EP0294683B1 - Method for recording ICR mass spectra and ICR mass spectrometer developed for performance of this method - Google Patents

Description

The invention relates to a method and a device for recording ICR mass spectra in each case according to the preambles of patent claims 1 and 4. Such a method is known from US Pat. No. 3,937,955.

Ion cyclotron resonance is an excellent method for mass spectroscopy because of its versatility, sensitivity and high resolution. Ions of different types contained in a gas sample can be excited simultaneously by a corresponding broadband pulse, so that a frequency mixture is present in the high-frequency signal induced by the excited ions after the end of the pulse. The components contained in the induction signal can then be resolved by a Fourier transform according to frequency and intensity.

However, ICR mass spectroscopy allows not only the analysis of substances and mixtures of substances, but also the observation of dynamic processes, such as the observation of the products of ion-molecule collisions and unimolecular fragmentations, through double resonance. In this double resonance, also known as an MS / MS experiment, all ions except the one type of ion that is to be investigated are first eliminated by the ions of the substance to be investigated, which are trapped in the measuring cell of an ICR mass spectrometer, by irradiation of appropriate cyclotron resonance frequencies. If necessary, a collision gas is then let into the measuring cell. The selected ion type is then excited to such an extent that there are collisions with one another or with the molecules of the collision gas and secondary fragments are formed by collision dissociation. The secondary ions formed are then analyzed by the usual ICR measurement cycle. If the original mass spectrum contains a number of N lines, a number N of such experiments is required for a complete analysis. This creates a number of new spectral lines for each line of the original spectrum, see above that a two-dimensional array of spectral lines is obtained if the original spectral lines are plotted along a coordinate direction and the secondary spectral lines assigned to these spectral lines are plotted along a second coordinate direction. Even if such an MS / MS experiment is carried out automatically, carrying out such an experiment requires a very long time and a considerable outlay on equipment. The automatic also fails if the spectra are very complex and have overlapping lines or if they contain weak lines lying on the detection line.

In the case of the method for recording ICR mass spectra known from the generic US Pat. No. 3,937,955, it can happen that closely located peaks of different productions may not be able to be resolved, since the known method uses only two excitation pulses separated by a delay time and this single delay time between the two excitation pulses is fixed and is not varied systematically.

The invention has for its object to further develop the generic method for recording ICR mass spectra so that it can be used with short measuring time even in complicated cases in which peaks lying close together must be resolved. Furthermore, it is an object of the invention to provide a device which is suitable for carrying out the method. This object is achieved by the features characterized in claims 1 and 4. The dependent claims 2, 3 and 5 characterize advantageous developments thereof.

The method according to the invention is accordingly comparable in some respects to the method of 2D exchange spectroscopy (NOESY) known from nuclear magnetic resonance, which serves to process dynamic processes such as chemical reactions, isomerization and the like. To be examined (see, for example, BH Meier and RR Ernst in J. Am. Chem. Soc. 101 (1979) 6441 and J. Jeener et al in J. Chem. Phys. 71 (1979) 4546). Nevertheless, it was not obvious to use an analog method in ICR spectroscopy, because there are fundamental differences between the transverse magnetization of the spins observed in NMR and the coherent resonance of the ions excited in ICR spectroscopy. In addition, the resonance frequencies that occur in NMR spectroscopy are very closely adjacent, so that they differ from each other by a few percent at most, whereas the resonance frequencies in cyclotron ion resonance are in a ratio of up to about 1 because of the greatly different charge / mass ratios : 50 can stand. In an ICR mass spectrometer in which the measuring cell is exposed to a static magnetic field of 3T, the resonance frequencies of substances of interest can vary from about 50 kHz to 2.6 MHz. However, the difficulties resulting therefrom can in an embodiment of the method according to the invention either are overcome in that the second RF pulse P ₃ has a different frequency than the two partial pulses P ₁ and P _2, or characterized also in that the high-frequency pulses broadband pulses given with a Range of varied frequency. Such broadband pulses are also referred to as "chirp pulses" (MB Comisarow and AG Marshall in Chem. Phys. Lett. 26 (1974) 489).

P₁

Erster Teilimpuls des ersten Hochfrequenzimpulses

P₂

Zweiter Teilimpuls des ersten Hochfrequenzimpulses

P₃

Zweiter Hochfrequenzimpuls

t₁

Variable Vorbereitungszeit (Zeitparameter in der ersten Dimension)

t₂

Meßzeit für das Interferogramm (Zeitparameter der zweiten Dimension)

τ_m

Reaktionszeit

The process according to the invention can therefore be described by the following sequence:
P ₁ - t ₁ - P ₂ - τ _m - P ₃ - t ₂

P ₁

First partial pulse of the first high-frequency pulse

P ₂

Second partial pulse of the first high-frequency pulse

P ₃

Second high frequency pulse

t ₁

Variable preparation time (time parameters in the first dimension)

t ₂

Measuring time for the interferogram (time parameter of the second dimension)

τ _m

reaction time

As mentioned above, the second sub-pulse P _{2 has} the same frequency and phase as the first sub-pulse P ₁ . If, at the end of the variable preparation time t _1, the ions have a phase that is opposite to the phase of the second partial pulse P ₂ , the second partial pulse P ₂ partially cancels the effect of the first partial pulse P ₁ . The effect of the second partial pulse therefore depends on the instantaneous phase of the movement of the individual ions after the first time t ₁ , which was therefore referred to as the preparation time. Therefore, the number of incoherent ions that are present after the end of the second partial pulse P ₂ and thus at the beginning of the reaction time τ _m is a function of the preparation time t ₁ . The events occurring within the reaction time τ _m , which depend on the number of excited ions, are therefore influenced accordingly. As a result, the induction recorded after the second excitation of the ions by the second high-frequency pulse P ₃ during the second measurement time t ₂ Depends on the duration of the preparation time t ₁ . According to the invention, the preparation time t _{1 is} varied systematically. The signals converted into the frequency domain with respect to the time axis t ₂ are converted into frequency-dependent signals with respect to the time axis t ₁ , so that a two-dimensional representation of the secondary effects which are conceivable for the primary ions is obtained. With a suitable choice of parameters and in the presence of a collision gas, spectra can be generated in this way, for example, which are comparable to the spectra obtained with the aid of the MS / MS experiment.

Fig. 1a

das als Funktion der Vorbereitungszeit t₁ modulierte ICR-Signal S(t₁, ω₂) von ⁸¹Br-Pyridin⁺,

Fig. 1b

die Fourier-Transformierte des ICR-Signals nach Fig. 1a,

Fig. 2

ein zweidimensionales Fourier-ICR-Spektrum von ⁸¹Br-Pyridin+ und

Fig. 3

das zweidimensionale ICR-Spektrum der Reaktion CH₃CO⁺ + CH₃COCH₃ → CH₃C⁺(OH)CH₃.

The invention is described and explained in more detail below on the basis of several exemplary embodiments of the method according to the invention and the spectra obtained thereby and shown in the drawing. Show it

Fig. 1a

the ICR signal S (t ₁ , ω ₂ ) of ⁸¹ Br-pyridine ⁺ modulated as a function of the preparation time t ₁ ,

Fig. 1b

the Fourier transform of the ICR signal according to FIG. 1a,

Fig. 2

a two-dimensional Fourier ICR spectrum of ⁸¹ Br-pyridine + and

Fig. 3

the two-dimensional ICR spectrum of the reaction CH ₃ CO ⁺ + CH ₃ COCH ₃ → CH ₃ C ⁺ (OH) CH ₃ .

The following experiments were carried out with a Spectrospin-ICR mass spectrometer type CMS-47, the superconducting Magnet generated a field of 3T, and evaluated with an Aspect 3000 computer.

• A = ⁸¹Br-Pyridin; m_A = 159 amu,
• f_A = 289.7 kHz; f_AH = 287.8 kHz

• B = ⁷⁹Br-Pyridin; m_B = 157 amu,
• f_B = 293.4 kHz; f_BH = 291.5 kHz

First, a mixture of ⁸¹ Br pyridine and ⁷⁹ Br pyridine was examined. These two substances are referred to below as A and B. Accordingly:

• A = ⁸¹ Br pyridine; m _A = 159 amu,
• f _A = 289.7 kHz; f _AH = 287.8 kHz

• B = ⁷⁹ Br pyridine; m _B = 157 amu,
• f _B = 293.4 kHz; f _BH = 291.5 kHz

sowie einen Protonentransfer vom Ion zu neutralen Teilchen, nämlich $${\text{A}}^{\text{+·}}{\text{oder B}}^{\text{+·}}{\text{+ A → AH}}^{\text{+}}\text{+ neutrale Produkte.}$$

As far as the processes relevant for this experiment are concerned, these substances can enter into the following reactions, namely a hydrogen transfer from neutral particles to the ion: $${\text{A}}^{\text{+ ·}}{\text{+ A or B → AH}}^{\text{+}}\text{+ neutral products,}$$

and proton transfer from ion to neutral particles, namely $${\text{A}}^{\text{+ ·}}{\text{or B}}^{\text{+ ·}}{\text{+ A → AH}}^{\text{+}}\text{+ neutral products.}$$

also immer dann, wenn die im Verlauf der Vorbereitungszeit t₁ sich entwickelnde Phasenverschiebung Ω_A t₁ gegenüber der Hochfrequenz-Schwingung des esten Teilimpulses P₁ eine Phasenverschiebung von 180° aufweist. Demgemäß erscheinen diese Spitzen in Zeitintervallen von 1,32 ms. Das bei der Aufnahme dieser Kurve verwendete Digitalisierungsintervall betrug Δ t₁ = 166 µs. Unter diesen Bedingungen hat der zweite Teilimpuls P₂ in der vorstehend erwähnten Sequenz die Wirkung einer "Aberregung" der Ionen, die ursprünglich von dem ersten Anregungsimpuls P₁ angeregt worden waren, so daß sie in dem Reaktionsintervall τ_m eine fast verschwindende kinetische Energie aufweisen und durch den zweiten Hochfrequenzimpuls P₃ in die Cyclotronbahnen zurückgebracht werden können, in denen sie beobachtet werden. Fig. 1b zeigt dann die Fourier-Transformierte des ICR-Signals nach Fig. 1a, in dem geradzahlige und ungeradzahlige Seitenbänder mit positiver bzw. negativer Amplitude erscheinen.The Br-pyridine was ionized at a pressure of 6.10 ^-8 mbar with a 20 ms pulse of 70 eV electrons. The duration of the excitation pulses was 20 µs and their amplitude 35 V _pp . The frequency f _{0 of} the excitation pulses was at a distance Ω _A / 2µ = 760 Hz from the frequency f _{A of} the ⁸¹ Br pyridine. The resulting spectral window was large enough to capture the signals from A ⁺ · and AH ⁺ , however the signals from BH ^{+ are} folded published. FIG. 1 shows the τ ₁ dependency of the signal of A ⁺ , which is caused by the measurement sequence treated at the beginning
P ₁ - t ₁ - P ₂ - τ _m - P ₃ - t ₂
is obtained. The sharp peaks in the t ₁ range always appear when $${\text{Ω}}_{\text{A}}{\text{t}}_{\text{1}}\text{= (2k + 1) π, k = 0.1.2 ...,}$$

that is, whenever the phase shift Ω _A t ₁ developing in the course of the preparation time t ₁ has a phase shift of 180 ° with respect to the high-frequency oscillation of the first partial pulse P ₁ . Accordingly, these peaks appear at time intervals of 1.32 ms. The digitization interval used when recording this curve was Δ t ₁ = 166 µs. Under these conditions, the second sub-pulse P ₂ in the above-mentioned sequence has the effect of "de-excitation" of the ions which were originally excited by the first excitation pulse P ₁ so that they have an almost vanishing kinetic energy in the reaction interval τ _m and can be brought back into the cyclotron orbits in which they are observed by the second high-frequency pulse P ₃ . FIG. 1b then shows the Fourier transform of the ICR signal according to FIG. 1a, in which even-numbered and odd-numbered sidebands with positive and negative amplitudes appear.

Fig. 2 shows the complete two-dimensional spectrum. The ω ₂ frequency axis corresponds to the Fourier transform with respect to the observation time t ₂ . The vertical ω ₁ range, which is determined by a real cosine transformation with respect to the Preparation time t _{1 was} obtained shows sideband families that are connected by arcs for clarity. The cross section, ie the column for ω ₂ = Ω _A , corresponds to the Fourier transform shown in FIG. 1b. The first sidebands in all families lie on one of the diagonals shown by dashed lines in Fig. 2, apart from the resonance at Ω _BH , which appears to be folded. The frequency origin at the intersection of the dashed diagonals corresponds to the RF carrier frequency f ₀ . The column at ω ₂ = Ω _AH contains not only a diagonal line with its series of sidebands, but also a cross line at ω ₁ = Ω _A and ω ₂ = Ω _AH with the associated sidebands, which are all highlighted by squares. These signals provide direct evidence of the above-mentioned reaction A ⁺ · → AH ⁺ · Because of their alternating signs, these lines can be identified unambiguously. The spectral width was 3000 Hz in both areas. The number of observed points was 240 x 2048 in the two time ranges t ₁ and t ₂ , which were filled up to zero with 256 x 2048 points before the Fourier transformation. There was a line broadening of 20 Hz in the ω ₂ range and 40 Hz in the ω ₁ range.

Despite the uniqueness of the lines, the interpretation of such two-dimensional spectra can be difficult due to the presence of both diagonal and cross lines with their associated sideband families. However, there is a possibility of the occurrence of sidebands. to be avoided if instead of a Fourier transformation the method of maximum entropy is used to convert the time-dependent resonance signals into frequency-dependent signals. However, the measuring method according to the invention remains unaffected by this, so that an example of using one has not been used the spectrum obtained using the maximum entropy method.

In order to illustrate the variant of the method, used in for the second RF pulse P ₃ is a different frequency than for the first two partial pulses P ₁ and P ₂ of the first high-frequency pulses, the following reaction has been selected: $${\text{CH}}_{\text{3rd}}{\text{CO}}^{\text{+}}{\text{+ CH}}_{\text{3rd}}{\text{COCH}}_{\text{3rd}}{\text{→ CH}}_{\text{3rd}}{\text{C.}}^{\text{+}}{\text{(OH) CH}}_{\text{3rd}}\text{.}$$

CH ₃ CO ^{+ has} the mass ratio m _c = 43 amu and a resonance frequency of f _c = 1071 kHz. The reaction product CH ₃ C ⁺ (OH) CH ₃ has the mass ratio m _D = 59 amu and the resonance frequency f _D = 779.9 kHz.

For the two partial pulses P ₁ and P _{2 of} the first high-frequency pulse, a frequency was chosen whose distance from f _{c was} 79 Hz, while for the second high-frequency pulse P ₃ a frequency was chosen whose distance from f _{D was} 100 Hz. The 2D ICR spectrum recorded in the manner described is shown in FIG. 3. The occurrence of a cross line in the ω ₂ range shown vertically here at ω ₂ / 2π = 100 Hz and in the ω ₁ range at ω ₁ / 2π = 79 Hz clearly proves that the above-mentioned reaction has taken place. The cross line is again accompanied in the horizontal ω ₁ range by a sideband family, the members of which appear in multiples of 79 Hz. The spectral width of the full matrix was 500 x 500 Hz, of which only 40% are shown. 56 x 4048 data points, filled up to 128 x 4048 data points with zeros, were processed. The line broadening was 30 Hz in the ω ₁ range and 20 Hz in the ω ₂ range.

Instead of using a different frequency for the second high-frequency pulse P ₃ than for the two sub-pulses P ₁ , P ₂ , the frequencies being matched to the resonance frequencies of the starting products and the end products, it is possible to use broadband pulses in the form of so-called chirp pulses. whose frequency is varied over a certain range, which includes the resonance frequencies of the starting materials and the expected reaction products. The use of such broadband pulses does not change the basic sequence of the method according to the invention.

As already mentioned above, the method according to the invention delivers essentially the same results as can also be obtained with an MS / MS experiment. Nevertheless, the method according to the invention has many advantages, which are particularly useful when complex networks are to be examined, in which a multiplicity of exchange processes take place simultaneously, all of which are recorded simultaneously using the method according to the invention, whereas in an MS / MS experiment all possible exchange processes have to be recorded by individual measurements to be carried out one after the other. The method according to the invention also allows the kinetics of reactions to be examined by observing the amplitude of the signals obtained as a function of the duration of the reaction interval τ _m or as a function of various manipulations to which the system under test is exposed during the reaction time τ _m , such as laser pulses, electron beam pulses or neutral gases introduced in the form of a pulse, the molecules of which give rise to collision reactions.

From the above it can be seen that the new method offers the person skilled in the art many possibilities for mass spectroscopic investigations offers that were difficult or impossible to implement with the previous methods.

Claims (5)

1. Method for recording ICR mass spectra with which:

   - ions and neutral molecules are present in the measuring cell of an ICR mass spectrometer,

   - the ions are excited into oscillation by a first radio frequency pulse (P₁, P₂) applied to the measuring cell,

   - can subsequently, during a predetermined mixing time (τ_m) , interact with the neutral molecules,

   - a second radio frequency pulse (P₃) then being irradiated and the resonance signals thereby induced being subsequently received during a predetermined measuring time t₂, recorded and converted into frequency dependent signals,

   characterized in that
   the first radio frequency pulse comprises two partial pulses (P₁, P₂) having the same frequency and whose time separation t₁ is systematically varied such that the amplitude of the induced resonance signal depends on t₁ and the set of resonance signals dependent on measuring time t₂ and the varied separation t₁ are converted into two dimensional frequency dependent signals with elimination of the measuring time dependence t₂ and the separation t₁.
2. Method according to claim 1, characterized in that the rf pulses have a frequency varied within a predetermined range.
3. Method according to claim 1, characterized in that the second high frequency pulse (P₃) has a different frequency than the two partial pulses (P₁, P₂) of the first radio frequency pulse.
4. ICR mass spectrometer for carrying out the method according to one of the preceding claims comprising a measuring cell, transmitter means connected thereto for generating rf signals, receiver means, which are likewise connected thereto, for the induced rf signals and a computer connected to the receiver means for transforming the received time-dependent rf signals into corresponding frequency dependent signals,
   characterized in that
   the transmitter means is adapted for generating two partial pulses, of equal frequency and separated by a time (t₁), of a first rf pulse comprising two partial pulses and a second rf pulse of equal or another adjustable frequency and comprises at least one time element by means of which the time interval (t₁) between the partial pulses can be progressively varied,
   and the receiver means is adapted for storing a plurality of time-dependent rf signals, and the computer is adapted for transforming the time-dependent rf signals to generate two-dimensional frequency-dependent signals from the sets of stored time-dependent rf signals.
5. ICR mass spectrometer according to claim 4, characterized in that the transmitter means is adapted for generating rf pulses with a frequency varying during their duration.

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