Patent Grant
6969848
The invention relates to mass spectrometry. More particularly, this invention relates to a method of and an apparatus for ionizing a sample in which vaporization and ionization of the sample are carried out separately.
Presently, known methods of creating ions from non-vaporizable molecules fall into two general categories: Electrospray Ionization (ESI) and desorption/ionization from a solid surface.
The ESI technique typically involves spraying a liquid, containing the sample molecules at atmospheric pressure, from a capillary which is at a high voltage relative to an orifice in a sampling plate. A high electric field at the capillary tip from which the liquid flows causes the liquid to become charged. This charged liquid eventually disperses into charged droplets which are drawn towards the sampling plate by the electric field. The region between the capillary tip and the sampling plate is at atmospheric pressure to provide energy to promote desolvation of the droplets. After evaporation of the solvent, either before or after the sampling plate, there are sample ions that may be singly or multiply charged (depending on the structure of the molecule). These sample ions are drawn through the sampling plate orifice into a reduced pressure region of a mass spectrometer due to the flow of background gas from the atmospheric pressure region, between the capillary tip and the sampling plate, to the sub-atmospheric pressure region in the mass spectrometer. Typically, the sub-atmospheric pressure region in the mass spectrometer is at a pressure of less than 10−5 Torr. The sample ions may pass through one or two chambers of intermediate pressure before reaching the high vacuum region of the mass spectrometer.
The most common of the desorption/ionization techniques is Matrix Assisted Laser Desorption Ionization (MALDI) which is most commonly used with a Time-Of-Flight (TOF) mass spectrometer. Typically, the sample and a matrix, such as 2,5-dihydroxybenzoic acid, are both dissolved in appropriate solvents, mixed and deposited on a solid probe surface. The probe effectively becomes an ion source. Once the liquid from the mixture has evaporated, the probe is inserted through vacuum locks into the high vacuum region of a mass spectrometer. A laser beam, often from a 337 nm nitrogen laser, is subsequently pulsed onto the probe surface vaporizing a small amount of dried matrix and sample molecules to form a plume or jet traveling out from the probe surface. The matrix material is specifically chosen to absorb the laser energy in order to rapidly heat and vaporize the sample molecules that it carries. Thus, ionization of at least some of the sample molecules occurs in the plume.
While the detailed mechanisms of vaporization and ionization are not fully understood, most currently accepted models propose that the matrix molecules become ionized in the plume or jet forming a micro-plasma. Neutral sample molecules which are carried away from the probe surface by the expanding micro-plasma then become ionized by charge transfer processes from the matrix ions in the micro-plasma. These processes occur in the micro-plasma only while the matrix ion and sample gas densities are high enough to allow interaction between the matrix ions and the sample molecules. Since the micro-plasma is generated by a laser pulse with a duration of a few nanoseconds focused to an area of less than 1 mm2, the micro-plasma region of each laser pulse is confined to a region very close to the probe surface. In a typical MALDI system, laser pulses are generated at a rate of a few Hz (i.e. 10 or fewer pulses per second). Each generated pulse of ions is accelerated into the TOF mass spectrometer, and a mass spectrum is generated by recording the arrival times of the ions. Usually, the spectra from many laser pulses are added together to create a mass spectrum which can be interpreted.
In the conventional MALDI-TOF configuration described above, the probe surface containing the sample/matrix mixture must be located in a high vacuum region with a typical pressure of 10−6 Torr and more often 10−7 Torr. This is because the TOF mass spectrometer requires high voltages in order to accelerate the ions. Accordingly, a high vacuum is required in order to prevent electrical breakdown in the instrument. In addition, it is very important that the sample ions, formed in the small region close to the probe surface, do not undergo any further collisions with neutral molecules after being accelerated by the high voltage since any further collisions tend to cause the ions to fragment which is undesirable.
A recent development by a group at the University of Manitoba (WO 99/38185) describes the operation of a MALDI ion source in a low vacuum region at a pressure of approximately 10 mTorr (or even up to 1 atmosphere if desired). Ions are generated from a probe surface, as in a conventional MALDI system, but the ions are allowed to collide, at low energies, with a background gas (typically nitrogen) before being introduced into the mass spectrometer. This interaction, often described as collisional cooling, allows the ions to achieve a quasi-thermal equilibrium with the gas which removes all of the original energy of the ions that was induced by the expanding plume from the probe surface. The collisional cooling process also completely decouples the mass spectrometer from the ion source such that ionization parameters such as laser power, the sample's position on the probe surface and the like do not affect the quality of the mass spectrum. The collisional cooling process also converts the pulsed ion stream, formed by the laser pulses of nanosecond duration, into a quasi-continuous ion stream since the ion pulses are stretched in time by collisions with the background gas. After the ions undergo collisional cooling, the ions can be analyzed by any mass spectrometer such as an orthogonal TOF mass spectrometer, a quadrupole or an ion trap.
In any conventional MALDI system that operates at a reduced pressure, the analyte ions must be introduced through a vacuum lock into the source region of a TOF mass spectrometer. However, the MALDI-TOF vacuum locks required for sample introduction add complication and cost. Laiko et al. (U.S. Pat. No. 5,965,884) avoids the problem of vacuum locks by performing the MALDI process at atmospheric pressure. However, this technique suffers ion losses of at least 99% while transferring ions from an atmospheric pressure region to a reduced pressure region.
In the Laiko technique, the surface containing the sample and matrix is located in a region at atmospheric pressure. The surface is also in front of a small orifice that provides a passage to the TOF mass spectrometer chamber. A laser pulse generates ions by the MALDI process at atmospheric pressure and the resulting ion plume is drawn into the TOF mass spectrometer region by a gas flow or an electric field. This technique avoids the necessity of introducing the sample molecules into the vacuum system, however only a small fraction of the sample ions are sampled through the orifice. There are two reasons for the small fraction of ions sampled. The first reason is that the high gas density in the atmospheric pressure region prevents opposite polarity charges, in the micro-plasma of the plume, from separating sufficiently quickly. These opposite charges then recombine which changes a sample ion to a sample molecule thereby reducing the sample ion intensity. The second reason is that the diameter of the orifice that connects the atmospheric pressure region to the vacuum region in the TOF mass spectrometer must be very small so that vacuum pumps can maintain the high vacuum necessary for the operation of the TOF mass spectrometer and pumping requirements are kept reasonable. Accordingly, the resulting poor sampling efficiency through this small orifice reduces the sensitivity of this method compared to the conventional MALDI process discussed above.
Although the details of the MALDI process are not fully understood, most researchers agree that the ionization efficiency is very low, i.e. only 0.1 to 0.01% of the deposited sample molecules are actually converted into ions in the laser-created plasma. It seems likely that many sample molecules are carried away from the probe surface as a neutral species and are simply pumped away by the vacuum pumps. Therefore, if these sample molecules could be ionized, the sensitivity of the method would be greatly increased.
Franzen et al. (U.S. Pat. No. 5,663,561) attempted to address the very low ionization efficiency of the MALDI process by using a laser to desorb the matrix/sample mixture in an atmospheric pressure region and separate, unipolar reagent ions from a corona discharge to subsequently chemically ionize these sample molecules at atmospheric pressure. It is known that conventional atmospheric pressure chemical ionization (APCI) efficiencies can approach nearly 100% (under favorable thermodynamic conditions). In a conventional APCI source, the sample to be ionized is in a gaseous form. The gaseous sample then flows through a region where reagent ions are created. Under conditions where the reagent ions and sample gas are well mixed and where the interaction time is relatively long (i.e. several milliseconds or longer), the ionization efficiency can be very high.
In particular, Franzen teaches that the material from the vaporized MALDI plume is drawn through a corona discharge region. The vaporized matrix/sample ions are mixed with the reagent ions from the corona discharge in a tube connected to a small hole in a sample plate. The resulting ions are then transferred into the vacuum region of the mass spectrometer. However, similarly to the Laiko method, ions must still be transferred through a small orifice into the vacuum chamber for analysis typically by a TOF mass spectrometer. This configuration results in poor sample ion transmission efficiency. As such an ion loss of up to 99% occurs which reduces the practical utility of this method for trace analysis.
Accordingly, it is desirable to provide a method and an apparatus that results in a more sensitive MALDI process that can be used with a mass spectrometer system.
The present inventors have realized that a more sensitive MALDI process can be achieved by separating the vaporization and ionization steps and increasing the ion sampling efficiency. More particularly, it is proposed to perform the steps of desorption of the sample from a matrix material by a laser beam followed by ionization of the sample molecules by a high intensity reagent ion beam within a sub-atmospheric system. The decoupling of ionization and vaporization allows each process to be separately optimized. Furthermore, the sampling efficiency of ions created in the sub-atmospheric pressure region can be significantly greater than if the ions were formed in an atmospheric region. In addition, it is expected that the sample specificity of the matrix will be reduced because ionization of the matrix ions is not required. The high intensity flux of reagent ions can be injected into the sub-atmospheric system which avoids the losses associated with transmitting sample ions from a weak plasma in an atmospheric pressure region through a small pinhole to a sub-atmospheric region.
In one embodiment, reagent ions may be generated in an atmospheric pressure discharge, with the ion source adjacent to an orifice, defining an atmospheric-to-vacuum region interface, so that most of the reagent ions are directed through the orifice by a gas flow. A laser-desorbed sample is then mixed with the high intensity flow of reagent ions just downstream of the orifice in a sub-atmospheric pressure region where the laser-desorbed sample is ionized by ion-molecule reactions. Ionized sample molecules in these sub-atmospheric pressure regions can then be more efficiently focused into a mass spectrometer.
In a first aspect, the present invention describes a method of generating sample ions from sample molecules having the steps of:
(1) vaporizing the sample molecules to generate substantially neutral molecules;
(2) separately generating reagent ions in a first region; and
(3) mixing the neutral molecules and the reagent ions in a vacuum chamber separate from the first region, wherein the vacuum chamber is at a substantially sub-atmospheric pressure to promote ionization of the neutral molecules to create sample ions, the sub-atmospheric pressure being approximately 10 Torr or less.
Alternatively, the vacuum chamber may be at a pressure of 10 mTorr or less. The method may also include the step of vaporizing the sample in a substantially atmospheric pressure region. Alternatively, the method may include vaporizing the sample in a sub-atmospheric pressure region. Furthermore, the method may include carrying out step (3) in a sub-atmospheric pressure region of a mass spectrometer.
The method may further comprise the step of:
In the method, vaporizing the sample molecules may be effected by providing the sample molecules on a support plate and irradiating the sample molecules with a laser beam.
Alternatively, vaporizing the sample molecules may be effected by providing the sample molecules on a heatable element and heating the element to vaporize the sample.
Vaporizing the sample molecules to generate substantially neutral molecules may be further effected by providing the sample in a matrix on the support plate and irradiating the sample and the matrix with a laser beam having a frequency selected to be absorbed by the matrix to effect matrix assisted laser desorption.
In another aspect, the method may further comprise:
Alternatively, the method may further comprise:
In another aspect, the method may further include:
Alternatively, the method may further include:
In another alternative, the method may further include:
In yet another alternative, the method may further include:
In another aspect, the present invention comprises an apparatus, for generating sample ions from sample molecules. The apparatus comprises a sample plate for supporting a sample comprising sample molecules for vaporization and means for vaporizing the sample molecules. The apparatus also comprises a reagent ion generation means for generating a stream of reagent ions in a first region, and a vacuum chamber separate from the first region, the vacuum chamber being at a substantially sub-atmospheric pressure connected to the means for vaporizing the sample molecules and the reagent ion generation means. In use, the vaporized sample molecules and reagent ions mix in the vacuum chamber to promote ionization of the sample molecules to create sample ions and the vacuum chamber includes means for maintaining the substantially sub-atmospheric pressure at approximately 10 Torr or less.
Alternatively, the vacuum chamber is maintained at a substantially sub-atmospheric pressure of 10 mTorr or less.
The means for vaporizing the sample molecules may include a laser for delivering laser beams. Alternatively, the sample plate may include means for heating the sample plate to vaporize a sample provided thereon.
In addition, the reagent ion generation means may include a central electrode with a sharp end and a tubular electrode with an outlet opening. The tubular electrode surrounds the central electrode and defines a conduit for gas flow. The reagent ion generation means also includes means for providing a potential between the central electrode and the tubular electrode to form a corona discharge between the sharp end of the central electrode and the outlet opening of the tubular electrode. The reagent ion generation means also has a gas supply for supplying gas to the duct of the tubular electrode to provide a gas flow through the outlet opening to entrain reagent ions.
Alternatively, the reagent ion generation means may include a central electrode with a sharp end and an open-ended tubular electrode. The open-ended tubular electrode surrounds the central electrode and the sharp end of the central electrode extends past the tip of the open-ended tubular electrode. The reagent ion generation means also includes means for providing a potential between the central electrode and the open-ended tubular electrode to form a corona discharge between the sharp end of the central electrode and the open-ended tubular electrode. The reagent ion generation means also has a plug in the tubular electrode to prevent gas flow through the tubular electrode.
In one embodiment of the apparatus, the sample plate is provided within the vacuum chamber and the vacuum chamber has means for collecting and focusing the sample ions.
Alternatively, the sample plate may be provided in a first chamber and the first chamber may be separated from the vacuum chamber by a skimmer cone. Pumping means are provided to maintain the first chamber at a higher pressure than the vacuum chamber. The first chamber is at the sub-atmospheric pressure and the reagent ions and the sample molecules mix to form sample ions. The skimmer cone includes an orifice to allow the sample ions to pass into the vacuum chamber and the vacuum chamber has means for collecting and focusing the sample ions.
Alternatively, the apparatus may further comprise a first chamber, a skimmer cone which separates the first chamber from the vacuum chamber and an electrode external to the first chamber to generate an electric field between the electrode and the support plate to generate reagent ions at atmospheric pressure. The sample plate is provided in the first chamber around a first orifice and the reagent ions pass through the first orifice into the first chamber. The first chamber is at the sub-atmospheric pressure and the reagent ions mix with the sample molecules to form sample ions. The skimmer cone has a second orifice to allow the sample ions to pass into the vacuum chamber and the vacuum chamber has means for collecting and focusing the sample ions.
The vacuum chamber may include means for collecting and focusing the sample ions and an orifice for receiving the sample molecules. The sample plate may be located in an atmospheric pressure region outside of the vacuum chamber immediately adjacent to the orifice and there may be a means for introducing a flow of reagent ions into the vacuum chamber adjacent to the orifice. In use, vaporized sample molecules pass through the orifice and expand in a free jet expansion into the vacuum chamber and simultaneously mix and react with the reagent ions.
For a better understanding of the present invention and to show more clearly how it may be carried into effect, reference will now be made, by way of example, to the accompanying drawings which show preferred embodiments of the present invention and in which:
a is a schematic view, on an enlarged scale, of the area indicated at III in
b is a schematic view, on an enlarged scale, of an alternate embodiment of the area indicated at III in FIG. 5.
Referring first to
A matrix and sample are deposited on a spot 20 on a sample support plate 12 that is used as an electrode to establish an electric field. A focused laser beam, such as a laser pulse 18, from a laser (not shown) is then focused on the spot 20 on the sample support plate 12 to vaporize the sample and the matrix. A reagent ion discharge source 13 provides reagent ions and is directed towards the spot 20. The reagent ions ionize the sample molecules which were separated from the spot 20. A DC electric field is provided between the sample support plate 12 and the multipolar RF ion guide 25, or alternatively any RF ion guide, to drive the sample ions towards a downstream mass spectrometer indicated by an arrow 28. The electric field can be adjusted to optimize the sample signal in the downstream mass spectrometer 28. The adjustment procedure is obvious to one skilled in the art.
If the sample support plate 12 is not electrically conductive, an electric field can be established by additional electrodes (not shown) located between the sample support plate 12 and the end of the vacuum chamber 22 indicated at 29. Other electrodes (not shown) could also be used to optimize the ion flux into the multipolar RF ion guide 25. These electrodes could be placed near the spot 20, as is familiar to those trained in the art.
It is also possible to replace the sample support plate 12 with a very fine conductive filament (not shown), or another heatable element, upon which the matrix and sample are deposited. A very brief large current is then pulsed through the filament causing it to heat rapidly. The typically solid sample is thereby quickly vaporized, after which the sample molecules are chemically ionized by the nearby reagent ion flux from the reagent ion discharge source 13.
A more detailed illustration of the reagent ion discharge source 13 contained within the dashed circle II of
For this invention, it is important to have the corona discharge occur in a region where there is a high gas flow velocity. The high gas flow velocity is needed to move a high proportion of the reagent corona ions into the region of the vaporized sample molecules so that the sample molecules may be chemically ionized by the reagent corona ions. The speed of this chemical ionization process will be affected by the local gas density in the vicinity of the spot 20. However, it is difficult to get a high local gas density due to the mechanical complexity required. For example, the well known free jet expansion theory predicts that for a gas at atmospheric pressure in the corona tube 14, and a diameter of 0.125 mm for the outlet opening 19, the local pressure will be 1/100th of an atmosphere 0.5 mm downstream of the opening of the corona tube 14 and dropping rapidly. At the same time, it is desirable to minimize the reagent gas flow 24 into the vacuum chamber 22 to minimize pumping requirements. The vacuum chamber 22 is usually maintained at a pressure in the range of 10 Torr to 10 mTorr. This can be done by reducing the pressure of the reagent gas in the corona tube 14 to below one atmosphere.
In order to control the average ion current into the mass spectrometer, it is also possible to pulse the corona discharge ions in concert with the pulsing of the laser vaporization. The gas(es) used with the corona discharge may also be selected for their ability to chemically ionize the particular sample molecules while not ionizing other molecules such as the matrix molecules. Accordingly, the matrix can be chosen to have an inability to be ionized by many different reagent corona ion species. For example, the matrix may be composed of a non-polar compound, which has a low proton affinity (i.e. gas phase basicity), so that it is not protonated by the reagent corona ions which will in general be a protonating species. Alternately, it may be advantageous to provide reagent ions which can act as charge transfer reagents. For example, benzene or toluene could be used as a reagent gas to form molecular M+ ions which can ionize certain species of sample molecules which have a lower ionization potential. Accordingly, the matrix may be selected to have a high ionization potential so that the matrix molecules are not ionized by the reagent ions. Furthermore, the laser pulse 18 should have a frequency such that the laser pulse 18 can be absorbed by the matrix to effect matrix assisted laser desorption. It is expected that characteristics such as laser energy, spot size and pulse frequency, can be optimized empirically in order to provide the best conditions for sample desorption and ionization by the reagent ions.
The configuration shown in
The advantage of the configuration shown in
Another alternate embodiment is shown in FIG. 4. In this configuration, the sample is deposited on the low pressure side of a sample support plate 12 in a region that surrounds an orifice 40. In a higher pressure region, at approximately atmospheric pressure for example, reagent corona ions, generated by an electric field between an electrode 42 and the sample support plate 12, are directed into a first vacuum chamber 44 through an orifice 40 by a gas flow and by the electric field between the electrode 42 and the outside of the sample support plate 12. Alternatively, an electric field may be used. The pressure on the low pressure side of the sample support plate 12 may be approximately 10 Torr, or as low as a few mTorr, depending upon the diameter of the orifice 40 and the size of the vacuum pump (not shown). A focused laser pulse 18 desorbs the deposited sample and matrix that are positioned in close proximity to the orifice 40. The configuration shown in
The material desorbed by the focused laser pulse 18 is immediately mixed with reagent corona ions which enter through the orifice 40. However, the sample and matrix must not block the orifice 40 through which the reagent corona ions enter. This may be ensured by placing a fine wire in the orifice 40 while the sample material is deposited and dried and then removing the wire before introducing the reagent corona ions through the orifice 40. This approach may also lend itself to a batch sample method in which there is a sample support plate containing multiple sample regions, each having its own orifice. The sample support plate could then be moved in front of the mass spectrometer to present each sample in turn to the laser desorption process and the reagent corona ions. Only one sample orifice would be open to the vacuum chamber 22 at one time while the other sample orifices would be blocked off.
The configuration of
Referring now to
a, and 6b show two variations for the dashed circular region III of
b illustrates an alternative for mixing the reagent corona ions with the neutral sample molecular flow of
A corona discharge can only be obtained if adequate pressures are present. For example, the suggested pressure of 10 mTorr in
As noted above, in
Although the embodiments described herein employ multipolar RF ions guides (typically understood to refer to RF quadrupoles, RF hexapoles, RF octapoles and the like), it should be understood that other RF ion guide devices, such as RF ring guides or tapered RF ring guides (sometimes referred to as ion funnels), could also be employed. The purpose of these devices is to provide ion confinement and collisional focusing, and their use is not fundamental to the invention, except as they provide higher sensitivity by means of improved ion transmission efficiency. Other ion focusing or transmission devices may be used to similar benefit.
It should be understood that various modifications can be made to the preferred embodiments described and illustrated herein, without departing from the present invention, the scope of which is defined in the appended claims.
| Number | Name | Date | Kind |
|---|---|---|---|
| 5663561 | Franzen et al. | Sep 1997 | A |
| 5961772 | Selwyn | Oct 1999 | A |
| 5965884 | Laiko et al. | Oct 1999 | A |
| 6335525 | Takada et al. | Jan 2002 | B1 |
| 6515280 | Baykut | Feb 2003 | B1 |
| 6627881 | Bertrand et al. | Sep 2003 | B1 |
| 20020145110 | Holle | Oct 2002 | A1 |
| Number | Date | Country |
|---|---|---|
| 0 560 537 | Sep 1993 | EP |
| WO 9938185 | Jul 1999 | WO |
| Number | Date | Country | |
|---|---|---|---|
| 20030111600 A1 | Jun 2003 | US |
| Number | Date | Country | |
|---|---|---|---|
| 60339465 | Dec 2001 | US |
