Patent Publication Number: US-2005116162-A1

Title: Tandem time-of-flight mass spectrometer with delayed extraction and method for use

Description:
RELATED APPLICATIONS  
      This is a continuation-in-part of patent application Ser. No. 90/020,142, filed on Feb. 6, 1998, the entire disclosure of which is incorporated herein by reference. 
    
    
     FIELD OF THE INVENTION  
      The invention relates generally to mass spectrometers and specifically to tandem mass spectrometers.  
     BACKGROUND OF THE INVENTION  
      Mass spectrometers vaporize and ionize a sample and determine the mass-to-charge ratio of the resulting ions. One form of mass spectrometer determines the mass-to-charge ratio of an ion by measuring the amount of time it takes a given ion to migrate from the ion source, the ionized and vaporized sample, to a detector, under the influence of electric fields. The time it takes for an ion to reach the detector, for electric fields of given strengths, is a direct function of its mass and an inverse function of its charge. This form of mass spectrometer is termed a time-of-flight mass spectrometer.  
      Recently time-of-flight (TOF) mass spectrometers have become widely accepted, particularly for the analysis of relatively nonvolatile biomolecules, and other applications requiring high speed, high sensitivity, and/or wide mass range. New ionization techniques such as matrix-assisted laser desorption/ionization (MALDI) and electrospray (ESI) have greatly extended the mass range of molecules which can be made to produce intact molecular ions in the gas phase, and TOF has unique advantages for these applications. The recent development of delayed extraction, for example, as described in U.S. Pat. Nos. 5,625,184 and 5,627,360, has made high resolution and precise mass measurement routinely available with MALDI-TOF, and orthogonal injection with pulsed extraction has provided similar performance enhancements for ESI-TOF.  
      These techniques provide excellent data on the molecular weight of samples, but little information on molecular structure. Traditionally tandem mass spectrometers (MS-MS) have been employed to provide structural information. In MS-MS instruments, a first mass analyzer is used to select a primary ion of interest, for example, a molecular ion of a particular sample, and that ion is caused to fragment by increasing its internal energy, for example, by causing the ion to collide with a neutral molecule. The spectrum of fragment ions is then analyzed by a second mass analyzer, and often the structure of the primary ion can be determined by interpreting the fragmentation pattern. In MALDI-TOF, the technique known as post-source decay (PSD) can be employed, but the fragmentation spectra are often weak and difficult to interpret. Adding a collision cell where the ions may undergo high energy collisions with neutral molecules enhances the production of low mass fragment ions and produces some additional fragmentation, but the spectra are difficult to interpret. In orthogonal ESI-TOF, fragmentation may be produced by causing energetic collisions to occur in the interface between the atmospheric pressure electrospray and the evacuated mass spectrometer, but currently there is no means for selecting a particular primary ion.  
      The most common form of tandem mass spectrometry is the triple quadrupole in which the primary ion is selected by the first quadrupole, and the fragment ion spectrum is analyzed by scanning the third quadrupole. The second quadrupole is typically maintained at a sufficiently high pressure and voltage that multiple low energy collisions occur. The resulting spectra are generally rather easy to interpret and techniques have been developed, for example, for determining the amino acid sequence of a peptide from such spectra. Recently hybrid instruments have been described in which the third quadrupole is replaced by a time-of-flight analyzer.  
      Several approaches to using time-of-flight techniques both for selection of a primary ion and for analysis and detection of fragment ions have been described previously. For example, a tandem instrument incorporating two linear time-of-flight mass analyzers using surface-induced dissociation (SID) has been used to produce the product ions. In a later version, an ion mirror was added to the second mass analyzer. U.S. Pat. No. 5,206,508 discloses a tandem mass spectrometer system, using either linear or reflecting analyzers, which is capable of obtaining tandem mass spectra for each parent ion without requiring the separation of parent ions of differing mass from each other. U.S. Pat. No. 5,202,563 discloses a tandem time-of-flight mass spectrometer comprising a grounded vacuum housing, two reflecting-type mass analyzers coupled via a fragmentation chamber, and flight channels electrically floated with respect to the grounded vacuum housing. The application of these devices has generally been confined to relatively small molecules; none appears to provide the sensitivity and resolution required for biological applications, such as sequencing of peptides or oligonucleotides.  
      For peptide sequencing and structure determination by tandem mass spectrometry, both mass analyzers must have at least unit mass resolution and good ion transmission over the mass range of interest. Above molecular weight 1000, this requirement is best met by MS-MS systems consisting of two double-focusing magnetic deflection mass spectrometers having high mass range. While these instruments provide the highest mass range and mass accuracy, they are limited in sensitivity, compared to time-of-flight, and are not readily adaptable for use with modern ionization techniques such as MALDI and electrospray. These instruments are also very complex and expensive.  
     SUMMARY OF THE INVENTION  
      The invention relates to tandem time-of-flight mass spectrometry including: (1) an ion generator; (2) a timed ion selector in communication with the ion generator (3) an ion fragmentation chamber in communication with the ion selector; and (4) an analyzer in communication with the fragmentation chamber. In one embodiment, the ion generator comprises a pulsed ion source in which the ions are accelerated so that their velocities depend on their mass-to-charge ratio. The pulsed ion source may comprise a laser desorption ionization or a pulsed electrospray source. In another embodiment, the ion generator comprises a continuous ionization source such as a continuous electrospray, electron impact, inductively coupled plasma, or a chemical ionization source. In this embodiment, the ions are injected into a pulsed ion source in a direction substantially orthogonal to the direction of ion travel in the drift space. The ions are converted into a pulsed beam of ions and are accelerated toward the drift space by periodically applying a voltage pulse.  
      In one embodiment, the timed ion selector comprises a field-free drift space coupled to the pulsed ion generator at one end and coupled to a pulsed ion deflector at another end. The drift space may include a beam guide confining the ion beam near the center of the drift space to increase the ion transmission. The pulsed ion deflector allows only those ions within a selected mass-to-charge ratio range to be transmitted through the ion fragmentation chamber. In one embodiment, the analyzer is a time-of-flight mass spectrometer and the fragmentation chamber is a collision cell designed to cause fragmentation of ions and to delay extraction. In another embodiment, the analyzer includes an ion mirror.  
      A feature of the present invention is the use of the fragmentation chamber not only to produce fragment ions, but also to serve as a delayed extraction ion source for the analysis of the fragment ions by time-of-flight mass spectrometry. This allows high resolution time-of-flight mass spectra of fragment ions to be recorded over their entire mass range in a single acquisition. Another feature of the present invention is the addition of a grid which produces a field free region between the collision cell and the acceleration region. The field free region allows the ions excited by collisions in the collision cell time to complete fragmentation.  
      The invention also relates to the measurement of fragment mass spectra with high resolution, accuracy and sensitivity. In one embodiment, the method includes the steps of: (1) producing a pulsed source of ions; (2) selecting ions of a specific range of mass-to-charge ratios; (3) fragmenting the ions; and (4) analyzing the fragment ions using delayed extraction time-of-flight mass spectrometry. In one embodiment, the step of producing a pulsed source of ions is performed by MALDI. In one embodiment, the step of fragmenting the ion is performed by colliding the ion with molecules of a gas. In one embodiment, the step of fragmenting the ion includes the steps of exciting the ions and then passing the excited ions through a nearly field-free region to allow the excited ions enough time to substantially complete fragmentation.  
      A method for high performance tandem mass spectroscopy according to the present invention includes selection of the primary ions. The parameters controlling the pulsed ion generator are adjusted so that the primary ions of interest are focused to a minimum peak width at the pulsed ion deflector. The deflector is pulsed to allow the selected ion to be transmitted, while all other ions are deflected and are not transmitted. The selected ions may be decelerated by a predetermined amount. The selected ions enter the collision cell where they are excited by collisions with neutral molecules and dissociate. The fragment ions, and any residual selected ions, exit the collision cell into a nearly field-free region between the cell and a grid plate maintained at approximately the same potential as the cell. The ion packet at this point is very similar to that produced initially by MALDI in that all of the ions have nearly the same average velocity with some dispersion in velocity and position.  
      An acceleration pulse of a predetermined amplitude is applied to the grid plate, after a short delay from the time the ions pass through an aperture in the grid plate, the spectrum of the product ions may be recorded and the precise masses determined. Theory predicts that resolution approaching 3000 for primary ion selection should be achievable with minimal loss in transmission efficiency The theoretical resolution for the fragment ions is at least ten times the mass of the fragment, up to mass 2000  
      It is therefore an objective of this invention to provide a high performance MS-MS instrument and method employing time-of-flight techniques for both primary ion selection and fragment ion determination. The invention is applicable to any pulsed or continuous ionization source such as MALDI and electrospray The invention is particularly useful for providing sequence and structural information on biological samples such as peptides, oligonucleotides, and oligosaccharides. 
    
    
     BRIEF DESCRIPTION OF THE DRAWINGS  
      This invention is pointed out with particularity in the appended claims. The above and further advantages of this invention may be better understood referring to the following description taken in conjunctions with the accompanying drawings, in which:  
       FIG. 1  is a block diagram of an embodiment of the invention;  
       FIG. 2A  is a schematic diagram of an embodiment of the invention of  FIG. 1 ;  
       FIG. 2B  is a graphical representation of the voltages present at each point of the embodiment of the invention shown in  FIG. 2A ;  
       FIG. 3  is a schematic diagram of an embodiment of the fragmentation chamber of  FIG. 2 ;  
       FIG. 4  is a schematic diagram of an embodiment of the pulsed ion deflector and associated gating potential of  FIG. 2 ;  
       FIG. 5  is a block diagram of an embodiment of the voltage switching circuits employed in the pulsed ion generator, the timed ion selector, and the timed pulsed extraction referenced in  FIG. 2 ;  
       FIG. 6  is a graph of the resolution versus mass-to-charge ratio for fragment ions resulting from fragmentation of a hypothetical ion of mass-to-charge ratio 2000 for the embodiment of the invention of  FIG. 2 ;  
       FIG. 7  is a schematic diagram of an embodiment of an ion guide comprising a stacked plate array that can be positioned in various field free regions of an embodiment of the invention of  FIG. 1 ;  
       FIG. 8  is a schematic diagram of another embodiment of the invention of  FIG. 1 ;  
       FIG. 9  is a schematic diagram of an embodiment of a collision cell as the fragmentation chamber for the embodiment of the invention shown in  FIG. 8 ;  
       FIG. 9A  is a cross section view of the collision cell in  FIG. 9 ;  
       FIG. 10  is a schematic diagram of an embodiment of a photodissociation cell as the fragmentation chamber of the embodiment of the invention shown in  FIG. 8 ;  
       FIG. 11  is a schematic diagram of an embodiment employing collisions of ions with solid or liquid surfaces in the fragmentation chamber of the embodiment of the invention shown in  FIG. 8 ; and  
       FIG. 12  is a schematic diagram of an embodiment of the invention of  FIG. 1  wherein a timed ion selector, ion fragmentation chamber and pulsed ion generator are contained within the same vacuum housing. 
    
    
     DETAILED DESCRIPTION OF THE INVENTION  
      Referring to  FIG. 1 , in brief overview, a tandem time-of-flight mass spectrometer  10  that uses delayed extraction according to the present invention includes: (1) a pulsed ion generator  12 , (2) a timed ion selector  14  in communication with the pulsed ion generator  12 , (3) an ion fragmentor or fragmentation chamber  18 , which is in communication with the timed ion selector  14 , and (4) an ion analyzer  24 . In operation, a sample to be analyzed is ionized by the pulsed ion generator  12 . The ions to be studied are selected by the timed ion selector  14 , and allowed to pass to the fragmentation chamber  18 . Here the selected ions are fragmented and allowed to pass to the analyzer  24 . The fragmentation chamber  18  is designed to function as a delayed extraction source for the analyzer  24 .  
      In more detail and referring to  FIG. 2A , an embodiment of a tandem time-of-flight mass spectrometer  10  using delayed extraction includes a pulsed ion generator  12 . The pulsed ion generator includes a laser  27  and a source extraction grid  36 . A timed ion selector  14  is in communication with the ion generator  12 . The ion selector  14  comprises a field-free drift tube  16  and a pulsed ion deflector  52 . The field-free drift tube  16  may include an ion guide as described in connection with  FIG. 7 .  
      An ion fragmentation chamber  18 , is in communication with ion selector  14 . The ion fragmentation chamber shown in  FIG. 2A  includes a collision cell  44 . However, the fragmentation chamber  18  may be any other type of fragmentation chamber known in the art such as a photodissociation chamber or a surface induced dissociation chamber. A small aperture  54  at the entrance to the pulsed ion deflector  52  allows free passage of the ion beam to the fragmentation chamber  18 , but limits the flow of neutral gas. The fragmentation chamber  18  is in communication with an ion analyzer  24 . A small aperture  58  at the exit of the fragmentation chamber  18  allows free passage of the ion beam, but limits the flow of neutral gas.  
      In one embodiment, a grid plate  53  is positioned adjacent to the collision cell  44  and biased to form a field free region  57 . The field free region  57  may include an ion guide  57 ′ which is shown as a box in  FIG. 2   a  and which is more fully described in connection with  FIG. 7 . A fragmentor extraction grid  56  is positioned adjacent to the grid plate  53  and to an entrance  58  to the analyzer  24 . In another embodiment, fragmentor extraction grid  56  is positioned directly adjacent to the exit aperture, eliminating the grid plate  53 . This embodiment is used for measurements where the fragmentation is substantially completed in the collision cell  44 . The analyzer  24  includes a second field-free drift tube  16 ′ in communication with an ion mirror  64 . The second field-free drift tube  16 ′ may include an ion guide as described in connection with  FIG. 7 . A detector  68  is positioned to receive the reflected ions.  
      The pulsed ion generator  12  and drift tube  16  are enclosed in a vacuum housing  20 , which is connected to a vacuum pump (not shown) through a gas outlet  22 . Also, the fragmentation chamber  18  and pulsed ion deflector  52  are enclosed in vacuum housing  19 , which is connected to a vacuum pump (not shown) through a gas outlet  48 . Similarly, the analyzer  24  is enclosed in a vacuum housing  26 , which is connected to a vacuum pump (not shown) through a gas outlet  28 . The vacuum pump maintains the background pressure of neutral gas in the vacuum housing  20 ,  19 , and  26  sufficiently low that collisions of ions with neutral molecules are unlikely to occur.  
      In operation, a sample  32  to be analyzed is ionized by the pulsed ion generator  12 , which produces a pulse of ions. In one embodiment, the pulsed ion generator  12  employs Matrix Assisted Laser Desorption/Ionization (MALDI). In this embodiment, a laser beam  27 ′ impinges upon a sample plate having the sample  32  which has been mixed with a matrix capable of selectively absorbing the wavelength of the incident laser beam  28 .  
      At a predetermined time after ionization, the ions are accelerated by applying an ejection potential between the sample  32  and the source extraction grid  36  and between the source extraction grid  36  and the drift tube  16 . In one embodiment, the drift tube is at ground potential. After this acceleration, the ions travel through the drift tube with velocities which are nearly proportional to the square root of their charge-to-mass ratio; that is, heavier ions travel more slowly. Thus within the drift tube  16 , the ions separate according to their mass-to-charge ratio with ions of higher mass traveling more slowly than those of lower mass.  
      The pulsed ion deflector  52  opens for a time window at a predetermined time after ionization. This permits only those ions with the selected mass-to-charge ratios, arriving at the pulsed ion deflector  52  within the predetermined time window during which the pulsed ion deflector  52  is permitting access to the collision cell  44 , to be transmitted. Hence, only predetermined ions, those having the selected mass-to-charge ratio, will be permitted to enter the collision cell  44  by the pulsed ion deflector  52 . Other ions of higher or lower mass are rejected.  
      The selected ions entering the collision cell  44  collide with the neutral gas entering through inlet  40 . The collisions cause the ions to fragment. The energy of the collisions is proportional to a difference in potential between that applied to the sample  32  and the collision cell  44 . In one embodiment, the pressure of the neutral gas in the collision cell  44  is maintained at about 10 −3  torr and the pressure in the space surrounding the collision cell  44  is about 10 −5  torr. Gas diffusing from the collision cell  44  through an ion entrance aperture  46  and ion exit aperture  50  is facilitated by a vacuum pump (not shown) connected to a gas outlet  48 . In another embodiment, a high-speed pulsed valve (not shown) is positioned in gas inlet  40  so as to produce a high pressure pulse of neutral gas during the time when ions arrive at the fragmentation chamber  18  and, for the remainder of the time, the fragmentation chamber  18  is maintained as a vacuum. The neutral gas may be any neutral gas such as helium, air, nitrogen, argon, krypton, or xenon.  
      In one embodiment, the grid plate  53  and the fragmentor extraction grid  56  are biased at substantially the same potential as the collision cell  44  until the fragment ions pass through an aperture  50 ′ in grid plate  53  and enter the nearly field-free region  59  between the grid plate  53  and the extraction grid  56 . At a predetermined time after the ions pass grid plate  53 , the potential on grid plate  53  is rapidly switched to a high voltage thereby causing the ions to be accelerated. The accelerated ions pass through the entrance  58  to the analyzer  24 , into a second field-free drift tube  16 ′, into the ion mirror  64 , and to the detector  68 , which is positioned to receive the reflected ions.  
      The time of flight of the ion fragments, starting from the time that the potential on the grid plate  53  is switched and ending with ion detection by the detector  68 , is measured. The mass-to-charge ratio of the ion fragments is determined from the measured time. The mass-to-charge ratio can be determined with very high resolution by properly choosing the operating parameters so that the fragmentation chamber  18  functions as a delayed extraction source of ion fragments. The operating parameters include: (1) the delay between the passing of the fragment ions through the aperture  50 ′ in grid plate  53  and the application of the accelerating potential to the grid plate  53 ; and (2) the magnitude of the extraction field between the grid plate  53  and the fragmentor extraction grid  56 .  
      In another embodiment, grid  53  is not used or does not exist. This embodiment is used for measurements where the fragmentation is substantially completed in the collision cell  44 . In this embodiment, the fragmentor extraction grid  56  is biased at substantially the same potential as the collision cell  44 . At a predetermined time after the ions exit the collision cell  44 , the high voltage connection to the collision cell  44  is rapidly switched to a second high voltage supply (not shown) thereby causing the ions to be accelerated. The accelerated ions pass through the entrance  58  to the analyzer  24 , into a second field-free drift tube  16 ′, into the ion mirror  64 , and to the detector  68 , which is positioned to receive the reflected ions.  
      The time of flight of the ion fragments, starting from the time that the potential on the collision cell  44  is switched and ending with ion detection by the detector  68 , is measured. The mass-to-charge ratio of the ion fragments is determined from the measured time. The mass-to-charge ratio can be determined with very high resolution by properly choosing the operating parameters so that the fragmentation chamber  18  functions as a delayed extraction source of ion fragments. The operating parameters include: (1) the predetermined time after the ions exit the collision cell  44  before the high voltage is rapidly switched to the second high voltage; and (2) the magnitude of the extraction field between the collision cell  44  and the fragmentor extraction grid  56 .  
       FIG. 2B  depicts the electric potential experienced by an ion in each portion of the embodiment of the tandem mass spectrometer illustrated in  FIG. 2A . A voltage  70  is applied to the sample  32  and a voltage  71  is applied to extraction grid  36 . Voltage  71  is approximately equal to voltage  72 . In response to the laser beam  28  impinging on the sample  32 , a pulse of ions is formed and emitted into a substantially field-free space  61  between sample  32  and the extraction grid  36 . The ions depart from the sample  32  with a characteristic velocity distribution which is nearly independent of their mass-to-charge ratio. As the ions drift in the nearly field-free space  61  between the sample  32  and the extraction grid  36 , the ions are distributed in space with the faster ions nearer to the extraction grid  36  and the slower ions nearer to the sample  32 . At a predetermined time after ionization, the voltage applied to the sample  32  is rapidly switched to higher voltage  72 , thereby establishing an electric field between the sample  32  and the extraction grid  36 . The electric field between the sample  32  and the extraction grid  36  causes the initially slower ion, which are closest to the sample  32 , to receive a larger acceleration than the initially faster ion.  
      The drift tube  16  is at a lower potential  73  than the extraction grid  36  and, therefore, a second electric field is established between the extraction grid and the drift tube. In one embodiment the voltage  73  is at ground potential. Thus, the ions are further accelerated by the second electric field. By appropriate choices of the voltages  71  and  72  and the delay time between formation of the ion pulse and switching on the extraction voltage  72 , the initially slower ions at  81  are accelerated more than the initially faster ions at  82  and, therefore, the initially slower ions eventually overtake the initially faster ions at a selected focal point  83 . The selected focal point  83  may be chosen to be at the pulsed ion deflector  52 , at the collision cell  44 , or any other point along the ion trajectory.  
      For the velocity distributions typical for production of ions by MALDI, the total time spread at the selected focal point  83  for ions of a specified mass-to-charge ratio is typically about one nanosecond or less. If the selected focal point  83  is chosen to coincide with the location of the pulsed ion deflector  52 , then the pulsed ion deflector  52  gate is opened for a short time interval corresponding to the time of arrival of ions of a selected mass-to-charge ratio and is closed at all other times to reject all other ions. The delayed extraction of the present invention is advantageous because the resolution of ion selection is limited only by properties of the pulsed ion deflector  52  since the time width of the ion packet can be made very small. Thus, high resolution ion selection is possible. In contrast, with continuous extraction, all of the ions receive the same acceleration from the electric fields and no velocity focusing occurs. Thus the time width of a packet of ions of a particular mass-to-charge ratio increases in proportion to the ion travel time from the sample to any point along the trajectory and the resolution of ion selection cannot be very high.  
      In operation, the pulsed ion deflector  52  establishes a transverse electric field that deflect low mass ions until the arrival of ions of a selected mass-to-charge ratio. At which time, the transverse fields are rapidly reduced to zero thereby allowing the selected ions to pass through. After passage of the selected ions, the transverse fields are restored and any higher mass ions are deflected. The selected ions are transmitted undeflected into the fragmentation chamber  18 .  
      A voltage  74  may be applied to the collision cell  44  to reduce the kinetic energy of the ions before they enter the collision cell  44  through the entrance aperture  46 . The energy of the ions in the collision cell  44  is determined by their initial potential  81  or  82  relative to voltage  74  plus the kinetic energy associated with their initial velocity. The energy with which ions collide with neutral molecules within the collision cell  44  can be varied by varying the voltage  74 .  
      When an ion collides with a neutral molecule within the collision cell  44 , a portion of its kinetic energy may be converted into an internal energy that is sufficient to cause the ion to fragment. Energized ions and fragments continue to travel through the collision cell  44 , with a somewhat diminished velocity, due to collisions in the cell  44  and eventually emerge through the exit aperture  50  within a still narrow time interval and with a velocity distribution corresponding to the initial velocity distribution, as modified by delayed extraction and by collisions.  
      In one embodiment, the voltage  74  applied to the grid plate  53  and the voltage  75  applied to the fragmentor extraction grid  56  are equal and, therefore, produce a field-free region between the collision cell  44  and the fragmentor extraction grid  56 . As the ions drift in the field-free region they are distributed in space with the faster ions nearer to the fragmentor extraction grid  56  and the slower ions nearer to the grid plate  53 .  
      After a predetermined time delay, the voltage applied to the grid plate  53  is rapidly switched to a higher voltage  76  thereby establishing an electric field between the grid plate  53  and the fragmentor extraction grid  56 . As a result, the initially slower ion receives a larger acceleration than the initially faster ion. In one embodiment, the grid plate  53  and the collision cell  44  are electrically connected so that both are switched simultaneously. In another embodiment, the voltage applied to the collision cell  44  is constant, and only the voltage applied to grid plate  53  is switched.  
      In another embodiment, the grid plate  53  is not used or does not exist. This embodiment is used for measurements where the fragmentation is substantially completed in the collision cell  44 . In this embodiment, there is no field-free region between the collision cell  44  and the fragmentor extraction grid  56 . After a predetermined time delay, the voltage applied to the collision cell  44  is rapidly switched to the higher voltage  76  thereby establishing an electric field between the collision cell  44  and the fragmentor extraction grid  56 . As a result, the initially slower ion receives a larger acceleration than the initially faster ion.  
      The ions are further accelerated in an electric field between the fragmentor extraction grid  56  and the drift tube  16 ′. In one embodiment, the voltage  77  may be at ground potential. By appropriately choosing the voltages  76  and  74  and the collision cell  44  switching time, the initially slower ions at  84  are sufficiently accelerated so that they at  85  overtake the initially faster ions at a selected focal point  89 .  
      In one embodiment, this focal point is chosen at or near the entrance  58  to the analyzer  24 . In this embodiment, the ions travel through a second field-free region  16 ′ and enter the ion mirror  64  in which the ions are reflected at voltage  79  and eventually strike the detector  68 . For a given length of the drift tube  16 ′ and length of the mirror  64 , the voltage  78  can be adjusted to refocus the ions, in time, at the detector  68 . In this manner, the fragmentation chamber  18  performs as a delayed extraction source for the analyzer  24  and high resolution spectra of fragment ions can be measured.  
       FIG. 3  is a schematic diagram of an embodiment of the fragmentation chamber  18  of  FIG. 2 . The collision cell  44  includes the gas inlet  40  through which gas is introduced into the collision cell  44  and entrance and exit apertures  46  and  50 , respectively, through which the gas diffuses (arrows D) out from the collision cell  44 . These apertures  46 ,  50  may be covered with highly transparent grids  47  to prevent penetration of external electric fields into the collision cell  44 . The gas which diffuses out is drawn off by the vacuum pump attached to the gas outlet  48  ( FIG. 2 ) of the fragmentation chamber  18 . In one embodiment, uniform electric fields are established between the collision cell  44  and entrance aperture  51  to fragmentation chamber  18 , and between fragmentor extraction grid  56  and entrance aperture  58  to the analyzer  24 .  
      A high voltage supply  92  is connected to extraction grid  56  and resistive voltage divider  53 ′. The voltage divider  53 ′ is attached to electrically isolated guard rings  55 , which are spaced uniformly in the space between extraction grid  56  and entrance aperture  58  to analyzer  24 , and between the collision cell  44  and the entrance aperture  51  to fragmentation chamber  18 . The voltage divider  53 ′ is adjusted to provide approximately uniform electric fields in these spaces. A high voltage supply  90  is electrically connected to the collision cell  44  and is set to voltage  74  ( FIG. 2B ). The voltage on the grid plate  53  is set by a switch  80  which is in electrical communication with high voltage supplies  90  and  91  that are set to voltages  74  and  76 , respectively ( FIG. 2B ).  
      The switch  80  is controlled by a signal from delay generator  87 . The delay generator  87  provides a control signal to the switch  80  in response to a start signal received from a controller (not shown), which in one embodiment is a digital computer. The delay is set so that ions of a selected mass-to-charge ratio pass through the aperture  50 ′ in the grid plate  53  shortly before the switch  80  is activated to switch the high voltage connection to the grid plate  53  from the voltage  74  produced by high voltage supply  90  to the voltage  76  produced by high voltage supply  91   
      Referring also to  FIG. 4 , in one embodiment, the pulsed ion deflector  52  includes two deflectors in series  100 ,  102  located between apertures  51  and  54  covered by highly transparent grids. Aperture  54  also serves as exit aperture from drift tube  16  and aperture  51  also serves as the entrance aperture  51  to the fragmentation chamber  18 . The gridded apertures  51  and  54  prevent the fields generated by the deflectors  100 ,  102  from propagating beyond the pulsed ion deflector  52 . The deflectors  100 ,  102  are located as close to the plane of the grids over the apertures  51 ,  54  as possible without initiating electrical breakdown.  
      In one embodiment, the deflector  100  closest to the sample  32  is operated in a normally closed (NC) or energized configuration in which the electrodes  101 A,  101 B of the deflector  100  have a potential difference between the electrodes. The second deflector  102  is operated in a normally open (NO) or non-energized configuration in which the electrodes  105 A,  105 B have no voltage difference between them. By correctly choosing the delay between the production of ions and time of arrival of ions of the desired mass-to-charge ratio at the deflector  100 , the entrance electrodes  101 A,  101 B may be de-energized to open just as the desired ions reach the deflector  100 , while the electrodes  105 A,  105 B of the second deflector  102  are de-energized to close just after the ions of interest pass deflector  102 . In this way, ions of lower mass are rejected by the first deflector  100  and ions of higher mass are rejected by the second deflector  102 . Ions are rejected by deflecting them through a sufficiently large angle to cause them to miss a critical aperture. In various embodiments ( FIG. 2 , for example), the critical aperture may coincide with the entrance aperture  46  to the collision cell  44 , to the entrance aperture  58  to the analyzer  24 , or to the detector  68 , whichever subtends the smallest angle of deflection.  
      The equations of motion for ions in electric fields allows time-of-flight for any ion between any two points along an ion trajectory to be accurately calculated. For the case of uniform electric fields, as employed in an embodiment depicted in  FIG. 2A  and B, these equations are particularly tractable, and provided that the voltages, distances, and initial velocities are accurately known, the flight time for any ion between any two points can be accurately calculated. Specifically, the time for an ion to traverse a uniform accelerating field is given by the equation:
 
 t =( v   2   −v   1 )/ a 
 
 where v 2  is the final velocity after acceleration, v 1  is the initial velocity before acceleration and t is the time that the ion spends in the field. The acceleration is given by
 
 a=z (V 1   −V   2 )/ md 
 
 where z is the change on an ion, m is the mass of the ion, V 1  and V 2  are the applied voltages, and d is the length of the field. In a field-free space, the acceleration is zero, and
 
t=D/v
 
 where D is the length of the field-free space and v is the ion velocity. 
 
      In conservative systems (i.e. no frictional losses), the sum of kinetic energy and potential energy is constant. For motion of charged particles in an electric field, this can be expressed as
 
 T   2   −T   1   =z ( V   1   −V   2 )
 
 where the kinetic energy T=mv 2 /2. This can be solved for v to give an explicit expression for the velocity of a charged particle at any point. 
 
      For ions traveling through a series of uniform electrical fields, the above equations provide exactly the time of flight as a function of mass, charge, potentials, distances, and the initial position and velocity of the ion. If the SI system is used, in which distance is expressed in meters, potentials in volts, masses in kg, charge in coulombs, and time in seconds, then no additional constants are required.  
      In some cases, all of the parameters may not be known a priori to sufficient accuracy, and it may be necessary in these cases to determine empirically, corrections to the calculated flight times.  
      In any case, the flight time for an ion of any selected mass-to-charge ratio can be determined with sufficient accuracy to allow the required time delays between generation of ions in the pulsed ion generator  12  and selection of ions in the timed ion selector  14  or pulsed extraction of ions from the collision cell  44  to be determined accurately.  
      Referring also to  FIG. 5 , in one embodiment, a four channel delay generator  162  is started by a start pulse  150  which is synchronized with production of ions in the pulsed ion generator  12 . In one embodiment, the start pulse is generated by detecting a pulse of light from the laser beam  28 . After a first delay period, a pulse  151  is generated by the delay generator  162 , which triggers switch  155  in communication with voltage sources providing voltages  70  and  72 , respectively.  
      Prior to receiving pulse  151 , the switch  155  is in position  160  connecting the voltage source for voltage  70  to sample  32 . Upon receiving pulse  151 , the switch  155  rapidly moves to position  161  which connects the voltage source for voltage  72  to sample  32 . The first delay is chosen so that ions of a selected mass-to-charge ratio produced by the pulsed ion generator  12  are focused in time at a selected point, for example, the pulsed ion deflector  52 .  
      After a second delay period, pulse  152  is generated which triggers switches  156  and  157 . Prior to receiving pulse  152 , switch  156  connects voltage source  120  to deflection plate  101 A, and switch  157  connects voltage source  121  to deflection plate  101 B. Upon receiving pulse  152 , the switches  156  and  157  rapidly move to connect both deflection plates  101 A and  101 B to ground.  
      Similarly, switches  158  and  159  initially connect electrodes  105 A and  105 B to ground, and in response to delayed pulse  153 , occurring after a third delay period, connect electrodes  105 A and  105 B to voltage sources  122  and  123 , respectively. In one embodiment, voltage sources  120  and  122  supply voltages which are equal and voltage sources  121  and  123  supply voltage sources which are equal in magnitude to the voltage supplied by voltage source  120  but of opposite sign. The second delay period is chosen to correspond to arrival of an ion of selected mass-to-charge ratio at or near the entrance aperture  54  of the pulsed ion deflector  52 , and the third delay period is chosen to correspond to arrival of an ion of selected mass-to-charge ratio at or near the exit aperture  51  of the pulsed ion deflector  52 .  
      After a fourth delay period, pulse  154  is generated which triggers switch  79 . Prior to receiving pulse  154 , switch  79  connects a voltage source supplying voltage  74  to grid plate  53 , and upon receiving pulse  154  switch  79  rapidly switches to connect voltage source supplying voltage  76  to grid plate  53 . The fourth delay period is chosen to correspond to arrival of an ion of selected mass-to-charge ratio at or near the aperture  50 ′ of grid plate  53 . With proper choice of the fourth delay period, the fragmentation chamber  18  acts as a delayed extraction source for analyzer  24 , and both primary and fragment ions are focused, in time, at the detector  68 . Each of the switches  79 ,  155 ,  156 ,  157 ,  158 , and  159  must be reset to their initial state prior to the next start pulse. The time and speed of resetting the switches is not critical, and it may be accomplished after a fixed delay built into each switch, or a delay pulse from another external delay channel (not shown) may be employed.  
      Referring also to  FIG. 6 , the resolution for fragment ions can be calculated for any instrumental geometry, such as the embodiment described in  FIG. 2 , with specified distances, voltages and delay times. The plots shown in  FIG. 6 , correspond to calculations of resolution as a function of fragment mass for an ion of mass-to-charge ratio (m/z) of 2000 produced in the pulsed ion generator  12  with a sample voltage  72  of 20 kilovolts and a collision cell voltage  74  of 19.8 kilovolts corresponding to an ion-neutral collision energy of 200 volts in the laboratory reference frame. ( FIGS. 2A  and B). At a delay of 858 nanoseconds after the primary ion of m/z 2000 was calculated to pass through the aperture  50 ′, the grid plate  53  was switched to the higher voltage  76 , which for purposes of this calculation was 25 kilovolts.  
      In one case (curve  111  in  FIG. 6 ), the voltage  75  applied to the fragmentor extraction grid  56  was also 19.8 kilovolts so that the region between the extraction grid  56  and the collision cell  44  was field-free. In another case (curve  112  in  FIG. 6 ), the voltage  75  applied to the fragmentor extraction grid  56  was 19.9 kilovolts, so that ions emerging from the exit  50  of the collision cell  44  were decelerated by a small amount. As can be seen from  FIG. 6 , the latter case  112  provides somewhat better resolution at lower fragment mass at the expense of slightly lower theoretical resolution at higher mass.  
      Referring also to  FIG. 7 , some embodiments of this invention include an ion guide  99  positioned in one or more field free regions. An ion guide may be positioned in at least one of the drift tube  16  or  16 ′ or the field free region  57  to increase the transmission of ions. Several types of ion guides are known in the art including the wire-in-cylinder type and RF excited multipole lenses consisting of quadrupoles, hexapoles or octupoles. One embodiment of the ion guide employs a stacked ring electrostatic ion guide. A stacked ring ion guide includes a stack of identical plates or rings  108 ,  108 ′, each with a central aperture  110 , stacked with uniform space between each pair of rings  108 . Every other ring  108 ′ is connected to a positive voltage supply  109 , and each intervening ring  108  is connected to a negative voltage supply  107 .  
      The end plates of the drift tube  16  in which the entrance  106  and exit  54  apertures are located, are spaced from the ends of stacked ring ion guide, by a distance which is one-half of the distance between the adjacent rings of the guide. To avoid perturbing the ion flight time through the ion guide  99 , it is important that the number of positively biased electrodes be equal to the number of negatively biased electrodes. By choosing an appropriate magnitude of the applied voltages from voltage supplies  107  and  109  relative to the energy of the incident ion beam, the ion beam is confined near the axis of the guide. The advantage of the stacked ring ion guide over, for example, the wire-in-cylinder ion guide, is that the ions are efficiently transmitted over a broad range of energy and mass without significantly perturbing the flight time of ions.  
       FIG. 8  is another embodiment of the invention. Referring also to  FIG. 8 , either a continuous or a pulsed source of ions  128  may be used to supply ions to the pulsed ion generator  12 . Any ionization techniques known in the art, including electrospray, chemical ionization, electron impact, inductively coupled plasma (ICP), and MALDI, can be employed with this embodiment. In this embodiment, a beam of ions  129  is injected into a field-free space between electrode  130  and extraction grid  36 , and periodically a voltage pulse is applied to electrode  130  to accelerate the ions in a direction orthogonal to that of the initial beam. Ions are further accelerated in a second electric field formed between extraction grid  36  and grid  136 .  
      Guard plates  134  are connected to a voltage divider (not shown) and may be used to assist in producing a uniform electric field between grids  36  and  136 . Ions pass through field-free space  16  and enter fragmentation chamber  18 . Within the fragmentation chamber  18 , ions enter collision cell  44  where they are caused to fragment by collisions with neutral molecules. In this embodiment, as discussed in more detail below, a pulsed ion deflector is located within the collision cell  44  and the fragmentation chamber  18  functions as a delayed extraction source for analyzer  24 . Ions exiting from the fragmentation chamber  18  pass through a field-free space  16 ′, are reflected by an ion mirror  64 , re-enter the field-free space  16 ′ and are detected by detector  68 .  
      Referring also to  FIG. 2B , this potential diagram also applies to an embodiment illustrated in  FIG. 8  with a few changes. Electrode  130  ( FIG. 8 ) replaces sample  32  ( FIG. 2 ) and pulsed ion deflector  52  is located within collision cell  44  ( FIG. 8 ). A beam of ions  129  produced in continuous ion source  128  enters the space between electrode  130  and extraction grid  36  between points  81  and  82 . Initially the voltage  70  on electrode  130  is equal to voltage  71  on extraction grid  36 , and periodically the electrode  130  is switched to voltage  72  to extract ions. The voltage difference between  70  and  72  is chosen so that ions in the beam are focused, in time, at or near the exit from the collision cell  44 . In one embodiment, the voltage  71  on extraction grid  36  is ground potential, and voltage  73  on drift tube  16  and  16 ′ is a voltage opposite in sign to that of ions of interest.  
      The energy of the ions in the collision cell  44  is determined by their initial potential  81  or  82  relative to voltage  74  plus the kinetic energy associated with their initial velocity. Thus the energy with which ions collide with neutral molecules within the collision cell  44  can be varied by varying the voltage  74 . In one embodiment, the voltage  71  and the voltage  74  are at ground potential. In this embodiment the extraction field between collision cell  44  and fragmentor extraction grid  56  is formed by switching voltage  75 , initially at or near ground, to a lower voltage.  
      Referring also to  FIG. 9 , in one embodiment, a pulsed ion deflector  52  is located within the collision cell  44 . Ions from the pulsed ion generator  12  ( FIG. 8 ) are focused at or near the exit  104  of collision cell  44 . At the time that a pulse of ions with a selected mass-to-charge ratio arrive at or near the entrance  103  to collision cell  44 , pulsed ion deflector  100  is de-energized to allow selected ions to pass undeflected. At the time that the pulse of ions with selected mass-to-charge ratio arrive at or near exit  104  to collision cell  44 , pulsed ion deflector  102  is energized to deflect ions of higher mass, which arrive later at pulsed deflector  102 . Thus, ions with lower mass-to-charge ratio are deflected by pulsed ion deflector  100  and ions with higher mass-to-charge ratio are deflected by pulsed ion deflector  102 , and ions within the selected mass-to-charge ratio range are transmitted undeflected. The voltages applied to the pulsed ion deflectors  100  and  102  are adjusted so that deflected ions and any fragments produced within collision cell are not transmitted through a critical aperture, which in this embodiment, is the entrance aperture  58  to the analyzer  24 .  
      In the embodiment illustrated in  FIG. 8 , the beam from the continuous ion source  128  is cylindrical in cross section and well collimated so that velocity components in the direction perpendicular to the axis of the beam are very small. As a consequence, the pulsed beam  39  generated by the pulsed ion generator  12  is relatively wide in the direction of ion travel from the continuous ion source  128 , but is narrow in orthogonal directions. That is, if the beam direction is along the x-axis, then the beam widths orthogonal to this will be narrow. The widths of the apertures  36 ,  136 ,  138 ,  103 ,  104 ,  56 , and  142  must be wide enough in the plane defined by directions of the continuous beam  129  and the pulsed beam  32  to allow essentially the entire pulsed beam to be transmitted, but may be narrow in the direction perpendicular to this plane. This is illustrated in  FIG. 9A  which shows a cross section through the collision cell  44 , wherein the exit aperture  104  is 25 mm long in the direction parallel to the beam from the continuous ion source  128 , and is 1.5 mm in the direction orthogonal to the plane defined by the beam from the continuous ion source  128  and the pulsed beam  39 . The other apertures  36 ,  136 ,  138 ,  103 ,  56 ,  142  may have similar dimensions. Also, the initial velocity of the continuous ion beam  129  adds vectorially to the velocity imparted by acceleration in the pulsed ion generator  12 . As a result, the trajectory of the pulsed ion beam  39  is at a small angle relative to the direction of acceleration and the slits are offset along their long direction so that the center of the pulsed ion beam  39  passes near the center of each aperture.  
      Referring also to  FIG. 10 , one embodiment of the invention employs a photodissociation cell  152  in fragmentation chamber  18 . In one embodiment, the photodissociation cell is similar to the collision cell  44 , but instead of an inflow of neutral gas through inlet  40 , a pulsed laser beam  150  is directed into the cell through aperture or window  160  and exits from the cell through aperture or window  161 . The laser pulse is synchronized with the start pulse and a delay generator (not shown) so that the laser pulse arrives at the center of the photodissociation cell at the same time as the ion pulse of a selected mass-to-charge ratio.  
      The wavelength of the laser is chosen so that the ion of interest absorbs energy at this wavelength. In one embodiment, a quadrupled Nd: YAG laser having a wavelength of the laser light of 266 nm is used. In another embodiment, an excimer laser having a wavelength of 193 nm is used. Any wavelength of radiation can be employed provided that it is absorbed by the ion of interest. The ion of interest is energized by absorption of one or more photons from the pulsed laser beam  150  and is caused to fragment. The fragments are analyzed with the fragmentation chamber  18  acting as a delayed extraction source for analyzer  24 , as described in detail above. The photodissociation cell  152  is also equipped with pulsed ion deflectors  100  and  102  to prevent ions of mass-to-charge ratios different from the selected ions from being transmitted to the analyzer  24 .  
      Referring also to  FIG. 11 , one embodiment of the invention employs a surface-induced dissociation cell  154  in fragmentation chamber  18 . In this embodiment, ions of interest are selected by pulsed ion deflector  52  and ions of other mass-to-charge ratios are deflected so that they do not enter the surface-induced dissociation cell  154 . A potential difference is applied between electrodes  158  and  156  to deflect selected ions so that they collide with the surface  159  of electrode  156  at a grazing angle of incidence. Ions are energized by collisions with the surface  159  and caused to fragment. In one embodiment, the surface  159  is coated with a high molecular weight, relatively involatile liquid, such as a perfluorinated, ether to facilitate fragmentation or to reduce the probability of charge exchange with the surface. The fragment ions are analyzed with the fragmentation chamber  18  acting as delayed extraction source for analyzer  24 .  
      Referring also to  FIG. 12  in one embodiment, the timed ion selector  14  and ion fragmentation chamber  18  are enclosed in the same vacuum housing  20  as the pulsed ion generator  12 . A pulsed ion extractor comprising the grid plate  53  and the fragmentor extraction grid  56  is located in vacuum housing  26  enclosing the analyzer  24 . A small aperture  58  located in the nearly field-free space  57  between the fragmentation chamber  18  and grid plate  53  allows free passage of the ion beam but limits the flow of neutral gas. In one embodiment, an einzel lens is located between the pulsed ion generator  12  and the timed ion selector  14  to focus ions through aperture  58 . In this embodiment, vacuum housing  19  ( FIG. 2 ) and its associated vacuum pump are not required. In one embodiment, collision cell  44  within fragmentation chamber  18  is connected to ground potential as is the fragmentor extraction grid  56 . Grid plate  53  is also held initially at ground, and switched to high voltage after ions of interest have reached the nearly field-free space  59  between the grid plate  53  and the fragmentor extraction grid  56 .  
      Having described preferred embodiments of the invention, it will now become apparent of one of skill in the art that other embodiments incorporating the concepts may be used. It is felt, therefore, that these embodiments should not be limited to disclosed embodiments, but rather should be limited only by the spirit and scope of the following claims.