Radiant energy – Ionic separation or analysis – Methods
Reexamination Certificate
2000-07-11
2002-06-25
Nguyen, Kiet T. (Department: 2881)
Radiant energy
Ionic separation or analysis
Methods
C250S292000
Reexamination Certificate
active
06410913
ABSTRACT:
The invention relates to the fragmentation of parent ions in ion traps filled with collision gas by exciting their axial oscillations in a dipolar excitation field with a frequency mixture which covers the oscillation frequencies of the parent ions.
The invention consists of ramping up the voltages of the frequency mixture for the dipolar excitation field, resulting in, surprisingly, approximately the same fragmentation in the same fragmentation times as with optimal voltage applied at a constant level, even for parent ions of different structures needing structurally specific fragmentation voltages.
PRIOR ART
Fragmentation of ions is necessary if daughter ion spectra have to be acquired. Daughter ions are nothing but charged fragments of the parent ions which result from them due to disintegration when energy is supplied into the ions.
The supplied energy can be radiation energy for instance; a new method here is BIRD=“Blackbody Infrared Radiation Dissociation”, which, however, only works in the case of large molecular ions and requires a considerable amount of apparatus. Generally the energy can be much more simply introduced to the ions by impact with a collision gas (abbreviated to CID=“Collisionally Induced Dissociation”; sometimes also called CAD=“Collisionally Activated Dissociation”).
Between two mass spectrometers arranged in series at a distance from each other one can shoot the ions through a cell containing collision gas between the spectrometers at relatively high levels of energy and then individual collisions already lead to spontaneous fragmentation. The daughter ions only lose a small amount of velocity; they can be fragmented into granddaughter ions in the same cell by another impact, and so on; these high-energy daughter ion spectra appear very different from low-energy daughter ion spectra due to the large number of descendant fragment ions.
In ion traps the ions cannot be accelerated such that an individual impact is sufficient for a fragmentation. By exciting the oscillations in the quasi potential well internal energy must be collected from a large number of low energy impacts until the ion ultimately breaks. Fractures are no longer spontaneous: the collected energy is statistically distributed over the entire ion, over all the possible oscillation states of the inner structure, and drifts to and fro inside of the ion due to coupled oscillations, until, due to random superimposition, a bond with low bonding energy collects sufficient energy for fracture.
The daughter ions have a different mass and thus a different oscillation frequency in the ion trap so they are no longer excited by the excitation frequency; on the contrary, they are cooled and decelerated by further impacts in the collision gas. Consequently, further granddaughter ions are not formed immediately; the spectra are pure daughter ion spectra.
Scanning granddaughter ion spectra calls for a two-stage fragmentation process: generation of initial substance ions, isolation of the selected parent ion type, fragmentation to form daughter ions, isolation of a selected daughter ion type, fragmentation to form granddaughter ions, and scanning the granddaughter ion spectrum. At the beginning of the process the ion trap has to be filled with sufficient ions - overloading generally causes no damage. Scanning great granddaughter spectra requires another intermediate step of isolation and fragmentation. Further descendant spectra can be scanned, the only prerequisite being that at the start the ion trap contains sufficient initial ions of the substance. The necessary filling can, from a series of spectra, be calculated by the method described in GB 2 322 961 (U.S. Pat. No. 5,936,241) and controlled. Commercially available ion traps permit generation of descendant spectra up to the tenth generation and above.
These non spontaneous fractures of the ions in ion traps are particularly informative about their structure because, firstly, they clearly relate to the bonds with the lowest levels of bonding energy and, secondly, because they indicate the relationship between the daughter and the mother. In high-energy impacts the relationship between the great granddaughter and the granddaughter, and also between the daughter and the mother, can no longer be read off in a simple manner. Only by complex methods of marking with enriched isotopes can the path of fraction and therefore the possible structure of the ion be clearly described.
Measurement of all the main daughter, granddaughter, and great granddaughter spectra in an ion trap therefore ideally reflects the fragmentation path and is of inestimable value in determining the structure and identity of the initial ion. Automatic measurement of all these ion spectra is desirable, but at the moment it is not practicable. It would be optimal if this automatic measurement could also be performed in the short time which is available in a separation of substances of a mixture, by liquid chromatography, for example, i.e. in about ten seconds.
The quantity of energy for fragmentation is, however, dependent on the structure of the ions. There are ion structures which already allow fragmentation by minimal energy imparted, and other ones which require much more energy. The optimal excitation voltage of an easily fragmentable ion and a poorly fragmentable ion of the same mass may well differ by a factor of 6. The optimum of the best excitation voltage is relatively sharp: slightly too little or too much voltage may even cause no fragmentation or—for reasons which are not fully understood yet no collectable daughter ions, possibly because the parent ions hit the end cap electrodes of the ion trap prior to their fragmentation.
Automation of the scanning of daughter spectra (and granddaughter spectra) in ion traps therefore becomes practically impossible, or at least exceptionally difficult. Graduated trial and error to achieve optimal fragmentation voltage is usually impossible because there is insufficient time.
Even within a group of similar substances—as for example the peptides—optimal fragmentation of the ions is still considerably dependent on the (usually still unknown) composition and structure of the ions. In addition, there is also an increase in the optimal excitation voltage as the mass of the ions increases, which superimposes itself on the structural difference. It can be explained by the fact that the energy is distributed over more degrees of freedom if the molecular ion is larger.
Moreover, the energy has to be supplied in a short time period because as the collected energy increases it is emitted by radiation or dissipated by very low-energy collisions (in the thermal range). Dissipation by radiation has a fixed time constant so the energy absorbed can only take on satisfactorily high levels if the supply of energy takes place within a short period of time. Before the ions of an ion type are fragmented, all the other ions which are in the ion trap are ejected from the trap by a special process so that these ions do not superimpose the spectrum of the daughter ions. This process is called isolation: only the ions to be fragmented are left in the ion trap. Since doubly charged parent ions can also form daughter ions whose mass-to-charge ratio m/e is larger than that of the parent ions, the ions not of interest which are heavier than the parent ions must also be removed, whereby the term “heavier” (as also the term “lighter” used in the following) relates to the mass-to-charge ratio m/e.
Scanning daughter ion spectra has proved particularly successful for investigating peptides in ionization by the electrospray method. The peptides usually stem from a digest of a larger protein due to an enzyme, trypsin for example. These digest peptides cover a mass range m of approx. 500 to approx. 4,000 atomic mass units. However, not only singly charged ions are generated but also ones with two, three, and even four charges. As a general rule of thumb one can say that the center of charge distribution increases by one charge per thousand mass units.
Brekenfeld Andreas
Franzen Jochen
Schubert Michael
Bruker Daltonik GmbH
Nguyen Kiet T.
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