Charge-stripping of multiply-charged ions

Abstract

A method of mass spectrometry or ion mobility spectrometry is disclosed wherein a sample is ionized by an electrified sprayer so as to produce multiply charged analyte ions of a first polarity in gas-phase. A reaction region is provided downstream of the electrified sprayer, wherein the reaction region is maintained substantially at atmospheric pressure and is maintained substantially free of electric-fields. A gas flow is provided from said electrified sprayer to said reaction region such that the gas flow carries the analyte ions from the electrified sprayer into the reaction region. Free electrons or reagent ions of a second polarity are generated in the reaction region, wherein the second polarity is opposite to said first polarity. The free electrons or reagent ions are then reacted with the analyte ions in the reaction region so as to reduce the charge state of the multiply charged analyte ions and thereby produce charge-reduced analyte ions.

Claims

1. A method of mass spectrometry or ion mobility spectrometry comprising: ionising a sample using an electrified sprayer so as to produce multiply charged analyte ions of a first polarity in gas-phase; providing a reaction region downstream of the electrified sprayer, wherein the reaction region is maintained substantially at atmospheric pressure, is maintained at a temperature of 80 C., and is maintained substantially free of electric-fields; providing a gas flow from said electrified sprayer to said reaction region such that the gas flow carries said analyte ions from the electrified sprayer into the reaction region; generating free electrons or generating reagent ions of a second polarity within the reaction region, wherein said second polarity is opposite to said first polarity; reacting the free electrons or reagent ions with the analyte ions in the reaction region so as to reduce the charge state of the multiply charged analyte ions and thereby produce charge-reduced analyte ions; and analysing the charge-reduced analyte ions.

2. The method of claim 1, comprising maintaining the temperature of the reaction region at a temperature selected from the group consisting of: 70 C.; 60 C.; 50 C.; 40 C.; 30 C.; 20 C.; 10 C.; or substantially at room temperature.

3. The method of claim 1, wherein substantially no fragmentation or dissociation of the analyte ions is caused by reacting the reagent ions with the analyte ions.

4. The method of claim 1, wherein said step of reacting the free electrons or reagent ions causes the analyte ions to reduce in charge state whilst maintaining the same polarity.

5. The method of claim 1, wherein the reaction region remains substantially free of electric fields whilst a voltage is applied to the electrified sprayer and/or whilst the sprayer is ionising the sample.

6. The method of claim 1, comprising generating the free electrons and/or reagent ions within the reaction region by photoionising molecules in the reaction region.

7. The method of claim 6, comprising introducing dopant molecules into the reaction region and photoionising the dopant molecules.

8. The method of claim 7, comprising introducing the dopant molecules into the gas flow from the electrified sprayer to the reaction region and photoionising the dopant molecules in the reaction region.

9. The method of claim 7, comprising varying the concentration of dopant in the reaction region with time so as to control the rate at which the free electrons and/or reagent ions are generated and hence control the rate at which the charge states of the analyte ions are reduced.

10. The method of claim 1, wherein the reagent ions are formed by providing free photoelectrons and neutral molecules in the reaction region such that the neutral molecules are ionised by the photoelectrons to form said reagent ions.

11. The method of claim 10, wherein the neutral molecules are oxygen molecules which react with the photoelectrons to form superoxide anions.

12. The method of claim 10, wherein the neutral molecules have a higher electron affinity than oxygen and are present in a concentration such that the neutral molecules react with the photoelectrons to form said reagent ions.

13. The method of claim 10, further comprising varying the concentration of said neutral molecules within said reaction region so as to vary the concentration of reagent ions generated and hence vary the level of charge state reduction of the analyte ions.

14. The method of claim 1, wherein the reaction region is arranged and configured such that electric fields generated by the electrified sprayer substantially do not enter the reaction region.

15. The method of claim 14, wherein a gas flow conduit is provided between the electrified sprayer and the reaction region for carrying said gas flow from the sprayer to the reaction region, and wherein a wire mesh is arranged in the conduit between the electrified sprayer and the reaction region so as to substantially prevent electric fields from the electrified sprayer from entering the reaction region.

16. The method of claim 14, wherein a gas flow conduit is provided between the electrified sprayer and the reaction region for carrying said gas flow from the sprayer to the reaction region, and wherein the conduit comprises one or more bends between the electrified sprayer and the reaction region so as to substantially prevent electric fields from the electrified sprayer from entering the reaction region.

17. The method of claim 14, wherein a gas flow conduit is provided between the electrified sprayer and the reaction region for carrying said gas flow from the sprayer to the reaction region, and wherein the diameter and length of the conduit between the electrified sprayer and the reaction region are such that electric fields from the electrified sprayer are substantially prevented from entering the reaction region.

18. The method of claim 1, wherein the reaction region is maintained substantially free of electric-fields for a first time period and an electric field is applied in said reaction region for a second time period.

19. The method of claim 18, wherein the electric field applied during the second time period is used to control the reaction rate at which the reagent ions are generated and/or to control the reaction rate between analyte ions and either the free electrons or reagent ions.

20. The method of claim 1, wherein the charge states of the analyte ions are reduced via proton transfer reactions.

21. The method of claim 1, wherein the analyte is a polyethylene glycol (PEG) or comprises at least one covalently bonded polyethylene glycol.

22. A mass spectrometer or ion mobility spectrometer comprising: an electrified sprayer configured to ionise a sample so as to produce multiply charged analyte ions of a first polarity in gas-phase; a reaction region arranged downstream of the electrified sprayer, wherein the reaction region is configured to be maintained substantially at atmospheric pressure, maintained at a temperature of 80 C., and maintained substantially free of electric fields; means for providing a gas flow from said electrified sprayer to said reaction region such that, in use, the gas flow carries said analyte ions from the electrified sprayer into the reaction region; means for generating free electrons or for generating reagent ions of a second polarity within the reaction region, wherein said second polarity is opposite to said first polarity, such that the free electrons or reagent ions react with the analyte ions in the reaction region to reduce the charge state of the multiply charged analyte ions and thereby produce charge-reduced analyte ions; and means for analysing the charge-reduced analyte ions.

23. The method of claim 1, wherein analysing the charge-reduced analyte ions comprises determining structural information for the charge-reduced analyte ions.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) The present invention will be further understood from the following description with reference to the accompanying drawing of a representative charge-stripping ion source according to the invention, in which all views are schematic and may not be to scale.

(2) FIG. 1 illustrates a schematic diagram of an embodiment of the present invention;

(3) FIG. 2 illustrates an embodiment of the apparatus of the present invention including a nanospray emitter;

(4) FIG. 3 illustrates an exemplary mass spectral trace of PEG 20K after charge-stripping, obtained using an embodiment of the present invention; and

(5) FIGS. 4A and 4B show mass spectral data obtained using FC-43 as a charge stripping agent.

(6) In the drawings, preferred embodiments of the charge-stripping ion source according to the invention are illustrated by way of example. It is to be understood that the description and drawings are only for the purpose of illustration and as an aid to understanding, and are not intended to be a constraint on the limits of the invention.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

(7) Referring to FIG. 1, there is illustrated a schematic diagram for an in-source atmospheric pressure charge-stripping method for mass spectrometric analysis of samples in accordance with an embodiment of the present invention. A liquid sample (2) is introduced into an electrified sprayer (4) by which gas-phase analyte ions having multiple charges (5) are produced. The gas-phase analyte ions (5) of the present example are positively charged, though the invention may alternately be used to generate negatively charged gas-phase analyte ions. The multiply charged analyte ions (5) are swept from the electrified sprayer (4) by a flow of gas (6) through a guide (8) for guiding the multiply charged analyte ions (5) towards a downstream reaction region (14) within the guide (8). A wire screen (10) is situated within the guide (8) between the electrified sprayer (4) and the reaction region (14) to shield the reaction region (14) from the electric field of the electrified sprayer (4). Bipolar primary reagent ion species (7) are generated using a bipolar primary ion reagent production means (12) comprised of a high energy photon source capable of photoionizing an ionizable species in the source, preferably a dopant mixed in the gas flow (6). The bipolar primary reagent ion production means (12) is situated downstream of the electrified sprayer (4) such that the bipolar reagent ion species (7) that are produced therefrom intersect the multiply charged ions (5) in the reaction region (14). The bipolar primary reagent ion species (7) are produced within the reaction region (14). The multiply charged ions (5) are mixed with the bipolar reagent ion species (7) in the reaction region (14) at or near atmospheric pressure. This mixing of these ionic species results in neutralization (charge-stripping) of a portion of the charge of the multiply charged ions, via gas-phase ion/ion reactions, to lower the charge state of the multiply charged ions (9) which are then passed into a mass analyzer (16) of a mass spectrometer. It is expressly understood that the arrangement of the elements of the method as depicted in FIG. 1 are for illustration only and should not be construed to limit the geometrical arrangement of the various elements of the invention. Various geometrical and spatial arrangements of the elements and the means of connecting the elements are possible.

(8) Referring to FIG. 2, an apparatus (21) in accordance with a preferred embodiment of the present invention is shown. The major features of the apparatus (21) comprise a nanospray emitter (34) for producing multiply charged analyte ions, a gas-discharge lamp (46) for producing bipolar primary reagents (radical cations and photoelectrons), a flow of gas (30) and a hollow guide (43) comprised of three connected sections each having a central channel, namely, a first guide section (28), a second guide section (36), and a third guide section (42), the hollow guide (43) for guiding the multiply charged analyte ions, and a high-transmission wire mesh (38) located between the first guide section (28) and the second guide section (36), said wire mesh (38) designed and configured to screen a reaction region (44) of the guide (43) from the electric field of the nanospray emitter (34). The reaction region (44) is located downstream of the nanospray emitter (34) within the central channel of the hollow guide (43).

(9) Now describing the apparatus (21) of FIG. 2 in detail, a liquid sample (20) is introduced into a stainless-steel union (22) for coupling the liquid sample (20) to the nanospray emitter (34). The union (22) allows for standard 1/16 outer diameter tubes to be joined on each side, with minimal dead-volume therebetween. The liquid sample (20) is delivered into the union (22) from the upstream side thereof, while the fused silica nanospray emitter (34) is fixed to the downstream side of the union (22). The union (22) is mounted and fastened within an electrically-insulating polyimide plug (26) which plug (26) is removably inserted into the central channel of the first section (28) of the stainless-steel guide (43) from the upstream end. The plug (26) is designed and configured to be removable from the first guide section (28) so as to provide easy access to the nanospray emitter (34) in case the nanospray emitter (34) must be replaced. The union (22), the plug (26) and the first guide section (28) are all mounted such that a substantially hermetic seal is maintained between the central channel of the first guide section (28) and the outside atmosphere, to prevent air from entering the guide (43) and to prevent the contents of the guide (43) from escaping. A stainless-steel electrode (24) connected to a first high voltage power supply (51) is held in electrical connection with the union (22) before the plug (26); the electrode (24) is provided simply as a means of connecting the first power supply (51) to the union (22). The liquid sample (20), the union (22) and the electrode (24) are all in electrical contact, so that the liquid sample (20) is electrified during transit through the union (22), which ultimately leads to the formation of multiply charged analyte ions at the exit of the nanospray emitter (34).

(10) A flow of gas (30), introduced and directed substantially perpendicularly to the hollow guide (43) is introduced into the first guide section (28) through a stainless-steel union (32) coupling the first guide section (28) and the source for the flow of gas (30). One end of the union (32) accepts a standard outer diameter tube used to deliver the flow of gas (30), while the other end is threaded for mating with a matching tapped hole in the first guide section (28). Multiply charged ions exiting the downstream end of the nanospray emitter (34) are guided through the first guide section (28) by the flow of gas (30). The gas (30) preferably consists of substantially pure nitrogen doped with a volatile photoionizable species such as acetone or toluene. As the gas (30) enters the guide (43), the gas (30) envelopes the nanospray emitter (34) within the first guide section (28) so that ions exiting the emitter are swept through the guide (43) by the gas (30). The inner diameter of the first guide section (28) is relatively large (10 mm in this embodiment) so that the velocity of the gas (30) at a given flow rate (typically around 10 l min.sup.1) around the nanospray emitter (34) is relatively low, which helps prevent the gas flow (30) from disrupting the electrospray plume at the tip of the emitter (34).

(11) A high-transmission wire mesh (38) is situated downstream of the nanospray emitter (34), between the first (28) and second (36) guide sections and in electrical connection therewith. The second guide section (36) is connected to a second high voltage supply (52). The first guide section (28), the wire mesh (38) and the second guide section (36) are all in electrical contact and are all held at the same electrical potential. The absolute value of the potential of the first high voltage power supply is greater than (and of the same polarity as) that of the second high voltage supply (52), to provide a strong electric field between the tip of the nanospray emitter (34) and the first section of the guide (28), as well as the wire mesh (38), and thereby to promote electrospray ionization of the liquid sample (20) as well as to assist in the delivery of multiply charged ions downstream. Openings in the wire mesh (38) permit multiply charged ions to be transmitted by the gas flow (30) into the downstream second (36) and third (42) guide sections. Because the wire mesh (38) and the surfaces of the neighbouring downstream guide sections (36, 42) are all at the same electrical potential, the reaction region (44) of the guide (43) is substantially field-free, effectively shielded from the electric field of the nanospray emitter (34).

(12) The second guide section (36) has a tapered entrance to reduce the internal diameter of its central channel (down to 7 mm in this embodiment) and thereby to increase the velocity of the gas flow (30) so that the residence time of multiply charged ions within the guide is decreased proportionally. It is desirable to minimize the residence time of multiply charged ions within the guide so that losses of ions due to diffusion to the walls of the guide are minimized (ions encountering the walls of the guide will be neutralized, preventing their detection by the mass spectrometer).

(13) A krypton discharge lamp (46), within an electrically-insulating cylindrical lamp holder made of polyimide (48), is mounted in the side of the second guide section (36) such that high energy photons generated in the lamp (46) are transmitted into the central channel of the second guide section (36) through an aperture in the wall of the second guide section (36). The lamp (46) receives power from a lamp power supply (53) electrically connected thereto. The negative high voltage outlet (54) of the lamp power supply (53) is in contact with an electrode (50) within the lamp holder (48) which is in electrical contact with the cathode of the lamp (46) via a metal spring. The high voltage return (55) of the lamp power supply (53) is in electrical communication with the second guide section (36) which is in communication with the anode of the face of the lamp (46) and the high voltage return (55) is also in electrical communication with the second high voltage power supply (52), effectively floating the guide (43), the lamp (46) and the lamp power supply (53) at the voltage of the second power supply (52).

(14) High energy photons from the lamp (46) intersect the gas flow (30) bearing the multiply charged analyte ions in a bipolar primary reagent generation region (40) within the central channel of the second guide section (36) where radical cations and photoelectrons are generated via photoionization of an ionizable species doped into the gas flow (30).

(15) Further, in the bipolar primary reagent generation region (40) any multiply charged analyte ions in the gas flow (30) commence reacting with the generated oppositely charged reagents resulting in charge-stripping from at least a portion of the analyte ions having multiple positive charges. The reaction mixture is guided from the bipolar primary reagent generation region (40) by the flow of gas (30) into the third and final guide section (42). The third guide section (42) also has a tapered entrance to reduce the diameter of its central channel and thereby increase the gas velocity and minimize ion losses due to diffusion. The inner volume of the third guide section (42) comprises the remainder of the reaction region (44) in which charge-stripping occurs. Upon exiting the guide (43) under the influence of the gas flow (30), ions are transferred into the mass analyzer of the mass spectrometer for mass analysis. This transfer is improved by maintaining the potential of the guide (43), as set by the second high voltage power supply (52), at a value suitable for directing the analyte ions towards the inlet of the atmosphere-vacuum interface of the downstream mass analyzer.

(16) Referring to FIG. 3, there is illustrated an exemplary mass spectrum of PEG 20K, a high-MW polymer representative of the type of sample to be analyzed by the present invention, obtained using an embodiment of the present invention. For this example, the charge-stripping ion source device was substantially the same as that depicted in FIG. 2, and the mass spectrometer used was a Synapt-G2S Q-TOF from Waters-Micromass (Manchester, UK). The spectrum of FIG. 3 clearly shows the peak envelopes due to the polymeric distribution of molecular masses for charge-states +4, +3, and +2, with the peak envelopes from the lower charge-states being well-resolved from those of the higher charge states, and thus the spectrum is capable of yielding the desired structural information for the sample. Significantly, without charge-stripping, the same sample yielded only ions of higher charge-states, with overlapping molecular mass peak envelopes, and so individual mass peaks could not be resolved and structural information for the sample was unattainable.

(17) FIGS. 4A and 4B show mass spectral data obtained using FC-43 as a charge stripping agent. FIG. 4B shows an expanded view of a portion of the spectrum shown in FIG. 4A. The FC-43 acts as a reagent to suppress ECD/ETD.

(18) Other variations and modifications of the invention are possible and aspects of some of these have been described above. For example, the liquid sample stream may be composed of a solution of sample in a solvent or solvent mixture, and the solvent or other additives may be used to provide a volatile component that is photoionizable to produce the gas phase bipolar primary reagents. In addition, a variety of electrified spray means may be employed in the practice of the invention. The electrified sprayer described above is but one of a number of different possible electrified spray means that can be employed in accordance with the invention. Electrified spray means include nanospray, electrospray, microspray, electrosonic spray and ionspray. All such modifications or variations and others that will occur to those skilled in the design of such systems are considered to be within the scope of the invention, as defined by the appended claims.