the CID cell 14 to improve the separation of the TOF 1.

Simply put, in operation, the pulsed ion source produces an ion pulse of the sample (parent) ion and is controlled by the voltage source 17, typically from 1.<u style="single">10</u>Ions are injected into TOF1 with reduced energy during eV. This is an important difference between this invention and the prior art. This is because TOF analyzers typically operate with energies between 3 and 30 keV.<u style="single">.. That is, it means that the ionic energy in TOF1 is 1/100 or less of the ionic energy in normal TOF2. T</u>Separation in OF1 typically takes a few milliseconds. As a guide, TOF1 = 8m effective length, average ion energy E = 3eV and ion mass m = 1000a.mu<u style="single">.</u>think of. In this example, the ion velocity is V = 800m / s and the flight time is 10ms.<u style="single">.. Time</u>The interleaved parent ion is controlled by the DC bias between TOF1 and the cell.<u style="single">Increased</u>The energy level is discharged from TOF1 to the CID cell in order. Energy collisions with carrier gas molecules transform parent ions into fragments. Subsequent gas collisions result in collision decay of fragment ions. Fragments move rapidly through cells and TOF2 analysis Injected into the meter. TOF2 separates fragment ions on a much shorter timescale between 10 and 100 μs<u style="single">.. That is, the flight time of 10 ms in TOF1 described above means that the flight time in TOF2 is at least 10 times larger. T</u>The dramatic difference in timescale between OF1 and TOF2 makes it possible to acquire data for multiple fragment spectra corresponding to different parent ions between source pulses. The special data acquisition system 16 acquires a plurality of fragment spectra in a time-nested manner, and the individual spectra are not mixed together. The fragment spectrum for each parent ion is integrated over several ion source pulses. The ion pulses generated by the ion source are used to obtain the complete set of MS-MS data for multiple parents without rejecting the ions at all stages.

With reference to FIG. 2, a typical time diagram shows the method of the invention, the synchronization of individual devices, and the principles of time-nested data acquisition. Graph 21 at the top shows the acquisition cycle, with ion injections every 10 ms, ie 100 times per second. The parent ion is separated by TOF1 within a time of 10 ms, and the CID cell receives a sequence of ion packets arranged according to the mass of the parent ion, graph 22. Parent ions are partially fragmented within the cell, and due to the short transmission time within the cell, the fragments reach TOF2 at about the same time as their parents, Graph 23. Each new lineage of ions (ie parent and daughter) is 10<u style="single">μ μ</u>Graph 24, which is orthogonally pulsed to high-energy TOF2 every s to generate a TOF2 spectrum for each parent's mass. Each TOF2 spectrum acquires the time tag of the TOF2 pulse with respect to the source pulse, i.e. the TOF1 time tag. A spectrum with the same TOF1 time tag is summed over multiple ion source pulses, as indicated by the dashed line connecting the two TOF2 spectra with the same TOF1 time tag.

In the above mode of operation, time-nested acquisition is done directly. The operating parameters of the equipment are the same regardless of the configuration of the ion beam from the ion source, and the data is acquired all the time. All information, such as parent ion spectra and fragment spectra for various parents, is extracted in subsequent data analysis.

In another mode of operation, which should be called "data-dependent acquisition," MS-MS analysis is performed in two steps. In the first step, the parent mass spectrum is obtained with TOF2, and the TOF1 and CID cells pass the ions continuously without fragmentation.<u style="single">.. No.</u>In step 2, the instrument operates as an MS-MS, i.e., TOF1 separates parent ions, fragmented cells form fragments, and TOF2 acquires fragment mass spectra in the form of time-nested data. Time-nested acquisition is improved by utilizing information about the mass of the parent ion and avoiding the acquisition of data in the blank time when the parent ion does not come. Any timed gate 19 may be used to improve TOF1 separation and chemical noise suppression. Naturally, the ion packet coming from TOF1 is expected to be shorter than the same ion packet at the exit of the CID cell. Timed gates only allow ions to enter through multiple narrow time windows that correspond to the arrival of the parent ion. Such gating suppresses ionic signals coming from chemical backgrounds and improves detection limits. Gate manipulation may be used to improve the separation of near-mass parent ion pairs at the expense of sensitivity. Several sets of MS-MS data are retrieved and the timed gate contains only the mass of one parent at a time.

Since the general method has been described for clarity, detailed examples will be given first at the level of the individual components and then as an example of an integrated TOF-TOF device. Some of the components used are well known in the art, but their components and parameters are modified to suit the purposes of the present invention. To understand the compromises chosen, we first consider the main challenges in TOF-TOF methods and equipment.

The method of the present invention is very counter-intuitive because it is said to be infeasible for multiple reasons. Those skilled in the art argue that the resolution of TOF1 is extremely low because the diffusion of ion energy at the source is comparable to that of ion energy at TOF1, and the resolution of TOF1 is at a weak acceleration field. Ion loss through TOF1 is due to the expected large length of TOF1 and the high branching of the slow ion beam at TOF1, which is also damaged by the time required (time diffusion caused by the initial velocity diffusion). Expected to be destructive, TOF1 vacuum stage and gas-filled CID cells should be separated by small openings, so ion loss is expected to be higher, 10-100<u style="single">μ μ</u>There seems to be no quick transmission through the CID cell on the s timescale. Most existing CID cells are 200-10,000<u style="single">μ μ</u>Has a time spread of s. Currently used in TOF technology<u style="single">Available</u>The data acquisition system cannot handle the expected data flow rate.

<u style="single">They are</u>It is mainly focused on TOF1 and comes from knowledge of existing TOF mass spectrometers operating at high energy. The inventor has found that multiple mechanisms of TOF1 allow low-speed separation at medium resolution.<u style="single">In some examples</u>The improvement in TOF1 resolution is achieved by using an ion mirror with a second-order potential distribution known to compensate for energy diffusion. This phenomenon is similar to elastic vibration, and the period does not depend on the vibration amplitude. The secondary field is well researched in the technical field of TOF.<u style="single">For example</u>, Int. J. of Mass Spectrom and Ion Process, v.146 / 147, 1995, pp.165-182, see Makarov et al. Unfortunately, such analyzers also introduce large beam bifurcations. The inventor has found that low energy TOFs can be improved by introducing radio frequency confinement of the ion beam in at least one direction. RF confinement branches the ion beam<u style="single">Reduction</u>It also eliminates critical surface charging for low energy devices. A new type of TOF has been found that combines RF confinement with an axial DC secondary potential.

With reference to FIG. 3, preferred embodiments of the novel low energy time-of-flight separator 31 include an RF-only multi-pole 32, two electrostatic mirrors 33 and a pulse generator 34. The mirror consists of a plurality of electrodes interconnected by a chain of split resistors 35. The external electrode of the mirror 33 is connected to the pulse generator 34, and the electrode in the middle of the mirror 33 is grounded. The end field is terminated by the opening 36 and the potential is regulated as part of the maximum potential of the pulse generator 34.

In operation, the RF field provides radial confinement as indicated by arrow 37 in FIG. Radial RF confinement does not affect the movement of ions along the axis. An axial parabolic electric field is formed by penetrating the field between the rods of multiple poles. The parabolic field is largely independent of the energy of the ion and provides axial reflection in the period proportional to the square root of the m / z of the ion. The pulse potential at the end of the mirror allows switching between ion implantation into TOF1, reflection 39 of ions within TOF1, and release of subsequent ions at the other end of TOF1. Valid flight path L1_EFFIs more than the length L of TOF1<u style="single">About Nπ + 1</u>Doubled, N is the number of full turns. Overall, RF confinement and multiple reflections allow extended time separation without ion loss, secondary potentials improve TOF1 resolution, and slow ions with high relative energy diffusion. Allows beam separation.

The ideal secondary mechanism is modified by the presence of free flight segments from and in the path to TOF1. According to the above-mentioned publication by Makarov et al. Even in the case of Yu flight, here, about 30% L<u style="single">1</u>_EEFIn the case of, a mass resolution of 2000 can be achieved for ion pulses of relative energy diffusion up to 50%. Free flight path 0.3L<u style="single">1</u>_EEFTo keep below, this mechanism requires at least 5 reflections corresponding to 2 full turns. This is L1_EFFHelps to increase to 7.3L, but reduces the mass range of the parent ion to a factor of 2, i.e. M_MAX/ M_MINSet 2.

See FIG. 4, another of the new low-energy time-of-flight separators 41.<u style="single">Executable</u>Examples include an RF channel 42 enclosed by a set of electrostatic electrodes 43, a termination electrode 44, and a deflector 45. The RF channel is formed by a plurality of rods 46 having alternating RF phases and aligned along the Y axis. The electrodes of the electrostatic mirror 43 are also aligned along the Y axis and connected via a chain of split resistors 47.

In operation, rods 46 with alternating RF potentials form an RF tunnel, trapping ions in the Z direction. The potentials of electrodes 43, 44 are dispersed by the strands of the resistor, forming a secondary potential along the smallest X-axis on the central plane of the TOF. The field of the external DC electrode penetrates the RF channel and provides a weak but still secondary potential distribution. If the fringe field is not taken into consideration, there is no field in the Y direction. Ions are injected at a small angle with respect to the X axis and deflected by the deflector 45, doubling the deflection angle for ions with average energy. Deflection reduces the Y-spatial diffusion caused by the diffusion of X-energy. Ion movement is a combination of slow drift along the Y direction and multiple reflections along the X direction. Overall, the orbit of the ion is corrugated and ends at the boundary of the RF tunnel. The ions gain some spatial diffusion at the exit of the TOF, which is partially compensated for by subsequent acceleration and lens focusing of the ions.

According to the SIMION simulation by the inventor, even with 50% energy diffusion, a 50x30 cm device allows pairs of reflections of N = 4 to 5 without mixing ions with adjacent turns. The effective flight path of the device is L^*<u style="single">π</u>^*Equal to N, L_EFFReach = 7.5m. The RF field does not limit the resolution of TOF1 to R = 1000. Obviously, a second type of TOF1, which can be called an RF confined W-type TOF, provides simpler operation and a longer flight path at TOF1 and is primarily due to the ratio of flight time between the two TOF analyzers. Improves resolution at limited TOF1. The complexity of TOF1 can be reduced by using a printed circuit board (PCB) assembly.

<u style="single">Above</u>For both TOF mass separators, the period of each reflection is largely independent of the ion's energy and is proportional to the square root of the ion's m / z. Ions are trapped by the RF field and ion loss is virtually eliminated. The introduction of a new low-energy TOF analyzer makes this invention practical and solves the above objections: a) High relative energy diffusion penetrates the DC electric field into multiple pole guides or tunnels. Compensated by the quadratic distribution of the potential of the ion mirror created by b) the ability of TOF1 to operate with high relative energy diffusion More, compared to traditional TOF, it can operate with lower ion energy and much longer time scale. As a result, the device allows longer ion pulses from the ion source and the duration is no longer an obstacle, c) dramatic differences in the TOF1 and TOF2 timescales allow time-nested data acquisition. D) Ion loss is substantially avoided by guiding the ions in the radio frequency guide or tunnel, e) Ion confinement by the RF field, and subsequent acceleration in front of the CID cell. F) Time diffusion in the CID cell is reduced by using a short high pressure cell with an additional axial DC field, which allows maximum transfer of the ion beam to the CID cell. G) Temporary recorders with large and fast average memory are now being introduced by the Swiss company Acquiris (www.acquiris.com).

A detailed description is continued at the level of the individual components, namely the pulsed ion source, the fragmented cells and TOF2 specially made for the purposes of the methods and devices of the present invention. Pay particular attention to the issue of time diffusion.

Referring to FIG. 5, the TOF-TOF method and apparatus of the present invention includes a source housing 52, a sample plate 53 with an analyzed sample 54, a pulsed laser 55, a low voltage power source 54, and an outlet opening 56. A pulsed MALDI ion source 51 containing is used.

In operation, the sample for analysis is prepared in a matrix known in the art and deposited on the sample plate 53. The pulsed laser 55 illuminates the sample and produces a short pulse of sample ions. Ions are known to be emitted at velocities of 300 to 600 m / s, which corresponds to the initial ion energy between .5 and 1.5 eV for 1 kD of ions. Ions are accelerated by a potential bias of a few volts. 1 kD ions can be expected to exit the ion source with diffusion over a period of a few microseconds and diffusion of energy less than 1 eV. The main drawback of the vacuum MALDI ion source is the temporary instability of the ions, well explained in conventional high energy MALDI. The present invention is applicable to softer MALDI ion sources using soft matrices or infrared lasers. Temporary stability of the ions is improved by collision cooling, which will be described later.

Referring to FIG. 6, the TOF-TOF method and apparatus of the present invention uses a gas-filled pulsed MALDI ion source 61. Source 61 includes features of a vacuum MALDI source such as source housing 62, sample plate 63 with sample 64 analyzed, pulsed laser 65, low voltage power supply 66, and opening 67A. The source 61 also includes a gas inlet 68 that supplies gas to the housing 62, an additional pump stage 69 that is terminated by an outlet opening 67B to reduce the gas load on the pump of the TOF1.

In operation, the source housing 62 is filled with buffer gas through the gas inlet 67. Gas pressure in the source housing is maintained between .01 and 1 torr to provide ion collision cooling (see Verentchikov et al. Of the ASMS conference at www. Asms.org). A differential pump system with two 1 mm openings 67A, B and two conventional 250 l / s turbopumps (one pumping the TOF1) maintains a better vacuum in the TOF1 than the 1E-6torr. To do. The laser pulse produces a rapid (1 to 3 ns) ion emission from the sample. Laser 65 is a high energy laser to improve ion generation. Collision with the buffer gas relaxes the internal energy of the ions. Collision with gas almost reduces the kinetic energy of ions to thermal energy, that is<u style="single">0.01</u>From<u style="single">0.1</u>It attenuates to eV, but still retains the pulse characteristics of the ion beam. Ions are sampled by the flow of gas through an opening supported by an approximately 1 V DC bias on the sample plate. The ions are then controlled by a DC bias between openings 67A, B<u style="single">necessary</u>It is accelerated to kinetic energy and exits the ion source. Internally cold ions are stable and long without ion decomposition Withstands separation within TOF1. Overall, gas attenuation at the MALDI source gives advantages to the TOF-TOF method of the present invention, while time and energy diffusion can be achieved for slow TOF1 separation 10<u style="single">μ μ</u>Within the boundaries of s and 1eV.

Referring to FIG. 7, the TOF-TOF method and apparatus of the present invention fills a continuous ion source with a soft ionization 72 with an outlet opening 73, and a gas enclosed in an additional pump stage 75. A pulse ion source 71 including the RF capture device 74 is used. The continuous ion source is as follows<u style="single">One of</u>That is, in one of electrospray (ESI), APCI, gas-filled MALDI, PI or CI.<u style="single">possible</u>.. The capture device is as follows<u style="single">One of</u>That is, one of a 3-D pole trap, a linear RF-only multi-pole with axial discharge, and a curved RF multi-pole with radial discharge.<u style="single">possible</u>.. A linear quadrupole ion trap with axial discharge is preferred.<u style="single">When used</u>The quadrupole 74 is surrounded by a DC electrode 76 and openings 73,77.

In operation, the quadrupole is filled with a buffer gas at a pressure of 1 to 100 mtorr. The differential pump system 75 reduces the gas load on the TOF1 pump. Ions are generated at the ion source 72 and continuously fill the RF-only quadrupole ion guide 74. The gas collision damps the kinetic energy of the ions and traps them along the axis of the quadrupole and at the bottom of the DC well made by the electrodes 76 and opening 77. Periodically, the potentials of the electrode 76 and the outlet opening 77 are adjusted to axially expel the accumulated ions to TOF1. This structure<u style="single">1e</u>Diffusion of energy less than V and 1<u style="single">0μ</u>Generate an ion pulse with diffusion for less than s.

<u style="single">In all the above examples, the pulsed ion source can generate an ion pulse with a diffusion of energy less than 1 eV and a time diffusion of less than 10 μs.</u>Mass decomposition of 300-500 desired TOF1 sufficient to separate isotope groups<u style="single">Noh</u>Is 600 to 1000<u style="single">time</u>Inter-decomposition<u style="single">Noh</u>Need. Initial time diffusion of 10 μs<u style="single">for,</u>The flight time for a 1 kD ion must be a few electron volts of ion energy and at least 10 ms reachable with an effective flight path of 5 to 10 m. The multi-turn TOF1 analyzer described above provides a 10 m effective path within a 5 to 1 m device. The next inevitable question is so that the primary separation is not destroyed<u style="single">10 μs</u>Is it possible to fragment the ions within?

Referring to FIG. 8, the TOF-TOF method uses a short, high gas pressure CID cell 81 for ion fragmentation. The CID cell 81 includes a vacuum housing 82, an inlet lens 83, a CID chamber 84 connected to the gas inlet 85, an RF focusing device 86 with any DC electrode 87 surrounded by the CID chamber, and an outlet ion lens 88. .. The CID cell also includes any timed ion selection gate 89. The gas inlet supplies buffer gas to the CID chamber. The CID chamber 83 includes openings 83A, B. The vacuum housing 82 includes openings 82A, B and vacuum pump 82C. The RF focusing device is preferably an RF-only quadrupole.

A conventional CID cell, typically 10 to 20 cm long, operates at a gas pressure of about 10 m Torr. To achieve rapid ion movement, the CID cell used in the present invention is much shorter, typically 5-10 mm, and operates at much higher gas pressures above 300 mTorr. The high pressure region is concentrated in chamber 84 and surrounded by an additional layer of differential pump. Openings 84A, B, typically 1.5 mm in diameter, approximately 0.1 torr the total flow of gas into the vacuum housing.^*Limit to L / s. Pump 82C with a pump speed of 300L / s exhausts the vacuum housing to about 3E-4Ttorr. The openings 82A, B are typically 1.5 mm in diameter, further reducing the flow of gas to TOF1 and TOF2,<u style="single">1E-6torr</u>Operates at lower gas pressures. To avoid gas release, the RF amplitude is<u style="single">Preferably peak to peak</u>Reduced to less than 300V<u style="single">Preferably</u>this Is accompanied by a decrease in frequency below 1MHz.

In operation, the ions are accelerated in front of the cell, accelerating to sufficient energy to fragment the ions, typically 50 eV / kDa. Ion packets enter the cell through openings 82A and 84A and are focused by lens 83. At a gas pressure of 300 mtorr, the gas density is n = 1E + 22m^-3Equal to<u style="single">σ = 100Å</u>^{<u style="single">2</u>}Ions with a mass of 1 kD with a cross section of<u style="single">λ = 1 / nσ = 0.1mm</u>Have. At a typical quadrupole length of L = 1 cm, the ions produce about 100 collisions. The number of collisions is three times greater than the ion / gas mass ratio, which is sufficient to ensure fragmentation with subsequent attenuation. The first energy collision converts the kinetic energy of the ion into the heating of the ion, resulting in ion fragmentation. Once the ions lose their kinetic energy, subsequent gas collisions stabilize the fragmented ions, further attenuating their kinetic energy and confining the ions to the axis by focusing on the RF field. The phenomenon of collision damping is Don Douglas<u style="single">And J. French</u>U.S. Patent by<u style="single">No. 4,963,736</u>Is well explained.

The time diffusion of the ion beam in the CID cell is an important concern of the present invention. The time traveled before the high pressure region is assumed when adjusting TOF1, which creates only a time delay, not a time spread. Gas collisions can cause large time diffusions even in short CID cells. To reduce diffusion, the passage of ions through the cell is assisted by an axial electrostatic field created by the DC potentials of openings 84A, B. In a typical quadrupole with an inscribed diameter D = 1 cm and length L = 1 cm, the fringe field penetrates the RF quadrupole and is suppressed less than twice. The 20V acceleration potential can provide ion dragging through the gas at a rate of about 500 m / s, maximizing transit time of 20.<u style="single">μ μ</u>Less than s, and spread over time 10<u style="single">μ μ</u>Limit to less than s. Controlling transit time helps to collect ions prior to injection into TOF2 (ie, compresses the duration of the ion pulse). The acceleration field in the CID cell is modulated and synchronized (with time-shifting) with the TOF2 injection pulse.

Referring to FIG. 9, the TOF-TOF method and apparatus of the present invention uses fragmented cells 91 with surface-induced dissociation (SID) for ion fragmentation. The SID cell 91 is a bunching (temporary focusing), spatial focusing and steering lens 92,<u style="single">Preferably</u>It includes a probe 93 coated with a single layer of fluorocarbon, a pulse generator 94 mounted on the probe, and a DC acceleration column 95 surrounded by a ground shield 96. The DC acceleration column includes a mesh 97 connected to a pulse generator 98.

In operation, the ion packet of the time-separated parent ion is pulse-accelerated to a specific energy of about 50 eV / kDa and collected by the lens 92. The bunching previously used in the magnet sector-TOF tandem has a duration of ion packets of dT &lt;1.<u style="single">μ μ</u>It is known to compress to less than s. The lens 92 focuses and steers the parent ion packet 99 at the center of the probe 93. The ion beam collides with the surface at an angle, for example 45 degrees. Medium energy collisions with the surface of a single layer of fluorocarbon are known to induce fragmentation of peptides and small molecular ions. Fragmented ions bounce off the surface at a rate of about 500 to 2000 m / s, dT &lt;1 for the duration of the primary ion packet.<u style="single">μ μ</u>Move less than 2mm within s. During the collision, a small deceleration potential is applied to the mesh 97 to prevent fragment ions from leaking into the TOF2 analyzer. After an appropriate delay corresponding to the collision of the entire primary ion packet, the pulse generators 94 and 98 are activated and an electrical pulse is applied to the probe 93 and mesh 97. Fragmented ions are pulse accelerated to the TOF2 analyzer.

Compared to the CID cell, the SID cell has the following advantages: it operates at low pressure and therefore eliminates the diffusion of time in the fragmentation step, which reduces the requirements for the pump system, the primary ion. It has the advantage of accepting a wider beam of.

The drawbacks of SID are the poorly characterized fragment pattern of medium mass ions, the higher energy diffusion of fragment ions that reduces the resolution of TOF2, and the disappearance of the metastable of fragment ions in the TOF2 analyzer. The CID cell is more suitable for inline TOF1 and the SID cell is more suitable for W-TOF1.

Referring to FIG. 10, the TOF-TOF method and apparatus of the present invention preferably uses a conventional orthogonal TOF 101 with a COD cell for mass spectrometry of fragment ions. o-TOF101 is an orthogonal pulse<u style="single">Accelerator</u>Includes 102, ion mirror 103, floating free flight zone 104, TOF detector 105 and in-line detector 106. Both detectors are connected to a data acquisition system that includes a fast temporary recorder 107. TOF analyzer 101 is a vacuum chamber 108<u style="single">Inside</u>It is surrounded by pump 109 and exhausted.

The operation of o-TOF is well described in the art. The continuous or pulsed ion beam is accelerated to about 10 eV and enters the acceleration region. Periodic pulses are orthogonal and accelerate the ions to about 3 keV into the TOF analyzer.<u style="single">Those</u>Inject. The ions are reflected by the ion mirror and hit the TOF detector 105. A portion of the original ion beam is acquired by the in-line detector 106. The o-TOF parameters are slightly modified to accommodate the rapid analysis of fragment ions. The analyzer is small, L = 10 to 20 cm, and operates with high TOF energy (5 to 15 kV) to accommodate high repetition rates of about 100 KHz. The small size of the analyzer allows operation at gas pressures just below 1E-5 Torr. Traditional TOF analyzers are also modified by using a high current secondary electron doubler (SEM) or hybrid MCP / PEM for the detector and a fast average temporary recorder for the data acquisition system. Will be done. Short lengths and short flight times limit the resolution of TOF2. To improve the resolution of TOF2, one of the following: 10 sandwiched between IMS scans<u style="single">μ μ</u>Using the time gate of s, and the slow pulse rate of TOF2, pulse TOF2 at a speed of 100KHz and divert the ions in TOF2 to some detectors, pulse TOF2 at a speed of 100KHz, at TOF2 The time window of ions that can enter can be limited while increasing the flight time within TOF2 by either using a position sensitive detector. The TOF2 may be equipped with an in-line detector to avoid acquiring signals in the blank time when ions do not come from the TOF1.

Referring to FIG. 11, the TOF-TOF method preferably uses the conventional reflective TOF111 as well as the SID cell for mass spectrometry of fragment ions. The TOF111 includes a built-in SID cell 91, an electrically floating free flight zone 112, a detector 114 with a detector shield 113, an ion mirror 115, a vacuum housing 116, a pump 117, and a temporary data acquisition. Includes recorder 118.

In operation, the pulse of fragmented ions is accelerated within the SID cell 91, flies through the fieldless region 112, reflected by the ion mirror 115 and hits the detector 114. The orbit of the ion is indicated by line 119. The signal from the detector is acquired by the temporary recorder 118. Again, for rapid data acquisition, the analyzer<u style="single">Relatively</u>It is short, L = 10 to 20 cm, and operates at high acceleration potentials to accommodate high repetition rates of 100 KHz.

Having described the individual components, it will be easier to understand the concepts and features of the integrated TOF-TOF method and equipment. Specific examples of the TOF-TOF tandem of the present invention are shown below, but the plurality of feasible combinations is not limited.

With reference to FIG. 12, a preferred embodiment of the TOF-TOF device 121 is a pulse source 71, a storage quadrupole 74, electrodes 76, 77, 2 provided with a continuous ion source 72 and connected in sequence. In-line time-of-flight mass spectrometer TOF1 31, surrounded by two pulse ion mirrors 33A and B and equipped with an RF-only quadrupole guide 32, short gas with RF quadrupole 86 surrounded by openings 84A and B It includes a filled collision CID cell 81, a second orthogonal time-of-flight mass spectrometer o-TOF2 101 with a pulse accelerator 102, and an analog data acquisition system 107. The individual components have already been described and are shown in FIGS. 3, 7, 8 and 10, and their previous numbers are retained in the description below.

In operation, the continuous ion source 71 supplies the parent ion to the accumulating quadrupole 74. Once every 10 to 20 ms, ions are expelled from the accumulating quadrupole by the pulse potential of the DC electrode 76 and the outlet opening 77. The ejected ion packet containing a plurality of different parent ions is less than 10 μs long and has an energy diffusion of less than 1 eV. The average energy of the emitted ion pulses is adjusted to about 2 eV by selecting the pulse potentials of electrodes 76 and 77. By lowering the potential of the first mirror 33A, the ions are placed in the TOF1 separator. Ions are captured radially by the quadrupole RF field, but move freely along the quadrupole axis. When the parent ion of all masses (limited to the ratio of Mmax / Mmin = 2) passes through the first mirror, the first mirror 33A is turned on. The second mirror 33B has been turned on in the previous cycle. The ions produce multiple reflections, preferably 5 reflections, between two mirrors with a quadratic potential distribution along the axis of TOF1. The period of vibration is largely independent of the energy of the ion and is proportional to the square root of the mass of the parent ion. The effective flight path of the analyzer is up to 2π + 1α = 7.3 times longer than the physical length of TOF1. Preferred number<u style="single">(Preferably 5 times)</u>After the reflection of, the ions are released from TOF1 by lowering the potential of the second mirror 33B. A sequence of time-separated ion packets goes into the CID cell. A typical time scale for time separation is on the order of 10 ms, measured as the flight time of 1 kDa ion, and the duration of each packet corresponding to the mass of the parent ion is approximately 10 μs. Parent ions are separated with a temporal resolution of about 100, which corresponds to a mass resolution of 500.

After leaving TOF1, each ion packet is accelerated to a specific energy of 50 eV / kDa sufficient to induce fragmentation in the gas collision. Ions are focused by the lens system and injected into the high pressure CID cell through openings 82A and 84A. The ions are fragmented in the cell, the fragmented ions are impact attenuated and trapped by the RF field. The cell is actively emptied by the pulse potentials of the two CID openings 84A, 84B that are synchronized and time-shifted with respect to the TOF2 pulse. The ions enter the orthogonal acceleration region 102, are injected into the TOF2 analyzer, time separated, and then mass spectrometrically analyzed in TOF2. Synchronized injection into TOF2 eliminates the time gap, i.e., almost no fragments are lost between pulses of TOF2. Synchronized injection also improves the TOF2 operating cycle. Most of the fragment ions are trapped in the acceleration region 102 at the time of the TOF2 pulse.

The TOF2 spectrum presents a fragment spectrum by mass of time-separated parent ions. Spectrums with the same TOF1 tag (ie, corresponding to the same m / z parent ion) are summed over injections from multiple sources. Within a second of acquisition, the data contains a spectrum of 100 fragments and is averaged over 100 source injections.

In the above device, three nearly equal (about 10)<u style="single">μ μ</u>s) There is a source of time diffusion, which reduces the resolution of TOF1 separation. That is, due to the time diffusion obtained at the ion source, the time diffusion at the CID cell, and the digitization of TOF2 (ie, the acquisition of the spectrum at discontinuous times). Assuming there is no interrelationship between those three sources, the overall time spread is 17<u style="single">μ μ</u>Estimated to be s (three square roots higher than each spread). The resulting resolution of TOF1 separation is equal to 300, which is still considered appropriate resolution for parent ion separation. In comparison, commercial MALDI TOF-TO The resolution of TOF1 at F is about 100, and the resolution of the Q-TOF quadrupole in high sensitivity mode is about 300. The resolution of the TOF1 of the present invention is one of the following means: increasing the length of the TOF1 to more than 1 m, optimizing the ion energy within the TOF1, multiple narrow outputs sandwiched between scans. It can be improved by either applying a timed gate with windows, pulsing TFO2 faster and deflecting ions to some detectors, or using position sensitive detectors in TOF2.

Referring to FIG. 13, another preferred embodiment of the TOF-TOF apparatus of the present invention includes a gas-filled pulsed MALDI ion source 61, a W-type TOF1 41, a SID cell 91 and a coaxial TOF2 111. The source 61 includes a gas-filled chamber 62, a sample plate 63, a laser 65, and a low voltage bias source 66 connected to the sample plate 63. The TOF1 41 includes a deflection plate 45, two static reflectors 43 with a termination plate 44, and a two-dimensional RF tunnel 42. The static reflector 43 surrounds the RF channel 42 to form a second-order potential distribution. The SID cell 91 includes a bunching and focusing lens 92, as well as a probe 93 coated with a single layer of fluorocarbon. TOF1 111 includes a secondary electron doubler SEM113 connected to a temporary recorder 114. Source 61 and SID cell 91 are offline to allow reflection of multiple ions within TOF1 41. The above-mentioned selected combinations of elements have been selected primarily to show interactions between elements not described in previous TOF-TOF embodiments.

In operation, the pulse of the laser 65 produces a short burst of primary ions from the sample plate 63 at a repetition rate of 50 to 100 Hz. The source chamber 62 is filled with gas to alleviate the internal energy of the ions and prevent the decomposition of the ions. Ions are sampled through a thin layer of gas by an electric field and gas flow,<u style="single">each</u>Ion packet is 10<u style="single">μ μ</u>It remains shorter than s and has a diffusion of energy less than 1 eV. Ion packets are a low voltage bias source 66<u style="single">Provided by</u>TOF1 41 with multiple reflections at a small angle to the Y-axis due to another few volt potential<u style="single">Accelerate to</u>.. The steering plate 45 doubles the angle to reduce spatial diffusion on the X axis with respect to energy diffusion on the Y axis. Ion movement within TOF1 has three independent components: oscillations in the confinement of the RF field in the Z direction, multiple reflections along the Y axis in a period that is largely independent of the ion's energy, and orthogonality. Has a slow drift along the X-axis. After some Y bounces, the ions leave TOF1 and enter the bunching lens 92 of SID cell 91, time-separated into a sequence of ion packets and aligned according to their m / z ratio. Multiple reflections with small ion energies allow extended time separation on the order of 10 ms. Since the secondary DC field at TOF1 compensates for the diffusion of ion energy, separation at TOF1 does not increase the diffusion of ion packets over the 10 us time. Therefore, after leaving TOF1, the parent ion is separated with a mass resolution of about 300-500.

Periodically, for example, 10<u style="single">μ μ</u>Once in s, the ion is about 1<u style="single">μ μ</u>It is time bunched into s packets and spatially focused to about 1 mm by the pulse lens 92. The pulse-focused ion packet hits the surface of the SID probe 93 coated with a single layer of fluorocarbon. Collision with the surface induces ion fragmentation. Fragments that move slowly from the surface are 1<u style="single">μ μ</u>It diffuses by about 1 mm within the time of s. The delayed electrical pulse applied to probe 93 accelerates the fragment ions and injects them into the second TOF2 111 analyzer. The initial parameters of the fragment ions (ie, the parameters prior to the probe pulse) are good enough to perform mass spectrometry within TOF2 with thousands of resolutions. The signal is detected by SEM114 in a high dynamic range. The signal is passed to the temporary recorder 113 and the data is acquired in a time-nested manner. The TOF2 transient currents representing the fragment spectra of the various parent ions are not mixed together. Each fragment mass spectrum acquires a TOF1 separation time tag measured as the time between the source pulse and the bunching lens pulse. TOF1 The time tag carries information about the m / z ratio of the parent ion. TOF2 spectra with the same TOF1 time tag are averaged over multiple laser pulses to improve the signal-to-noise ratio.

The comprehensive TOF-TOF method of the present invention is feasible using a simple static TOF1.<u style="single">Is recognized</u>.. Below are some examples of static separators. Retention of the ion beam in a static field<u style="single">100 ~ 200eV</u>It requires operation with relatively high energy before and after. Millisecond separation times are achieved by extending the flight path and using specially designed electrostatic field focusing properties.

Referring to FIG. 14, another embodiment of the low energy time-of-flight separator 121 is an electrostatic lens 122, a deflector 123, and an inlet unit 124, two coaxial electrodes to which a DC voltage is applied. Includes an analyzer consisting of 125 and 126, as well as an outlet unit 127 followed by a deflector 128 and a lens 129. The device described above is known as the "Spiratron" and is described in Bakker IMB, The Spiratron-In: Adv. In Mass spectrom, London, 1971, V.5, pp. 278-280. Novelty is introduced by using this device as a low energy separator in a tandem TOF system.

In operation, the ion beam from the pulsed ion source 71 is transformed by the lens 122 into a wide beam with proportionally lower angular diffusion (quasi-parallel beam). This beam is deflected by the deflector 123 to provide a controlled tilt angle α with respect to the axes of electrodes 125 and 126. For example, the same effect can be achieved by positioning the electrodes 125 and 126 at a fixed angle. The ion beam enters the radial electrostatic field between the electrodes 125 and 126 through the opening of the inlet unit 124. A preferred embodiment of the inlet unit 124 is three double-sided printed circuit boards (PCBs).<u style="single">including</u>.. The outer surface of these circuit boards faces the deflector 123 and has a metal coating over them to create equipotential surfaces. Of these circuit boards<u style="single">Opposite</u>The face faces the gap between electrodes 125 and 126 and includes a set of metal-coated strips. These bands are connected to a resistive voltage divider that provides a voltage distribution that matches the ideal logarithmic voltage distribution between the electrodes 125 and 126, minimizing perturbations in this field along the orbit of the ions. The outlet unit 127 may have a similar structure.

After the ions pass through the inlet unit 124, they begin to move along a spiral orbit, wrap around the electrode 125 and separate in flight time according to their mass-to-charge ratio. Rhino ion beam to minimize's, the spiral should be circular. This is because the voltage U between electrodes 125 and 126<u style="single">As specified by the following formula</u>Average ion energy V₁It is realized when it corresponds to.

<maths num="1"><img file="JP4435682B2_D0001.tif" /></maths>

Where r1 and r2 are<u style="single">Respectively</u>The radii of the electrodes 125 and 126. After some rotation, the ions drift a distance H along the axis and exit the field through the exit unit 127. The structure of the outlet unit 127 is similar to that of the injection unit 124. The maximum number of rotations is mainly limited by the diffusion Δα at the maximum angle of the ion beam (Δα &lt;&lt; 1), which is<u style="single">As specified by the following formula</u>Limited by the effective temperature kT of the initial ion beam.

<maths num="2"><img file="JP4435682B2_D0002.tif" /></maths>

Where M is the magnification of lens 122 and the coefficient p depends on the level of confidence required (p for 95% ions).<u style="single">~</u>P for 4,99% ions<u style="single">~</u>P for 5,99.9% ions<u style="single">~</u>6.6)。<u style="single">The decomposition power of the spyratron is limited by both the maximum number of revolutions and the diffusion of energy at levels of 50-70.</u>

<maths num="3"><img file="JP4435682B2_D0003.tif" /></maths>

<u style="single">For example, if the length is H = 0.5m, kT = 0.05eV, V1 = 100V, m = 5, the total flight path is L1 22m. The time scale ratio between TOF1 and TOF2 is specified by the above equation. For the above parameters, the effective path length of TOF2 is L2 = 0.5m, the average acceleration voltage V2 = 5000V, and the ratio 150. That is, the flight path in TOF1 is at least 30 times larger than that in TOF2.</u>Although inferior in resolution to the TOF1 analyzer described above, the Spiratron device has the advantages of simplicity, higher operating energy and works without the strobe technology prior to TOF2.

The new static low energy TOF is fragmented as described above.<u style="single">system</u>And can be combined with either a TOF2 analyzer or fragment analysis. Referring to FIG. 14, TOF1 121 is combined with CID cell 81 and orthogonal TOF101. The main challenge of this combination is to focus the primary beam at the entrance of the CID cell. The ion beam has a high energy of 100 eV and the beam is wide at the exit, but the beam is large and parallel and can be well focused to a small opening by a conventional lens.

Referring to FIG. 15, Electrostatic multipath separator<u style="single">Also known as</u>, Another embodiment of the first (ie, TO1) time-of-flight separator 151 of the present invention comprises a free flight channel 152, and a focusing electrode 154 and a reflecting electrode 155.<u style="single">Linear 153 and</u>Includes two electrostatic mirrors. Free flight channel 152 has entrance and exit windows 156. All electrodes extend along the Y axis and the electrostatic field is two-dimensional in the area of the ion path. The pulsed ion beam is introduced into the multi-turn electrostatic TOF 151 via a set of spatial focusing lenses 157 and steering plates 158A, B. The ionic pathway of the ion is indicated by line 159. A typical axial potential distribution U (x) is shown by graph 160.

In operation, the ion pulse is the lens<u style="single">157</u>Focused on a parallel beam by the plate<u style="single">158A, B</u>Steered by. The beam is introduced into the separator 151 through the entrance window 156 at a small angle with respect to the X axis. Ions drift slowly along the Y-axis, producing multiple reflections along the X-axis. After multiple full turns (each full turn is formed by a pair of reflections), the ions are separated through the exit window 157.<u style="single">151</u>Is time-separated according to their m / z ratio. Number of full turns,<u style="single">Therefore, the flight length is</u>It depends on the injection angle, which is adjustable by the potential of the steering plate.

Electrostatic mirror is gridless TOF<u style="single">apparatus</u>Designed similar to the mirror of. The electrostatic potential applied to the mirror electrode is adjusted to satisfy the conditions of spatial focusing and flight time focusing. Graph 160 shows the types of axial potential distribution U (x) that meet these requirements. To provide spatial focusing along the Z direction, each of the electrostatic mirrors 153 forms a focused lens near the central plane of the free flight zone (indicated by the dashed line). The ion beam (line 159) starts as a parallel beam at the entrance window 156. After the first reflection on the right mirror, the beam is focused on a point in the middle plane. It should be noted that the focusing of all ions is shown in the drawing by the orbit of a single ion intersecting the axis. After reflection on the left-hand mirror, the beam is converted back into a parallel beam.

According to the inventor's simulation of the ion optics using the SIMION program, the spatial focusing at a particular TOF 1 151 is compatible with at least the first-order flight time focusing, that is, orthogonal to the original energy. The first derivative of flight time in displacement is equal to zero. The ion beam is confined only if the initial spatial diffusion is less than 5% of the width of TOF1 and the angular diffusion is less than 2 degrees. With energy diffusion below 3%, TOF1 has a flight time resolution of over 10,000. Such an initial condition is after pulse ejection from a linear accumulating quadrupole.<u style="single">Approximately 30 eV</u>It is realistic for an ion beam that is accelerated to.

Operation at relatively high energies (30-100 eV) compared to other examples requires a long ion path (30-100 m) at TOF1 to achieve millisecond timescale separation at TOF1. And. The ionic pathway is easily extendable due to the low design complexity of TOF1 and its static operation. A 1 m long device with about 20 full ion turns corresponds to an effective flight path of at least 50 m.

Referring to FIG. 16, another embodiment of the present invention is a modified electrostatic multipath separator formed by folding a two-dimensional field into a cylindrical field.<u style="single">Use</u>.. In this embodiment, it is called a cylindrical multipath separator 161 for its compact design, and each elongated electrode is converted into a pair of coaxial cylinders, i.e. a pair of inner and outer cylinders. Separator 161 includes a free flight channel formed by cylinders 162, 163, and two electrostatic mirrors including focusing cylinder 164 and reflecting cylinder 165. The outer cylinder of the free flight channel 162 has inlet and exit windows 166 and comprises a beam deflector 170. The pulsed ion beam enters through a pair of spatially focused lenses 167 and steering plate 168. It is introduced into the separator 161 through the window 166 and the deflector 170. The ionic pathway is indicated by line 169.

In operation, the cylindrical separator is very similar to the two-dimensional electrostatic multipath separator described above. The ions are spatially focused by the electrodes of the lens while causing multiple bounces between the mirrors. Additional potentials are applied between the external and internal cylinders 162 and 163 to keep the ions close to orbits of the same radius. Radial deflection potentials may be applied between the outer and inner cylinders of electrodes 164 and 165.

Ion inlets and outlets can be organized in multiple ways. Figure<u style="single">16</u>Shows an example of ion introduction through a slit-shaped window 166B with a subsequent horizontal deflection that aligns the ion beam along the X axis. The deflector 170B is surrounded by a mesh to reduce the fringe field. Figure<u style="single">16</u>Is an example of the introduction of ions along the X axis through the cutout of the entire segment of a cylindrical analyzer.<u style="single">Also</u>Shown. The beam is injected into the analyzer after horizontal deflection by plate 170C. Field distortion is minimized by using double-sided PCBs, equipotentials within the cutout, and has a dispersed potential on the side oriented towards the cylindrical analyzer. The electrostatic multipath separators described above include the pulse ion sources, fragmentation cells, and high speed described above.<u style="single">TOF2</u>Proposed for use in the comprehensive tandem TOF analyzer of the present invention in various combinations with.

The illustrated examples of TOF1 separators, including separators with RF confinement, spiralons and static multipath separators, provide all possibilities for TOF1 to retain an ion beam while providing extended time separation. Rather, it demonstrates the feasibility of the general method of comprehensive tandem TOF mass spectrometry of the present invention.

The tandem TOF analyzer of the present invention described above improves the speed and sensitivity of analysis as compared with existing TOF-TOF mass spectrometers. This improvement is achieved by using the principle of time-nested acquisition, which applies to tandem TOFs for the first time. Ion pulses from ion sources are fully utilized and multiple parent ions are analyzed per single source pulse. The present invention also improves the speed of MS-MS information as compared to the closest basic form of IMS-TOF, which also uses time-nested acquisition. Improvements are made by gaining higher resolution in the parent ion separation step, resulting in more complex mixture analysis.<u style="single">To.</u>

Tandem MS-MS analysis<u style="single">Provided by</u>High speed combines multi-step liquid phase separation with tandem MS analysis on a realistic timescale<u style="single">big</u>Provide an opportunity. Such separation technology<u style="single">Ion exchange separation</u>, Binding separation, liquid phase chromatography (LC) and capillary electrophoresis (CE).<u style="single">short</u>High-speed LC and CE separation on a time scale is common in LC-MS analysis. However, LC-MS-MS analysis is usually slowed down by the low speed of the MS-MS stage, but the comprehensive TOF-T of the present invention. Not applicable after introducing OF methods and equipment.

Some examples of combining useful elements<u style="single">With</u>Of this invention<u style="single">Various</u>Having described the embodiments, it will be apparent to those skilled in the art that other embodiments incorporating this concept may also be used. Therefore, these examples should not be limited to the disclosed examples, but only by the spirit and scope of the claims.

<figref num="1">It is a block diagram which shows the method of this invention.</figref><figref num="2">It is a timing diagram of the operation of the tandem TOF-TOF mass spectrometer.</figref><figref num="3">It is a schematic diagram of a new inline TOF1.</figref><figref num="4">It is a schematic diagram of a new W type TOF1.</figref><figref num="5">It is the schematic of the vacuum pulse MALDI ion supply source.</figref><figref num="6">It is the schematic of the pulse MALDI ion supply source using collision attenuation.</figref><figref num="7">It is a schematic diagram of the continuous ion supply source provided with a pulse accumulation quadrupole.</figref><figref num="8">It is a schematic diagram of a CID cell.</figref><figref num="9">It is a schematic diagram of a SID cell.</figref><figref num="10">It is a schematic diagram of orthogonal TOF2.</figref><figref num="11">It is a schematic diagram of the coaxial TOF2.</figref><figref num="12">It is a schematic diagram of TOF-TOF provided with in-line TOF1 and CID cell.</figref><figref num="13">It is a schematic diagram of TOF-TOF provided with W type TOF1 and SID cell.</figref><figref num="14">It is a schematic diagram of a TOF-TOF equipped with a static coaxial TOF1.</figref><figref num="15">It is the schematic of the electrostatic multipath TOF1 of a plane.</figref><figref num="16">It is the schematic of the cylindrical electrostatic multi-pulse TOF1.</figref>