WO2002017349A1

WO2002017349A1 - Two-dimensional tofms for desorption ion sources

Info

Publication number: WO2002017349A1
Application number: PCT/AU2001/001049
Authority: WO
Inventors: Michael Guilhaus; Mark James Lewin
Original assignee: Unisearch Ltd
Current assignee: Unisearch Ltd
Priority date: 2000-08-24
Filing date: 2001-08-23
Publication date: 2002-02-28
Anticipated expiration: 2003-02-24
Also published as: AUPQ963600A0

Abstract

A reflecting MALDI oa-TOFMS instrument is provided, having a pulsed correcting lens to reduce expansion of the ion plume desorbed from the target prior to orthogonal acceleration. The Mass Spectrometer has a MALDI Source including a target (11), upon which samples are deposited, an orthogonal accelerator (12) and a mirror (16) located to deflect ions towards the detector (17). A mask (18), is located between the MALDI Sample Target (11) and the orthogonal accelerator (12), to limit the spread of ions in the orthogonal acceleration direction in the orthogonal acceleration chamber prior to the acceleration pulse being applied. A pulsed lens (19) is included between the MALDI Sample Target (11) and the mask (18). The lens comprises a plurality of cylindrical elements located axially along a path of desorbed ions travelling from the sample target (11) to the orthogonal accelerator (12). A high voltage pulse is applied between the elements of the lens as the plume travels through the lens to produce a gradient field which acts todifferentially accelerate the ions in the plume to reduce expansion of the plume prior to entry into the orthogonal accelerator.

Description

Two-dimensional TOFMS for desorption ion sources

Introduction

The present invention relates generally to the field of Mass Spectrometers and in particular, the invention provides a new pulsed source Mass Spectrometer configuration.

Embodiments of the invention offer potential strategic advantages of higher mass accuracy, simplified mass calibration and facilitate improved compactness in bench top instrumentation for highly topical and economically significant genomic and proteomic applications, as well as other applications that require accurate mass measurement of involatile compounds.

Biotechnology companies increasingly recognise that mass spectrometry is the best analytical solution to the problem of measuring the purity of their products, but are reluctant to invest in the current electrospray and MALDI instruments because they are cumbersome and require skilled operators. Background to the Invention

The Time-of-Flight Mass-Spectrometer (TOFMS) of the present invention provides an alternative approach to currently available instrumentation such as conventional delayed extraction MALDI TOFMS and electrospray MS for the analysis of a wide range of materials particularly biopolymers.

In recent years, Matrix Assisted Laser Desorption/Ionisation (MALDI) has become an accepted tool for the bioanalytical chemist. It is also well suited to many other types of molecules outside the realm of biochemistry. While the advent of delayed extraction (DE) approaches has brought exceptional improvements in resolving power and sensitivity to MALDI, there remain some areas where further improvements are highly desirable. One such area is that of the predictability of the time-to-m/z relationship which, with DE MALDI is subject to the specific conditions of laser desorption energy and delay because the desorption axis and TOF axis are the same. This is most clearly evident in the need, for high mass accuracy, to calibrate the instrument with internal standards or at least place the calibrant compound in close proximity to the sample on the probe and alternate between acquiring the spectrum of the sample and that of the standard. This significantly complicates the analysis and decreases confidence of the measurement and, most importantly, is a limiting factor when large numbers of samples need to be analysed quickly.

In MALDI it is often difficult to analyse mixtures and the mixing of internal standard and sample can lead to suppression of sample signals and increased heterogeneity of the sample target. The resolution of reflecting DE MALDI instruments is affected by large desorption velocity spread and the corresponding effectiveness (or limit in effectiveness) of the ion mirror to deal with it. To this end a number of groups have reported some limited success in decoupling of the desorption and TOF velocities by employing the principles of orthogonal acceleration to the MALDI ion source. Early attempts to do this yielded very poor results in terms of resolution and sensitivity.

Researchers at UNS W were first to demonstrate a linear Orthogonal Acceleration Time of Flight (oa-TOF) instrument with a resolving power similar to that attainable with comparable linear DE MALDI instruments (m/Δm = 4000) V. Mlynski and M. Guilhaus, Rapid Commun. Mass Spectrom., 10, 1524, (1996).

However this was limited in mass range due to the low acceptance angle of the small detector and the low ion energy used. Another group are known to have pursued an approach involving collisional focusing to reduce the desorption velocity spread. Summary of the Invention

The present invention consists in a Time of Flight Mass Spectrometer (TOFMS) comprising a pulsed ion source which projects a packet of ions along a first path toward an orthogonal acceleration means, packet conditioning means for conditioning the spatial distribution of the ions, the orthogonal acceleration means being arranged to impart an accelerating force on the packet of ions in a second direction orthogonal to a direction of the first path, a flight path extending from the orthogonal accelerating means to a detector, the flight path originating from the orthogonal accelerating means in a second direction defined by the vector sum of an initial average velocity component in the direction of the first path of the packet when it enters the orthogonal accelerating means and an average orthogonal velocity component imparted to the packet by the orthogonal accelerating means, and time of flight monitoring means to monitor a time of flight of ions from the orthogonal accelerating means to the detector, the TOFMS characterised in that the conditioning means is located between the source and the orthogonal acceleration means and includes a pulsed lens for reducing or reversing the expansion of the packet of ions in the direction of the first path.

In a preferred embodiment of the present invention, the conditioning means applies a force to each ion in the packet which is a function of the distance travelled from the source in the first direction, such that those ions that have travelled furthest from the source will be advanced the least and those ions that have travelled the shortest distance from the source will be advanced the most, relative to a notional centre of the packet of ions, to thereby reduce or reverse the expansion of the packet of ions.

Preferably, the conditioning means also includes means for limiting the spread of ions about an axis in the direction of the first path.

Preferably also, embodiments of the 2D-TOFMS will include spatial focusing to limit the effect of small deviations of ions from the axis of the first path when the orthogonal acceleration means is activated.

In a preferred embodiment, the pulsed lens includes a planar element adjacent to the source with an orientation normal to the first path, the planar element having an aperture through which the path extends, a first cylindrical element having a central passage extending there through, the central passage being coaxial with the first path and having one end located substantially adjacent to the planar element, the first cylindrical element being held at a reference potential (typically ground potential), and the planar element being pulsed with a voltage to differentially accelerate a packet of ions created at the source. The preferred embodiment may also have a second cylindrical element having a central passage extending there through, the central passage being coaxial with and extending from the open end of the first element towards the orthogonal acceleration means, and a third cylindrical element having a central passage extending there through, the central passage being coaxial with the central passage of the first and second cylindrical elements and extending from an open end of the second cylindrical element furthest from the source, the central passage of the third cylindrical element converging in width with increasing distance from the source, the third cylindrical element being held at the reference potential and the second cylindrical element having a voltage applied to redirect divergent ions in the packet of ions travelling from the source so that their trajectories enter the orthogonal acceleration means.

Preferably, the diameter of the aperture in the planar element is smaller than the diameter of the passage in the first cylindrical element. Preferably also, the first cylindrical element includes an inwardly directed flange located at an end of the passage closest to the planar element. Preferably, the diameter of the passages in the first and second cylindrical elements are substantially the same and the passage in the third cylindrical element has a first diameter at an end closest the second cylindrical element which is substantially the same as the diameter of the first cylindrical element and decreases towards its other end.

In a second, less preferred embodiment, the pulsed lens comprises a first cylindrical element substantially toroidal in cross-section and coaxial with the first path and having one end, located, substantially adjacent to the source, a second cylindrical element substantially toroidal in cross section and coaxial with and extending towards the orthogonal acceleration means from an open end of the first element, a pulsed voltage source being connected between the two cylindrical elements to differentially accelerate a packet of ions travelling from the source. The first cylindrical element in the second embodiment includes an inwardly extending flange located at an end closest the source and defining an aperture through which the ion packet is projected, the diameter of the aperture being smaller than the diameter of the passage through the first element. Preferably, the diameters of the passages in the first and second cylindrical elements are substantially equal.

It is also possible to implement the invention using other configurations that are not necessarily cylindrical or coaxial in configuration.

Examples of other possible configurations include, multipole lenses and planar lenses having a slit geometry.

The preferred embodiment will also include hybrid detection recording systems connected to the detection means, including an integrating transient recorder and a time-to-digital recorder.

Any discussion of documents, acts, materials, devices, articles or the like which has been included in the present specification is solely for the purpose of providing a context for the present invention. It is not to be taken as an admission that any or all of these matters form part of the prior art base or were common general knowledge in the field relevant to the present invention as it existed in Australia before the priority date of each claim of this application. Throughout this specification the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps. Brief Description of the Drawings

Embodiments of the invention will now be described by way of example, with reference to the accompanying drawings in which:

Figure 1 is a block schematic diagram showing a MALDI oa-TOF (a) without 2D-TOF correction and (b) with 2D-TOF correction with a pulsed lens;

Figure 2 is a graphical representation of a non-linear potential function along the axis of the pulsed lens of figure 1(b);

Figure 3 is a MALDI oa-TOF mass spectrum of Myoglobin by (a) 100 shot acquisition without a 2D-TOF pulsed lens, and (b) 50 shot acquisition with a 2D-TOF pulsed lens;

Figure 4 is a more detailed schematic diagram of the 2D-TOF system;

Figure 5 is a detailed schematic diagram of a preferred embodiment of the 2D-TOF pulsed lens of the system of figure 4;

Figure 6 is a detailed schematic diagram of a simplified embodiment of the 2D-TOF pulsed lens of the system of figure 4;

Figure 7 is a MALDI oa-TOF mass spectra of melittin without using a 2D-TOF pulsed lens;

Figure 8 is a MALDI oa-TOF mass spectra of insulin without using a 2D-TOF pulsed lens; and Figure 9 is a timing diagram showing the relationship between the voltage pulses that drive the lens and orthogonal accelerator in the 2D-TOFMS of figure 4. Detailed Description of the Preferred Embodiments

Referring to figure 1, two reflecting MALDI oa-TOFMS instruments are illustrated. Figure 1(a) shows a simple reflecting MALDI oa-TOF instrument, while Figure 1(b) shows an oa TOF instrument with a 2D-TOF having a pulsed correcting lens by way of comparison.

The Mass Spectrometers of figures 1(a) and 1(b), each have a MALDI Source including a target 11 upon which samples are deposited, an orthogonal accelerator 12 comprising a pulsed plate 13 and a series of grids 14, a mirror 16 located to deflect ions towards the detector 17. A mask 18, is located between the MALDI Sample Target 11 and the orthogonal accelerator 12, to limit the spread of ions in the orthogonal acceleration direction in the orthogonal acceleration chamber prior to the acceleration pulse being applied. In the device of Figure 1(b), a pulsed lens 19 is also included between the MALDI Sample Target 11 and the mask 18.

The Figure 1(a) instrument has been shown to give close to the predicted limit of resolving power of about m/Δm = 10000. However it was limited at higher mass with velocity correction (albeit a much higher mass than earlier linear oa-TOF instruments). The restricted sensitivity at high mass was attributed to the large velocity spread acquired between the MALDI Sample Target 11 and the orthogonal acceleration chamber 12 by ions much heavier than the matrix. As a result of this observation, a solution to the coupling of MALDI with oa-TOF has been devised using the pulsed lens 19. The approach that is shown in Figure 1(b), does not require collisional focusing. Narrowing the Velocity Spread in the Desorption Axis

For analyte ions of a particular mass, a substantial spread of desorption velocities usually arises from ion/molecule collisions in the expanding plume of the desorbed matrix. The associated spread in analyte ion kinetic energy increases with mass if the analyte ions adopt a velocity spread similar to that of the lighter matrix molecules. The expansion of the plume of analyte ions in the desorption axis may be large enough, e.g., for high mass ions, so that substantially less than 100% of the ions fall on the detector after they pass through the oa-TOF mass analyser. It is therefore highly desirable if this energy spread could be minimised and the mean energy controlled at a value that gives rise to a spontaneous drift trajectory that is incident upon the chosen detector (M. Guilhaus, Spontaneous and Deflected Drift-Trajectories in Orthogonal Acceleration Time-of-Flight Mass Spectrometry, J. Am. Soc. Mass Spectrom., 5 (1994) 588-595). This is a well-known problem in oa- TOFMS with atmospheric pressure ion sources, which similarly suffer from the effects of ion/molecule collisions.

A popular solution to the problem of primary beam velocity spread is to use a collisional focusing device such as an RF-only multipole operating in a region of elevated collision gas pressure. Such devices, while generally effective, require a more complicated differentially pumped vacuum system as well as RF power supplies and carefully designed optics at the boundaries of the device to transfer ions into and away from the collision gas region. Embodiments of the present invention employ a different method of reducing the desorption velocity spread for a finite range of m/z. The method is based on the principle that ions desorbed at the same time will separate spatially according to their desorption velocities. This same technique is used in delayed extraction (DE) MALDI TOFMS, primarily to improve resolving power. In DE MALDI the correlation is used to bring ions to a sharp time-focus at a TOF detector. By contrast, in embodiments of the present invention, the correlation is used with a specifically shaped accelerating field to bring ions of a particular m/z to a final kinetic energy that is nearly independent of the desorption velocity. Such a condition leads to higher duty cycle efficiency because all or most of the ions will have the correct trajectory in the oa-TOF mass analyser to reach the detector. With moderately large detectors, as are typically used in oa-TOFMS instruments, the condition set for ions of a particular m/z will provide significant duty cycle advantages over a larger range of masses. With such an approach the sensitivity of the oa-TOF MALDI combination is improved and it is possible to take advantage of the mass accuracy and resolving power advantage of the oa-TOF mass analyser. Unlike DE MALDI, the desorption and mass analysis velocities remain essentially decoupled and this provides a basis for better mass accuracy especially at high mass. The independence for desorption and TOF velocity simplifies calibration needs which, in many cases, will not require an internal mass calibrant.

Embodiment of the present invention make use of an arrangement which provides simultaneous combination of a crude TOF analysis in the desorption direction with high-resolution TOF analysis in the orthogonal direction. For simplicity, this approach has been referred to as two dimensional (2D) TOFMS. Figure 1 illustrates this principle by showing the effect of velocity spread on a packet of high mass ions with and without the

2D-TOF delayed acceleration.

Implementation of 2D-TOF with a Non-Linear Field

The principle of narrowing the velocity spread is based on a delayed acceleration of desorbed ions in a non-linear field. For example, with reference to Figure 2, an ion of mass m and charge q desorbs with velocity u₀ and kinetic energy U₀ and travels a distance s_x along a field-free desorption axis in time τ_v At time τ_x a voltage V_x0 is applied to the first element of the extraction lens 19 and an associated electric field is generated along the desorption axis. The lens 19 is engineered so the potential, V_x, along the desorption axis is given approximately by

^sι and the initial energy of the ion is

T T _L 2 t ₀ = 2 ^mU0 while the position of the ion along the desorption axis will be

s_x = u₀τ_l The potential to which the ion is elevated when the field is created is

and the energy gained after τ , as the ion is accelerated to a field-free region at zero potential, is qV_x and thus the total kinetic energy of the ion will be

U_λ = ^ m ² ₀ + qV_x which, upon substitution and rearrangement may be written as

C _t = ^_o +( -^)^ (1) Thus, setting the condition that the coefficient of u₀ ² is zero in equation 1 gives rise to a final kinetic energy that is independent of u₀ and equal to qV_P. This condition is met by setting the delay time according to

, - ^y (2) Equation 2 shows that the correction is mass dependent but it is not a dependence that is critical. That is, when the condition is set, the transmission of the ions to the detector 17 is enhanced over a range of mass that is reasonably large and scales with the size of the detector. Moreover, if necessary, it is relatively simple to acquire data in ranges where each is optimised for maximum transmission. However, the most frequent and important experiments targeted by the preferred embodiments are for the accurate mass measurement over a relatively small mass range.

The creation of the shaped field is relatively simply achievable and will be described with reference to a preferred embodiment illustrated in Figure 5. This embodiment includes a planar element 125 located adjacent to the sample target 11, and having an aperture 150 substantially coaxial with a nominal first ion path 52 extending from the sample target 11. A first cylindrical element 123 is located adjacent to the planar element 125 and has a passage 151 extending through its centre substantially coaxially with the first ion path 52 and the aperture 150. The first cylindrical element includes an inwardly extending annular flange 124 which partially closes the passage 151 at an end close to the sample target 11. The first cylindrical element 123 is held at the system reference potential (typically ground), and a voltage pulse V_P is applied between the first cylindrical element 123 and the planar element 125 to preserve the state of compression of the packet of ions travelling from the sample target 11 along the path 52. The voltage pulse V_P is applied after a delay τ_x from the firing of the laser such that the ions desorbed from the sample target 11 have sufficient time to travel into the lens 19 before the field caused by the pulse V_P is created

The focusing pulses N_p and N₂ of the lens 19, and the pulse produced by the power supply 27 for the orthogonal accelerator 12, are usually of the same polarity as that of the ions being examined. That is, for positive ions the focusing pulses and the orthogonal accelerating pulse are positive and for negative ions the pulses are negative.

The ions desorb (initially) in field free space, and when the focusing pulses are applied, a field rapidly propagates, such that the ions are subjected to a descending potential function. Therefore, for a positive focusing pulse, positive ions will be accelerated towards the orthogonal accelerator 12, but to different degrees depending on how high up the potential slope they are located. For negative ions, a negative pulse is used and therefore all of the potentials are inverted to produce the same effect. Referring to Figure 9 a timing diagram is illustrated in which the beginning of the initiation period 160 initiates the laser shot 161, slightly delayed due to laser characteristics. The delay τ_x is commenced by the same initiating signal that initiates the laser and at the end of the delay τ_x the focusing pulse 162 is generated by the power supply 28. The delay τ₂ is also initiated at the same time as the laser firing and the orthogonal acceleration pulse 163 is initiated at the end of this delay.

While the lens 19 can be designed to provide the correct non-linear potential function along its symmetry axis, a fortuitous advantage exists as a result of the associated potential function off the axis. The curvature here assists with the direction focusing of the diverging plume of ions from the MALDI event. This adds to the advantage of narrowing the velocity spread but can introduce increased spatial spread and velocity spread in the TOF direction. This will manifest in some reduction in resolving power however, the effect can be kept small, with optimisation of the pulsed lens 19.

To this end, further focusing is provided by the second and third cylindrical elements 122, 121 which each also have a passage 153, 154 through their respective centres, coaxial with the passage 151 through the first cylindrical element 123 and the first ion path 52. The passage 153 in the second cylindrical element 122 is of the same or similar diameter to the widest portion of the passage 151 in the first cylindrical element 123. The third cylindrical element 121 is longer (in the axial direction of the passage) than the first and second cylindrical elements 123, 122 and the passage 154 in the third cylindrical element reduces in diameter with the increasing distance from the sample target 11. In the preferred embodiment, the passage 154 is approximately 10% greater in diameter than the diameter of the passage 153 in the second cylindrical element, at its end closest the second element, and approximately 40% of the diameter of the passage 153 at its end furthest from the second element. The passage 154 is of substantially constant diameter for approximately 10% of its length at its narrowest end.

The third cylindrical element 121 is held at the same system reference potential as the first cylindrical element 123, whereas the second cylindrical element has a voltage N₂ applied to it. The combination of the shape of the passage 153 in the third cylindrical element 121 and the characteristics of the voltage applied between the second and third cylindrical elements 122, 123 serves to limit the spread of ions away from the axis of the lens. A positive pulse is applied to the direction focusing electrode 122 (for the case of positive ions) to redirect ions that are diverging back towards the average (i.e. central) path. In a second embodiment, illustrated in figure 6, a simplified arrangement is illustrated in which a first cylindrical lens element 22 extends from the sample target 11 and has a passage 51 extending through its centre. One end of the passage 51 closest to the sample target 11 is partially closed by an annular flange 24. The passage 51 is substantially coaxial with the nominal first ion path 52 extending from the source target 11. The open end 53 of the first cylindrical element 22 is positioned adjacent to a second cylindrical 21 element which also has a passage 54 extending through its centre, coaxial with the first cylindrical element 22. The second cylindrical element 21 is held at a reference potential for the system (typically, ground potential) while a voltage pulse V_P is applied to the first cylindrical element 22.

Referring to Figure 4, the preferred embodiment of the present invention is a reflecting TOFMS. The entire optics of the instrument is 85 cm in length. It is thus a relatively compact device. The resolving power increases with m/z due to the (un-correlated) finite detector pulse width and jitter but an intrinsic instrument resolution (perfect detector and/or extremely high m/z) is about 10,000 (fwhm). The instrument gives, for ions up to about 5,000 Da, excellent sensitivity and resolution without 2D-TOFMS applied. This is because the large detector accommodates the energy spread. Figures 7 and 8 show typical results that exemplify this performance. Note that the resolution indicated for the relatively low mass of Melittin is an impressive 9100 fwhm for a relatively small instrument.

The environment in which the preferred embodiment of the invention has been tested is an experimental test bed and the following details are given by way of example only and are not crucial to the invention. The chamber is pumped by a 240 Ls^"1 turbomolecular pump. The probe port (not shown) allows 25 mm diameter devices 25 having a target disk 26 to be introduced and rotated. The CCD camera 41 has a long focal length zoom lens which provides a 3mm field of view. This enhances the ability to select and target suitable sample regions 11 on the target disk 26. The push-out pulse driver 27, is a 1200 V monopolar device and a similar device 28 is used to generate the approximately, 200N V_P pulses with separate controlled amplitudes to the lens element 123, and the target 11 in the 2D-TOF mode. A further power supply (not shown) is used to supply a voltage to the lens elements 121, 122. The detector assembly 17 contains a 70 mm diameter non-imaging focal plane detector 29 operating with a 3 kN bias for fast electron transfer to the grounded anode. The anode is butted onto a 50Ω transmission line 31 creating a deliberate impedance mismatch that generates ringing frequencies well above the bandwidth of the measuring device. Single or multiple firing of the laser and the τ₂ delay 62 are controlled by a software 32 developed with LabNIEW and utilising National Instruments I/O and IEEE boards (not shown) in a 300 MHz desktop computer 33 (note, the r₂ delay is provided by a function within the desktop computer). The IEEE interface supports control and data acquisition via a LeCroy 9384 integrating transient recorder 35 capable of digitising at rates up to 4

Gsample s^"1 (250 ps resolution) with a bandwidth of 1GHz. The software facilitates a range of data processing and presentation functions including: raw time spectrum acquisition; calibration and conversion to mass spectrum; filtering; scaling; mass range selection; zooming; integration of selected peak areas; and export of data in a variety of formats for external graphing and further manipulation.

The τ_t delay 61 is generated with a TTL pulse generator controlled by a precision timing device. Signals from the detector are measured simultaneously with the LeCroy ITR system 35 and a Fast ComTec time-to- digital converter 34.

Most MALDI instruments use integrating transient recorders. These are limited in dynamic range and are generally inefficient under conditions of low count-rates per time-bin. Here the internal noise of the digitiser adds nearly as quickly as the signal and no real S/N advantage is obtained by signal averaging. At high mass, the natural isotopic dilution of the TOF signal (many combinations of isotopic mass for the same chemical structure) together with the broadening of the TOF peaks over an increasing number of time-bins, leads to a progressive decline in 'detectability' of high mass species. This is not helped by the usual decrease in detector efficiency as mass increases. Under low count-rate conditions, the current best technological solution for ion detection is to use a time-to-digital converter. Such devices record only the time of ion arrival events along with the time-bin. Multiple ion events are seen as one (hence they are useful only at low counts/bin). However, these devices are null-data suppressing and are available with excellent time-resolution. A TDC based system is entirely inappropriate for MALDI at low mass where the ion count rates per bin are very high. This creates a serious dynamic range problem - ITRs are unsuitable at high mass while TDCs are not useful at low mass. The approach used in the preferred embodiment makes use of an ITR and a TDC in parallel and provides improved dynamic range in the difficult time frame of the ion abundant measurement window produced by a MALDI TOF device.

As the TDC 34 demands pulses higher in amplitude than the raw (c.lOmN) pulses from a plate detector, a preamplifier 36 is provided to boost the signal from the detector. As the ITR 35 will also benefit from higher signal levels, a dual output preamplifier has been chosen.

The timing of the firing of the laser 37 to initiate desorption of the sample and output of pulse generators 27 and 28 to control travel of the desorbed ions to the detector 17, is controlled by the instrument control software 32 running on the computer 33. The laser beam 38, 138 is directed onto the target 26 via an attenuator 69 and a mirror 39 having a directional characteristic (similar to that of a half silvered mirror) such that the laser light is reflected on to the target, but, most of the light from the target passes through the mirror to the CCD camera 41 located behind the mirror 39 enabling the camera 41 to view the target, and thereby enabling accurate placement of the laser beam 38 onto specific regions of a sample 11 located on the target 26.

When a packet or plume of ions (63 in Figure 1) is desorbed from the sample 11 by a laser pulse, it drifts through the lens 19, where spatial focusing is performed as described above, and into the orthogonal accelerator 12. When it reaches the desired location 64 in the orthogonal accelerator 12 a push-out pulse is applied to electrode 13 of the orthogonal accelerator and the packet of ions is accelerated along a second path 65 towards an ion mirror 16. The packet is reflected by the mirror 16 along a third path 66 towards the target 17. In some instances it is desirable to perform measurements without the mirror 16 active, in which case a second target 67 is employed to detect the ions after they pass through the mirror 16. The second target 67 is also connected to the pre-amplifier 36 via a transmission line 68. Experimental Results

As stated earlier, MALDI oa-TOFs have previously been critically limited in sensitivity at high m/z because of the large desorption velocity spread of high mass ions. Prior to implementation of the present invention, and despite many arduous attempts, it has not been possible to observe good signals for the reference protein Myoglobin (Mass= c. 17,000 Da) using the MALDI oa-TOF to which the present invention has been applied. Figure 3a shows the best signal obtained without the implementation of the 2D-TOF technique. The signal is just discernible above the noise and would by most definitions be below the detection limit of 3 standard deviations of the noise. Figure 3b shows the corresponding data obtained using an embodiment of the present invention. The signal-to-noise ratio is excellent for a few tens of laser shots and this represents a dramatic improvement on what hitherto was more-or-less not detectable.

It will be appreciated by persons skilled in the art that numerous variations and/or modifications may be made to the invention as shown in the specific embodiments without departing from the spirit or scope of the invention as broadly described. The present embodiments are, therefore, to be considered in all respects as illustrative and not restrictive.

Claims

CLAIMS:

1. A Time of Flight Mass Spectrometer (TOFMS) comprising a pulsed ion source which projects a packet of ions along a first path toward an orthogonal acceleration means, packet conditioning means for conditioning the spatial distribution of the ions, the orthogonal acceleration means being arranged to impart an accelerating force on the packet of ions in a second direction orthogonal to a direction of the first path, a flight path extending from the orthogonal accelerating means to a detector, the flight path originating from the orthogonal accelerating means in a second direction defined by the vector sum of an initial average velocity component in the direction of the first path of the packet when it enters the orthogonal accelerating means and an average orthogonal velocity component imparted to the packet by the orthogonal accelerating means, and time of flight monitoring means to monitor a time of flight of ions from the orthogonal accelerating means to the detector, the TOFMS characterised in that the conditioning means is located between the source and the orthogonal acceleration means and includes a pulsed lens for reducing or reversing the expansion of the packet of ions in the direction of the first path.

2. The mass spectrometer as claimed in claim 1 or 2 wherein, the conditioning means applies a force to each ion in the packet which is a function of the distance travelled from the source in the first direction, whereby those ions that have travelled furthest from the source will be advanced the least and those ions that have travelled the least distance from the source will be advanced the most, relative to a notional centre of the packet of ions, to thereby reduce or reverse the expansion of the packet of ions.

3. The mass spectrometer as claimed in claim 1 or 2 wherein, the conditioning means further comprises means for limiting the spread of ions about an axis in the direction of the first path.

4. The mass spectrometer as claimed in claim 1, 2 or 3 further comprising spatial focusing means to limit the effect of small deviations of ions from the axis of the first path when the orthogonal acceleration means is activated.

5. The mass spectrometer as claimed in any on of the preceding claims wherein, the conditioning means comprises a pulsed lens including a planar element adjacent to the source with an orientation normal to the first path, the planar element having an aperture through which the path extends, a first cylindrical element having a central passage extending there through, the central passage being coaxial with the first path and having one end located substantially adjacent to the planar element, the first cylindrical element being held at a reference potential, and the planar element being pulsed with a voltage to differentially accelerate a packet of ions created at the source.

6. The mass spectrometer as claimed in claim 5 wherein, the reference potential is ground potential.

7. The mass spectrometer as claimed in claim 5 or 6 wherein, the diameter of the aperture in the planar element is smaller than the diameter of the passage in the first cylindrical element.

8. The mass spectrometer as claimed in claim 5, 6 or 7 wherein, the first cylindrical element has an inwardly directed flange located at an end of the passage closest to the planar element.

9. The mass spectrometer as claimed in claim 5, 6, 7 or 8 wherein, the pulsed lens further comprises a second cylindrical element having a central passage extending there through, the central passage being coaxial with and extending from the open end of the first element towards the orthogonal acceleration means, and a third cylindrical element having a central passage extending there through, the central passage being coaxial with the central passage of the first and second cylindrical elements and extending from an open end of the second cylindrical element furthest from the source, the central passage of the third cylindrical element converging in width with increasing distance from the source, the third cylindrical element being held at the reference potential and the second cylindrical element having a voltage applied to redirect divergent ions in the packet of ions travelling from the source so that their trajectories enter the orthogonal acceleration means.

10. The mass spectrometer as claimed in claim 9 wherein, the diameter of the passages in the first and second cylindrical elements are substantially the same and the passage in the third cylindrical element has a first diameter at an end closest the second cylindrical element which is substantially the same as the diameter of the first cylindrical element and decreases towards its other end.

11. The mass spectrometer as claimed in any one of claims 1 to 4 wherein, the conditioning means comprises a the pulsed lens including a first cylindrical element substantially toroidal in cross-section and coaxial with the first path and having one end, located, substantially adjacent to the source, a second cylindrical element substantially toroidal in cross section and coaxial with and extending towards the orthogonal acceleration means from an open end of the first element, a pulsed voltage source being connected between the two cylindrical elements to differentially accelerate a packet of ions travelling from the source.

12. The mass spectrometer as claimed in claim 11 wherein, the first cylindrical element has an inwardly extending flange located at an end closest the source and defining an aperture through which the ion packet is projected, the diameter of the aperture being smaller than the diameter of the passage through the first element.

13. The mass spectrometer as claimed in claim 11 or 12 wherein, the diameters of the passages in the first and second cylindrical elements are substantially equal.

14. The mass spectrometer as claimed in any one of the preceding claims wherein, the time of flight monitoring means comprises a hybrid detection recording system connected to the detector, the hybrid detection recording system comprising an integrating transient recorder and a time-to-digital recorder.