Exact mass determination with maldi time-of-flight mass spectrometers

A method relates to exact mass determination of analyte ions in time-of-flight mass spectrometers using an ionization of analyte substances on sample supports by matrix-assisted laser desorption (MALDI), and an improvement in mass resolution by time-delayed ion acceleration in the field between the sample support and an intermediate acceleration electrode. It particularly relates to methods for the stabilization of a once calibrated mass scale when there are unwanted changes in the distance of the sample support from the intermediate acceleration electrode.An unknown change of this distance can be compensated for by a coupled change of both total accelerating voltage and partial acceleration voltage between sample support and intermediate electrode in a simple manner by adjusting the time of flight of ions from a reference substance to the value given by the calibrated mass scale. Oligomeric ions from the matrix of the MALDI method serve very well as reference ions. Furthermore, if the range of optimum focus is shifted by a change in the time delay, the calibrated mass scale can be kept valid for all masses through a simultaneous change of the accelerating voltage.

The invention relates to exact mass determination of analyte ions in 
time-of-flight mass spectrometers using an ionization of analyte 
substances on sample supports by matrix-assisted laser desorption (MALDI), 
and an improvement in mass resolution by time-delayed ion acceleration in 
the field between the sample support and an intermediate acceleration 
electrode. It particularly relates to methods for the stabilization of a 
once calibrated mass scale when there are unwanted changes in the distance 
of the sample support from the intermediate acceleration electrode. 
An unknown change of this distance can be compensated for by a coupled 
change of both total accelerating voltage and partial acceleration voltage 
between sample support and intermediate electrode in a simple manner by 
adjusting the time of flight of ions from a reference substance to the 
value given by the calibrated mass scale. Oligomeric ions from the matrix 
of the MALDI method serve very well as reference ions. Furthermore, if the 
range of optimum focus is shifted by a change in the time delay, the 
calibrated mass scale can be kept valid for all masses through a 
simultaneous change of the accelerating voltage. 
PRIOR ART 
The exact determination of ion masses is becoming more and more important, 
especially for macromolecular ions of biogenic materials. Thus the 
molecular masses of glycoproteins, glycolipids or oligosaccharides alone, 
for example, provide information about structures as well, since the 
genesis of biomolecules by prescribed formation mechanisms greatly limits 
the possibly extremely large structural variety. Mass determination of 
fragment ions supports and expands these claims. But also in many other 
biochemical and medical applications, the mass determination of ions from 
biogenic materials is moving to the foreground. 
Among the methods for ionization of macromolecular substances on sample 
supports, matrix-assisted desorption by a laser flash (MALDI) has found 
the widest acceptance. After leaving the surface, the ions generally have 
a substantial average velocity which is the same to a large extent for 
ions of all masses, and a strong spread around the average velocity. The 
average velocity leads to a non-linear relationship between the flight 
time and root of the mass, and therefore the mass scale. The spread leads 
to bad mass resolution and thus to an uncertainty when measuring the 
signals of individual ion masses, however there are methods which improve 
mass resolution. 
For ionization by matrix-assisted laser desorption (MALDI), the large 
analyte molecules are stored on a sample support in or on a layer of 
low-molecular matrix substance. A laser light pulse of a few nanoseconds 
duration, focussed onto the sample surface, vaporizes a small amount of 
the matrix substance in a quasi-explosive process, whereby the analyte 
molecules are also transferred into the initially tiny vapor cloud. 
The vapor cloud expanding into the vacuum not only accelerates the 
molecules and ions of the matrix substance through its a diabetic 
expansion, but also the molecules and ions of the analyte substance 
through viscose entrainment, which thereby receive higher kinetic energies 
than would correspond to thermal equilibrium. Even without an accelerating 
field, the ions attain average velocities of about 500 to 1,000 meters per 
second, dependent upon the energy density of the laser beam; the 
velocities are to a large extent independent of the mass of the ions, 
however they have a large spread of velocity which ranges from about 200 
up to 2,000 meters per second. It must be assumed that the neutral 
molecules of the cloud also attain these velocities. 
The ions are accelerated in the ion source with electrical fields at 
energies of around 10 to 30 keV, are shot into the flight path of the mass 
spectrometer and detected with high time resolution at the end of the 
flight path. From their flight time, their mass-to-charge ratio can be 
determined. Since this type of ionization practically supplies only singly 
charged ions, the term "mass determination " will be used in the 
following, not the more exact term "determination of the mass-to-charge 
ratio". Flight times are converted into mass via a calibration curve, the 
acquisition of this calibration curve being described as "calibration of 
the mass scale" of the time-of-flight spectrometer. The calibration curve 
can be stored in table form as a sequence of pairs of mass and flight time 
values in the memory of the data processing system, or in the form of 
parameter values for a mathematical function describing the mass relative 
to the flight time. 
During formation of the vapor cloud, a small part of both the matrix and 
analyte molecules are ionized. During expansion of the vapor cloud, 
continuous ionization of the larger analyte molecules takes place through 
further ion-molecule reactions at the cost of the smaller matrix ions. 
The large spread of velocities and the formation process of the ions which 
is smeared over time, limit the mass resolution both in both linear as 
well as energy-focussing, reflecting time-of-flight mass spectrometers. A 
spread of initial velocities alone could be focused out using the 
energy-focussing reflector, however the time-smearing of the formation of 
the ions cannot. 
A method for the improvement of mass resolution under these conditions has 
been known for some time. The ions of the cloud are first allowed to fly a 
brief time .tau. in a drift region without any electrical acceleration. 
The faster ions thereby distance themselves further from the sample 
support electrode than the slow ions, and the distribution of the ions' 
velocities results in a spatial distribution. Only then the acceleration 
of the ions is switched on. The acceleration field normally is homogeneous 
and shows a linearly declining accelerating potential. When the 
acceleration field is switched on, the faster ions have a wider distance 
from the sample support electrode and therefore are located at a somewhat 
reduced accelerating potential. This gives them a somewhat lower final 
velocity for the drift region of the time-of-flight spectrometer than the 
initially slower ions. With correct selection of the time lag .tau. and 
potential drop (i.e. the strength of the acceleration field), the 
initially slower, but after acceleration faster ions catch up again with 
the initially faster, but after acceleration slower ions exactly at the 
detector. In this way, ions are dispersed at the location of the detector 
relative to the mass, but if of equal mass, are focussed in first order 
relative to the flight time. In this way, a sufficiently high mass 
resolution is achieved in a linear time-of-flight mass spectrometer. There 
is a similar method for time-of-flight spectrometers with reflectors, by 
which even second order focussing is achieved. 
Delayed ion acceleration need not be connected to a switching of the entire 
accelerating voltage U. Switching of such high voltages in extremely short 
times of a few nanoseconds is still almost unattainable today and 
associated with high costs. Switching of a partial accelerating voltage V 
is sufficient, if an intermediate electrode is installed in the 
acceleration path. Then only the space between the sample support 
electrode and the intermediate electrode, which have a relatively low 
distance d from one another, need be field-free at first and then switched 
over after a delay into an acceleration field with a strength of V/d. The 
distance d of the sample support to the intermediate electrode should be 
as small as possible in order to switch the lowest possible voltages V. 
There is a lower limit of about 1 mm for this distance which is hardly 
realizable however for practical designs of ion sources. In practice, this 
distance d is about three millimeters. 
The desire for a good mass resolution essentially has the purpose of 
achieving a good mass determination. However, since introduction of this 
method, it has been seen that the possibility in principle of good mass 
determination does not always lead to correct mass determination. The 
function which describes the mass relative to the time of flight, i.e. the 
calibrated mass scale, often leads to erroneous mass determinations during 
MALDI ionization. For an ion with a mass of 5,000 atomic mass units, the 
result of the mass calculation from scan to scan can fluctuate between 
several mass units in extreme cases. 
It has therefore become customary for exact mass determinations to correct 
the masses of the analyte ions to be determined by simultaneous measuring 
the flight time of ions from added known substances (so-called "internal 
reference substances"). As the simplest method, the mass of the analyte 
substances was corrected by linear extrapolation based on a relationship 
between the flight time and the root of the mass assumed to be linear. As 
reference masses, the known ions of the matrix, particularly their dimeric 
ion, were taken. This method leads to greatly improved accuracy in mass 
determination, on the order of magnitude of about 200 ppm. In this way, 
however, there is still an uncertainty of one mass unit for the ion with a 
mass of 5,000 u. 
Even the method for improvement of mass resolution through delayed 
acceleration has a decisive disadvantage for exact mass determination: it 
provides the optimum mass resolution only in a narrow range of the mass 
scale. In the other mass ranges, resolution is still considerably 
improved, but not up to its optimum value. This range of optimum 
resolution may be adjusted to any desired position on the mass scale by 
changing the time lag .tau., to a smaller degree also by changing the 
partial accelerating voltage V, but if this is done the calibration of the 
mass scale is thereby lost. 
According to DIN, the term "precise" is used for high repeatibility, and 
the term "accurate" for correct determination of mass with an as small as 
possible deviation between the corrected measurement value and the true 
value of the mass. 
OBJECTIVE OF THE INVENTION 
It is the objective of the invention to find an electrical compensation for 
a changed distance d between sample support and intermediate electrode 
which keeps valid the calibrated mass scale within narrow error tolerances 
and, at the same time, retains the optimum focus. It is a further 
objective of the invention to find a method by which the optimum focus can 
be adjusted to the analyte ions to be measured, whereby again the once 
calibrated mass scale should remain valid within narrow error tolerances. 
It is also the objective of the invention to maintain the validity of the 
mass scale even with superimposition of the displacement of the focus and 
compensation for the distance d. It is finally the objective of the 
invention to find a method by which even changes in the distance d of an 
unknown size may be compensated. 
BRIEF DESCRIPTION OF THE INVENTION 
It is a basic idea of the invention to retain the calibration of the mass 
scale by compensating for an unknown change in the distance d in such a 
way that both voltages V and U are commonly changed (coupled change), 
until the flight time of a known reference ion mass takes on exactly the 
value given by the calibration of the mass scale. If V is changed by the 
relative amount p=.DELTA.V/V, the total voltage U must then be changed by 
the relative amount c.sub.1 .times.p.times.U, c.sub.1 being an instrument 
constant which must be determined only once for one type of mass 
spectrometer. 
It is another idea of the invention to retain the calibration of the mass 
scale during shifts of the optimum mass resolution. If the time delay 
.tau. is changed to shift the point of best mass resolution in the 
spectrum, the acceleration voltage U has to be changed in a partial linear 
proportion with an instrumental constant c.sub.2. Even both changes can be 
compensated for in a single correction if a third instrumental constant 
C.sub.3 is introduced. 
It is a further idea of the invention to maintain the calibration of the 
mass scale for large sample support plates by a three-point compensation 
with further linear interpolation of the compensation control.

DETAILED DESCRIPTION OF THE INVENTION 
Surprisingly, it has been shown in experiments that electrical compensation 
for the change in the distance d is indeed possible in contrast to the 
impression given by equation (2). An experimentally introduced change in 
distance d of 10% was already compensated for in the first approximation 
by a change just in the partial accelerating voltage V between the sample 
support and intermediate electrode (at a constant total accelerating 
voltage U), so that the calibrated mass scale became valid once again with 
a maximum error of about 30 ppm of mass over a wide mass range of 500 u to 
2,500 u. However, the voltage V had to be changed by more than just 10%, 
an overcompensation of about 14% was necessary. At the same time however, 
degradation of the resolution, i.e. the sharpness of the mass signals, 
occurs. 
Degradation of the resolution can however be avoided if, through a change 
in the partial accelerating voltage V which essentially restores only the 
field strength in the first acceleration path between the sample support 
and intermediate electrode, and through a further compensation of the 
distance change by the total accelerating voltage U. V is therefore 
changed in proportion to the change in distance d, and the remainder of 
the compensation is effected by a change in the voltage U. Surprisingly, 
the mass scale is retained over the entire calibrated mass range with good 
accuracy through this coupled change in both accelerating voltages U and 
V. 
It is surprising that the accelerating voltage U must also be changed 
linearly to the change of d, however not completely proportionally, but 
rather only linearly proportional at a small fraction. For a 
time-of-flight mass spectrometer with a distance d=3 millimeters, a second 
acceleration path of 30 millimeters, and a flight path of 1.6 meters, this 
fraction c.sub.1 amounts to about 1/130. Therefore, if d is changed by 
10%, V must also be changed by 10%, however the accelerating voltage U is 
only changed by 0.075%. The mass scale is then valid once again in a very 
large mass range of 500 to 10,000 atomic mass units, with a maximum error 
of 1 ppm of flight time or 2 ppm of the mass. These results exceed the 
measurement accuracy achieved experimentally and were gathered through 
computer simulations, however they were also verified on the 
time-of-flight spectrometer within the scope of measurement accuracy 
available there of about 10 ppm. 
If the distance d therefore changed by a relative amount .DELTA.d/d, the 
accelerating voltage V must also be changed by, this amount 
V.times..DELTA.d/d, the total accelerating voltage on the other hand by 
the amount c.sub.1 .times.U.times..DELTA.d/d whereby the fraction c.sub.1 
=0.0075 applies for the type of time-of-flight spectrometer described 
above. For types of mass spectrometers which are geometrically different, 
similar results can be achieved with different values for the constant 
c.sub.1. 
If we introduce the index c for the parameters used during the calibration 
of the mass scale, and the index s for the parameter values changed 
through the control, and if we use the abbreviation 
EQU p=.DELTA.V/V=(V.sub.s -V.sub.c)/V.sub.c =(d.sub.s -d.sub.c)/d.sub.c(3) 
then the following equations apply: 
EQU V.sub.s =V.sub.c .times.(1+p) and (4) 
EQU U.sub.s =U.sub.c .times.(1+c.sub.1 .times..DELTA.V/V)=U.sub.c 
.times.(1+c.sub.1 .times.p)=U.sub.c +U.sub.c .times.c.sub.1 .times.p.(5) 
It is therefore a basic idea of the invention to compensate for a change in 
the distance d by a coupled change in the accelerating voltages U and V in 
such a way that the calibration of mass scale is retained. An unknown 
change in the distance d can be compensated by a variation of p until the 
flight time of a known reference ion takes on exactly the value calibrated 
for it. If V is changed by the relative amount p=.DELTA.V/V, U must then 
be changed by the relative amount c.sub.1 
.times..DELTA.V/V.times.U=c.sub.1 .times.p.times.U, c.sub.1 being an 
instrument constant which must be determined only once for one type of 
mass spectrometer. 
From this a control system may be constructed which influences the 
parameter p via measurement of the flight time t of a reference ion mass 
and a comparison with the correct value for this flight time long enough 
until the measured flight time t takes on the correct set value. As 
already described above, it has been found through computer simulations 
(and also experimentally within the scope of accuracy) that the mass scale 
is then valid over a very large range, for example for the range from 500 
u to 10,000 u with a maximum error below 2 pate per million (ppm) of the 
mass m (1 ppm of the flight time t). Even at a mass of 10,000 u, the mass 
could still be determined exactly to within 0.02 u if the measuring 
accuracy would allow it. At the same time, the adjusted resolution remains 
optimal. 
The value of the parameter p can be calculated in a first approximation, 
with the help of equations (1) to (5) and with a reasonable assumption for 
the average start velocity v, from the deviation of the flight time of the 
reference ions. 
For these measurements, the flight time t for small ions must be determined 
exactly to within about two hundredths of a nanosecond, which is not yet 
entirely possible today. The centroid of the line profile is normally used 
for the measurements. The line profile is scanned according to current 
technology using a transient recorder with 1 or 2 gigahertz. Transient 
recorders with a 4 gigahertz scanning rate are in the introductory phase. 
Generally, the measurements from several measurement cycles are cumulated 
before the centroid is created. 
The method for improvement of the mass resolution through delayed start of 
acceleration provides, as already mentioned above, the optimum mass 
resolution only in a narrow range in the mass scale. In the other mass 
ranges, the resolution is still considerably improved, however not to its 
optimum value. The optimum resolution can particularly be adjusted to any 
desired ion mass through changes in the time lag .tau. (but also through 
the accelerating voltages U and V). 
If the mass of the reference ions is much smaller than that of the analyte 
ions, as for example when the always present matrix ions are used as 
reference ions, a special method for improvement of the mass accuracy can 
be applied: one can temporarily shift the range of optimum resolution to 
the range of the light reference ion masses by changing the time lag 
.tau., and then adjust the distance d using the flight time of these light 
reference ions. An extremely good focussing results for light ions. The 
width of the mass signal amounts to only one or two nanoseconds for light 
ions of about 500 atomic mass units, therefore it is possible to make a 
very precise adjustment here. The flight time t of the reference ions must 
be adjusted in this case to a value which corresponds to a different 
calibration curve for the mass scale with this adjusted focus range. After 
shifting the optimum focus range back to the analyte ions whose mass is to 
be measured, the mass of these analyte ions can then be correctly measured 
since the change in the distance d has been compensated for by changes in 
the accelerating voltages. 
For this, it is not even necessary to have calibrated the entire mass scale 
for the case of adjusted focus range; it is sufficient to know the value 
of the correct flight time t.sub.c for the matrix ions. If only a small 
number of different matrix ions are used, their flight times t.sub.c can 
easily be measured and stored at the same time using the calibration of 
the mass scale. The voltages can be set so precisely nowadays that voltage 
inaccuracies are unimportant. 
If, however, reference ions should be used which are different from sample 
to sample, this method is not applicable since their flight times must be 
measured at the same time as the one-time calibration of the mass scale. 
It is therefore a further surprising finding of our experiments that the 
range of optimum resolution can be shifted in such a way that the 
calibrated mass scale remains valid within very narrow margins of error. 
In this way, the optimum focus range for the light reference masses can be 
shifted in such a way that the flight time t can be adjusted for that set 
value which is prescribed by the calibrated mass scale for these reference 
ions. 
If the mass scale is to remain valid, the range of optimum focus can 
therefore not be adjusted by a change in voltages alone. This always leads 
to a tipping of the mass scale and cannot be corrected electrically. It is 
necessary to adjust the focus range via the time lag .tau.. By changing 
.tau. alone, the mass scale moves slightly, however this movement can be 
compensated for by a slight co-control of the total accelerating voltage 
U. Surprisingly, the accelerating voltage can be changed again linearly in 
a simple manner with .tau.. If .tau. is changed by the relative amount 
.DELTA..tau./.tau., the accelerating voltage U must be changed by the 
factor (1+c.sub.2 .times..DELTA..tau./.tau.). Using the example of the 
above given mass spectrometer, c.sub.2 =0.00299. 
This constant c.sub.2 can also be determined once and re-used again and 
again. If now analog to above, the abbreviation 
EQU q=.DELTA..tau./.tau.=(.tau..sub.s -.tau..sub.c)/.tau..sub.c(6) 
is introduced, the accelerating voltage must be controlled as follows: 
EQU U.sub.s =U.sub.c .times.(1+C.sub.2 .times..DELTA..tau./.tau.)=U.sub.c 
.times.(1+c.sub.2 .times.q)=U.sub.c +U.sub.c .times.C.sub.2 .times.q.(7) 
In this way a control can be designed which places the range of optimum 
focus at any desired position on the mass range, without allowing the 
calibrated mass scale to become invalid. The relationship between q and 
the mass where the optimum focus lies, can easily be determined 
experimentally and stored in a control table. It can again be shown 
through computer simulations (and also experimentally within the scope of 
measurement accuracy present there), that the mass scale remains valid 
over a very large range, for example for the range from 500 u to 10,000 u 
with a maximum error of below 2 ppm of mass (1 ppm of flight time). Even 
for a mass of 10,000 u, the mass could still be determined exactly to 
within 0.02 u if the measuring accuracy would allow it. 
For superimposed adjustments in the distance and the focus range, a further 
element p.times.q with a new constant C.sub.3 is added for optimum 
maintenance of the mass scale: 
EQU U.sub.s =U.sub.c +U.sub.c .times.C.sub.1 .times.p+U.sub.c .times.C.sub.2 
.times.q+U.sub.c .times.c.sub.3 .times.p.times.q= (8) 
EQU =U.sub.c +U.sub.c .times.C.sub.1 .times.(V.sub.s -V.sub.c)/V.sub.c +U.sub.c 
.times.C.sub.2 .times.(.tau..sub.s -.tau..sub.c)/.tau..sub.c +U.sub.c 
.times.C.sub.3 .times.(V.sub.s -V.sub.c)/V.sub.c .times.(.tau..sub.s 
-.tau..sub.c)/.tau..sub.c 
For the above described time-of-flight mass spectrometer, c.sub.1 =0.0075, 
c.sub.2 =0.00299 and C.sub.3 =0.00015. 
With a control according to equation (8) the mass scale remains exact up to 
a maximum error of flight times of about 2 ppm, of mass determination of 4 
ppm. In this way, an ion with a mass of 10,000 u can still be measured 
exactly to 0.04 atomic mass units with an incorrectly adjusted distance of 
d =3.30 millimeters, even though, for example, the mass scale was 
calibrated at an optimum focus for the mass 1,000 u and at a distance of d 
=3.00 millimeters. Equation (8) includes the above given equations (5) and 
(7), and is therefore a comprehensive equation for the co-control of 
U.sub.s according to this invention. 
The compensation described by equation (8) can be further improved by the 
addition of quadratic terms p.sup.2 and q.sup.2 (with the constants 
C.sub.4 and c.sub.5), though this improvement is hardly necessary in the 
scope of tasks proposed here. The precision of flight time determination 
from the ion current signal, which is dependent on the number of ions 
measured, does not normally attain the above given accuracies of mass 
determination and therefore creates a stronger limitation than the 
remaining impression of the equation (8). Complete utilization of the 
accuracy given by the equation (8) already requires the accelerating 
voltage U.sub.s to be set with a digital-to-analog converter with 20 bit 
control accuracy. 
In the case of lighter reference ions, for example when using the dimeric 
or trimeric matrix ions, the focus can therefore first be relocated to 
these ions by means of parameters q (under co-control of U.sub.s according 
to equation (8)), without allowing the mass scale to become invalid. Using 
these ions, a possible change in distance is compensated for electrically 
by the control parameter p (under co-control of U.sub.s), until the flight 
time of the light reference ions is at the value of the calibration. Then 
the range of optimum focus is shifted back to the analyte ions by means of 
the parameter q (under co-control of U.sub.s), whereby the control 
parameter p is maintained. The masses of the analyte ions can now be 
measured very exactly with a scan. 
For sample supports with a small sample support surface, only one single 
control process is normally required, since the parallelism of the sample 
surface to the intermediate electrode is generally guaranteed sufficiently 
well by the mounting. 
For MALDI sample substances of varying thicknesses, there must be a 
compensation for the distance for every measurement of a sample. To do 
this it is necessary to always be able to measure ions of a reference 
sample at the same time as measurement of the analyte ions. In many cases, 
ions of the matrix can be used for this, for example the frequently 
occurring dimeric or trimeric ions of the matrix substance. For this, the 
above described special method with an adjustment of the focal range can 
be used. In other cases, an appropriate reference substance must be added 
to the analyte substance. 
The goal of automatic measurement of thousands of samples makes ever larger 
sample supports necessary. These can certainly be created so flat that the 
effects which deviations in flatness have on the distance of the sample 
support may be disregarded. The samples may also be applied very uniformly 
thin, and also in this way hardly any deviations result. But exact 
positioning of the distance inside the vacuum system is difficult since 
neither lubricating greases nor very narrow sliding tolerances can be 
used. When introducing the sample support into the holder and during 
parallel movement of the sample support, changes in the distance from the 
intermediate electrode occur very easily and these must be readjusted 
according to this invention. 
This invention provides a particular advantage here since the change in 
control parameter p is directly proportional to the change in the distance 
d. So if the electrical compensation by parameter p is known at some 
positions on the sample support, the optimum values of p can be linearly 
interpolated at other positions on the sample support. 
For these large sample supports, at least three reference samples are 
necessary which should be applied as far from one another as possible on 
the edge of the sample support. Once the distances d at the large sample 
support plate are compensated on three positions by determination of the 
control parameter values p, the samples from all remaining positions can 
automatically be measured by interpolating p from the position in the 
known manner. 
If the analyte substance is unevenly thick, or if a wavy matrix film is 
stuck onto the sample support, electrical compensation is necessary for 
each individual sample. For this, a simultaneous reference measurement is 
again always necessary. 
It is a further particular advantage of the invention that electrical 
compensation for the distance d also restores the second order focussing 
in a time-of-flight spectrometer with reflector. 
Particularly Favorable Embodiments 
The method presented here for precise mass determination according to this 
invention is based upon a linear time-of-flight mass spectrometer, as 
shown in FIG. 1. The method for co-control of U.sub.s according to 
equation (8) is however also utilizable with other values for the 
constants c.sub., c.sub.2 and C.sub.3, for time-of-flight spectrometers 
with energy-focussing reflectors. 
When using delayed acceleration as described above, the intermediate 
electrode 2 is initially at potential U of the sample support 1, and is 
switched down after a time lag .tau. of several tens to hundreds of 
nanoseconds to the potential U-V. An operation is also possible in which 
both the sample support and intermediate diaphragm are at the potential 
U-V, whereby sample support 1 is raised to potential U after time lag 
.tau.. 
With this arrangement for a time-of-flight mass spectrometer, spectra of 
analyte substances can be scanned as usual. Scanning begins with 
ionization of the analyte substances 8 on the sample support 1, as in the 
MALDI method of ionization described here. The ions are generated by a 
light flash of about 3 to 5 nanoseconds duration from laser 5. Usually, UV 
light with a wavelength of 337 nanometers is used from a moderately priced 
nitrogen laser. The light flash is focussed through lens 6 as convergent 
light beam 7 onto the sample 8 on the surface of the sample support 1. The 
ions formed in the vapor cloud, which is generated by the laser focus, are 
accelerated, after the time lag .tau., first in the electrical field 
between sample support 1 and intermediate electrode 2, and then in the 
electrical field between intermediate electrode 2 and base electrode 3. 
The ion beam slightly defocussed in the electrode arrangement is focussed 
onto the detector 10 at the beginning of the flight path in the Einzel 
lens 4. The flying ions form a strongly time variable ion current 9, which 
is measured at the end of the flight path by ion detector 10 with high 
temporal resolution. 
The time variable ion current provided by the ion beam is usually measured 
and digitalized at the detector with a scanning rate of 1 or 2 gigahertz. 
Transient recorders with even higher temporal resolution will soon be 
available. Usually, the concurrent measuring values from several scans are 
cumulated before--by means of data evaluation--the mass lines in the 
stored data are sought and transformed from the time scale into mass 
values via the mass calibration curve. 
The polarity of the high voltage used for the ion acceleration must be the 
same as the polarity of the ions being analyzed: positive ions are 
repelled and accelerated by a positively charged sample support, negative 
ions by a negatively charged sample support. 
Of course, the time-of-flight mass spectrometer can also be operated in 
such a way that the flight path is in a tube (not shown in FIG. 1), which 
is at accelerating potential -U, while the sample support 1 is at base 
potential. In this special case, the flight tube is at a positive 
potential if negatively charged ions are to be analyzed, and vice versa. 
This operation simplifies the design of the ion source, since the 
isolators for the holder of the exchangeable sample support 1 are no 
longer necessary. However, there are disadvantages in other respects. 
There are two types of problems which have occurred up to now in precise 
and exact mass determination using the MALDI ionization method: 
(a) During calibration of the mass scale, the range of optimum focus must 
be set permanently at an arbitrary mass. This may however be very 
different from the analyte mass which must later be measured and which is 
unknown during the calibration. In this way conditions for the mass 
determination are not ideal due to the poor resolution. 
(b) Through the technique of exchangeable sample supports, which must be 
brought through a lock into the vacuum and held there, a constancy of the 
distance d to the intermediate diaphragm is not guaranteed with the 
tolerance of less than 10 micrometers necessary for a precise mass 
determination. This problem becomes more acute if large surface sample 
supports are used which must be moved on two coordinates parallel to the 
surface of the sample support. Through changing distances d, no 
reproducible mass determination is possible. 
Both of these problems are solved by this invention. 
The focus range can be adjusted by control of the time lag .tau..sub.s as 
desired, whereby the mass scale remains valid if the accelerating voltage 
U.sub.s is also adjusted in accordance with equation (8). A change in the 
distance d can be compensated for by control of the partial accelerating 
voltage V.sub.s while retaining the focus range, whereby the mass scale 
remains valid if the accelerating voltage U.sub.s is also adjusted 
according to equation (8). An unknown change in the distance d can be 
compensated by bringing the flight time of a reference ion mass back to 
the value valid for the calibration through coupled control of V.sub.s and 
U.sub.s according to equation (8). 
Calibration of the mass scale can also benefit from the invention. 
Calibration is normally undertaken using a carefully produced mixture of 
calibration substances. This mixture contains a number of substances, the 
ion masses of which fully cover the mass range to be calibrated. Therefore 
very small as well as very large molecular weights must be present in the 
mixture. This mixture is ionized in a MALDI process and the flight times 
of the individual ions are measured as precisely as possible. From the 
flight times and the corresponding, known masses, the calibration curve 
for the masses (the "mass scale") is put together. In order to determine 
the individual flight times as precisely as possible, the optimum focus 
according to this invention can now be adjusted to the individual 
calibration masses by control of the time lag .tau..sub.s under co-control 
of the accelerating voltage U.sub.s according to equation (8). In this way 
the mass scale can now be calibrated more precisely. 
If a mass to be measured is placed exactly in the center of the focus 
range, the mass signal is thus often slightly asymmetrical. This is 
because both the faster as well as the slower ions than those of the 
average velocity v branch off on the same side of the mass line. Through 
slight shifting of the optimal focus range, the mass line can be made 
symmetrical to a large extent without at first noticeably changing the 
line width. In this way, measurement of the exact mass is simplified, 
which can then be done using a centroid, for example. 
The method of this invention also aims at arriving at more reproducible 
flight times of the ions. As described above, the lack of reproducibility 
is due to changes in the distance d of the exchangeable sample support 
from the intermediate diaphragm. This distance can be measured in 
principle and changes compensated for with a coupled control of U and V 
according to this invention. Direct measurement of the distance d would 
however require additional installation of very precise measurement 
systems in the ion source of the time-of-flight spectrometer. These 
installations are however in principle unnecessary, since a compensation 
of unknown distances is possible according to this invention. 
This method consists of first checking the distance d before a mass 
determination of analyte ions using the measurement of flight times of a 
reference mass and, if necessary, to compensate with a coupled control of 
U and V according to this invention. To do this, the flight time of the 
reference ions is measured in a first sample measurement and compared with 
the correct flight time during calibration. If there is a deviation, the 
distance d must be electrically compensated. The compensation can be 
calculated from the above given equations. For very exact measurements, a 
repetition of the reference measurement with a second electrical 
compensation is appropriate. 
The parameter p can in principle be calculated if a suitable value for the 
initial average velocity v can be assumed: 
##EQU3## 
with .DELTA.t being the flight time difference t.sub.s -t.sub.c of the 
reference ions, m and q mass and charge of the reference ions, d.sub.c the 
correct distance during calibration, and v the initial average velocity of 
the ions at the start of the acceleration. However, because the initial 
average velocity v is essentially unknown, a cyclic approximation method 
should be applied. 
It is convenient when testing the distance d to use the ions of the matrix 
since no special reference substance then needs to be added. It has become 
apparent that monomer ions are not well suited due to their much too high 
intensity and the resulting overloading of the measurement device. 
Furthermore their mass is so far down on the bottom margin of the usable 
mass range that extrapolation into the desired mass range is unfavorable. 
In most spectra however, there are dimeric ions in the correct intensity 
range, sometimes even trimeric or even oligomeric ions. These lines are 
very sharp and more suitable due to their higher mass. The masses of these 
ions are still very small however compared to heavy analyte ions. They are 
generally in the mass range of 300 u to 800 u. It is therefore appropriate 
to use the above mentioned method for adjustment of the optimum focus 
range. 
For large, although flat, sample supports which can hold thousands of 
samples, it is practical to test the distance at several, or at least 
three positions, before the analyte samples are measured. The reference 
samples for the distance test must be applied as closely to the margin as 
possible. With the knowledge of the three control parameter values p to 
compensate for the distance changes, the control values for all other 
sample positions can then be calculated using linear interpolation. This 
method however requires that the sample cannot wobble in its holder, but 
rather be moved reproducibly by the movement device. 
For automatic mass determination of thousands of samples, the nature of the 
analytical problem is often such that the only question to answer is 
whether the sample has a previously known molecular weight or not. The 
expected molecular weight can thereby be very different from sample to 
sample. For this type of task, this invention can be very helpful since 
the range of optimum focus for every sample can be adjusted to the 
expected value being tested. 
The considerations discussed here for linear mass spectrometers also apply, 
as any specialist can appreciate, to time-of-flight mass spectrometers 
with energy-focussing reflectors. Here the reflector voltages must be 
co-controlled in the same manner as the accelerating voltages U. For this 
type of mass spectrometer, different apparatus constants c.sub.1, c.sub.2 
and C.sub.3 then apply. 
The method of precise mass determination given here with a time-of-flight 
spectrometer according to this invention can of course be varied in many 
ways. The specialist in development of mass spectrometers and their 
measurement methods can easily realize these variations.