Radiant energy – Ionic separation or analysis – Ion beam pulsing means with detector synchronizing means
Reexamination Certificate
1999-06-21
2002-10-15
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
06465777
ABSTRACT:
FIELD OF THE INVENTION
This invention relates to time-of-flight mass spectrometers which must keep ass flight times highly constant to preserve calibrated mass scaling, even under varying ambient temperatures.
BACKGROUND OF THE INVENTION
Compared to other mass spectrometers, the functioning of time-of-flight mass spectrometers can be very easily understood, even though in detail this category of instruments is similarly complicated. The ions of the analyte substance to be analyzed, formed by pulse type ion source in a very short time span of only a few nanoseconds, are all accelerated to the same energy for each ion charge within relatively short acceleration fields. They then fly through a field-free flight path and are measured at its end by an ion detector with high time resolution as a temporally variable ion current. The flight time of the various ion types can be determined from the measurement signals of the ion current.
Using the very simple basic equation for the kinetic energy of ions with e as the elementary charges:
E=
½
m v
2
/e,
(1)
the ratio m/e from mass m to charge e can be determined at equal energy E of all ions from their velocity v. The velocity v of the ions is obtained, as indicated above, in a flight tube of length L by measurement of the flight times t of the ions using the equation:
v=L/t.
(2)
It is therefore a simple matter to calculate the ratio of mass m to charge e from the flight time:
m/e=
2
Et
2
/L
2
=c×t
2
. (3)
For very precise determination of the ion mass, the above indicated equations become more complicated since the ions in the ion source unavoidably receive initial energies from the ionization process before their electrical acceleration. These energies change the equation (3) slightly, but very decisively for precise mass determinations. In this way, the relationship between the mass m and the square of the flight time t
2
becomes slightly nonlinear. This relationship is therefore normally determined by experiment and saved on the spectrometer's computer for future mass determinations as a so-called “mass scale.”
The term “mass scale” is understood here to mean the assignment by a connected computer system of the flight times determined from the measurement signals to the masses of the ions (more precisely: the mass-to-charge ratios). This mass scale is calibrated using a special method by means of precisely known reference substances and should remain stable for as long as possible without recalibration.
A large number of influences generally affect the stability of the calibrated mass scale: inconstancy of the high voltages for acceleration of the ions, changing distances between the acceleration diaphragms in the ion source caused by mounting of the sample support introduced into the vacuum, changing initial energies of the ions caused by the ionization process and, not least, the thermal alterations in the length of the flight path.
Therefore, for highly precise measurements of the masses of an analyte substance, the mass of a reference substance is also measured within the same mass spectrum. The reference substance must be added to the analyte substance (so-called measuring method with an “internal reference”). If the calculated mass of the reference substance deviates from the true known value, the mass calculated for the analyte ions can then be corrected in a known manner (refer for example to application DE 196 35 646).
Unfortunately, the various influences on the mass determination nevertheless end up with different correction functions for the mass. Alterations of the high voltage, for example, cause a proportional change in the energy E of the ions, which according to equation (3) enter into the mass calculation linearly, i.e. mass proportionally. Changes to the flight length L, however, enter into the mass calculation according to equation (3) proportional to the root of the mass. If the reference mass and analyte mass are very different, it is no longer possible to make a successful correction without precise knowledge of the type of influence. For very similar masses of analyte and reference substance, it is still possible to make corrections with relatively good success. Today, mass accuracies of about 30 parts per million (ppm) are achieved using high performance time-of-flight mass spectrometers in respect to reference substances not contained in the analyte sample (“method with external reference”). Using reference substances which are added to the analyte sample (“internal reference”), accuracies of 10 ppm are achieved. Today, however, mass accuracies of 1-5 ppm are the target of protein chemists and other users, and are correspondingly demanded by the manufacturers of mass spectrometers.
The stainless steel flight tubes standard today, which determine the distance between the ion source and ion detector, have linear coefficients of thermal expansion of about &agr;=13×10
−6
K
−1
. The more seldomly used Duralumin even demonstrates an expansion of &agr;23×10
−6
K
1
. Since the relationship
dm/m=−
2
dL/L
(4)
can be derived from equation (3), a temperature change produces an apparent change in mass of about 26 ppm per centigrade (or Kelvin) due to the expansion of the stainless flight tube. Compared to the target of 1-5 ppm for the mass accuracy, that is an extremely large amount. Even if mass reference substances contained in the sample are used, the targeted accuracies cannot be achieved. Therefore today, in the case of highest demands on the accuracy of the mass determination, a temperature-dependent mass calibration is required which is, however, very complicated to perform and demands very precise temperature measurement at a good constancy of ambient temperature.
The ambient temperature in non-air-conditioned rooms varies by more than 10 degrees Celsius. These temperature fluctuations are transferred relatively quickly to the, in most cased openly exposed flight tubes of relatively thin-walled stainless steel, but due to the flange-mounted pumps, temperature distribution is very irregular along the length of the flight tube.
However, even greater difficulties are, caused by today's strict requirements for electromagnetic compatibility (EMC). This, in conjunction with necessary pulse methods for ion generation and pulse-switched high voltages, means that at least parts of the mass spectrometer's flight tube;, including the electronics, have to be installed in a hermetically sealed housing. Through the heating of the vacuum pumps and the electronics, flight tube temperature increases of up to 20 degrees Celsius must be reckoned with in spite of fan cooling. Without appropriate consideration or compensation, this corresponds to an apparent mass change of about 500 ppm for measurements during the start-up phase of the instrument. But even if equilibrium is achieved, thermal fluctuations remain within the range of about 10 Kelvin and correspond to apparent, relative mass changes of 260 ppm. On the other hand, the use of cooling water is undesirable today for ecological and financial reasons. Even for measuring methods using an internal reference, difficulties arise here in deciding on application of the right correction.
For routine analyses with tens of thousands of samples daily, such as are expected for DNA analyses, a mass determination with internal reference is too complex, since each individual sample requires the addition of reference substances which are similar in their mass to the mass of the analyte to be measured. For these methods (which are not however subject to the extreme demands on the accuracy of mass determination cited above), the target is to keep all operating parameters as constant as possible so as to perform the mass determination without internal reference substances and achieve a long period of validity for mass calibration.
Controlled temperature stabilization of the flight tube, including the ion source and detector, is an apparently
Bruker Daltonik GmbH
Lee John R.
Vanore David A.
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