Radiant energy – Ionic separation or analysis – Ion beam pulsing means with detector synchronizing means
Reexamination Certificate
2001-03-14
2004-08-24
Lee, John R. (Department: 2881)
Radiant energy
Ionic separation or analysis
Ion beam pulsing means with detector synchronizing means
C250S281000, C250S282000, C250S288000
Reexamination Certificate
active
06781121
ABSTRACT:
FIELD OF INVENTION
This invention relates to time-of-flight mass spectrometers, in which the mass/charge ratio (m/z) of a number of ions can be deduced from the time taken for the ions to be propelled a given distance by a suitable accelerating field such as an electrostatic field.
BACKGROUND TO THE INVENTION
One example of a time of flight spectrometer is a Matrix Assisted Laser Desorption Ionisation Time of Flight (MALDI-TOF) spectrometer.
Such a spectrometer is commonly used to analyse high molecular weight substances, particularly biochemicals, and uses a short pulse of laser energy to eject and ionise molecules of an analyte from crystals which are held within a matrix formed from small organic molecules absorbent to the incident laser radiation. The matrix resonantly absorbs this radiation which accordingly causes an ablation of a region of the matrix, analyte mixture, and the formation of an expansion jet above the surface within which jet ion/molecule reactions take place. Molecules and ions can be ejected from the matrix with various different kinetic energies.
The ions so created are then accelerated towards a detector, normally by an electrostatic field. The kinetic energy KE, of a particle of mass m travelling at a velocity v is given by the equation:
KE
=
1
2
mv
2
.
Thus, each particle travels with the velocity rated to its mass by equation:
v
=
2
KE
m
Accordingly, if a pulse of ions is formed, defining time zero, and travels some distance to the detector, then the lighter the ion the earlier it will arrive at the detector. Consequently, the detector signal as a function of time will represent a m/z spectrum of particles of a given charge.
Time of flight mass spectrometers have two important advantages over other types of mass spectrometer:
1. They have a very high sensitivity because most of the ions produced will be detected at the detector, in contrast to scanning spectrometers in which only ions of a given m/z are focussed on the detector at any one time; and
2. A very large mass range, limited only by the efficiency of the detection of high mass ions, can be achieved by a TOF spectrometer.
However, one of the most important factors which limits the accuracy of mass measurements obtained by a MALDI-TOF spectrometer is the variation in ion extraction times from the region of the source. This is believed to arise from variations, from pulse to pulse of the laser, in the initial velocities of ions and positions at which the ions are formed following the laser pulses.
One way of attempting to reduce the effect of this problem is to use very high extraction fields to accelerate the ions towards the detector. However, this does not remedy the problem itself and mass accuracy and mass resolution is still limited.
It is known to equip a spectrometer with a reflectron to increase the mass resolution of the spectrometer. The reflectron reflects the ions to increase the path lengths from source to detector whilst causing the more energetic ions (of a given m/z) to travel a further distance than the less energetic ions to compensate for the differing ion velocities. The reflectron can therefore position the faster moving ions behind the ions with slower velocities. If this happens, the faster ions will eventually catch up with and overtake the slower moving ions at a temporal focal point of the spectrometer.
In addition, it is known to use delayed extraction techniques, whereby a delay is introduced between the firing of the laser pulse and the application of the accelerating field so that when the field is applied, ions with a higher initial velocity will have drifted further away from the sample plate than those with a lower initial velocity.
The latter ions are thus accelerated to a greater degree than those which originally had a higher energy. The ions with the lower initial velocities will then catch up to and eventually overtake the other ions at another temporal focal point.
There has also been reported a variation on delayed extraction where there is a small retarding field between the sample plate and the first extraction plate during the delay period, prior to application of the extraction pulse. (U.S. Pat. No. 5,625,184).
However, even when these techniques are combined, the variations in initial kinetic energies can still significantly reduce the mass accuracy of the spectrometer. Mass accuracy is also affected by variations in power supply voltages (from which the accelerating voltages are derived), temperature drift and other factors which may influence flight times of ions to the detector.
A known way of further improving the mass accuracy of the spectrometer is to use internal standards. An internal standard is a known compound (or number of compounds) which is mixed with the sample to be analysed and is ionised with the analyte. It is believed that the same variations in initial conditions will be experienced by both the known compound(s) and the analyte, so that the internal standard can then be used to recalibrate the mass spectrum obtained from the detector. However, it can be difficult to cause the internal standard to co-crystallise uniformly with the compounds being analysed.
SUMMARY OF THE INVENTION
According to the invention, there is provided a time of flight mass spectrometer for measuring characteristics of the m/z ionised particles, the spectrometer comprising acceleration means for accelerating the particles along at least two paths and two detectors which are situated one in each respective path and are operable to detect particles travelling therealong, wherein the length of the path leading to the first detector differs from that of the path leading to the second detector to a sufficient extent to enable the difference in the detection times of corresponding particles at the two detectors to be used to provide a measurement of said characteristics.
It will be appreciated that, for the purposes of this specification, the characteristics to be measured, may for example, comprise charge to mass ratio or its reciprocal.
Variations in the initial velocities or ionisation times of the particles will affect the outputs of both detectors. However, these variations will have a similar effect on the detector outputs so that one detector can, in effect, be used to calibrate or correct the output of the other detector. Similarly, variations of any other parameters such as accelerating voltages will affect the outputs of both detectors.
Typically, the output of each detector will have one or more peaks. Consequently, the analysis of the detector outputs could involve identifying corresponding peaks in the detector outputs, and calculating the difference in their respective times of occurrence.
Preferably, the spectrometer includes temporal focusing means for at least partially compensating for any spread in the initial kinetic energies of particles of a given m/z so as to provide two temporal focal points, wherein each detector is situated at a respective temporal focal point. The focusing means can function in one or more of a number of ways. For example, particles with higher kinetic energies can be caused to travel along longer paths than those with lower kinetic energies, and/or can be accelerated to a lesser extent than the slower particles.
Preferably, the spectrometer is operable to create a beam of said particles, said beam containing both of said paths.
In this case, the focusing means may to advantage comprise reflection means for reflecting the particles in the beam in such a way that the higher the kinetic energy of particles of a given charges and mass, the longer the path of those particles through the reflection means, the reflection means being situated in the path of the beam between the two detectors.
Preferably, the focusing means comprises further reflection means positioned in the path of the beam between the sample and first of the detectors so that the beam is of a generally serpentine shape.
The spectrometer conveniently includes a laser for releasing said ionised particles from the sample. Alternatively, the spect
Davis Stephen Charles
Hoffman Andrew David
Makarov Alexander Alekseevich
Gurzo Paul M.
Haynes and Boone LLP
Lee John R.
Thermo Finnigan LLC
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