Patent Application
20100176289
The invention relates to methods for the acquisition of mass spectra in ion cyclotron resonance mass spectrometers, in particular to methods for exciting the ions to cyclotron trajectories. In ion cyclotron resonance mass spectrometers (ICR-MS), the charge-related masses m/z of the ions are determined by measuring their orbital frequencies in a homogeneous magnetic field with high field strength. The orbital motion is essentially a cyclotron motion on which a smaller magnetron motion is usually superimposed. The magnetic field is normally generated by superconducting magnet coils cooled with liquid helium. Nowadays, commercial mass spectrometers provide ICR measuring cells with usable diameters of up to approximately 6 centimeters at magnetic field strengths of between 7 and 15 tesla.
In ICR measuring cells, the orbital frequency of the ions is measured in the most homogeneous part of the magnetic field. The ICR cells normally comprise four longitudinal electrodes, which are parallel to the magnetic field lines and surround the interior of the measuring cell like a cylinder jacket. Cylindrical measuring cells are usually used, as shown in
The introduction of the ions into the measuring cell, ion excitation and ion detection are carried out in successive phases of the method. Various methods are available to introduce the ions into the ICR measuring cell and, in particular, for their capture, for example the “side-kick” method or the method of dynamic capture with a steady increase of the potential, but they will not be discussed further here. Those skilled in the art are familiar with these methods.
The ions are excited by absorbing energy in a dipolar alternating electric field between the two excitation electrodes. The frequency of the field must resonantly coincide with the cyclotron frequency of an ion species. The cyclotron frequency of the ions is inversely proportional to their mass m/z. Since the ratio m/z of the mass m to the number of elementary charges z of the ions (referred to below simply as “charge-related mass”, and sometimes simply as “mass”) is unknown before the measurement, the ions are excited by as homogeneous a mixture as possible of all the excitation frequencies for a desired mass range. This mixture can be a temporal mixture with frequencies linearly increasing or decreasing with time (called a “chirp”), or it can be a synchronous, computer-calculated mixture of all frequencies (a “sync pulse”). Commercial mass spectrometers usually operate with chirps; their initial and final frequencies, duration and voltage are chosen so that they lift ions of a selected mass range uniformly to a cyclotron trajectory with desired radius.
The ion image currents that are induced in the detection electrodes by the orbiting ion clouds form a co-called “transient” as a function of time. The transient is a “time-domain signal”. It usually starts with initially large ion image currents, which decrease during the measuring time to such a degree that only noise remains. The useful length of the transient up to the informationless noise is usually a few seconds, but in correctly adjusted ICR cells with compensation electrodes, as shown in
The ion image currents of the transients are amplified, digitized and analyzed by Fourier analysis to determine the orbital frequencies of the ion clouds occurring therein; the ion clouds each consist of ions of different masses orbiting in phase. The Fourier analysis transforms the sequence of the original ion image current values of the transient from the “time domain” into a sequence of frequency values in a “frequency domain”. ICR is therefore also called Fourier Transform Mass Spectrometry (FTMS), although it should be noted that, today, there are other types of FTMS which are not based on the orbiting of ions in magnetic fields.
The frequency signals of the various ion species, which can be recognized as peaks in the frequency domain, are then used to determine their charge-related masses m/z and their intensities. The high stability of the magnetic fields used and the high accuracy for frequency measurements make it possible to achieve an extraordinarily accurate mass determination. Fourier transform ICR mass spectrometers (FT-ICR-MS) are currently the most accurate of all types of mass spectrometer, with accuracies far better than one millionth of the mass for masses in the range up to around one thousand atomic mass units. FT-ICR-MS also provides the best mass resolutions, which are usually above one million for lighter ions, but which decline in inverse proportion as the mass of the ions increases. The mass resolution essentially depends on the number of ion orbits which can be detected by the measurement.
The transient usually looks like a very noisy signal which decreases roughly exponentially in time. The noise is only apparent; the signal very reproducibly consists of the superimposition of the many ion image current frequencies.
If, however, a particular mixture of ions consists of a larger number of ion species whose masses differ by the same mass difference in each case, the ion image current transient looks completely different. When the ions are excited by a standard chirp, so-called “beats” are formed in the transient when the image currents are measured. The ion clouds jointly lifted onto the cyclotron trajectory are initially all close together and produce the strong image currents of a first beat. The ion clouds of the slightly different masses, having slightly different speeds, then increasingly separate, however, and spread almost uniformly over the whole orbit over a long period; their image current signals appear to almost cancel each other out, as happens with an interference. Only when the ion clouds come close together again after many orbits is there a next “beat” of the image current. This process repeats periodically. The number of orbits nb between two beats is nb=M/ΔM, where M is the mass of the first ion of the group and ΔM is the mass difference between the different ions of the mixture.
These beats are especially common if one investigates organic substances with very high molecular weights. The ions of these substances are usually produced by electrospraying, which creates a broad distribution of multiply charged ions for large molecules. As an example,
With heavy organic substances, the ions of one charge state form an isotope group with often far more than a hundred isotope satellites. Since the ions of this isotope group each differ by one atomic mass unit (to be more precise, by the mass difference between 12C and 13C in each case), they constitute a very uniformly structured ion mixture, which forms a transient with pronounced beats on being excited with a chirp, as can be seen in
The information contained in the transients is not only found in the beats, but also in the spaces between the beats, which visually appear to be almost empty. In these spaces, the image current frequencies are superimposed in a similar way to the “normal” transient of
The mass spectrum of the isotope signals of the BSA ions with 49 charges, which is shown in
As stated above, the beats are produced by an interference behavior of the ions during their orbits. The excitation lifts the ions to a cyclotron trajectory where all the ion clouds are initially very close together and result in a strong ion image current signal, the first beat. Then the ion clouds, which each differ by a tiny fraction of their relative mass and thus by a tiny fraction of their speed, slowly drift apart and distribute themselves almost evenly over the complete cyclotron orbit. When the distribution is even, however, the ion image current signals almost cancel each other out; the intensity of the signals is very low and can hardly be measured next to the intense beats. In the case of BSA, all the ions then come together again after 66,389 orbits of the monoisotopic ions; the ions of the first 13C satellite mass pass through one orbit less than the monoisotopic ions, the ions of the second satellite two fewer orbits, the ions of the third satellite three fewer orbits, etc. This produces the second beat. The ion species then spread out again until they meet up once again after a further 66,389 orbits to form a third beat.
This process continues periodically but the beats become smaller and smaller because, although the mass differences are identical over the whole mixture, this is not the case for the differences of the speeds, which are reciprocal to the masses, as can easily be mathematically verified. Since the differences in the speeds are only equal in the first approximation, albeit a very good approximation, the ions meet up less and less after each successive 66,389 orbits, and their beat becomes smaller.
The chirps used in the current prior art have a linear frequency function with the same amplitude for all frequencies, as is shown in
To describe the effect of a chirp, we will now assume it to be a chirp with linearly increasing frequency. For this chirp with linearly increasing frequency function, the ions are excited in a sequence from heavy to light masses. If the increasing frequencies of such a linear chirp cover the cyclotron resonance frequencies of all the ions of an ion mixture in roughly ten milliseconds, the lightest ions reach their cyclotron orbit some ten milliseconds later than the heaviest ions. The temporal separations of the ions for reaching the orbit are proportional to their mass difference.
If the ion mixture consists of ions with the same mass differences throughout, all ions catch up with the heaviest ions simultaneously because the light ions fly slightly faster on their orbit and because the temporal separations between the lighter ions and the heavier ones are inversely proportional to their speed on reaching the orbit. All the light and heavy ions will therefore come together at the same time, resulting in the first beat.
The image currents are measured using amplifiers which offer a wide range of amplification adjustment, and analog-to-digital converters (ADC) with 16 to 20 bit conversion width. The latter determine the dynamic measuring range with which a transient can be measured.
Where the term “acquisition of an ICR mass spectrum” or similar wording is used below, this encompasses the complete sequence of steps: filling the ICR measuring cell with ions, exciting the ions to cyclotron trajectories, measuring the image current transient, digitizing, Fourier transformation, determining the frequencies of the individual ion species and, finally, calculating the charge-related masses m/z and intensities of the ion species which constitute the mass spectrum, as is known by anyone skilled in the art.
In accordance with the principles of the invention ions in an ICR measuring cell are excited by a chirp with a nonlinear frequency function to, at least, strongly reduce, if not completely prevent, the generation of beats. When a nonlinear function of the frequency vs. time is used in the chirp, once the ions have each reached their orbit, the orbital separations between the ions are no longer proportional to their speeds. It is therefore no longer possible for all the ions to meet at the same point in time. The more distant the ions are from each other at the closest convergence, the more effectively the generation of a beat is prevented.
Any continuous and, preferably, also continuously differentiable nonlinear function can be selected as the frequency function. It is advantageous, for example, to select a quadratic dependence of the frequency on time, or a root function.
For a nonlinear frequency function with constant amplitude of the excitation voltage, the ions of different mass are excited for slightly different durations and thus lifted to cyclotron trajectories of slightly different radii; it is therefore advantageous to change the amplitude with the frequency function in such a way that the ions are lifted to the same cyclotron trajectories.
a shows the corresponding narrowband mass spectrum, which was measured with a resolution of R=2,500,000. The monoisotopic signal of the doubly charged molecular ions and three 13C satellites are visible in the mass spectrum.
a shows the narrowband mass spectrum with the complete isotopic group of the ions with 49 charges; below it, in
c, a further enlarged section, which extends over only 0.030 atomic mass units, but still contains 15 ion signals of the individual isotope satellites. The mass resolution amounts to R=800,000. It was only possible to acquire such good mass spectra because, in this case, 200 individual measurements of the transient in
While the invention has been shown and described with reference to a number of embodiments thereof, it will be recognized by those skilled in the art that various changes in form and detail may be made herein without departing from the spirit and scope of the invention as defined by the appended claims.
The invention consists in using nonlinear chirps to excite the ions in an ICR measuring cell. The term “nonlinear chirp” here means a chirp with nonlinear frequency function. The nonlinear frequency function for the excitation can, at least, greatly reduce, and usually even completely prevent, the generation of beats.
Chirps can be used with increasing or decreasing frequency function. Any continuous and, preferably, also continuously differentiable nonlinear function can be selected for the nonlinear frequency function. It is advantageous, for example, to select a quadratic dependence of the frequency on time, or a root function. But higher power functions, an exponential function or logarithmic function can also be used.
With a nonlinear frequency function in the chirp, the time differences and also the separations of the ions on reaching the orbit are nonlinearly stretched. The separations of the ion clouds are no longer proportional to their speeds, which are determined by the cyclotron frequencies of their ions and do not change. Since the starting points of the ions in the orbit are stretched in a nonlinear way, the ions can no longer meet up at a single point in time. The further the ions are from each other at a closest convergence, the more effectively the generation of a beat is prevented.
A nonlinear frequency function with constant amplitude of the excitation voltage no longer excites the different ion species of different masses in the same way. Since the speed of change of the frequencies is no longer constant, some ion species are excited for a slightly longer time than others, because their resonant frequency is supplied for a somewhat longer time. The varying length of excitation causes different ion species to be lifted to cyclotron trajectories of slightly different radii. The change in speed corresponds to the gradient of the frequency function or the first derivative of the frequency as a function of time.
In order to lift all the ions to the same orbit, the amplitude must be changed with the frequency. It is advantageous to change the amplitude proportionally to the gradient of the frequency function.
The excitation of the ions in the ICR measuring cell is effected by two excitation electrodes (or two series of excitation electrodes) which are located opposite each other, and to which the voltages of the chirp are applied. This generates a somewhat distorted dipole field. The distortion has proven to be almost completely irrelevant.
For maximum resolution, ICR mass spectrometers are always operated in a so-called “narrowband mode”, which measures only a small section of a full mass spectrum at any one time, as is familiar to those skilled in the art. All commercial ICR mass spectrometers offer this narrowband mode in addition to a broadband mode, thus making it possible to measure mass spectra over varying mass ranges. Transients with beats predominantly occur when acquiring narrowband spectra.
The sync pulses which are sometimes used instead of the chirps lift the ion clouds of different masses synchronously into their orbit. In this case, a beat is always initially produced until the ions have spread out and distributed themselves statistically over the orbit. If one wants to achieve the same effect with a sync pulse as with a nonlinear chirp, one has to depart from the basic idea of a sync pulse and temporally distribute the frequencies nonlinearly within the sync pulse. If one pursues this idea logically, one ends up with a nonlinear chirp.
All measurements described below were conducted in a magnetic field of a mere seven tesla; in currently available superconducting magnets with a magnetic flux density of 11 and 15 tesla, correspondingly higher mass resolutions could be achieved.
a shows the narrowband mass spectrum which was derived from the transient of
b is an enlarged section of
For those substances with a very much higher mass in the order of several ten thousand atomic mass units, it is usual to follow a broadband acquisition of an overview spectrum with a further acquisition of a narrowband mass spectrum which only shows the ions of one charge state at maximum resolution. A broadband mass spectrum of BSA is shown in
For heavy molecules, the ions of one charge state form an isotope group with often far more than a hundred isotope satellites. The problem with the beats occurs in this case. Since the ions of this isotope group each differ by one atomic mass unit (to be more precise: by the mass difference between 12C and 13C in each case), they constitute a mixture of many ions with the same mass differences, which, in a narrowband measurement, forms a transient consisting of a series of individual “beats”, as can be seen in
The formation of these beats impairs the resolution of the mass spectrum derived from it because the beats require a very high dynamic measuring range for the measurement of the image currents. The beats exceed the transient itself in the spaces by a factor of 100 or more. The measurements of the image currents of normal transients require a measuring range of about 1:106; the occurrence of beats means that a measuring range of 1:108 would be required. This measuring range can hardly be provided by the electronics, and so the prior art overcomes this problem by using a very high number of repeat measurements. Hundreds of image current measurements are performed and added together, but this requires exceedingly long measuring times.
In
It should be noted here that a successful summation of 200 individual transients requires the electronics to have an unusually high degree of stability, which is rarely the case. Furthermore, a long measuring time of about one hour is required, which is not available for analyses in routine laboratories.
From mass spectra of the type shown in
The invention thus offers the advantage of performing measurements of uniformly structured ion mixtures in a significantly shorter time, yet providing mass spectra of the same quality. It is even to be expected that mass spectra with still higher quality, for example better resolution and higher mass accuracy, can be measured. The use of nonlinear chirps does not have to be restricted to uniformly structured ion mixtures, but can be used for all types of spectral acquisitions, effectively as the basic setting.
| Number | Date | Country | Kind |
|---|---|---|---|
| 10 2008 064 610.5 | Dec 2008 | DE | national |
