EP0333912A2 - Method for the evaporation of a sample - Google Patents

Method for the evaporation of a sample Download PDF

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Publication number
EP0333912A2
EP0333912A2 EP88111565A EP88111565A EP0333912A2 EP 0333912 A2 EP0333912 A2 EP 0333912A2 EP 88111565 A EP88111565 A EP 88111565A EP 88111565 A EP88111565 A EP 88111565A EP 0333912 A2 EP0333912 A2 EP 0333912A2
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Prior art keywords
sample substance
matrix material
molecules
laser beam
sample
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EP88111565A
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German (de)
French (fr)
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EP0333912A3 (en
EP0333912B1 (en
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Edward Prof. Dr. Schlag
Josef Lindner
Ronald C. Dr. Beavis
J. Dr. Grotemeyer
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Bruker Daltonics GmbH and Co KG
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Bruken Franzen Analytik GmbH
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    • H—ELECTRICITY
    • H05—ELECTRIC TECHNIQUES NOT OTHERWISE PROVIDED FOR
    • H05H—PLASMA TECHNIQUE; PRODUCTION OF ACCELERATED ELECTRICALLY-CHARGED PARTICLES OR OF NEUTRONS; PRODUCTION OR ACCELERATION OF NEUTRAL MOLECULAR OR ATOMIC BEAMS
    • H05H3/00—Production or acceleration of neutral particle beams, e.g. molecular or atomic beams
    • H05H3/02—Molecular or atomic beam generation

Definitions

  • the invention relates to a method for vaporizing a sample substance consisting of large molecules, in which the sample substance is exposed to laser beam pulses of high energy, so that the molecules located on the surface of the sample substance are desorbed by the energy of the laser beam pulses.
  • the molecules of the sample substance are effectively cooled, by means of which Decomposition should be largely excluded.
  • the ionization of the sample molecules required for mass spectroscopic analysis takes place within the beam of the carrier gas at a later point in time.
  • the invention has for its object to provide a method for vaporizing large molecules, in which the risk of destruction of the molecules by the Energy supplied to evaporation is greatly reduced, if not completely switched off.
  • sample substance is mixed with a matrix material which decomposes easily under the influence of the laser beam pulses and the mixture consisting of the sample substance and the matrix material is exposed to the laser beam pulses.
  • the energy supplied by means of the laser beam pulses is distributed to the sample substance and the matrix material and is primarily used to cause the matrix to disintegrate.
  • an effective destruction of the material takes place in the vicinity of the sample molecules embedded in the matrix substance, with the result that the sample molecules lose the connection to the surface and thus also to other molecules and are thus thrown away from the surface of the sample substance .
  • This process could be called a "local explosion". Therefore, when using the method according to the invention, the sensitive molecules of the sample substance are detached from the sample surface without having to absorb a very high energy themselves.
  • the disintegration of the matrix material creates a kind of "own jet", which is directed away from the sample surface and whose gas particles effectively cool the desorbed sample molecules before they reach a supersonic jet, for example, in which further cooling takes place in the manner described above.
  • a matrix material which consists of at least one compound which easily decomposes into gas molecules. It is advantageous for effective protection of the sample substance if a mixture is used in which the number of molecules of the matrix material is greater than the number of molecules of the sample substance.
  • the proportion of the sample substance in the mixture can be 10 to 40% by weight, depending on the type of sample substance on the one hand and the type of compounds used for the matrix on the other hand.
  • the method according to the invention is particularly effective when a matrix material is used which contains at least one compound which absorbs light with the wavelength of the laser beam pulses.
  • a matrix material which contains at least one compound which absorbs light with the wavelength of the laser beam pulses.
  • organic and inorganic compounds are sugar, in particular pentose or hexose, but also polysaccharides such as cellulose. These compounds decompose thermally to CO2 and H2O, so that they do not form residues that could lead to chemical reactions.
  • An inorganic In particular, ammonium nitrate, which disintegrates practically without residues, should be mentioned.
  • metal dust preferably gold or silver dust with a grain size of less than 40 ⁇ m
  • matrix materials can also be used which are not thermally decomposed as a result of the absorption of the laser radiation.
  • plasma waves are generated on the surface of the metal particles, which propagate as shock waves and on the surface of the matrix material cause the matrix to tear and thus release the embedded molecules.
  • the use of a polyethylene as the matrix material has proven to be particularly suitable for this variant of the method according to the invention.
  • polyethylene has the particular advantage that this material is already used as a matrix material in infrared spectroscopy and therefore tried and tested materials and devices for embedding the sample substance in such polyethylene are available.
  • pellets can be formed from the matrix material and the sample substance and exposed to the laser beam pulses.
  • the method according to the invention was used for the vaporization of organic compounds which vary greatly in their chemical composition. So it can easily be used for molecules that have strongly polar groups, as well as for non-polar molecules. Connections are among the first with an acidic and / or basic character, such as peptides, amino acids and dyes, while the latter include aromatic and non-aromatic hydrocarbons. It has been found to be particularly advantageous that the total yield of desorbed sample molecules can be increased by a factor of 4 to 10 compared to evaporation without mixing with a matrix material, depending on the type of sample substance.
  • pellets made of a spectroscopic polyethylene which is transparent to radiation in the range of 10 ⁇ m wavelength are used in a proportion of about 10 ⁇ 1 to 10 ⁇ 2 Gew.T. the sample substance and about 10 ⁇ 1 to 10 ⁇ 2 Gew.T. made of gold or silver dust and exposed to the radiation of a CO2 laser.
  • a spectroscopic polyethylene which is transparent to radiation in the range of 10 ⁇ m wavelength
  • a sample which was located on a sample holder arranged a few millimeters below a supersonic jet nozzle was used to carry out the method according to the invention irradiated with an IR laser beam pulse, the energy of which was 50 mJ and the duration of which was 20 ⁇ s.
  • the supersonic gas jet was switched on in each case after an IR laser beam pulse, so that the gaseous products generated by the laser beam pulse were carried away by the supersonic gas jet and cooled when the gas jet expanded.
  • the gas jet was then passed through means for removing any cations so that only neutral molecules enter a subsequent ionization area in which a UV laser beam cuts the gas jet.
  • Laser beam pulses of 5 ns duration with an energy of 300 ⁇ J were generated by the UV laser.
  • the cations generated in this way were fed to a time-of-flight mass spectrometer and detected with a multi-channel plate arrangement.
  • the time-of-flight mass spectrometer used was that of Anal. Instrum., 16, 151 (1986).
  • the typical mass resolution is in the range from 6000 to 10000 according to the FWHM definition.
  • sample substances examined with the described device were dipeptides. About 1 mg of the peptide was slurried in 50 ul water and then 20 ul of this slurry was applied to the sample holder. In most of the spectra obtained, about 10% of the substance applied to the sample carrier was used to generate the spectrum.
  • FIG. 1 shows the mass spectrum of the pure peptide leucine tryptophan obtained in the manner described above.
  • the spectrum shows a further line 2 of a substance of the mass M-18.
  • FIG. 2 shows the spectrum of the same peptide leucine-tryptophan, however after embedding the peptide in a glucose matrix in a ratio of 1 mg glucose per 1 mg peptide. Mixing with the glucose results in almost complete suppression of the M-18 line, which is due to the destruction of part of the peptide molecules during evaporation.
  • pellets were produced from 5 mg of powdered polyethylene, about 0.1 mg of powdered silver or gold and the specified amount of the sample substance. These pellets were exposed to the radiation of a scanning TEA laser with a wavelength of 10.6 ⁇ m and a pulse power of 10 mJ.
  • the intensity of the trailing edge was only about half the intensity of the sharp one Top.
  • the molecules of the sample substance desorbed by the laser beam pulses entered a gas jet generated by a supersonic nozzle, which was located at a distance of 1 to 2 mm from the desorption point.
  • the back pressure of the jet was 1 to 2 bar.
  • the molecules of the sample substance were distributed over the gas jet after a flight of 80 mm in the direction of the ionization area.
  • the mass spectrometer used was the same as in the previous examples.
  • 5 and 6 illustrate the significant increase in sensitivity that can be obtained by embedding the substance to be examined in a matrix of polyethylene with an admixture of silver.
  • 10 mg of powdered Leu-Tyr-Leu results in a line with an intensity that is only slightly greater than the intensity of the line obtained from only 100 ng of Leu-Tyr-Leu embedded in polyethylene with silver, that is, around 10 ⁇ 5 lesser amount.
  • the reason for this is that the evaporation of the Leu-Tyr-Leu embedded in the polyethylene matrix with the addition of silver powder takes place practically without any destruction of the molecules, while the substance without a protective matrix by bombardment with the laser beam to a high degree gets destroyed.
  • FIG. 7 to 9 illustrate the spectra of substances from which no signal could be obtained at all up to now, ie without embedding according to the invention in a matrix material.
  • the spectrum of Fig. 7 shows the line of thymine, which was obtained from only 50 ug of the substance in a matrix of polyethylene with silver.
  • the spectrum according to FIG. 8 was even contained in a gold dust using only 10 ⁇ g adenosine received matrix.
  • Fig. 9 shows the spectrum of tris-Ru-bipyridyl acetate. The amount used was only 20 yg in a matrix containing gold.

Abstract

During the evaporation of a sample substance consisting of large molecules, in particular for mass spectroscopy investigations, the energy supplied for the evaporation can bring about a thermolytic decomposition of the sample substance. According to the invention, in order to prevent such a decomposition, the sample substance is mixed, before irradiation, with a matrix material which consists of a compound which readily decomposes into gaseous molecules on exposure to the laser beam. This material may be one which absorbs the radiation, readily decomposes thermolytically or, alternatively, is transparent to the laser radiation but is mixed with a metal powder. If the mixture is exposed to laser beam pulses, the unstable matrix material first decomposes and thereby releases the embedded molecules of the sample substance. In particular, in combination with a cooling gas jet, a destruction of the molecules of the sample substance can be avoided almost completely in this way. Suitable compounds for the matrix are, in particular, sugar, cellulose and NH4NO3, and polyethylene with gold or silver dust added.

Description

Die Erfindung betrifft ein Verfahren zum Verdampfen einer aus großen Molekülen bestehenden Probensubstanz, bei dem die Proben­substanz Laserstrahlimpulsen hoher Energie ausgesetzt wird, so daß die sich an der Oberfläche der Probensubstanz befindenden Moleküle durch die Energie der Laserstrahlimpulse desorbiert werden.The invention relates to a method for vaporizing a sample substance consisting of large molecules, in which the sample substance is exposed to laser beam pulses of high energy, so that the molecules located on the surface of the sample substance are desorbed by the energy of the laser beam pulses.

Für die massenspektroskopische Untersuchung ist es erforderlich, feste Probensubstanzen in einen gasförmigen Zustand zu über­führen. Ein solcher Vorgang ist dann mit erheblichen Schwierig­keiten verbunden, wenn die Probensubstanz aus sehr großen Mole­külen besteht, die durch die Zufuhr der zum Verdampfen erforder­lichen Energie leicht zersetzt werden können. Aus der DE-OS 32 24 801 ist ein Verfahren zum Verdampfen einer aus großen Mole­külen bestehenden Probensubstanz bekannt, bei dem die Proben­substanz Laserstrahlimpulsen ausgesetzt wird, deren Energie und Dauer so bemessen ist, daß die Probensubstanz schneller ver­ substanz Laserstrahlimpulsen ausgesetzt wird, deren Energie und Dauer so bemessen ist, daß die Probensubstanz schneller ver­dampft als sie sich zersetzt. Die dabei entstehenden neutralen Moleküle werden dem Strahl eines Trägergases beigemischt, der durch Expansion adiabatisch gekühlt wird. Indem die neutralen Moleküle in einen Bereich des Strahles eingeführt werden, in dem dieser zu expandieren beginnt, und dieser Bereich auf einer Temperatur gehalten wird, die wesentlich geringer ist als die Zersetzungstemperatur der Probensubstanz, erfolgt eine effektive Kühlung der Moleküle der Probensubstanz, durch die deren Zer­setzung weitgehend ausgeschlossen werden soll. Die zur massen­spektroskopischen Untersuchung erforderliche Ionisierung der Probenmoleküle erfolgt innerhalb des Strahles des Trägergases zu einem späteren Zeitpunkt.For mass spectroscopic analysis, it is necessary to convert solid sample substances into a gaseous state. Such a process is associated with considerable difficulties if the sample substance consists of very large molecules which can easily be decomposed by the supply of the energy required for the evaporation. From DE-OS 32 24 801 a method for vaporizing a sample substance consisting of large molecules is known, in which the sample substance is exposed to laser beam pulses, the energy and duration of which is dimensioned such that the sample substance ver ver faster Substance is exposed to laser beam pulses, the energy and duration of which is such that the sample substance evaporates faster than it decomposes. The resulting neutral molecules are mixed into the jet of a carrier gas, which is cooled adiabatically by expansion. By introducing the neutral molecules into a region of the beam in which it begins to expand and this region is kept at a temperature which is substantially lower than the decomposition temperature of the sample substance, the molecules of the sample substance are effectively cooled, by means of which Decomposition should be largely excluded. The ionization of the sample molecules required for mass spectroscopic analysis takes place within the beam of the carrier gas at a later point in time.

Obwohl das bekannte Verfahren bei vielen Substanzen mit Erfolg angewendet werden kann, haben die massenspektroskopischen Unter­suchungen solcher Substanzen gezeigt, daß sich im Spektrum Linien befinden, die als Zerfallsprodukte der Probensubstanz betrachtet werden können. Eingehende Untersuchungen haben ge­zeigt, daß diese Zerfallsprodukte beim Verdampfen der Proben­substanz und nicht bei der späteren Ionisierung entstehen. Diese Zerfallsprodukte verhindern zwar nicht die spektrometri­sche Feststellung der Probensubstanz, vermindern jedoch die Ausbeute an unversehrten Molekülen und führen zu störenden Linien im Spektrum.Although the known method can be successfully applied to many substances, the mass-spectroscopic examinations of such substances have shown that there are lines in the spectrum which can be regarded as decomposition products of the sample substance. In-depth studies have shown that these decay products arise when the sample substance evaporates and not during later ionization. Although these decay products do not prevent the spectrometric determination of the sample substance, they reduce the yield of undamaged molecules and lead to disruptive lines in the spectrum.

Demgemäß liegt der Erfindung die Aufgabe zugrunde, ein Verfahren zum Verdampfen von großen Molekülen zur Verfügung zu stellen, bei dem die Gefahr einer Zerstörung der Moleküle durch die zum Verdampfen zugeführte Energie stark reduziert, wenn nicht sogar gänzlich ausgeschaltet ist.Accordingly, the invention has for its object to provide a method for vaporizing large molecules, in which the risk of destruction of the molecules by the Energy supplied to evaporation is greatly reduced, if not completely switched off.

Diese Aufgabe wird nach der Erfindung dadurch gelöst, daß die Probensubstanz vor dem Bestrahlen mit einem unter dem Einfluß der Laserstrahlimpulse leicht zerfallenden Matrixmaterial ver­mischt und das aus der Probensubstanz und dem Matrixmaterial bestehende Gemisch den Laserstrahlimpulsen ausgesetzt wird.This object is achieved according to the invention in that the sample substance is mixed with a matrix material which decomposes easily under the influence of the laser beam pulses and the mixture consisting of the sample substance and the matrix material is exposed to the laser beam pulses.

Durch das Einbetten der Probensubstanz in ein leicht zerfallen­des Matrixmaterial wird die mittels der Laserstrahlimpulse zugeführte Energie auf die Probensubstanz und das Matrixmaterial verteilt und in erster Linie dazu verbraucht, einen Zerfall der Matrix zu bewirken. Durch diesen Zerfall des Matrixmaterials in Gasmoleküle findet in der Umgebung der in die Matrixsubstanz eingebetteten Probenmoleküle eine effektvolle Zerstörung des Materials mit dem Ergebnis statt, daß die Probenmoleküle die Verbindung zur Oberfläche und damit auch zu anderen Molekülen verlieren und dadurch von der Oberfläche der Probensubstanz weggeschleudert werden. Diesen Vorgang könnte man als "lokale Explosion" bezeichnen. Daher werden bei Anwendung des erfin­dungsgemäßen Verfahrens die empfindlichen Moleküle der Proben­substanz von der Probenoberfläche gelöst, ohne daß sie selbst eine sehr hohe Energie aufnehmen müßten. Zugleich entsteht durch den Zerfall des Matrixmaterials eine Art "Eigenjet", der von der Probenoberfläche weggerichtet ist und dessen Gasteilchen die desorbierten Probenmoleküle schon wirksam abkühlen, bevor sie beispielsweise einen Überschallstrahl erreichen, in dem in der oben beschriebenen Weise eine weitere Abkühlung stattfindet.By embedding the sample substance in a readily disintegrating matrix material, the energy supplied by means of the laser beam pulses is distributed to the sample substance and the matrix material and is primarily used to cause the matrix to disintegrate. As a result of this disintegration of the matrix material into gas molecules, an effective destruction of the material takes place in the vicinity of the sample molecules embedded in the matrix substance, with the result that the sample molecules lose the connection to the surface and thus also to other molecules and are thus thrown away from the surface of the sample substance . This process could be called a "local explosion". Therefore, when using the method according to the invention, the sensitive molecules of the sample substance are detached from the sample surface without having to absorb a very high energy themselves. At the same time, the disintegration of the matrix material creates a kind of "own jet", which is directed away from the sample surface and whose gas particles effectively cool the desorbed sample molecules before they reach a supersonic jet, for example, in which further cooling takes place in the manner described above.

Bei einer Variante des erfindungsgemäßen Verfahrens wird ein Matrixmaterial verwendet, das aus mindestens einer thermolytisch leicht in Gasmoleküle zerfallenden Verbindung besteht. Hierbei ist es für einen wirksamen Schutz der Probensubstanz vorteil­haft, wenn ein Gemisch verwendet wird, in dem die Anzahl der Moleküle des Matrixmaterials größer ist als die Anzahl der Moleküle der Probensubstanz. Dabei kann der Anteil der Proben­substanz im Gemisch, je nach Art der Probensubstanz einerseits und der Art der für die Matrix verwendeten Verbindungen anderer­seits, 10 bis 40 Gew. % betragen.In a variant of the method according to the invention, a matrix material is used which consists of at least one compound which easily decomposes into gas molecules. It is advantageous for effective protection of the sample substance if a mixture is used in which the number of molecules of the matrix material is greater than the number of molecules of the sample substance. The proportion of the sample substance in the mixture can be 10 to 40% by weight, depending on the type of sample substance on the one hand and the type of compounds used for the matrix on the other hand.

Das erfindungsgemäße Verfahren ist dann besonders wirksam, wenn ein Matrixmaterial verwendet wird, das mindestens eine Verbindung enthält, die Licht mit der Wellenlänge der Laser­strahlimpulse absorbiert. In diesem Fall ist besonders gut gewährleistet, daß der wesentliche Teil der durch die Laser­strahlimpulse zugeführten Energie von dem Matrixmaterial ab­sorbiert wird und die Moleküle der Probensubstanz durch die in ihrer Umgebung in gasförmige Moleküle zerfallenden Verbindungen des Matrixmaterials freigesetzt werden.The method according to the invention is particularly effective when a matrix material is used which contains at least one compound which absorbs light with the wavelength of the laser beam pulses. In this case it is particularly well ensured that the major part of the energy supplied by the laser beam pulses is absorbed by the matrix material and the molecules of the sample substance are released by the compounds of the matrix material which break down into gaseous molecules in their environment.

Die oben genannte Bedingung, daß die das Matrixmaterial bil­denden Verbindungen thermolytisch leicht in gasförmige Moleküle zerfallen, wird sowohl von organischen als auch anorganischen Verbindungen erfüllt. Besonders geeignete organische Verbin­dungen sind Zucker, insbesondere Pentose oder Hexose, aber auch Polysaccharide wie Cellulose. Diese Verbindungen zerfallen thermolytisch zu CO₂ und H₂O, so daß sie keine Rückstände bil­den, die zu chemischen Reaktionen führen könnten. An anorgani­ schen Verbindungen ist insbesondere Ammoniumnitrat zu nennen, das praktisch rückstandsfrei zerfällt.The above-mentioned condition that the compounds forming the matrix material easily decompose into gaseous molecules is met by both organic and inorganic compounds. Particularly suitable organic compounds are sugar, in particular pentose or hexose, but also polysaccharides such as cellulose. These compounds decompose thermally to CO₂ and H₂O, so that they do not form residues that could lead to chemical reactions. An inorganic In particular, ammonium nitrate, which disintegrates practically without residues, should be mentioned.

Bei einer anderen Variante des erfindungsgemäßen Verfahrens wird in das Matrixmaterial Metallstaub, vorzugsweise Gold- oder Silberstaub mit einer Korngröße von weniger als 40 µm, eingebettet. In diesem Fall können auch Matrixmaterialien ver­wendet werden, die nicht infolge der Absorption der Laserstrah­lung thermolytisch zersetzt werden. Obwohl diese Theorie nicht vollständig gesichert ist, kann angenommen werden, daß an der Oberfläche der Metallteilchen Plasmawellen entstehen, die sich als Schockwellen ausbreiten und an der Oberfläche des Matrixma­terials ein Zerreißen der Matrix und damit wiederum ein Frei­setzen der eingebetteten Moleküle bewirken. Als besonders geeig­net hat sich für diese Variante des erfindungsgemäßen Verfahrens die Verwendung eines Polyethylens als Matrixmaterial erwiesen. Die Verwendung von Polyethylen hat den besonderen Vorteil, daß dieses Material bereits in der Infrarot-Spektroskopie als Ma­trixmaterial Material verwendet wird und daher erprobte Mate­rialien und Geräte zur Einbettung der Probensubstanz in solches Polyethylen zur Verfügung stehen.
So können insbesondere aus dem Matrixmaterial und der Proben­substanz Pellets geformt und den Laserstrahlimpulsen ausgesetzt werden.In another variant of the method according to the invention, metal dust, preferably gold or silver dust with a grain size of less than 40 μm, is embedded in the matrix material. In this case, matrix materials can also be used which are not thermally decomposed as a result of the absorption of the laser radiation. Although this theory is not fully supported, it can be assumed that plasma waves are generated on the surface of the metal particles, which propagate as shock waves and on the surface of the matrix material cause the matrix to tear and thus release the embedded molecules. The use of a polyethylene as the matrix material has proven to be particularly suitable for this variant of the method according to the invention. The use of polyethylene has the particular advantage that this material is already used as a matrix material in infrared spectroscopy and therefore tried and tested materials and devices for embedding the sample substance in such polyethylene are available.
In particular, pellets can be formed from the matrix material and the sample substance and exposed to the laser beam pulses.

Das erfindungsgemäße Verfahren wurde zur Verdampfung von orga­nischen Verbindungen angewendet, die in ihrer chemischen Zu­sammensetzung stark variieren. So läßt es sich ohne weiteres bei Molekülen, die stark polare Gruppen haben, als auch bei unpolaren Molekülen anwenden. Zu den ersten gehören Verbindungen mit acidischem und/oder basischem Charakter, wie z.B. Peptide, Amminosäuren und Farbstoffe, während zu den letzten aromatische und nicht aromatische Kohlenwasserstoffe zählen. Dabei hat sich als besonders vorteilhaft herausgestellt, daß die Total­ausbeute an desorbierten Probenmolekülen gegenüber der Ver­dampfung ohne die Vermischung mit einem Matrixmaterial, je nach der Art der Probensubstanz, um einen Faktor 4 bis 10 erhöht werden konnte.The method according to the invention was used for the vaporization of organic compounds which vary greatly in their chemical composition. So it can easily be used for molecules that have strongly polar groups, as well as for non-polar molecules. Connections are among the first with an acidic and / or basic character, such as peptides, amino acids and dyes, while the latter include aromatic and non-aromatic hydrocarbons. It has been found to be particularly advantageous that the total yield of desorbed sample molecules can be increased by a factor of 4 to 10 compared to evaporation without mixing with a matrix material, depending on the type of sample substance.

Bei einer besonders bevorzugten Ausführungsform des erfindungs­gemäßen Verfahrens werden Pellets aus einem spektroskopischen Polyethylen, das für Strahlung im Bereich von 10 µm Wellenlänge durchlässig ist, mit einem Anteil von etwa 10⁻¹ bis 10⁻² Gew.T. der Probensubstanz und etwa 10⁻¹ bis 10⁻² Gew.T. an Gold- oder Silberstaub hergestellt und der Strahlung eines CO₂-Lasers ausgesetzt. Auf diese Weise ist es gelungen, nicht nur die Empfindlichkeit des erfindungsgemäßen Verfahres erheblich zu steigern, sondern auch Moleküle der Massenspektroskopie zuzu­führen, deren massenspektroskopische Untersuchung bisher unmög­lich schien, nämlich Nukleotide.In a particularly preferred embodiment of the process according to the invention, pellets made of a spectroscopic polyethylene which is transparent to radiation in the range of 10 μm wavelength are used in a proportion of about 10⁻¹ to 10⁻² Gew.T. the sample substance and about 10⁻¹ to 10⁻² Gew.T. made of gold or silver dust and exposed to the radiation of a CO₂ laser. In this way, it was not only possible to significantly increase the sensitivity of the method according to the invention, but also to supply molecules to mass spectroscopy, the examination of which by mass spectroscopy previously seemed impossible, namely nucleotides.

Die Erfindung wird im folgenden anhand einiger Beispiele näher beschrieben und erläutert, deren Ergebnisse durch die in den Fig. 1 bis 9 der Zeichnung dargestellten Diagramme wiedergegeben werden.The invention is described and explained in more detail below with the aid of a few examples, the results of which are reproduced by the diagrams shown in FIGS. 1 to 9 of the drawing.

Bei den durch die Fig. 1 bis 4 veranschaulichten Beispielen wurde zur Durchführung des erfindungsgemäßen Verfahrens eine Probe, die sich auf einem wenige Millimeter unterhalb einer Überschall-Strahldüse angeordneten Probenträger befand, mit einem IR-Laserstrahlimpuls bestrahlt, dessen Energie 50 mJ und dessen Dauer 20 µs betrug. Der Überschall-Gasstrahl wurde je­weils nach einem IR-Laserstrahlimpuls eingeschaltet, so daß die durch den Laserstrahlimpuls erzeugten gasförmigen Produkte von dem Überschall-Gasstrahl mitgenommen und bei der Expansion des Gasstrahles gekühlt wurden. Der Gasstrahl wurde dann durch Einrichtungen zum Entfernen jeglicher Kationen geführt, so daß in einen folgenden Ionisationsbereich nur neutrale Moleküle eintreten, in dem ein UV-Laserstrahl den Gasstrahl schneidet. Von dem UV-Laser wurden Laserstrahlimpulse von 5 ns Dauer mit einer Energie von 300 µJ erzeugt. Die dadurch erzeugten Kationen wurden einem Flugzeit-Massenspektrometer zugeführt und mit einer Mehrkanal-Plattenanordnung detektiert. Das verwendete Flugzeit-Massenspektrometer war von der in Anal. Instrum., 16, 151 (1986) beschriebenen Art. Die typische Massenauflösung liegt im Bereich von 6000 bis 10000 nach der FWHM-Definition.In the examples illustrated by FIGS. 1 to 4, a sample which was located on a sample holder arranged a few millimeters below a supersonic jet nozzle was used to carry out the method according to the invention irradiated with an IR laser beam pulse, the energy of which was 50 mJ and the duration of which was 20 μs. The supersonic gas jet was switched on in each case after an IR laser beam pulse, so that the gaseous products generated by the laser beam pulse were carried away by the supersonic gas jet and cooled when the gas jet expanded. The gas jet was then passed through means for removing any cations so that only neutral molecules enter a subsequent ionization area in which a UV laser beam cuts the gas jet. Laser beam pulses of 5 ns duration with an energy of 300 µJ were generated by the UV laser. The cations generated in this way were fed to a time-of-flight mass spectrometer and detected with a multi-channel plate arrangement. The time-of-flight mass spectrometer used was that of Anal. Instrum., 16, 151 (1986). The typical mass resolution is in the range from 6000 to 10000 according to the FWHM definition.

Bei den mit der beschriebenen Einrichtung untersuchten Proben­substanzen handelte es sich um Dipeptide. Es wurden etwa 1 mg des Peptids in 50 µl Wasser aufgeschlämmt und es wurden dann 20 µl dieser Aufschlämmung auf den Probenträger aufgebracht. Bei den meisten der erhaltenen Spektren wurden etwa 10% der auf den Probenträger aufgebrachten Substanz zur Erzeugung des Spektrums verbraucht.The sample substances examined with the described device were dipeptides. About 1 mg of the peptide was slurried in 50 ul water and then 20 ul of this slurry was applied to the sample holder. In most of the spectra obtained, about 10% of the substance applied to the sample carrier was used to generate the spectrum.

In gleicher Weise wurden Mischungen von Dipeptiden und Matrix­materialien hergestellt. Es wurde 1 mg des Peptids in 50 ml einer wässrigen Lösung der gewünschten Matrixverbindung aufge­schlämmt und es wurden dann 20 ml der resultierenden Aufschläm­mung auf dem Probenträger aufgebracht. In beiden Fällen wurde das Wasser einfach durch Trocknen an der Luft entfernt. Als Matrixverbindungen wurden Sucrose und Glucose verwendet. Das verwendete Wasser war dreifach deionisiert.Mixtures of dipeptides and matrix materials were prepared in the same way. 1 mg of the peptide was slurried in 50 ml of an aqueous solution of the desired matrix compound, and then 20 ml of the resulting slurry was applied to the sample holder. In both cases the water is simply removed by air drying. Sucrose and glucose were used as matrix compounds. The water used was triple deionized.

Fig. 1 zeigt das auf die vorstehend beschriebene Weise erhaltene Massenspektrum des reinen Peptids Leucin-Tryptophan. Neben der Linie 1 für das reine Peptid mit der sich aus der auf der Abs­zisse aufgetragenen Flugzeit ergebenden Masse M zeigt das Spek­trum eine weitere Linie 2 einer Substanz der Masse M - 18. Fig. 2 zeigt das Spektrum des gleichen Peptids Leucin-Trypto­phan, jedoch nach Einbetten des Peptids in eine Glucosematrix im Verhältnis 1 mg Glucose pro 1 mg Peptid. Die Vermischung mit der Glucose hat eine fast vollständige Unterdrückung der Linie M - 18 zur Folge, die auf eine Zerstörung eines Teiles der Peptid-Moleküle bei der Verdampfung zurückzuführen ist.1 shows the mass spectrum of the pure peptide leucine tryptophan obtained in the manner described above. In addition to line 1 for the pure peptide with the mass M resulting from the flight time plotted on the abscissa, the spectrum shows a further line 2 of a substance of the mass M-18. FIG. 2 shows the spectrum of the same peptide leucine-tryptophan, however after embedding the peptide in a glucose matrix in a ratio of 1 mg glucose per 1 mg peptide. Mixing with the glucose results in almost complete suppression of the M-18 line, which is due to the destruction of part of the peptide molecules during evaporation.

Ähnlich wie die Fig. 1 und 2 zeigen auch die Fig. 3 und 4 das Spektrum eines reinen Peptids bzw. eines in eine Sucrose-Matrix eingebetteten Peptids. Als Peptid findet diesmal Methionin-­Tyrosin Verwendung. Auf der Abszisse der Diagramme nach den Fig. 3 und 4 ist diesmal das Massen/Ladungs-verhältnis M/Z aufgetragen, während die Koordinate wiederum die Intensität der Linien wiedergibt. Bei der Ionisation der Substanz entstand nur das A₁ Fragment mit M/Z = 104. Die Bezeichnung A-Fragment beruht auf der Roepstroff-Fohlman-Nomenklatur [Biodmed. Mass Spectrom. 11,601 (1984)].Similar to FIGS. 1 and 2, FIGS. 3 and 4 show the spectrum of a pure peptide or a peptide embedded in a sucrose matrix. This time methionine tyrosine is used as the peptide. This time, the mass / charge ratio M / Z is plotted on the abscissa of the diagrams according to FIGS. 3 and 4, while the coordinate again shows the intensity of the lines. When the substance was ionized, only the A 1 fragment with M / Z = 104 was formed. The designation A fragment is based on the Roepstroff-Fohlman nomenclature [Biodmed. Mass Spectrom. 11,601 (1984)].

Ähnlich wie bei dem durch die Fig. 1 und 2 veranschaulichten Versuch tritt auch hier bei der Verdampfung des reinen Peptids eine Fragmentation des Peptids ein, die zu der Linie mit der Massenzahl M - 18 führt. Dagegen verschwindet diese Linie voll­kommen, wie aus Fig. 4 ersichtlich, wenn das Peptid in eine Sucrosematrix eingebettet wird. Es ist ohne weiteres verständ­lich, daß das erst nach der Verdampfung der Peptidmoleküle bei der Ionisation entstehende A₁-Fragment auch bei dem Verdampfen des Peptids in einer Sucrosematrix erhalten bleibt.Similar to the experiment illustrated by FIGS. 1 and 2, evaporation of the pure peptide also occurs here fragmentation of the peptide leading to the M-18 mass number line. In contrast, this line disappears completely, as can be seen from FIG. 4, if the peptide is embedded in a sucrose matrix. It is readily understandable that the A 1 fragment which arises only after the evaporation of the peptide molecules during the ionization is retained even in the evaporation of the peptide in a sucrose matrix.

Es sei noch erwähnt, daß an den Proben, die zu den vorstehend behandelten Spektren geführt haben, die Pyrolyse der Zucker­matrix als Schwärzung der Probe durch die Einwirkung der wieder­holten Laserstrahlimpulse erkennbar war. Eine solche Schwärzung trat bei den Proben, welche die reinen Peptide enthielten, nicht ein. Es ist anzunehmen, daß die Zersetzung der Zucker eine pyrolytische Dehydratation der Peptide verhindert, weil die Pyrolyse des Zuckers zu einem Überschuß an Wasser in der Umgebung der Peptid-Moleküle führt, wodurch die Dehydratations-­Reaktion der Peptide in die andere Richtung getrieben wird.It should also be mentioned that on the samples which led to the spectra treated above, the pyrolysis of the sugar matrix was recognizable as blackening of the sample by the action of the repeated laser beam pulses. No such darkening occurred in the samples which contained the pure peptides. It is believed that the decomposition of the sugars prevents pyrolytic dehydration of the peptides because the pyrolysis of the sugar leads to an excess of water in the vicinity of the peptide molecules, which drives the dehydration reaction of the peptides in the other direction.

Für die durch die Diagramme nach den Fig. 5 bis 9 veranschau­lichten Beispiele wurden, soweit nichts anderes angegeben, aus 5 mg pulverförmigem Polyethylen, etwa 0,1 mg pulverförmigem Silber oder Gold und der angegebenen Menge der Probensubstanz Pellets hergestellt. Diese Pellets wurden der Strahlung eines getasteten TEA-Lasers mit einer Wellenlänge von 10,6 µm und einer Pulsleistung von 10 mJ ausgesetzt. Der von dem Laser erzeugte Puls war bimodal und hatte eine kurze, scharfe Spitze von 2 µs Dauer (d.h. FWHM = 2 µs) und eine breite Rückflanke von 20 µs Dauer (d.h. FWHM = 20 µs). Die Intensität der Rück­flanke betrug nur etwa die Hälfte der Intensität der scharfen Spitze. Die durch die Laserstrahlimpulse desorbierten Moleküle der Probensubstanz gelangten in einen von einer Überschall-­Düse erzeugten Gasstrahl, die sich in einem Abstand von 1 bis 2 mm von der Desorptionsstelle befand. Der Staudruck des Strahls betrug 1 bis 2 bar. Die Moleküle der Probensubstanz verteilten sich über den Gasstrahl nach einem Flug von 80 mm in Richtung auf den Ionisationsbereich. Das verwendete Massenspektrometer war das gleiche wie bei den vorhergehenden Beispielen.Unless otherwise stated, for the examples illustrated by the diagrams according to FIGS. 5 to 9, pellets were produced from 5 mg of powdered polyethylene, about 0.1 mg of powdered silver or gold and the specified amount of the sample substance. These pellets were exposed to the radiation of a scanning TEA laser with a wavelength of 10.6 µm and a pulse power of 10 mJ. The pulse generated by the laser was bimodal and had a short, sharp peak of 2 µs duration (ie FWHM = 2 µs) and a broad trailing edge of 20 µs duration (ie FWHM = 20 µs). The intensity of the trailing edge was only about half the intensity of the sharp one Top. The molecules of the sample substance desorbed by the laser beam pulses entered a gas jet generated by a supersonic nozzle, which was located at a distance of 1 to 2 mm from the desorption point. The back pressure of the jet was 1 to 2 bar. The molecules of the sample substance were distributed over the gas jet after a flight of 80 mm in the direction of the ionization area. The mass spectrometer used was the same as in the previous examples.

Die Fig. 5 und 6 veranschaulichen die bedeutende Steigerung der Empfindlichkeit, die durch das Einbetten der zu untersuchen­den Substanz in eine Matrix aus Polyethylen mit einer Beimengung von Silber erhalten werden kann. So ergeben 10 mg pulverförmiges Leu-Tyr-Leu eine Linie mit einer Intensität, die nur wenig größer ist als die Intensität der Linie, die von nur 100 ng in Polyethylen mit Silber eingebettetes Leu-Tyr-Leu erhalten wird, also von einer um 10⁻⁵ geringeren Menge. Der Grund dafür besteht darin, daß die Verdampfung des in die Polyethylen-Matrix mit der Beimengung von Silberpulver eingebetteten Leu-Tyr-Leu prak­tisch ohne jegliche Zerstörung der Moleküle erfolgt, während die Substanz ohne schützende Matrix durch den Beschuß mit dem Laserstrahl in einem hohen Maße zerstört wird.5 and 6 illustrate the significant increase in sensitivity that can be obtained by embedding the substance to be examined in a matrix of polyethylene with an admixture of silver. For example, 10 mg of powdered Leu-Tyr-Leu results in a line with an intensity that is only slightly greater than the intensity of the line obtained from only 100 ng of Leu-Tyr-Leu embedded in polyethylene with silver, that is, around 10 ⁻⁵ lesser amount. The reason for this is that the evaporation of the Leu-Tyr-Leu embedded in the polyethylene matrix with the addition of silver powder takes place practically without any destruction of the molecules, while the substance without a protective matrix by bombardment with the laser beam to a high degree gets destroyed.

Die Fig. 7 bis 9 veranschaulichen die Spektren von Substanzen, von denen bisher, d.h. ohne die erfindungsgemäße Einbettung in ein Matrixmaterial, überhaupt kein Signal erhalten werden konn­te. Das Spektrum nach Fig. 7 zeigt die Linie von Thymin, die von nur 50 µg der Substanz in einer Matrix aus Polyethylen mit Silber erhalten wurde. Das Spektrum nach Fig. 8 wurde unter Einsatz von sogar nur 10 µg Adenosin in einer Goldstaub enthal­ tenden Matrix erhalten. Endlich zeigt Fig. 9 das Spektrum von tris-Ru-Bipyridylacetat. Die eingesetzte Menge betrug nur 20 yg in einer Gold enthaltenden Matrix.7 to 9 illustrate the spectra of substances from which no signal could be obtained at all up to now, ie without embedding according to the invention in a matrix material. The spectrum of Fig. 7 shows the line of thymine, which was obtained from only 50 ug of the substance in a matrix of polyethylene with silver. The spectrum according to FIG. 8 was even contained in a gold dust using only 10 μg adenosine received matrix. Finally, Fig. 9 shows the spectrum of tris-Ru-bipyridyl acetate. The amount used was only 20 yg in a matrix containing gold.

Claims (12)

1. Verfahren zum Verdampfen einer aus großen Molekülen bestehenden Probensubstanz, bei dem die Probensubstanz Laserstrahlimpulsen hoher Energie ausgesetzt wird, so daß die sich an der Oberfläche der Probensubstanz befindenden Moleküle durch die Energie der Laserstrahlimpulse desorbiert werden,
dadurch gekennzeichnet, daß
die Probensubstanz vor dem Bestrahlen mit einem unter dem Einfluß der Laserstrahlimpulse leicht zerfallenden Matrixmaterial vermischt und das aus der Probensubstanz und dem Matrixmaterial bestehende Gemisch den Laserstrahlimpulsen ausgesetzt wird.1. A method for vaporizing a sample substance consisting of large molecules, in which the sample substance is exposed to laser beam pulses of high energy, so that the molecules located on the surface of the sample substance are desorbed by the energy of the laser beam pulses,
characterized in that
prior to irradiation, the sample substance is mixed with a matrix material which decomposes easily under the influence of the laser beam pulses and the mixture consisting of the sample substance and the matrix material is exposed to the laser beam pulses. 2. Verfahren nach Anspruch 1, dadurch gekennzeichnet, daß ein Matrixmaterial verwendet wird, das aus mindestens einer thermolytisch leicht in Gasmoleküle zerfallenden Verbindung besteht.2. The method according to claim 1, characterized in that a matrix material is used which consists of at least one thermolytically easily decomposing into gas molecules compound. 3. Verfahren nach Anspruch 1 oder 2, dadurch gekennzeichnet, daß ein Gemisch verwendet wird, in dem die Anzahl der Moleküle des Matrixmaterials größer ist als die Anzahl der Moleküle der Probensubstanz.3. The method according to claim 1 or 2, characterized in that a mixture is used in which the number of molecules of the matrix material is greater than the number of molecules of the sample substance. 4. Verfahren nach Anspruch 2, dadurch gekennzeichnet, daß der Anteil der Probensubstanz im Gemisch 10 bis 40 Gew.% beträgt.4. The method according to claim 2, characterized in that the proportion of the sample substance in the mixture is 10 to 40 wt.%. 5. Verfahren nach einem der vorhergehenden Ansprüche, dadurch gekennzeichnet, daß ein Matrixmaterial verwendet wird, das mindestens eine Verbindung enthält, welche Licht mit der Wellenlänge der Laserstrahlimpulse absorbiert.5. The method according to any one of the preceding claims, characterized in that a matrix material is used which contains at least one compound which absorbs light with the wavelength of the laser beam pulses. 6. Verfahren nach einem der vorhergehenden Ansprüche, dadurch gekennzeichnet, daß als eine das Matrixmaterial bildende Verbindung ein Zucker, insbesondere eine Pentose oder Hexose, verwendet wird.6. The method according to any one of the preceding claims, characterized in that a sugar, in particular a pentose or hexose, is used as a compound forming the matrix material. 7. Verfahren nach einem der Ansprüche 1 bis 5, dadurch gekennzeichnet, daß als eine das Matrixmaterial bildende Verbindung ein Polysaccharid, insbesondere Cellulose, verwendet wird.7. The method according to any one of claims 1 to 5, characterized in that a polysaccharide, in particular cellulose, is used as a compound forming the matrix material. 8. Verfahren nach einem der Ansprüche 1 bis 5, dadurch gekennzeichnet, daß als eine das Matrixmaterial bildende Verbindung Ammoniumnitrat verwendet wird.8. The method according to any one of claims 1 to 5, characterized in that ammonium nitrate is used as a compound forming the matrix material. 9. Verfahren nach einem der vorhergehenden Ansprüche, dadurch gekennzeichnet, daß in das Matrixmaterial Metallstaub, vorzugsweise Gold- oder Silberstaub mit einer Korngröße von weniger als 40 µm, eingebettet wird.9. The method according to any one of the preceding claims, characterized in that in the matrix material metal dust, preferably gold or silver dust with a grain size of less than 40 microns, is embedded. 10. Verfahren nach Anspruch 9, dadurch gekennzeichnet, daß als eine das Matrixmaterial bildende Verbindung ein Polyethylen verwendet wird.10. The method according to claim 9, characterized in that a polyethylene is used as a compound forming the matrix material. 11. Verfahren nach einem der vorhergehenden Ansprüche, dadurch gekennzeichnet, daß aus dem Matrixmaterial und der Probensubstanz und ggf. dem Metallstaub Pellets geformt und den Laserstrahlimpulsen ausgesetzt werden.11. The method according to any one of the preceding claims, characterized in that pellets are formed from the matrix material and the sample substance and possibly the metal dust and are exposed to the laser beam pulses. 12. Verfahren nach den Ansprüchen 9 bis 11, dadurch gekennzeichnet, daß die Pellets aus einem spektroskopischen Polyethylen, das für Strahlung im Bereich von 10 µm Wellenlänge durchlässig ist, mit einem Anteil von etwa 10⁻⁴ bis 10⁻⁵ Gew.T. der Probensubstanz und etwa 10⁻⁴ bis 10⁻⁵ Gew.T. an Gold- oder Silberstaub hergestellt und der Strahlung eines CO₂-Lasers ausgesetzt werden.12. The method according to claims 9 to 11, characterized in that the pellets of a spectroscopic polyethylene which is transparent to radiation in the range of 10 microns wavelength, with a proportion of about 10⁻⁴ to 10⁻⁵ wt. the sample substance and about 10⁻⁴ to 10⁻⁵ Gew.T. made of gold or silver dust and exposed to the radiation of a CO₂ laser.
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DE3809504C1 (en) 1989-09-21
US5062935A (en) 1991-11-05
EP0333912B1 (en) 1995-06-28

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