Radiant energy – Ionic separation or analysis
Reexamination Certificate
2003-03-10
2004-06-15
Lee, John R. (Department: 2881)
Radiant energy
Ionic separation or analysis
Reexamination Certificate
active
06750448
ABSTRACT:
BACKGROUND OF THE INVENTION
This invention is in the field of mass spectrometry and more specifically relates to the separation, collection and quantification of components of mixtures using a magnetic analyzer coupled with a collection array. The method is of particular application to separation of biologically-active components from mixtures from biological samples such as natural products, peptides, polynucleotides, proteins and polysaccharides. Biological samples include various types of samples (gas, liquid, or solid) from various biological environments, e.g., various human or veterinary medical samples (blood, urine, etc.), samples of bacteria, fungi or other microorganisms; water, soil of air samples, etc.)(gas, liquid, or solid.) The method is also useful for the separation of synthetic organic components from complex mixtures, such as combinatorial libraries.
Mass spectrometers use various combinations of electric and magnetic fields to achieve spatial or temporal separation of ions in the rarefied gas phase (1). In addition to the analytical utility of mass spectrometry, spatial separation of ions by mass spectrometric methods has been considered previously in conjunction with preparative separation of selected components of mixtures. For example, mass spectrometers were used in the Manhattan Project for the separation of the
235
U isotope from the much more abundant
238
U isotope (2-4). The mixture of isotopes was atomized and ionized in an efficient ion source, separated by a homogeneous magnetic field and landed on a collector. This was done under destructive conditions that excluded survival of molecular species because of the ionizing conditions and the high kinetic energies with which the ions impinged on the collector (3,4). Recently several attempts have been made to soft land gas phase ions following mass separation by a mass spectrometer (5-17). The term “soft landing” usually refers to and is used herein to refer to, non-destructive capture of a gas-phase ion on a target, such that it can be retrieved from the vacuum system of the mass spectrometer and identified or otherwise analyzed or used. Soft landing is not always required for identification of a mixture component, but is essential for efficient, high-yield preparative separation of mixture components for further analysis, functional assays or use. Mass separation or separation by mass refers to separation of ions possessing different mass to charge ratios (m/z). When the ions generated are singly charged, m/z values can be replaced by and referred to as masses.
Examples of soft landing of ions include a polypropylene glycol oligomer (5), chlorobenzyl ions (6), sulfonium ions (7), a mixture of multiply charged DNA fragments (8), CO
2
(9) and inorganic metal clusters (10-12). The targets used for soft landing of ions include metal surfaces (5, 7), inert gas matrices (9-12), nitrocellulose membranes (8) and self-assembled monolayers (6,13,14). Mass separation in these examples was achieved by mass spectrometers including quadrupole mass filters (5,6,9,13-16), a sector instrument (7), and an ion-cyclotron resonance instrument (8).
These references all exemplify single-channel ion collection and isolation. In single-channel collection, ions are mass selected by tuning a mass spectrometer to a selected mass and collecting only ions having the selected mass. Collection of a second ion requires retuning of the mass spectrometer to select the mass of the second ion and collection of the second ion. The application of single-channel ion collection to component separation can be prohibitively time consuming for practical application, particularly when separation of multiple components of complex mixtures is desired.
Thus, prior art efforts to achieve separation can be characterized as single-channel isolation of mass-selected ions in slightly modified commercial or existing mass spectrometers. The yields of soft-landed ions using such methods have not been quantified. Feng et al. (8) estimate capture of attomole amounts of DNA, while Geiger et al. (7) report reanalysis of a soft-landed sample collected “overnight” by fast-atom bombardment mass spectrometry, which typically requires at least picomole amounts of sample. Thus, while the prior art suggests that soft landing of a variety of mass-selected gas phase ions is possible, the implementation of component separation using soft landing of ions lacks practical implementation.
There remains a significant need in the art for improved mass spectrometer-based methods for separation of components of mixtures that are sufficiently efficient and high yield for practical application.
SUMMARY OF THE INVENTION
The present invention provides an instrument and methods for the preparative separation of components of mixtures using mass spectrometric methods. Nondestructive ionization methods are employed to generate ionized components of a mixture, the ionized components are spatially separated by mass and the mass-separated ion components are trapped. The ion source and mass spectrometric techniques employed allow the generation of large ion currents of ion components, on the order of nanoamps, which facilitate rapid accumulation of nanomole quantities of mass-separated components in relatively short times (minutes to hours).
The method of this invention can, for example, provide several nanomoles of a compound of interest for 10 h of collection of ions generated at 10 nA ion current by electrospray ionization. The amount of time needed to accumulate a nanomole of material depends on the abundance of the component in a mixture (e.g., its molarity) and the ionization efficiency of the component (e.g., its electrospray ionization efficiency). In order to obtain 10 picomol of a biological sample for biological testing, the collection time would be about 100s for an ion generated at about 10 nA ion current and about 1000s for an ion generated at about 1 nA ion current. Products can be collected at a rate of about 10 picomole/h or more and, preferably, at a rate of about 50 picomole/h or more. Note that the collection time for multiple components from the same sample is significantly decreased in the method of this invention because multiple components can be mass dispersed and collected simultaneously. A plurality of ion components from a mixture can typically be collected in less time than has been needed in prior art methods to collect a single ion component.
Typical samples for preparative electrospray mass spectrometry are in the range of about 5×10
−5
to about 1×10
−4
M/component. Samples for preparative electrospray mass spectrometry are typically solutions in volatile water-miscible solvents, such as water, volatile alcohols (methanol, ethanol, etc.) acetonitrile, nitromethane, tetrahydrofuran, and volatile organic acids (formic acid, acetic acid, propionic acid, etc.).
Mixtures are subject to non-destructive ionization, preferably using atmospheric pressure ionization techniques, such as electrospray ionization or atmospheric pressure chemical ionization techniques, to generate ionized components of the mixture. These ionized components are transmitted into a high vacuum region, where they are accelerated to high kinetic energy (on the order of kilo electron volts, keV). The ions generated in the ion source at higher pressures (about 1 Torr) are transported employing ion lensing and ion guiding to the high vacuum region (about 10
−6
Torr). Accelerated ions are energy selected in an electrostatic analyzer and passed into a magnetic analyzer where they are dispersed by mass. The mass-dispersed ions are decelerated to low kinetic energy (about 15 eV or less) and trapped on a collector array where the location of trapping on the array depends on the mass of the trapped ion. Ions can, for example, be collected according to mass into an array of collector compartments or bins. Bins or compartments are sized, spaced and arrayed along the length of the collector each to receive ionic species of different m/z or to receive ionic species h
Olney Terry
Patek Marcel
Scheidemann Adi
Schirlin Daniel
Schumacher Frank J.
Greenlee Winner and Sullivan P.C.
Lee John R.
Smith II Johnnie L
University of Washington
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