EP0503748B1 - Method for generating ions, specially for a mass spectrometer such as a time-of-flight mass spectrometer, from thermally instable, non-volatile, large molecules - Google Patents

Description

The invention relates to a method for generating ions, in particular for a mass spectrometer, such as time-of-flight mass spectrometer, from thermally unstable, non-volatile large molecules, in which a sample substance containing the molecules is exposed to energy pulses, by which molecules are released from the sample substance, and in which the released substances Molecules are taken along by a jet of a carrier gas and cooled during its expansion and then ionized in an ionization space.

DE-A-38 00 504 discloses a method of the generic type in which the molecules are desorbed by a laser beam. It is used to convert, in particular, large molecules into the gas phase, before the molecules are brought into a chemical state by means of a subsequent ionization process, in which they are only accessible for mass spectrometric analysis. This takes advantage of the fact that the internal energy absorbed by the molecules through the desorption in the carrier gas jet is greatly reduced, so that the molecules are strongly cooled and their thermal decomposition is largely prevented. This desorption method is suitable for liquid and solid sample substances, and it has proven to be advantageous to accommodate the molecules of the sample substance in a matrix that decomposes easily thermolytically.

A device with which this desorption process can be carried out is described in the magazine "ANGEWANDTE CHEMIE" 1988, pages 461 ff. The sample substance is placed in front of the opening of a nozzle from which the carrier gas emerges. The molecules of the sample substance are desorbed into the expanding beam of the carrier gas by using infrared laser light. This cools the internal degrees of freedom of the molecules and the molecules are transported further by the carrier gas jet. Usually this is Device operated as a pulsed system, which consists of a pulsed valve for generating the carrier gas jet and a laser for desorbing the neutral molecules. Since the molecules are transported on as a beam, or in the pulse mode as a particle packet, it is possible to keep this desorption process spatially separate from the ionization process that now follows.

In the publication "Laser desorption jet-cooling of organic molecules" (Applied Physics B, Volume B41, No. 6, pages 395 - 403) the cooling effect of the carrier gas jet on the desorbed molecules is examined. The detection sensitivity of the system used, in which a laser is used to ionize the molecules, is also measured.

Single-photon or multiphoton ionization has proven itself for the mass spectrometric analysis of the large molecules under consideration. Since the wavelength of the irradiated photons can be matched to the energy difference between the ground state and an excited state of the neutral molecule, it is possible to selectively ionize only the molecules to be examined; the carrier gas particles remain in their neutral state and do not influence the subsequent test results.

Although multiphoton ionization mass spectrometry is operated successfully, it cannot be used for some problems, since often only a selective excitation of the neutral molecules cannot provide enough information for the desired structural elucidation of the molecule, because the unknown excitation wavelength is not sufficient for unknown molecules can be predetermined. It has also been found that some substances are difficult to ionize in the manner shown.

Ionization processes are known which act non-selectively. This includes electron impact ionization. However, such methods cannot be used for large molecules, as in the present case, since they lead to severe fragmentation of the molecule. In addition, the carrier gas particles would also be ionized, which would lead to saturation effects, electrostatic repulsion and thus to poor resolution and insufficient sensitivity of the analysis. Such influences cannot be neglected, because the carrier gas particles are at least a thousand times higher than the molecules to be examined.

The use of electron impact ionization is known from DE-A-873 765, however the combination of this procedure with a method as is known from DE-A-36 19 886 only leads to highly fragmented ions in the low mass range. so that there are no large, thermally labile, non-volatile molecules such as B. peptides.

In the publication "Analytical Instrumentation", Volume 16, No. 1, 1987, pages 93a to 115 describe a method for laser desorption with ionization in one step. This process is made possible by the modification of an apparatus for the ionization of volatile substances through electron collisions. The modified ionization method described above can thus also be carried out with such a modified apparatus.

The object of the invention is to further develop the generic method with an ionization process that is separate from the desorption process in such a way that ions can be provided from thermally unstable, non-volatile large molecules, a non-selective ionization method being able to be used.

According to the invention, this object is achieved in a further development of the generic method in that the molecules are ionized by electron impact; that the radiation density of the electrons used for the ionization is selected so that a potential well is created in the focus of the electron beam, the depth of which is greater than the translation energy of the molecular ions in the carrier gas stream; that the molecular ions generated by the electron impact ionization are collected in the potential well for a certain period of time; and that the molecular ions collected in each case in the potential well are pulsed out of the ionization chamber.

Particularly preferred embodiments of the method according to the invention are the subject of the corresponding subclaims.

The invention is based on the surprising finding that, contrary to the widespread prejudice of the professional world, it is also possible to ionize unstable molecules by means of electron impact, this possibility being created in that the "jet" beam, which is generated by means of the generic method, is a such cooling of the heavy molecules which only carry out very small relative movements within the beam and which are later to be ionized, leads to the fact that they do not disintegrate during electron impact ionization. The additional measure of the pulsed removal of the ionized molecules, which is made possible by the generation of the potential well of adjustable depth, promotes the detection sensitivity in the mass spectrometer and thus the resolution in a manner essential to the invention.

According to the invention, molecules are thus ionized by electron impact, helium and / or neon preferably being used as the carrier gas; the energy of the electrons in the electron beam is of course preferably below the ionization energy of the carrier gas.

With the method according to the invention, the entire advantages of the known technique of laser evaporation with subsequent cooling can be used to investigate thermally unstable, non-volatile molecules that would otherwise not be accessible to such analysis methods. Since the internal energies of the molecules are significantly reduced by the cooling, the subsequent electron impact ionization also leads to fewer fragments than is usually expected. The advantages of the spatial separation of evaporation and ionization can be maintained - flexibility in the design of the desorption and ionization system without the need for structural compromises; no staining of the ion source by desorbed sample material; Ions formed during desorption (in contrast to neutral sample molecules) do not reach the ion source - whereby good yields can be achieved. By using the noble gases helium or neon as carrier gas, the generation of carrier gas ions is prevented or at least very strongly suppressed. Both noble gases have a very high ionization potential (24.6 eV or 21.6 eV), so that they are practically not ionized at electron energies below these ionization potentials, as are preferably used. The molecules to be investigated, with an ionization potential of the order of about 10 eV, are already very well ionized at the electron energies mentioned.

A mass spectrometric detection method that uses the pulse structure of ion generation is time-of-flight (TOF) mass spectrometry. Basically, a time-of-flight mass spectrometer has the advantage that a complete mass spectrum is registered with each pulse. In addition, time-of-flight mass spectrometry has a physical property that makes it particularly suitable for the study of large molecules. The resolution increases with increasing mass.

However, a good resolution when examining the molecular ions with a time-of-flight mass spectrometer can only be achieved if the molecular ions start at a point (t <5ns) that is as precisely defined as possible in the smallest possible space (<1mm). In general, it is therefore not possible to use the entire sample contained in a gas jet.

However, it has been shown that the sensitivity of the arrangement can be increased if the radiation density of the electrons used for the ionization is chosen such that a potential well is generated in the focus of the beam. The neutral molecules to be examined fly into the focus of the electron beam, are ionized there, but then - in the ionized state - can no longer leave the focus. They are therefore collected in a spatially limited volume over a relatively long period of up to 100 µs. The ionized molecules collected in this way can each be subtracted as a "package" with a precisely defined start time. B. 50 ns after the end of the collection (switching off the electron beam) the end plate is switched to 0 V and thereby the ionized molecules are accelerated into the mass spectrometer. This pulsed "packet transmission" enables a good yield with high resolution (as is customary for laser ionization) and at the same time high sensitivity (as is characteristic of electron impact ionization) to be achieved.

Light pulses generated by laser can also be used as energy pulses for desorption; continuously operating lasers are also suitable. The wavelengths used are in the range from micrometers down to a few tens of nanometers. The energy pulses can also be applied by bombardment with ions or neutral particles.

In a special embodiment of the invention, the sample is optionally ionized by electron impact or by photon excitation in the same ionization space. The sample to be examined therefore only has to be prepared once and let into the ionization space and can then be examined using the advantages of both ionization methods. This ensures that one and the same sample is examined using both measurement methods. It is advantageous if both the photon beam is pulsed and the gaseous sample is supplied in a pulsed manner, the ionization processes having to be switched in a correspondingly synchronized manner. The switching frequency is only limited by the time required to record the spectrum and evaluate it.

In contrast to the prior art, as stated, an electron source and a photon source are provided for one and the same ionization chamber and can be operated optionally for ionizing the gaseous sample within the ionization chamber, the ionization preferably taking place in a pulsed manner. In the apparatuses known hitherto, the photon ionization was carried out in a constant electric field, the necessary precisely defined starting time of the ions being determined by means of laser pulses which were measured over time. Such a procedure is impractical for electron impact ionization. If one would like to switch easily between electron impact ionization and photon ionization, this can only be done by carrying out the photon ionization not in a standard manner but in a manner adapted to the apparatus for electron impact ionization.

For the switchover, it is also important that the adjustment and thus also the mass calibration of the downstream mass spectrometer do not have to be changed. Only like that Repetition rates of the order of 20 Hz can be achieved, so that it is possible to switch over from the molecules to be examined for each pulse packet. To achieve this, the ions must be ionized at exactly the same time, in the same volume and at exactly the same potential. In addition to carrying out the ionization within an ionization chamber for the different ionization processes, it is advantageous that the electron beam emitted by the electron source and the photon beam emitted by the photon source are focused essentially on the same area of the ionization chamber. If the electron beam and the laser beam are set up to be adjustable, the required settings can be made relatively easily during the operation of the device.

It has already been stated above that the photon ionization is also carried out under the same conditions as the electron impact ionization. For this purpose, the ionization chamber is advantageously an ionization chamber set to a positive potential, the end plate of which, ie the area in which the ionized molecules leave the ionization chamber, can be acted upon separately. The start time for the ions released from the ionization chamber is then determined by the fact that this end plate is at ground potential, i.e. is switched to 0 V. Of course, it is also possible within the concept of the invention to switch the end plate to a potential other than 0 V, provided that this is only chosen so that it is suitable for accelerating the ions into the mass spectrometer. At the time of switching the end plate to the acceleration potential, the ions start their flight in the acceleration field thus created in the mass spectrometer connected downstream of the ionization chamber.

According to a preferred embodiment of the invention Electron source and / or photon source, as already explained, operated in pulsed mode, so that a time-of-flight mass spectrometer can be used as the mass spectrometer, with which a complete mass spectrum can be recorded for each ion packet.

Fig. 1

den schematischen Aufbau einer Vorrichtung zur Durchführung eines ersten Ausführungsbeispieles des erfindungsgemäßen Verfahrens;

Fig. 2

eine Detailansicht der Vorrichtung im Bereich der zu verdampfenden Probensubstanz;

Fig. 3

ein Intensitäts-Zeit-Diagramm für den verwendeten Elektronenstrahl;

Fig. 4

ein Spannungs-Zeit-Diagramm für die Beschleunigungsplatte aus der Vorrichtung nach Figur 1;

Fig. 5

ein Rohdatenspektrum der nichtflüchtigen Substanz Mesoporphyrin, wobei dem Trägergasstrahl (Helium) zu Testzwecken Luft und Benzol beigemischt wurde;

Fig. 6

das Rohdatenspektrum des nicht-flüchtigen, thermisch instabilen Peptids Trp-Met-Asp-Phe-NH₂;

Fig. 7

den schematischen Aufbau einer Vorrichtung zur Durchführung einer weiteren Ausführungsform des erfindungsgemäßen Verfahrens;

Fig. 8

mit der Vorrichtung von Fig. 7 erhaltene Rohdatenspektren für das thermisch instabile Peptid Trp-Pro-Leu-Gly-Amid, wobei sowohl das Multiphotonen-Ionisationsspektrum (MPI) als auch das Elektronenstoß-Ionisations-Spektrum (EI) dargestellt sind; und

Fig. 9

ein Rohdatenspektrum, erhalten mit der Vorrichtung von Fig. 7, des thermisch instabilen Peptides Pro-Phe-Gly-Lys-Acetat, wobei wiederum sowohl das Multiphotonen-IonisationsSpektrum (MPI) als auch das Elektronenstoß-Ionisations-Spektrum (EI) dargestellt sind.

Further features and embodiments of the invention result from the claims and from the following description, in which exemplary embodiments are explained in detail with reference to the schematic drawing. It shows:

Fig. 1

the schematic structure of a device for performing a first embodiment of the method according to the invention;

Fig. 2

a detailed view of the device in the region of the sample substance to be evaporated;

Fig. 3

an intensity-time diagram for the electron beam used;

Fig. 4

a voltage-time diagram for the acceleration plate from the device of Figure 1;

Fig. 5

a raw data spectrum of the non-volatile substance mesoporphyrin, air and benzene being added to the carrier gas jet (helium) for test purposes;

Fig. 6

the raw data spectrum of the non-volatile, thermally unstable peptide Trp-Met-Asp-Phe-NH ₂ ;

Fig. 7

the schematic structure of a device for performing a further embodiment of the method according to the invention;

Fig. 8

7 for the thermally unstable peptide Trp-Pro-Leu-Gly-Amide, obtained with the device from FIG. 7, both the multiphoton ionization spectrum (MPI) and the electron impact ionization spectrum (EI) being shown; and

Fig. 9

a raw data spectrum obtained with the device of Fig. 7, the thermally unstable Peptides Pro-Phe-Gly-Lys-Acetate, again showing both the multiphoton ionization spectrum (MPI) and the electron impact ionization spectrum (EI).

A device for generating ions from thermally unstable, non-volatile large molecules according to the method according to the invention, which is shown in FIG Vacuum emerges. When helium is used as the carrier gas, a gas pulse with a length of 1 µs to 10 ms is generated, a pulse length of 500 µs or less being optimal for most purposes. A helium pre-pressure of about 2 x 10 ⁵ Pa (2 bar) is set on the high pressure side of the valve; In principle, it can be advisable to keep the pressure between 2 x 10 ⁴ - 2 x 10 ⁷ Pa (0.2 bar to 200 bar) depending on the requirements. The nozzle 10 has an opening with a diameter of 0.2 mm, which can also be varied in the size range from 0.01 to 1 mm. The valve or nozzle 10 is opened electromagnetically.

A gas pulse generated in this way is also called a supersonic jet. The carrier gas atoms move at approximately the same speed, the relative thermal movement of the atoms being relatively low. As a result, the beam has a low temperature of the order of 1 K.

In the immediate vicinity of the opening of the nozzle 10 there is a sample holder 3 with a sample attached to it, which sample can either be solid or liquid, it also being possible to incorporate this sample into a matrix. Pulsed infrared light is emitted from a suitable light source, for example from a CO ₂ laser, approximately perpendicular to the beam emerging from the nozzle 10 as a photon beam 2 onto the sample carrier 3 or the sample located thereon.

A lens 20 is provided for focusing the photon beam 2. The pulse of this photon beam 2 is synchronized in time with the pulse of the carrier gas emerging from the nozzle 10. A suitable pulse length for a CO ₂ laser with a wavelength of 10.6 µm is 10 µs. The material to be examined is preferably desorbed into the space in front of the nozzle 10 by the incident photon beam 2. First of all, all degrees of freedom of the molecules, namely degrees of freedom of rotation, vibration and translation, are excited; the energy contained therein will then cool down sharply in the particle beam, the supersonic jet. This largely prevents the thermally unstable molecules from decaying.

The molecules desorbed from the sample carrier 3 are now in the gaseous state and are largely in the carrier gas jet emerging from the nozzle 10. Together with the carrier gas, the molecules are transported as a particle beam 4 to a stripper or skimmer 5, which only allows the central area of the particle beam 4 to pass. The "peeled" portion of the particle beam 4 must be pumped out for reasons of vacuum technology and is therefore no longer available for analysis.

The skimmer 5 consists essentially of a hollow cone placed on a flat wall 50, the tip of which is formed into an opening 51, the diameter of which is selected in accordance with the cross section of the particle beam 4 to be masked out. It is thus achieved that a particle beam aligned almost exactly in a preselected direction finally enters the ionization region.

The ionization takes place within an ionization chamber 7. The front wall 70 of the ionization chamber 7 is aligned with the nozzle 10 and the opening 51 of the skimmer 5, an inlet opening 71 through which the particle beam 4 enters. Impinging the particle beam 4 perpendicularly, a pulsed electron beam 6 is introduced into the ionization chamber 7, the focus 61 of which is set such that it lies on the path of the particle beam 4. The electron beam is pulsed in time with a length of 10 ns to 100 μs, the pulse being synchronized with the time span in which the particle "packet" is flying by. The ionization chamber 7 is at a positive potential of approximately 1000 V.

The energy of the electrons introduced in the electron beam 6 can be regulated from a few eV to 100 eV. These electrons now ionize the molecules to be examined by electron impact. If the energy of the electrons is selected in the order of magnitude of 25 eV, the particles of the carrier gas are not ionized, so that there is no falsification of the result later in the mass spectrometric analysis.

The radiance of the electrons is so high that in the focus 61 of the electron beam 6 a potential well or potential can build up that is deep enough for the ionized molecules, i.e. molecular cations, which initially move at the speed of the particle beam 4 for a short time catch. In this way, the molecular ions to be examined are collected in a spatially limited volume.

The pulse duration of the electron beam 6 is adjusted so that the pulse is ended when the collection is also finished. A few 10 ns later, the end plate 73 closing the ionization chamber 7 is switched to 0 V in less than 5 ns. At this time, the molecular ions start their flight in the resulting acceleration field from the collection point in focus 61 to the outlet opening 72 in the end plate 73 Time of flight mass spectrometer.

Figure 2 shows the space in front of the nozzle 10 of the device for generating a carrier gas jet. The jet 4 emerges as a pulse packet from the nozzle 10 and passes through the sample substance 30 on the sample carrier 3 from the molecules to be examined. Synchronized with the carrier gas pulse package, a photon pulse 2 is irradiated, which causes the molecules to be desorbed from the sample substance or from the sample carrier 3. The molecules diffuse into the particle beam and are carried by it in the direction of the skimmer or the ionization chamber.

FIG. 3 shows the intensity-time diagram of the electron beam which effects the ionization of the molecules in the ionization chamber. The pulse has steep edges and is kept constant over the period of time required for ionization.

After switching off the electron beam pulse (Figure 3), the potential of the end plate of the Faraday cage, as shown in Figure 4, is switched off within a very short time, so that there is also a steep-edged pulse, which over a period of z. B. 20 µs is kept at 0 V, which is sufficient to generate the field required for the acceleration of the molecular ions; of course, the end plate can also be switched to another potential suitable for accelerating the molecular ions instead of to 0 V.

FIG. 5 shows a raw data spectrum of the non-volatile substance mesoporphyrin. Air and benzene are mixed with the carrier gas helium (with the mass / charge ratio m / z = 4). These admixtures result in peaks in the range of m / z = 28 and a relatively precisely defined peak at m / z = 78.

Although usually a high fraction of fragments occurs during electron impact ionization, while the ionized molecule cannot usually be observed, a well-formed peak is obtained here at m / z = 566.3, the existence of which is due to the cooling of those to be investigated previously Molecules is evoked. The mass spectrum (isotope distribution) in the vicinity of the molecular ion peak is shown in higher resolution of the same figure in detail.

FIG. 6 shows a raw data spectrum of the thermally unstable peptide Trp-Met-Asp-Phe-NH ₂ . Here, too, a peak can be found for the corresponding molecular ion (m / z = 596.4), which has never occurred before without the combination of electron impact ionization with previous cooling.

The device shown in FIG. 7 for a further exemplary embodiment of the invention comprises an ionization chamber 7, the front wall or plate 70 of which is provided with an inlet opening 71 through which the molecules 4 to be examined can enter in the form of a continuous beam or as a particle packet. An end plate 73 is provided opposite the front plate 70 and has an outlet opening 72 aligned with the inlet opening 71.

As soon as the molecules to be examined are in the ionization chamber 7, the ionization process is initiated.

For example, ionization by electron impact can initially respectively. For this purpose, an electron beam 6 is spatially focused on the center of the ionization chamber, the energy of the electrons being adjustable from a few eV up to 100 eV.

If the inlet of the molecules to be examined takes place in a pulsed manner, the electron beam 6 is also switched on in pulsed operation, the pulse duration being from 10 ns to approximately 100 μs.

In order to achieve a good resolution when examining the molecular ions with a time-of-flight mass spectrometer, the molecular ions must start at a point (t <5 ns) that is as precisely defined as possible in a space that is as small as possible (<1 mm). In general, it is not possible to meet these conditions and to use the entire sample contained in a gas jet. However, it has been shown that the sensitivity of the arrangement can be increased if the radiation density of the electrons used for the ionization is selected such that a potential well is generated in the focus 61 of the beam. The neutral molecules to be examined fly into the focus of the electron beam 6, are ionized there, but then - in the ionized state - can no longer leave the focus 61. They are therefore collected in a spatially limited volume over a relatively long period of up to 100 µs.

After the electron impact ionization has ended, the end plate 73 of the ionization chamber 7 is switched to 0 V approximately 10 ns later, this switching taking place in less than 5 ns. This provides the starting pulse for the ions for their flight in the time-of-flight mass spectrometer.

A few microseconds later, the ionization chamber 7 is again placed at positive potential overall, for example at 600 V. The photon ionization can then be carried out.

For this purpose, a pulsed laser beam 4a is irradiated into the ionization chamber 1. The laser pulses used have a typical duration of 5 ns.

In the case of photon ionization, the short duration of the laser pulses alone would ensure a precisely defined starting time for the ions, so that the ionization chamber 7 with the closing plate 73, which can be acted upon separately, would not be required. However, it would not be possible to switch easily between electron impact ionization and photon ionization if one had to use different spatial arrangements for both ionization methods. It would also be possible per se to keep the end plate at a constant potential in the case of photon ionization, but in practice there are variable potential distributions which make it necessary to readjust the mass spectrometer. Therefore, the start pulse for the ionized molecules is also given for the photon ionization by switching the end plate 73 to 0 V, which takes place under the same conditions as previously described in connection with the electron impact ionization.

In the device according to FIG. 1, the focus of the electron beam 6 and the focus of the photon beam 4a coincide in a region 61 which lies on the path of the molecules to be examined.

8 shows raw data spectra for the thermally unstable peptide Trp-Pro-Leu-Gly-Amid. The multiphoton ionization spectrum (MPI) shows a well-formed peak at the corresponding molecular ion (m / z = 447.4), which is less developed in the electron impact ionization spectrum (EI). The two opposing spectra clearly show that different fragments are obtained in different proportions. Both spectra were under exact same experimental conditions recorded with the same sample, the rapid switching according to the invention between photon ionization and electron impact ionization being carried out. Laser desorption in a supersonic jet, which is suitable for thermally unstable molecules, was used as the inlet system.

9 shows the raw data spectra of Pro-Phe-Gly-Lys acetate, the spectra in turn being recorded on the one hand by multi-photon ionization (MPI) and on the other hand by electron impact ionization (EI). The same experimental conditions were observed using the same sample. Again it was quickly switched between photon ionization and electron impact ionization. It can be seen that smaller fragments are obtained with the electron impact ionization, so that it becomes clear here that the two spectra obtained with different ionization methods complement one another advantageously.

REFERENCE SIGN LIST

1

Submission to generate a carrier gas jet

2nd

Photon beam

3rd

Sample holder

4th

Particle beam

4a

Photon beam

5

Scraper

6

pulsed electron beam

7

Ionization chamber

10th

pulsed valve with nozzle

20th

lens

30th

sample

50

wall

51

opening

61

focus

70

Front wall

71

Entrance opening

72

Outlet opening

73

End plate

Claims (10)

1. A method of generating ions from thermally unstable non-volatile large molecules, particularly for a mass spectrometer such as a time-of-flight mass spectrometer, wherein a sample substance comprising the molecules is exposed to energy pulses which liberate molecules from the sample substance and wherein the liberated molecules are entrained by a jet of a carrier gas and cooled on expansion thereof and subsequently ionised in an ionisation chamber, characterised in that the molecules are ionised by electronic impact; the radiation density of the electrons used for ionisation is chosen so that a potential trough is generated in the focus of the electron jet, the depth of the trough being greater than the translation energy of the molecule ions in the carrier-gas jet; the molecule ions generated by electronic impact ionisation are collected in the potential trough for a given time and the molecule ions collected in the potential trough are accelerated in pulsed manner out of the ionisation chamber.
2. A method according to claim 1, characterised in that the energy of the ionising electrons is chosen lower than necessary for ionising the carrier gas.
3. A method according to claim 1 or 2, characterised in that the carrier gas used is helium and/or neon.
4. A method according to any of the preceding claims, characterised in that the energy pulses used for liberating the molecules from the sample substance are laser-generated light pulses.
5. A method according to any of claims 1 to 3, characterised in that the energy pulses used for liberating the molecules from the sample substance are supplied by bombardment with ions or neutral particles.
6. A method according to any of claims 1 to 5, characterised in that the molecules are periodically ionised by photon energisation in the same ionisation chamber (7), the photons being delivered in pulsed manner.
7. A method according to any of the preceding claims, characterised in that the electrons are delivered in pulsed manner.
8. A method according to claim 6 or 7, characterised in that the sample molecules are supplied in pulsed manner.
9. A method according to any of claims 6 to 8, characterised in that the molecule ions are accelerated by electronic impact and/or photon ionisation from the ionisation chamber (7) at the same rhythm.
10. A method according to any of claims 6 to 9, characterised by use of multi-photon excitation.

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