Ion analysis method and ion analysis device

Abstract

An ion analysis method according to one embodiment of the present invention comprises: an ionization step in which an object to be measured is ionized; an ion introduction step in which the ion obtained in the ionization step or an ion derived from the ion, i.e., a target ion, is introduced into a movement space (30) that is adjusted to a predetermined air pressure within a range in which the particles can perform Brownian motion and on which gravity and an electric field can act; a measurement step for acquiring first information relating to a motion caused by gravity and an electric field and acquiring second information relating to a Brownian motion with respect to the target ion introduced into the movement space; and a calculation step in which the mass or charge of the target ion is obtained on the basis of the first information and the second information obtained in the measurement step. As a result, the mass or charge of ions such as huge molecules or viruses can be measured with high accuracy and high efficiency.

Description

Technical Field

[0001] This invention relates to a method and apparatus for analyzing ions derived from various components in a sample. Background Technology

[0002] As a method for measuring the charge of particles, Millikan's oil drop experiment disclosed in Non-Patent Document 1 is known. In Millikan's oil drop experiment, the motion of charged particles such as charged oil droplets, determined by the gravity and Coulomb force acting on a DC electric field, is measured, and the mass or charge of the charged particles is calculated based on the measurement results.

[0003] Existing technical documents

[0004] Non-patent literature

[0005] Non-Patent Document 1: "Measurement of 9-Elementary Charges - Explanation of the Millikan Elementary Charge Measurement Principle -", [Online], [Searched November 28, 2023], Shiga Prefectural General Education Center, Internet<URL:https: / / www.shiga-ec.ed.jp / www / contents / 1440578636412 / files / kiki_phys_09.pdf>

[0006] Non-patent literature 2: NCContino et al., “Chargedetection mass spectrometry for single ions with a limit of detection,” International Journal of Mass Spectrometry, Vol. 345-347, 2013, pp. 153-159.

[0007] Non-Patent Document 3: “Charge Detection”, [Online], [Searched November 28, 2023], Megadalton Solutions, Internet <URL: https: / / megadaltonsolutions.com / charge-detection / >

[0008] Non-Patent Document 4: Kenichiro Aoki, "To Those Who Want to Study Modern Physics - From Atoms to the Universe", Keio University Press, May 20, 2011, pp. 30-32.

[0009] Non-patent literature 5: Furuhashi et al., “Development of a digital ion trap mass analysis device”, Shimadzu Review, Vol. 62, No. 3-4, 2005.

[0010] Non-Patent Document 6: "Principle of Dynamic Light Scattering DLS Measurement", [Online], [Searched November 28, 2023], Consortium for Measurement Solutions Supporting the Manufacturing Industry (COMS), Internet <https: / / unit.aist.go.jp / mcml / coms / nano-dls.html> Summary of the Invention

[0011] The technical problem that the invention aims to solve

[0012] In Millikan's oil drop experiment, particles of a material with a known density are used as the measurement object, and the mass or charge of the charged particle is calculated using this known density. Therefore, in the case of particles with an unknown material density, it is impossible to determine the mass or charge of the charged particle.

[0013] On the other hand, as a method for measuring the mass of molecules / particles ions, there is an analytical method called CDMS (Charge Detection on Mass Spectrometry) (Non-Patent Literature 2, 3). In CDMS, the molecular ion to be observed is introduced into a reciprocating reflective ion trap, causing it to repeatedly reciprocate within the trap. A detection unit is installed in the ion trap to detect the moving ion using electrostatic induction. The induced charge signal obtained from the detection unit is processed by Fourier transform in the data processing unit. Through this Fourier transform, the mass-to-charge ratio (m / z: strictly speaking, italicized "m / z", but here it is represented by a non-italicized "m / z"). Simultaneously, the valence of the ion can be determined from the amplitude of the signal, and the mass of the ion can be calculated based on its m / z and valence.

[0014] Thus, while CDMS can determine the mass or charge of ions with unknown densities, it may yield erroneous results if the ion concentration is not at the single-particle level. For example, if two ions with the same m / z pass near the detector simultaneously or very close in time, they will have a larger induced charge and thus a larger amplitude than a single particle. If this is interpreted as a single particle, an incorrect valence z will be given, resulting in an incorrect mass m. To avoid erroneous interpretation, the ion concentration needs to be reduced to the single-particle level, especially when measuring ions originating from various components contained in the sample, which reduces the measurement throughput.

[0015] This invention was made to solve such technical problems, and its purpose is to provide a new ion analysis method and ion analysis device that can measure the mass or charge of ions in molecules using a method completely different from CDMS.

[0016] Solution to the above technical problems

[0017] The first aspect of the ion analysis method of the present invention comprises:

[0018] The ionization step ionizes the object being measured;

[0019] The ion introduction step involves introducing the ions obtained in the ionization step or the ions derived from those ions, i.e., the target ions, into a moving space. This moving space is adjusted to a specified pressure within the range where the particles can undergo Brownian motion and where gravity and an electric field can act.

[0020] The measurement steps involve acquiring first information related to the motion caused by gravity or the action of gravity and electric field for the target ion introduced into the moving space, and acquiring second information related to Brownian motion.

[0021] The calculation step involves determining the mass or charge of the target ion based on the first and second information obtained in the measurement step.

[0022] A first aspect of the ion analysis apparatus of the present invention is an apparatus for implementing the ion analysis method of the first aspect described above, comprising:

[0023] The ionization section ionizes the object being measured.

[0024] The analysis chamber has a movement space inside, which is adjusted to a specified air pressure within which particles can undergo Brownian motion and where gravity acts.

[0025] An electric field forming unit selectively forms an electric field in the moving space;

[0026] An ion input unit inputs a target ion into the moving space. The target ion is an ion generated by the ionization unit or an ion originating from the ion.

[0027] The measurement execution unit acquires first information related to the motion caused by gravity and electric field and second information related to Brownian motion for the target ion that has been introduced into the moving space.

[0028] The processing unit calculates the mass or charge of the target ion based on the first information and the second information.

[0029] The second aspect of the ion analysis method of the present invention has the following features:

[0030] The ionization step ionizes the object being measured;

[0031] In the ion introduction step, the ions obtained in the ionization step or the ions derived from the ions, i.e. the target ions, are introduced into a moving space. The moving space is adjusted to a specified gas pressure and gravity acts therein, and an electric field is formed that acts on the ions in a direction orthogonal or oblique to the direction of gravity.

[0032] The separation step utilizes the movement of ions in the moving space, which reflects the differences in size and mass of ions due to gravity and the movement of ions, which reflects the differences in charge of ions due to the electric field, to spatially separate the target ions introduced into the moving space from other ions.

[0033] The detection step detects at least a portion of the target ions separated in the separation step.

[0034] A second embodiment of the ion analysis apparatus of the present invention is an apparatus for implementing the ion analysis method of the second embodiment described above, comprising:

[0035] The ionization section ionizes the object being measured.

[0036] The analysis chamber has a movable space inside that is adjusted to a specified air pressure and subjected to gravity;

[0037] An electric field forming unit selectively forms an electric field within the moving space that acts on ions in a direction orthogonal or oblique to the direction of gravity.

[0038] An ion input unit inputs a target ion into the moving space. The target ion is an ion generated by the ionization unit or an ion originating from the ion.

[0039] The detection unit detects at least a portion of the target ions that are spatially separated from other ions by the movement of ions in the moving space due to the movement of ions reflecting differences in size and mass caused by gravity and the movement of ions reflecting differences in charge caused by the electric field.

[0040] Invention Effects

[0041] In the ion analysis method and apparatus of the first scheme, the Brownian motion of ions and the motion of ions caused by gravity or the combined effects of gravity and an electric field are observed, and the mass or charge of the ion is calculated based on information related to these motions. Therefore, according to the ion analysis method and apparatus of the first scheme, for ions of large molecules, either or both of their mass and charge can be determined without prior substantial ion separation.

[0042] On the other hand, in the ion analysis method and apparatus of the second scheme, even ions of large molecules can be separated and detected based on their mass and charge by utilizing both the effect of gravity and the effect of an electric field acting in a direction different from that of gravity. Therefore, according to the ion analysis method and apparatus of the second scheme, similarly to the ion analysis method and apparatus of the first scheme, for ions of large molecules, it is possible to determine either or both of their mass and charge without prior substantial ion separation. Attached Figure Description

[0043] Figure 1 This is a schematic diagram of an ion analysis apparatus according to one embodiment of the present invention.

[0044] Figure 2 This is a schematic diagram of an ion analysis apparatus according to another embodiment of the present invention.

[0045] Figure 3 yes Figure 2 A schematic diagram of a modified example of the ion analysis apparatus shown.

[0046] Figure 4 yes Figure 2 A schematic diagram of a modified example of the ion analysis apparatus shown.

[0047] Figure 5 yes Figure 2 A schematic diagram of a modified example of the ion analysis apparatus shown.

[0048] Figure 6 yes Figure 1 A schematic diagram of a modified example of the ion analysis apparatus shown.

[0049] Figure 7 This is a schematic diagram illustrating the principle of the ion analysis method of the present invention.

[0050] Figure 8 This is an explanatory diagram illustrating the method for calculating the migration velocity of ions when the effects of gravity and electric field, as well as diffusion, are simultaneously present.

[0051] Figure 9 This is an illustration of an example of a method for calculating the valence of ions.

[0052] Figure 10 It is shown Figure 9 A graph showing the relationship between the valence of ions and the horizontal distance they move. Detailed Implementation

[0053] [1] The principle of the ion analysis method in this invention

[0054] [1-1] Previous reference methods and their technical problems

[0055] When identifying a substance, the molecular weight of its constituent components is crucial. Ion analysis devices, which include mass analysis equipment, aim to determine the mass of ions originating from these components. However, measuring the ions of large molecules, typically with a mass of MDa (=10), using existing general mass analysis techniques presents challenges. 6 One problem with ions of molecules at the Da level (hereinafter, the ions of viruses and these giant molecules will be collectively referred to as "giant molecule ions." The term "giant molecule ions" also includes adduct ions generated during ionization, fragment ions, and product ions generated by CID, etc.) is that the distribution of valence numbers is too broad to determine the valence. Sometimes, even determining only the valence, i.e., the charge, is sufficient as a measurement result. Therefore, this study considers obtaining either or both of the mass and charge of giant molecule ions.

[0056] Ions are a type of particle, and the ions of giant molecules can be considered particles. Therefore, the inventors of this application initially conceived the idea of ​​applying Millikan's oil drop experiment to measure the charge of ions in giant molecules. However, for the following reasons, the charge measurement of charged oil droplets in Millikan's oil drop experiment cannot be directly applied to the measurement of the charge of ions.

[0057] In Milli Kan's oil drop experiment, the relationship between the falling velocity *v* of a charged oil droplet and the intensity of the electric field *E* was measured and expressed by the linear equation *v = αE - v0*. That is, even with an increased number of measurement points, the information obtained is only the slope *α* of the linear equation and the *-v0*, which serves as the Y-intercept. On the other hand, the motion of the particles observed in Milli Kan's oil drop experiment involves the friction coefficient *k*, which is related to air resistance. Therefore, there are three unknowns to be determined (charge *q*, mass *m*, and friction coefficient *k*). In other words, the amount of information obtained through measurement is still one less unknown than the number of unknowns. In fact, in Milli Kan's oil drop experiment, the material of the particles is known, so their density *ρ* is known. The radius *r* of the particles is obtained from the mass *m*, and the friction coefficient *k* can be calculated from this radius *r* according to Stokes' law of fluid dynamics. Thus, in Milli Kan's oil drop experiment, by reducing one unknown, the mass *m* and charge *q* are determined separately. In other words, in Milli Kan's oil drop experiment, it is impossible to determine the mass of the object being measured when the material is unknown (i.e., the coefficient of friction k is unknown).

[0058] [1-2] Diffusion of ions

[0059] In contrast, the inventors of this application have noticed that when ions of large molecules fall under the influence of gravity, these ions, as particles in a gas, undergo diffusion motion in addition to the motion caused by gravity. Specifically, Brownian motion, resulting from collisions between a gas at a known temperature and the particles, is utilized. The diffusion coefficient can be determined by observing the Brownian motion of the ions as they fall in a space with a gravitational field or both a gravitational and electric field acting on them, and the mass of the ions can be determined based on the result. Furthermore, in actual measurements, since diffusion motion and drift motion caused by the gravitational and / or electric fields occur simultaneously, the motion of the ions needs to be treated as a superposition of diffusion and drift.

[0060] Typically, the diffusion coefficient D of a particle in Brownian motion in a gas is expressed by Einstein's relation (non-patent document 4), which is the earliest example in the history of the Fluctuation-Dissipation Theorem. This relation is shown in (1). Here, k B is Boltzmann's constant, and T is the absolute temperature of the gas. Furthermore, μ is called the mobility, equivalent to the velocity produced by a unit force. Therefore, μ is related to the coefficient of friction k based on air resistance as μ = 1 / k.

[0061] D = μk B T = k B T / k…(1)

[0062] If equation (1) is transformed, it becomes equation (1A) below.

[0063] kD = k B T = [constant]...(1A)

[0064] Equation (1A) implies that a larger friction coefficient k results in a smaller diffusion coefficient D, and that a smaller friction coefficient k is needed to increase diffusion, which aligns with physical intuition. Furthermore, Equation (1A) does not contain information about the atoms and molecules that constitute the gas, but this is presumably because the fluctuation dissipation theorem is a rule that holds true over a sufficiently long timescale compared to the collision times of gases and particles. In this case, it can be inferred that it is likely to hold true for lighter gases such as helium (He) and hydrogen (H2).

[0065] Here, since the diffusion and drift of particles under reduced pressure (but in the presence of gas) are also considered, it is necessary to know in advance the change of the friction coefficient k with changes in pressure p (the dependence of the friction coefficient k on pressure p). Although this relationship is not directly proportional, it can be inferred that it is a monotonically increasing relationship where the friction coefficient increases as the pressure increases. However, it is assumed that when the pressure approaches 1 atmosphere, the friction coefficient saturates and hardly changes. That is, if the pressure p increases and the mean free path of the gas is smaller than the particle size and can be considered as a fluid, then according to Maxwell's kinetic theory of gases, the friction coefficient is presumed to be constant. On the other hand, it is also obvious that k = 0 when p = 0.

[0066] While the pressure dependence of the friction coefficient k is ideally to be investigated experimentally, it can be inferred based on the results of particle trajectory simulations. In this simulation, by assuming a value for the friction coefficient k, the diffusion coefficient D can be determined according to equation (1), and by assigning an appropriate initial value, the equation of motion with random walk can be solved, thereby predicting the particle trajectory. That is, by adjusting the friction coefficient k as a parameter in the simulation in a way that reproduces experimental results, the value of the friction coefficient k, which is considered to depend on pressure and gas type, can be inferred.

[0067] Next, the possibility of realizing the measured diffusion coefficient D based on observations under a microscope, such as those used in the Millikan oil drop experiment, will be described.

[0068] In Perrin's experimental verification of Brownian motion, the motion of particles (radius: 213 nm) in solvents such as water was observed under a microscope. The observed diffusion distance was approximately 9 μm over 30 seconds. Based on this result, the extent to which particles of the same size diffuse in a gas over a measurement time of 0.3 seconds was estimated. Theoretically, the diffusion distance x over time t is x = √(2Dt), and the diffusion coefficient in a liquid is 10 times that in a gas. -4 This is several times larger, therefore it should be approximately 90 μm. This is a size that can be adequately detected optically. In particles smaller than viruses, the diffusion distance should be even longer.

[0069] As an example, consider microscopic observation. If only diffusion occurs strictly in the horizontal direction (the direction orthogonal to gravity), then the average distance x that the particle moves in the horizontal direction during the time period Δt is represented by the following equation (2).

[0070] x=√(2DΔt)…(2)

[0071] Therefore, by investigating the distance the particle moves horizontally at certain time intervals Δt, we can obtain x1, x2, x3, ..., x nGiven these measurement results, the squared average x av 2 It is expressed by equation (3).

[0072] x av 2 =(x1) 2 +x2 2 +x3 2 +…+x n 2 ) / n→2DΔt(n→∞)…(3)

[0073] That is, by repeatedly measuring the distance the particles move, the measurement accuracy can be improved.

[0074] In conclusion, it is entirely feasible to determine the diffusion coefficient D based on information obtained by observing the Brownian motion of ions in macromolecules. In particular, it can be said that the accuracy of the diffusion coefficient D can be improved by repeatedly measuring the distance the ions travel over a certain period of time.

[0075] [1-3] Utilizes the measurement of mass of diffusion motion and drift motion

[0076] Next, the method for measuring the mass m of an ion in the presence of the aforementioned diffusion motion and drift motion caused by gravity and electric field will be described. First, consider the case where the direction of the electric field is opposite to the direction of gravity.

[0077] First, consider the drift motion of ions caused by the gravitational field. The vertical terminal velocity v0 of the particle when the electric field is zero is [air resistance kv0] = [gravity mg], and is therefore expressed by the following equation (4).

[0078] v0=mg / k…(4)

[0079] If we combine it with the fluctuation dissipation theorem, we get equation (5).

[0080] m = v0k B T / (Dg)…(5)

[0081] Equation (5) means that the mass m can be known from the measurable quantities v0 and D. As mentioned above, the diffusion coefficient D can be known based on the observation of the horizontal motion of the ion (i.e., diffusion motion only), and the terminal velocity v0 can be known based on the vertical motion of the ion generated at the same time (i.e., diffusion motion and motion caused by the gravitational field). From this, the mass m of the ion can be calculated.

[0082] Furthermore, in order to calculate the accurate mass m according to equation (5), the value of gravitational acceleration g also needs to be accurate. As is well known, gravitational acceleration g varies greatly depending on the latitude and altitude of the measurement location, but an accurate value can be used depending on the measurement location. In addition, for example, if the m / z value of the target ion (where the valence z is unknown) can be obtained using other mass analysis devices, even if the accuracy of the mass m value is low, the value of z can be estimated from the low-precision mass m and the accurate m / z value, and the accurate mass m can be calculated.

[0083] In the above method for measuring the mass m of an ion, apart from the terminal velocity v0 of the ion, it is sufficient to measure the diffusion coefficient D or the friction coefficient k of the ion. Therefore, the necessary prerequisites are as follows.

[0084] • The temperature in the space where ions move is constant and in thermal equilibrium.

[0085] • If the speed of particles in the moving space is slow enough, the ratio of friction to speed holds true.

[0086] Provided the above premise holds true, there are no device-related limitations or constraints, such as the charge resolution limitation caused by ion orbitals in CDMS.

[0087] Here, as an example, a method for improving measurement accuracy during microscopic observation is described. In this measurement method, to improve measurement accuracy, it is desirable for the particles to remain in the microscope's field of view for as long as possible. The first reason is that if the fall time is too short, it is difficult to improve the measurement accuracy of the terminal velocity v0 when the electric field is zero. Furthermore, the second reason is that if the dwell time is short, the number of data points used to calculate the diffusion coefficient D is small, making it difficult to improve its accuracy. That is, in this measurement method, from the viewpoint of measurement accuracy, it is desirable for the particle's fall velocity to be as small as possible. Of course, in practical terms, when the fall velocity is small, the measurement throughput decreases accordingly, so a balance between accuracy and throughput needs to be considered. To reduce the fall velocity, the strength of the electric field induced vertically upwards by the ions can be adjusted to roughly counteract the effect of gravity.

[0088] In a state where the effect of gravity is completely canceled by the electric field, i.e., in a virtual weightless (suspended) state, the following equation (6) holds true.

[0089] 0=mg+qE0→m / q=-E0 / g…(6)

[0090] That is, at this point, the specific charge m / q can be known.

[0091] When an electric field of intensity E (≈E0) is formed, reducing the falling velocity of the ions, equation (7) holds true.

[0092] kv=mg+zE→m=vk B T / (g a D)…(7)

[0093] Here, g a =g(1-E / E0) is the apparent effective gravitational acceleration.

[0094] Furthermore, when the electric field strength E = 0, equation (7) becomes

[0095] kv0=mg→m=v0k B T / (gD)

[0096] This is consistent with equation (5) above. By setting v << v0, that is, by spending more time measuring, the measurement accuracy can be improved. This is the same as CDMS.

[0097] Furthermore, in the weightless (suspended) state shown in equation (6), the movement of ions is caused only by diffusion, thus allowing the application of Dynamic Light Scattering (DLS) as described in Non-Patent Document 6, etc. DLS is one of the common methods for measuring particle size, which involves irradiating a particle suspended in a solution with a laser to measure the particle size. Specifically, the laser is incident at a predetermined angle relative to the particle's movement space, and the scattered light from the particle exiting the movement space at a certain angle is detected using photon counting, recording the time change of its intensity. Then, the autocorrelation function is obtained based on the time change of the scattered light intensity, and the diffusion coefficient is calculated based on its relaxation velocity (the reciprocal of the relaxation time). Finally, the hydrodynamic particle size is calculated using the Stokes-Einstein formula, which represents the relationship between the diffusion coefficient and the particle size. In principle, the diffusion distance required for measurement is the wavelength of the laser, so compared to directly observing Brownian motion, the movement space is smaller, and the measurement time can be shortened.

[0098] The above description assumes the electric field acts in the opposite direction to gravity, but in reality, the direction of the electric field is not limited to this. That is, the angle θ between the direction of the electric field and the direction of gravity is arbitrary, ranging from 0 to 180°. This will be explained further. Now, the gravitational acceleration g↑ (here, *↑ represents the vector of *) acts in the direction of gravity, and the electric field E↑ acts in the direction of the electric field. g↑ and E↑, as vectors, form an arbitrary angle θ.

[0099] Under the stable conditions of the aforementioned gravitational and electric fields, the ion possesses a certain velocity (vector) v↑. At this time,

[0100] kv↑=qE↑+mg↑…(8)

[0101] Therefore, we can find the following equation (9).

[0102] v↑=(q / k)E↑+(m / k)g↑…(9)

[0103] As mentioned above, the unknowns we want to know are three (q, m, k). In contrast, in equation (9), only the two parameters (q / k) and (m / k) appear. As mentioned above, if the diffusion coefficient D is unknown, then q and m are uncertain.

[0104] Therefore, the ion velocity v↑ and diffusion coefficient D are calculated as follows.

[0105] Assuming the drift motion caused by gravity and electric field is uniform linear motion, the velocity vector v↑ that matches the measured data is determined using the least squares method as described below. Figure 8 This is a diagram illustrating an example of the trajectory of an ion. (For example...) Figure 8 As shown, ions move at certain time intervals Δt in the sequence [1]→[2]→[3]→[4]→…. Assuming the velocity vector v↑, calculate the residual vector for each time interval Δt as follows.

[0106] Δr1↑=r1↑-v↑Δt

[0107] Δr²↑=r²↑-v↑Δt

[0108] Δr3↑=r3↑-v↑Δt

[0109] …

[0110] Calculate the sum of squares R of the absolute values ​​of the above residual vectors. 2 Explore how to make the sum of squares R 2 The minimum velocity vector v↑.

[0111] R 2 =│Δr1↑│ 2 +│Δr2↑│ 2 +│Δr3↑│ 2 +…

[0112] In this case, the relationship between velocity and residual is linear, therefore, as the velocity vector v↑, a unique solution can be determined.

[0113] If the velocity vector v↑ is determined, then the diffusion coefficient D is calculated.

[0114] The residual vectors Δr1↑, Δr2↑, Δr3↑, ... obtained as a result of the least squares method retain only the random components, which can be regarded as random walks. Therefore, the diffusion coefficient D can be analytically determined from these residual vectors.

[0115] Specifically, when denoted as Δr i↑=(Δx i Δy i When ), its probability density function should follow a two-dimensional normal distribution {exp[-(Δx)}. i ) 2 / (4DΔt)]}×{exp[-(Δy i ) 2 / (4DΔt)]}。 Therefore, in the simplest way, it is possible to make |Δr1↑| 2 The average value is 4DΔt, from which the diffusion coefficient D can be obtained.

[0116] In this way, regardless of the direction of the electric field, the velocity and diffusion coefficient of the ions can be determined based on the measurement data obtained from observing the ion's trajectory, and the mass m can be calculated.

[0117] As described above, the terminal velocity v0 of the ion can be determined by (a1) direct observation of the ion using a microscope and (a2) position measurement using a laser. The diffusion coefficient D can be determined by (b1) direct observation using a microscope and (b2) DLS. Of course, the terminal velocity v0 and diffusion coefficient D of the ion can also be determined by methods other than those mentioned above, and the mass m of the ion can be calculated using the terminal velocity v0 and diffusion coefficient D obtained by any combination of methods.

[0118] [1-4] Utilizing the measurement of charge in the horizontal electric field

[0119] In the above measurement method, an electric field is used to adjust the falling velocity of ions, but it is also possible to more actively use the electric field to separate ions based on charge or valence. This is the second ion analysis method. In this method, for example, a mass analysis unit such as a digital ion trap (described later) is used to determine the m / z value of the target ion. However, when the target ion is a multivalent ion, the valence z is unknown, and therefore the mass m is uncertain. If the valence z or charge q of the target ion can be determined by a method different from mass analysis, the accurate mass m can be determined. Therefore, as a method derived from the first ion analysis method, a method that extends the method in Millikan's oil drop experiment in two dimensions is used.

[0120] Typically, in this second analytical method, in a gaseous atmosphere, the ions being observed are made to fall vertically downwards at a uniform velocity (terminal velocity) due to gravity, and an electric field is formed inducing the ions in the horizontal direction, causing them to also move at a uniform velocity in the horizontal direction. He is preferred as the gas used here. The reasons are as follows.

[0121] The two electrons of He are strongly bound by the gravitational pull of the atomic nucleus, thus the change in the wave function caused by the charge of nearby ions is small. Therefore, it is appropriate to assume that the friction coefficient k generated by the collision between He and ions is unlikely to change with the valence z of the ions (Millikan himself also calculated the elementary charge under the premise that the friction coefficient k does not depend on the valence z of the ions). If the friction coefficient k is constant regardless of the valence z, then the horizontal velocity v of the ions... h It becomes the following formula (10).

[0122] kv h =zeE→v h =zeE / k…(10)

[0123] That is, ensure the speed v h Take discrete values ​​at equal intervals.

[0124] Figure 7 This is a diagram that roughly illustrates the behavior of ions in this second ion analysis method. As mentioned above, ions move at a constant speed in the vertical direction (Z-axis direction) solely under the influence of gravity, i.e., they fall. The velocity at this point depends on the particle size and weight, and therefore can be separated solely by particle size and weight. Specifically, ions that are heavier (i.e., subject to greater gravity) and larger (i.e., have relatively less friction) fall at a greater speed. On the other hand, ions move at a constant speed in the horizontal direction (here, the Y-axis direction) according to their valence z or charge q. Therefore, they are separated by discreteness corresponding to their valence z. That is, as... Figure 7 As shown, if the falling ions have the same mass, they will reach different positions in the horizontal direction depending on their valence z. Specifically, the larger the valence z, the farther the ion will reach in the horizontal direction. Therefore, target ions with various valence z can be separated according to their valence based on their falling position or their trajectory. If the purpose is simply to separate ions by valence, or to determine whether ions with different valences exist, it is not necessary to calculate the valence of each separated ion.

[0125] One method for determining the valence of each separated ion is to conduct preliminary experiments to investigate the relationship between the ion's mass *m*, valence *z*, the electric field strength, and the ion's falling position. Using this information, the valence *z* of the target ion can be calculated based on its falling position. Furthermore, different falling positions mean that the angle between the line drawn by the ion's trajectory and the Z-axis varies depending on the valence *z* or charge *q*. Therefore, for example, the valence *z* of the target ion can be estimated based on the ion's falling position, as shown below. This calculation is possible only if multiple ions originating from the same composition and whose valences differ by one are observed.

[0126] Now, as a single example, such as Figure 9 As shown, we consider the case where we obtain the observation that ions of the same composition (i.e., the same mass m1) fall to two close positions. In this case, since it is reasonable to assume that the valences of the ions falling to different positions differ by 1, we presume that the valence of the ion on one side (the side closer to the vertical falling position) is z, and the valence of the ion on the other side is z+1.

[0127] As mentioned above, if we base our assumption that the coefficient of friction k is unlikely to vary with the valence z of the ion (even if the coefficient of friction k depends on the valence z of the ion, its variation is quite smooth and can be considered substantially unchanged), then for the same particles, even if the valence z is different, the gravitational force and frictional force experienced in the vertical direction Z are the same. Therefore, they should reach the same vertical coordinate Z in the same falling time. On the other hand, regarding the horizontal coordinate Y, if we assume the coefficient of friction to be the same based on the above assumption, then they should reach a horizontal position proportional to the valence z.

[0128] Therefore, if we graphically represent the relationship between the valence (charge) z of an ion and the horizontal distance y it moves, then as follows: Figure 10 As shown. According to Figure 10 It can be seen that, using the two points P1 and P2 corresponding to z1 and z+1, plus the point P0 where z=0, the quadratic function y=az should be uniquely determined. 2 +bz+c (where c = 0 automatically because it passes through the origin). Under the above premise, the valence z is obtained by making the quadratic function as close to a straight line as possible. It can be considered that the valence z1 that minimizes the absolute value of the coefficient |a| of the quadratic term of the quadratic function is the most appropriate estimate. This z1 is of course a natural number, so it is obviously very easy to find. In this way, the valence z of an ion can be estimated based on the falling positions of two ions whose valences differ by 1.

[0129] When more than three ions with valences differing by 1 are observed (i.e., more than three falling positions), the above approach can be extended for calculation. One considered most universal method is to use spline curves instead of quadratic functions. In this case, the condition of obtaining the smoothest curve from two or more data points is guaranteed by minimizing the elastic energy. Therefore, it is believed that by using spline curves, the optimal valence z1 can be easily explored.

[0130] Furthermore, in cases with multiple drop locations and various particle types, the intervals of the bright spots in the observations showing the particle positions appear non-fixed, making charge determination based on spline curves potentially difficult. However, the Fourier transform method can be used as a powerful and high-speed analytical method even under such complex conditions.

[0131] use Figure 9To explain, on a constant horizontal line (Y), for the same particles with different valence numbers (z), the bright spots should be arranged at approximately equal intervals (ΔZ) in the Z direction. Therefore, if a Fourier transform is performed on the intensity distribution of the bright spots along the constant horizontal line (Y), peaks appear at discrete frequencies such as 2π / ΔZ, 4π / ΔZ, 6π / ΔZ, ... . Thus, not only can the value of ΔZ be determined, but also the bright spots that contribute significantly to the Fourier transform peaks can be identified, allowing spline curves to be used based on these bright spots. Furthermore, if the position of the constant horizontal line (Y) is shifted vertically (up and down), the value of ΔZ should also change linearly. Therefore, the charge of the target particle (ion) can be determined by maximizing the use of the two-dimensional information from the image obtained by observing the bright spots.

[0132] [1-5] Research on appropriate analytical conditions

[0133] In the first and second ion analysis methods described above, analytical conditions that are advantageous in improving measurement accuracy were investigated.

[0134] Now, the distance the target ion moves with a drift velocity v during time t is vt, and the distance the ion moves through diffusion during the same time t is √(2Dt). Therefore, the ratio of these two, R (=[drift distance] / [diffusion distance]), is given by the following equation (11).

[0135] R=vt / √(2Dt)=(v√t) / √(2D)=(mg a √t) / (k√(2D))=(mg a √t) / √(2kk B T)…(11)

[0136] Therefore, in the first ion analysis method, the condition that is conducive to improving accuracy is the direction that increases the diffusion distance and R→0, thus increasing the apparent effective gravitational acceleration g. a Approaching zero (approaching zero gravity, a state of suspension) is crucial. On the other hand, in the second ion analysis method, the condition that is conducive to improving accuracy is that the drift distance increases in the direction R→∞, so reducing the friction coefficient k, i.e., reducing the gas pressure (increasing the vacuum level), is effective.

[0137] Next, the configuration and operation of the ion analysis apparatus that utilizes the first and second ion analysis methods described above will be explained.

[0138] [2] Ion analysis apparatus of the first embodiment

[0139] [2-1] Composition

[0140] Figure 1This is a schematic diagram of one embodiment of an ion analysis apparatus utilizing the first ion analysis method.

[0141] The ion analysis device includes an ion supply unit 1, an ion trap 2, an ion drift unit 3, a detection unit 4, a voltage generation unit 5, and a data processing unit 10. Although not shown, at least the ion trap 2 and the ion drift unit 3 are disposed within a vacuum chamber, the interior of which is evacuated to a predetermined pressure by a vacuum pump. The ion trap 2 and the ion drift unit 3 can be configured to be disposed in separate vacuum chambers with different vacuum levels.

[0142] For ease of explanation, Figure 1 In this design, the three mutually orthogonal axes X, Y, and Z are defined in space. The Z-axis is the direction of gravity, i.e., the vertical direction.

[0143] The ion supply unit 1 generates ions that are the target of measurement, and typically includes an ion source that ionizes the components in the sample. The ionization method in the ion source is not limited to a specific method. For example, when the sample is a liquid sample, methods such as electrospray ionization, atmospheric pressure chemical ionization, and probe electrospray ionization can be used to ionize the sample components at atmospheric pressure. Furthermore, in addition to the ion source, the ion supply unit 1 may also include a collision chamber plasma dissociation unit that dissociates the ions generated by the ion source using appropriate methods, such as collision-induced dissociation.

[0144] Ion trap 2 has the function of temporarily capturing the ions of the observed object and specifying their initial position when they fall. Furthermore, although not strictly necessary, ion trap 2 can, as needed, have the function of distinguishing ions based on their m / z, thereby limiting the m / z or m / z range of the falling ions. This m / z-corresponding ion discrimination function is important when measuring complex molecules with various ion masses m and charges q.

[0145] Ion trap 2 only needs to achieve the above functions, but here it is a three-dimensional quadrupole ion trap (also called a Paul trap). This three-dimensional quadrupole ion trap includes: an annular electrode 22 having a hyperboloid of revolution extending along the Z-axis as its axis of symmetry; and a pair of end cap electrodes 20 and 21 arranged vertically opposite each other across the annular electrode 22. An ion incident opening 20a is provided through the upper incident-side end cap electrode 20, and an ion exit opening 21a is provided through the lower exit-side end cap electrode 21. However, in such a three-dimensional quadrupole structure, it is known that the annular electrode 22 can be replaced with a cylindrical electrode, and the end cap electrodes 20 and 21 can be replaced with flat plate electrodes, and such a simpler ion trap structure can also be used.

[0146] Furthermore, the type and method of the ion trap 2 are not limited to a specific type or method; in addition to a three-dimensional quadrupole ion trap, a two-dimensional ion trap can also be used. Moreover, here, a digital ion trap using a rectangular wave-shaped RF voltage as the RF voltage for forming the trapping electric field is used (see Non-Patent Document 5, etc.), but a simulated ion trap using a sinusoidal wave-shaped RF voltage can also be used. However, in order to efficiently trap ions of large molecules, it is desirable to use a digital ion trap and minimize the frequency of the RF voltage as much as possible. The advantage of a digital ion trap is that, by utilizing coarse isolation and forward / reverse scanning, ions in a specific m / z range can be selected at high resolution in a short time. Furthermore, as described later, in order to limit the initial position of the ions before they fall, it is desirable to use a three-dimensional ion trap capable of confining the ions to a narrow region.

[0147] The ion drift section 3 provides a space for ions to fall under the influence of gravity and move through Brownian motion. The air pressure (or vacuum) and temperature of this space are managed with high precision. Here, the ion drift section 3 includes a pair of flat plates 31 and 32 arranged at a predetermined interval, forming an ion movement space 30 between the flat plates 31 and 32 for ion movement. On the upper flat plate 31, an ion passage opening 31a is provided at a position corresponding to the ion emission opening 21a of the ion trap 2.

[0148] The detection unit 4 is an optical detection unit capable of detecting ions moving in the ion movement space 30. When viruses or large molecular ions are being observed, a method can be used to irradiate the object with light of a specified wavelength and detect the scattered light. Furthermore, the size of the detectable particles depends on the wavelength of the light used; therefore, by using a light source that generates light with a shorter wavelength (e.g., a 265nm LED specifically used for disinfection), even smaller particles of the object can be detected. Additionally, when observing a large number of ions simultaneously over a wide area, a sheet laser or similar device for fluid visualization can be used.

[0149] The voltage generating unit 5 includes: a trap voltage generating unit 50, which applies a predetermined voltage to the annular electrode 22 of the ion trap 2; an end cap voltage generating unit 51, which applies predetermined voltages to the end cap electrodes 20 and 21 respectively; and a drift unit voltage generating unit 52, which applies a predetermined voltage to the plate-shaped electrode 32 of the ion drift unit 3. Figure 1 In this case, the plate-shaped electrode 31 is grounded and has a zero potential, but a specified voltage other than zero may also be applied to the plate-shaped electrode 31.

[0150] The data processing unit 10 is configured, for example, around a computer including a CPU, and processes the detection results (observation image data, measurement data) obtained by the detection unit 4.

[0151] [2-2] Analyze the action

[0152] This illustrates an example of the analytical operation of the ion analyzer.

[0153] The ion supply unit 1 ionizes the component molecules contained in the provided sample. Here, the component molecules are imagined to be, for example, viruses or macromolecules. The ions generated by the ion supply unit 1 are introduced into the internal space of the ion trap 2 through the ion injection opening 20a via an ion guide (not shown). When the ions are introduced, a DC voltage of the same polarity as the ions is applied to the ejection-side end cap electrode 21. As a result, the ions introduced into the internal space are subjected to a force approximately in the negative direction (i.e., upward) of the Z-axis by the electric field formed in the internal space of the ion trap 2, preventing them from being discharged from the ion ejection opening 21a. Furthermore, at the same time as or with a time delay compared to the ion introduction, a rectangular wave RF voltage of a predetermined frequency is applied to the annular electrode 22 from the trap voltage generation unit 50. As a result, the ions are captured by the internal space by the ion trapping electric field formed in the internal space.

[0154] After ions are introduced into the internal space of ion trap 2 and captured by an electric field, a gas supply unit (not shown) introduces a specified inert gas as a cooling gas into the internal space. The captured ions lose kinetic energy upon contact with the cooling gas and concentrate near the center of ion trap 2. After a specified cooling time, the trap voltage generating unit 50 stops applying a rectangular wave RF voltage to the annular electrode 22. As a result, the electric field acting on the ions concentrated near their center in the internal space of ion trap 2 disappears, and the ions fall due to gravity, passing sequentially through the ion emission opening 21a and through the opening 31a into the ion movement space 30. At this time, a DC voltage can also be applied to the electrode to form a DC electric field between the two end cap electrodes to accelerate the ions. In a digital ion trap, by appropriately controlling the phase of the voltage when the voltage for stopping capture is applied, the initial positions of the ions inside ion trap 2 can be made consistent or set to the optimal initial positions.

[0155] A high-purity, specified gas is introduced into the ion movement space 30, and its pressure, i.e., the density of gas molecules, is precisely controlled. Furthermore, the temperature of the ion movement space 30 is also precisely controlled. The ions introduced into the ion movement space 30 repeatedly come into contact with the gas molecules, whose density and temperature are precisely controlled. Therefore, each introduced ion diffuses while undergoing Brownian motion. The detection unit 4 acquires data containing information indicating the position of each ion at regular time intervals. Based on this data, the data processing unit 10 calculates the horizontal movement distance of each ion within a specified unit of time. From this, the average square of the horizontal movement distance x caused by the Brownian motion as described above is calculated, and the diffusion coefficient D of the ion is calculated based on this information.

[0156] Furthermore, gravity acts on the ions within the ion movement space 30, causing each ion to move (fall) along the Z-axis. That is, the ions undergo drift motion based on the gravitational field. When the voltage applied from the drift voltage generator 52 to the plate-shaped electrode 32 is zero, the electric field acting on the ions within the ion movement space 30 is zero. As described above, the vertical terminal velocity v0 of the ions at this time can be calculated based on information obtained by the detection unit 4, for example, showing the position of each ion at regular time intervals. Thus, the data processing unit 10 calculates the terminal velocity v0 and diffusion coefficient D of each ion based on the measurement data obtained by the detection unit 4, and then calculates the mass m of the ion using the above equation (6) based on these values.

[0157] In addition, the value of gravitational acceleration g used for mass calculation can be set to any of the following: a preset default value, a value selected based on gravity-related information such as the longitude and altitude of the measurement location input by the user, or a value input by the user.

[0158] The measurement of the mass m of the ions described above in the ion analysis apparatus of this embodiment can be performed without forming an electric field in the ion movement space 30. Furthermore, as described above, by applying an appropriate voltage from the drift voltage generating unit 52 to the plate-shaped electrode 32, an electric field that substantially counteracts gravity is formed in the ion movement space 30, thereby reducing the ion falling velocity and improving measurement accuracy.

[0159] Furthermore, by applying an appropriate voltage from the drift voltage generating unit 52 to the plate-shaped electrode 32, a predetermined electric field is formed in the ion movement space 30, and the charge q (or valence z) of the ion being observed can be determined. That is, in the absence of an electric field, only the mass m of the ion can be determined, but by comparing the cases with and without an electric field, the charge q of the ion can also be determined.

[0160] For example, if the velocity of the vertical ion in the absence of an electric field is set as v0, and the velocity in the presence of an electric field is set as v, then the following equation (12) holds true.

[0161] mg=kv0=kv-qE→q=k(v-v0) / E=(v-v0)k B T / (DE)…(12)

[0162] The charge q can be obtained using equation (12). That is, in this ion analysis device, in addition to being able to determine the mass m of the ion of the object under observation without forming an electric field in the ion movement space 30, the charge q (or valence z) of the ion can also be determined by using the observation results of the ion when an appropriate electric field is formed in the ion movement space 30.

[0163] Furthermore, as described above, the velocity and diffusion coefficient of an ion can be calculated, and the mass m of the ion can be calculated, based on the observation of the ion's trajectory in a state where an electric field is formed in the ion movement space 30.

[0164] Furthermore, by combining the aforementioned ion analysis apparatus with other existing mass analysis apparatuses, a more accurate mass m can be determined.

[0165] Specifically, using other mass analysis devices, the m / z of the target ion originating from a large component molecule is determined. However, the valence z is unknown at this time, so m cannot be determined from m / z. On the other hand, when using the aforementioned ion analysis device to measure ions originating from the same component molecule as described above, even if the value is not accurate (at least less accurate than the other mass analysis devices mentioned above), the mass (approximate value) m is determined. An estimated value of m / z, assuming a value of z, can be obtained from this estimated value of m / z. Therefore, the value of valence z can be determined from this estimated value of m / z and the accurate value of m / z where the valence z is unknown. Thus, the accurate mass m can be determined from the accurate value of m / z.

[0166] Furthermore, in the ion analysis apparatus of this embodiment, the detection unit 4 uses multiple cameras arranged in a manner capable of capturing a wide range of images within the ion movement space 30 from various angles. By investigating the correlation between multiple observation images obtained from these multiple cameras, information related to the movement status and positional changes of each ion can be obtained. This allows for high-precision tracking of the trajectories of multiple ions falling simultaneously, facilitating comprehensive measurement of a large number of ions. Moreover, by also capturing observation images of ions within the internal space of the ion trap 2, differences in initial positions during descent can be corrected, thereby improving measurement accuracy.

[0167] [3] Ion analysis apparatus of the second embodiment

[0168] Figure 2 This is a schematic diagram of one embodiment of an ion analysis apparatus utilizing the second ion analysis method.

[0169] In the ion analysis apparatus of the first embodiment described above, in order to determine the diffusion coefficient D of the ions, it is necessary to observe the Brownian motion of the ions falling due to gravity or the combined effects of gravity and an electric field. In contrast, in the ion analysis apparatus of the second embodiment, ions are separated in space based on charge or valence using an electric field.

[0170] exist Figure 2In this second embodiment, the same reference numerals are used for components that are identical or substantially identical to those in the ion analysis apparatus of the first embodiment, and detailed descriptions are omitted. In this second embodiment of the ion analysis apparatus, the ion drift unit 6 includes a pair of plate-shaped electrodes 61 and 62 arranged at a predetermined interval in the Y-axis direction (more precisely, in the direction placed on the XY plane), and an ion movement space 60 is formed between the plate-shaped electrodes 61 and 62. One of the plate-shaped electrodes 61 is grounded, and when a predetermined voltage is applied from the drift unit voltage generating unit 53 to the other plate-shaped electrode 62, an electric field that induces ions in the horizontal direction (in this example, the Y-axis direction) is formed in the ion movement space 60.

[0171] A plurality of auxiliary electrodes 63 are disposed between a pair of plate-shaped electrodes 61 and 62. By applying a step voltage (smaller in absolute value) to each auxiliary electrode 63 than the voltage applied to the plate-shaped electrodes 62, the horizontal intensity of the electric field formed in the ion movement space 60 can be easily kept approximately constant. However, the auxiliary electrodes 63 are not an essential component.

[0172] In this ion analysis apparatus, similar to the ion analysis apparatus of the first embodiment, ions are sufficiently cooled in the ion trap 2. When the voltage applied to the annular electrode 22 of the ion trap 2 is stopped, the ions fall from the ion trap 2 into the ion movement space 60. For each ion introduced into the ion movement space 60, gravity acts vertically downwards, and the force generated by the electric field acts horizontally. Although the pressure is reduced within the ion movement space 60, gas molecules exist at a suitable density, so the ions fall at a speed corresponding to their mass and size. At the same time, the force generated by the electric field of a certain intensity acts horizontally on each ion, so the larger the charge of the ion, the longer the distance it travels in the horizontal direction. Therefore, for example, even ions with the same mass m originating from the same molecule will have different trajectories and reach different positions in the horizontal direction if their charge q, i.e., valence z, is different.

[0173] The detection unit 4X is a detector capable of detecting the arrival of ions. As the detection unit 4X, a detector based on the same method as the detection unit 4 in the ion analysis apparatus of the first embodiment, namely optical detection, can be used. In particular, the aforementioned detector based on a sheet laser is useful for detecting ions over a wider area. However, since it is not necessary to observe ion movement as strictly as Brownian motion, a simpler detector can also be used.

[0174] When the electric field in the horizontal direction is weak, the ions travel a short distance in the horizontal direction. Therefore, although the separation ability based on valence is low, a relatively wide overall image of the valence z distribution can be obtained based on the detection results of the detection unit 4X. Based on this result, the dependence of the friction coefficient k on the valence z can also be analyzed. On the other hand, if the electric field in the horizontal direction is strengthened, it becomes difficult to obtain an overall image of the valence distribution, but the separation ability corresponding to the valence is high. That is, separation of ions based on valence is possible. Therefore, by setting holes or slits at the bottom of the ion movement space 60, i.e., at specific positions where the falling ions arrive, it is possible to filter only ions with specific valences.

[0175] [4] A variation of the ion analysis apparatus of the second embodiment

[0176] Figure 3 This is a schematic diagram of a modified example of the aforementioned ion analysis apparatus. Figure 3 In this drawing, the same reference numerals are used for components that are the same or substantially the same as those in the ion analysis apparatus of the second embodiment, and detailed descriptions are omitted.

[0177] like Figure 3 As shown, in this ion analysis apparatus, an ion drift section 7 with the same configuration is provided after the ion drift section 6, which forms an electric field that induces ions in the horizontal direction. That is, the two ion drift sections 6 and 7 are cascaded. In the upper-stage ion drift section 6, ions are separated approximately according to their valence by a weak electric field in the horizontal direction. The separation capability here is relatively low, so the ions are not completely separated according to their valence. Ions within a specific valence range are introduced into the lower-stage ion drift section 7 through the aperture opening 64a formed in the orifice plate 64 provided between the upper-stage ion drift section 6 and the lower-stage ion drift section 7.

[0178] In the lower-level ion drift section 7, ions introduced are separated according to their valence with high separation capability by a strong horizontal electric field. Therefore, even for multivalent ions with high valence, it is possible to selectively detect only ions with specific valences.

[0179] Figure 4 yes Figure 3 A schematic diagram of another variation of the ion analysis apparatus shown.

[0180] In this modified example, another ion trap 8, with the same configuration as ion trap 2, is disposed between the two cascaded ion drift sections 6 and 7. Ions of a specific valence range separated and filtered by the upper-level ion drift section 6 are temporarily captured and cooled within the internal space of the ion trap 8. As a result, the diffusion of ions caused by diffusion during drifting in the ion movement space 60 of the ion drift section 6 is reversed, thus enabling more precise separation of ions according to valence in the lower-level ion drift section 7. Consequently, the selectivity of the target valence ion is improved.

[0181] In addition, Figure 3 , Figure 4 In the example, the direction of the electric field in both ion drift sections 6 and 7 is set to the Y-axis direction, but this does not need to be the same.

[0182] In addition, it is obviously possible to use a configuration in which three or more ion drift sections are cascaded together.

[0183] Figure 5 yes Figure 3 A schematic diagram of another variation of the ion analysis apparatus shown.

[0184] In this modified example, only ions with a specific valence selected in the ion drift section 6 are introduced into the quadrupole mass separator 9. The quadrupole voltage generating section 58 applies voltage to the quadrupole mass separator 9 in such a way that only ions with a specific m / z value pass through the quadrupole mass separator 9, while other ions diverge midway. As described above, if the ions are of the same size and weight, only ions with a specific valence z are selected by the ion drift section 6 and pass through the aperture opening 64a. However, when ions of various sizes and weights are introduced into the ion drift section 6, ions other than those with the target mass m and valence z may also pass through the aperture opening 64a. In contrast, in Figure 5 In the modified example shown, the quadrupole mass separator 9 can be used to select and detect only ions with a specific mass m and a specific valence z among the ions that have passed through the pore opening 64a.

[0185] Ions selected by the quadrupole mass separator 9 can be detected using the detection unit 4, which is configured to optically detect ions passing through the space near the outlet within the quadrupole mass separator 9. In this case, it can be ensured that the space for detecting ions is relatively long in the direction of ion passage, thus enabling more reliable ion detection.

[0186] [5] Ion analysis apparatus of the third embodiment

[0187] In the ion analysis apparatus of the first embodiment described above, the ion trap 2 and the ion drift section 3 for introducing ions into the ion movement space 30 are set as separate units. However, if they are integrated, the configuration of the apparatus can be further simplified. Figure 6 This is a schematic diagram of the ion analysis apparatus of the third embodiment with such a configuration.

[0188] In this ion analysis apparatus, an observation opening 22a is provided on a portion of the annular electrode 22, allowing observation of the internal space from the detection unit 4 located externally. Ions of the observed object are captured and cooled within the internal space of the ion trap 2, and the application of the capture voltage is stopped after ions are collected near the center of the internal space. As a result, the ions diffuse within the internal space via Brownian motion and fall due to gravity. By appropriately adjusting the voltage applied to the end cap electrodes 20 and 21, an electric field can be formed in a manner approximately balanced with gravity. The diffusion motion of the ions and the drift motion caused by gravity and the electric field are observed through the observation opening 22a, and the diffusion coefficient D and terminal velocity v0 are calculated based on the observation results, from which the mass m is determined. By adjusting the voltage applied to the end cap electrodes 20 and 21, ions that have fallen a predetermined distance within the internal space of the ion trap 2 can be lifted back to their original position (near the center of the internal space).

[0189] In this ion analysis device, the process of ion capture → cooling → observation of Brownian and drift motions in suspension → cooling → … can be repeated multiple times. By increasing the number of repetitions, the measurement accuracy can be improved. Furthermore, when a large number of ions are simultaneously captured inside the ion trap 2, the influence of Coulomb repulsion between the ions needs to be considered, but numerical simulations incorporating this repulsion can also be performed. Therefore, by comparing the simulation results with experimental results beforehand to obtain correction calculations related to the Coulomb repulsion, the mass and / or charge with sufficient accuracy can be calculated based on the measurement results.

[0190] [6] Other variations

[0191] The above-described ion analysis method and ion analysis apparatus can be further modified as follows.

[0192] In the aforementioned ion analysis method, a gas with a pressure lower than atmospheric pressure is introduced into the ion movement space, and the collisions between the ions to be measured and this gas are used for measurement. The falling trajectory of the ions in the gas varies depending on the geometry of the falling particles, thus it can be said to utilize gas chromatography. If different types of gases are used, for example, the interaction with the falling particles changes when the gas is N2 versus He. Furthermore, if the molecular structure of the observed object is different, the interaction with different types of gases will also be different, thus resulting in differences in the terminal velocity v0. Therefore, by comparing the results of measuring the ions of the observed object using different gases, it is possible to identify ions with the same mass but different molecular structures.

[0193] Furthermore, while optical methods such as detecting scattered light are used in the aforementioned ion analysis methods to observe or detect ions, fluorescent substances can be pre-attached to the particles being measured for more accurate and efficient detection. In this way, a bandpass filter can be used, for example, to selectively detect light at wavelengths corresponding to the fluorescent substance in the detection unit, thereby improving the signal-to-noise ratio (SN ratio) of the detection signal and enhancing the accuracy and efficiency of the measurement.

[0194] Furthermore, the above-described embodiments and variations are merely examples of the present invention. Therefore, any appropriate modifications, additions, or deletions made within the scope of the present invention are naturally included in the claims of this application.

[0195] <Various Options>

[0196] Those skilled in the art will understand that the above exemplary embodiments are specific examples of the following solutions.

[0197] (Item 1) One aspect of the ion analysis method of the present invention comprises:

[0198] The ionization step ionizes the object being measured;

[0199] The ion introduction step involves introducing the ions obtained in the ionization step or the ions derived from those ions, i.e., the target ions, into a moving space. This moving space is adjusted to a specified pressure within the range where the particles can undergo Brownian motion and where gravity and an electric field can act.

[0200] The measurement steps involve acquiring first information related to the motion caused by gravity or the action of gravity and electric field for the target ion introduced into the moving space, and acquiring second information related to Brownian motion.

[0201] The calculation step involves determining the mass or charge of the target ion based on the first and second information obtained in the measurement step.

[0202] (Item 2) In the ion analysis method described in Item 1 above, it can be configured such that, in the measurement step, second information related to the Brownian motion of the target ion in a direction different from the direction of gravity is obtained.

[0203] (Item 3) Furthermore, in the ion analysis method described in Item 1 above, it can be configured such that, in the calculation step, the velocity of the target ion is determined based on the first information, the diffusion coefficient of the target ion is determined based on the second information, and the mass or charge of the target ion is calculated based on the velocity and the diffusion coefficient.

[0204] (Item 20) One aspect of the ion analysis apparatus of the present invention is an ion analysis apparatus for implementing the ion analysis method described in item 1, comprising:

[0205] The ionization section ionizes the object being measured.

[0206] The analysis chamber has an internal movement space that is adjusted to a prescribed air pressure where particles can undergo Brownian motion and where gravity acts.

[0207] An electric field forming unit selectively forms an electric field in the moving space;

[0208] An ion input unit inputs a target ion into the moving space. The target ion is an ion generated by the ionization unit or an ion originating from the ion.

[0209] The measurement execution unit acquires first information related to the motion caused by gravity and electric field and second information related to Brownian motion for the target ion that has been introduced into the moving space.

[0210] The processing unit calculates the mass or charge of the target ion based on the first information and the second information.

[0211] In the ion analysis method described in item 1 and the ion analysis apparatus described in item 20, the Brownian motion of ions and the motion of ions caused by gravity or the combined effects of gravity and an electric field, typically falling motion, are observed. The mass or charge of the ion is calculated based on information related to these multiple motions. As mentioned above, in CDMS, to detect the induced charge when ions of the observed object pass through, it is necessary to ensure that multiple ions do not pass near the detection unit simultaneously or in extremely close time. However, in the ion analysis method described in item 1 and the ion analysis apparatus described in item 20, information related to the motion of each ion can be obtained well as long as the ion density is not extremely high. Therefore, for ions of large molecules, it is possible to know either or both of their mass and charge without prior substantial ion separation. Furthermore, in the ion analysis method described in item 1 and the ion analysis apparatus described in item 20, the measurement is performed in the presence of a gas of a certain density, similar to the Brownian motion of particles, in the analysis chamber. Therefore, extremely high vacuum is not required, which helps to reduce analysis costs.

[0212] (Item 4) In the ion analysis method described in Item 1, it can be set to further have:

[0213] The m / z measurement step involves measuring the m / z value of the target ion when the valence z is undetermined.

[0214] The second calculation step involves determining a more accurate mass value than the mass value obtained in the calculation step, or narrowing down the m / z value, based on the mass or charge value obtained in the calculation step and the m / z value obtained in the m / z measurement step.

[0215] Here, the "m / z measurement step" does not necessarily have to be the step of calculating the m / z value itself, but also includes the step of screening ions with specific m / z values. Specifically, for example, it can include operations such as selecting only ions with specific m / z values ​​in the ion trap and keeping them inside the ion trap, or conversely, selecting only ions with specific m / z values ​​and removing them from the ion trap. According to the ion analysis method described in item 4, even if the accuracy of the mass or charge calculated in the computational step is not sufficiently high, an accurate mass can be obtained.

[0216] (Item 5) In the ion analysis method described in Item 1, it can be configured such that, in the measurement step, the first information is acquired in a state where the electric field is zero.

[0217] Although it is impossible to obtain information about the charge of an ion without using an electric field, the mass of the ion can be calculated using the first and second information.

[0218] (Item 6) In the ion analysis method described in Item 1, it can be configured such that, in the measurement step, in a state where an electric field is substantially present, first information relating to the motion of the target ion under an effective gravitational acceleration that is apparent to be adjusted by the action of the electric field is obtained.

[0219] (Item 7) In the ion analysis method described in Item 6, it can be configured such that, in the measurement step, the first information is acquired in the presence of an electric field that counteracts the effect of gravity.

[0220] In the ion analysis methods described in items 6 and 7, for example, the falling velocity of ions can be reduced by decreasing the apparent effective gravitational acceleration. This increases the residence time of ions in the moving space, i.e., the time for observing the movement of the target ion, thus improving the accuracy of the observation of this movement, and consequently improving the accuracy of the mass or charge calculated based on the observation results.

[0221] (Item 8) In the ion analysis method described in Item 1, it can be configured such that, in the measurement step, an optical method is used to acquire first information related to the motion caused by gravity or the action of gravity and electric field for a target ion introduced into the moving space, and second information related to Brownian motion is acquired.

[0222] Here, optical methods for obtaining information related to the movement of target ions include, for example, using one or more cameras to acquire microscopic images of a defined range containing one or more ions. Specifically, this is particularly useful for detecting the scattered light of ions relative to irradiating light and detecting the fluorescence emitted by ions in response to excitation light. Alternatively, dynamic light scattering methods, which are used in particle size measurement of microparticles in liquids, can also be employed.

[0223] (Item 9) In the ion analysis method described in Item 8, it can be configured such that, in the measurement step, light is irradiated onto the ions in the moving space, and the light scattered in the ions or the light emitted from the ions is detected, thereby determining the movement of the ions.

[0224] Here, the method for detecting scattered light naturally includes dynamic light scattering (DLS). According to the ion analysis method described in item 9, the movement of individual ions in a moving space can be accurately determined. In particular, by irradiating ions with short-wavelength light and detecting their scattered light, not only large ions but also relatively small ions can be detected, thus expanding the range of m / z values ​​for the ions being measured.

[0225] (Item 10) In the ion analysis method described in Item 1, it can be configured such that, in the ion introduction step, after the target ion is temporarily held in an ion holding section provided outside the moving space, the ion is released at a predetermined time, thereby introducing the target ion into the moving space.

[0226] (Item 11) In the ion analysis method described in Item 10, the ion holding part can be configured to be an ion trap consisting of a plurality of electrodes, wherein ions can be held in the space surrounded by the plurality of electrodes by applying a rectangular wave voltage to at least one of the plurality of electrodes.

[0227] According to the ion analysis methods described in items 10 and 11, multiple ions can be introduced into a moving space approximately simultaneously from approximately the same position. This allows for efficient measurement. Furthermore, since measurements can be repeated at regular time intervals, it facilitates the measurement of ions from continuously supplied samples. Moreover, according to the ion analysis methods described in items 9 and 10, ions can fall from a state where their initial energy in the direction of gravity is approximately zero, thus enabling high-precision measurements.

[0228] (Item 12) In the ion analysis method described in Item 10, it can be configured such that, in the ion introduction step, after identifying the ions held in the ion trap based on the m / z value, the ions remaining after the identification are introduced into the moving space.

[0229] According to the ion analysis method described in item 12, even when ions originating from various components are generated during the ionization step, it is possible to pinpoint ions originating from a specific component and measure the mass or charge of those ions. This improves the accuracy of mass or charge measurement.

[0230] (Item 13) Another aspect of the ion analysis method of the present invention has the following features:

[0231] The ionization step ionizes the object being measured;

[0232] In the ion introduction step, the ions obtained in the ionization step or the ions derived from the ions, i.e. the target ions, are introduced into a moving space. The moving space is adjusted to a specified gas pressure and gravity acts therein, and an electric field is formed that acts on the ions in a direction orthogonal or oblique to the direction of gravity.

[0233] The separation step utilizes the movement of ions in the moving space, which reflects the differences in size and mass of ions due to gravity and the movement of ions, which reflects the differences in charge of ions due to the electric field, to spatially separate the target ions introduced into the moving space from other ions.

[0234] The detection step detects at least a portion of the target ions separated in the separation step.

[0235] (Item 21) Furthermore, another aspect of the ion analysis apparatus of the present invention is an apparatus for implementing the ion analysis method described in item 13, comprising:

[0236] The ionization section ionizes the object being measured.

[0237] The analysis chamber has a movable space inside that is adjusted to a specified air pressure and subjected to gravity;

[0238] An electric field forming unit selectively forms an electric field within the moving space that acts on ions in a direction orthogonal or oblique to the direction of gravity.

[0239] An ion input unit inputs a target ion into the moving space. The target ion is an ion generated by the ionization unit or an ion originating from the ion.

[0240] The detection unit detects at least a portion of the target ions that are spatially separated from other ions by the movement of ions in the moving space due to the movement of ions reflecting differences in size and mass caused by gravity and the movement of ions reflecting differences in charge caused by the electric field.

[0241] According to the ion analysis method described in item 13 and the ion analysis apparatus described in item 21, even large molecular ions can be separated and detected based on their mass and charge by means of both the action of gravity and the action of an electric field acting in a direction different from that of gravity. Therefore, with this ion analysis method and apparatus, for large molecular ions, it is possible to know either or both of their mass and charge without prior physical ion separation. Furthermore, particularly in this ion analysis method and apparatus, ions are spatially, i.e., physically, separated based on their mass and charge, making ion detection easy and enabling selective extraction of a portion of the separated ions (e.g., ions within a specific valence range) for more detailed measurements.

[0242] (Item 14) In the ion analysis method described in Item 13, it can be set such that the electric field formed in the moving space is a DC electric field acting on the ions in a direction orthogonal to the direction of gravity.

[0243] According to the ion analysis method described in item 14, ions with the same mass will reach different positions within the movement space based on their valence through falling motion. This makes valence identification easy and valence calculation simple.

[0244] (Item 15) In the ion analysis method described in Item 13, it can be set to further have:

[0245] The m / z measurement step involves measuring the m / z value of the target ion when the valence z is undetermined.

[0246] The calculation step involves determining the charge q or valence z of the target ion based on the detection result in the detection step, and calculating the mass of the target ion based on this result and the m / z value obtained in the m / z measurement step.

[0247] According to the ion analysis method described in item 15, the accurate mass of the target ion can be easily determined.

[0248] (Item 16) In the ion analysis method described in Item 13, it can be set up such that information related to the slope of the falling trajectory of the target ion is obtained through the detection step, and the valence z of the target ion is obtained based on the information.

[0249] According to the ion analysis method described in item 16, the valence can be easily calculated, for example, by analyzing the captured microscopic images.

[0250] (Item 17) In the ion analysis method described in Item 13, it can be configured such that, in the ion introduction step, after the target ion is temporarily held in an ion holding section provided outside the moving space, the ion is released at a predetermined time, thereby introducing the target ion into the moving space.

[0251] (Item 18) In the ion analysis method described in Item 17, the ion holding part can be configured to be an ion trap consisting of a plurality of electrodes, wherein ions can be held in the space surrounded by the plurality of electrodes by applying a rectangular wave voltage to at least one of the plurality of electrodes.

[0252] According to the ion analysis methods described in items 17 and 18, multiple ions can be introduced into a moving space approximately simultaneously from approximately the same position. This allows for efficient measurement. Furthermore, since measurements can be repeated at regular time intervals, it facilitates the measurement of ions from continuously supplied samples. Moreover, according to the ion analysis methods described in items 16 and 17, ions can fall from a state where their initial energy in the direction of gravity is approximately zero, thus enabling high-precision measurements.

[0253] (Item 19) In the ion analysis method described in Item 18, it can be configured such that, in the ion introduction step, after identifying the ions held in the ion trap based on the m / z value, the ions remaining after the identification are introduced into the moving space.

[0254] According to the ion analysis method described in item 19, even when ions originating from various components are generated during the ionization step, it is possible to pinpoint ions originating from a specific component to measure the valence of that ion or investigate the distribution of valence.

[0255] Explanation of reference numerals in the attached figures

[0256] 1 Ion Supply Department

[0257] 2. 8-ion trap

[0258] 20 Incident Side End Cap Electrodes

[0259] 20a ion injection opening

[0260] 21. Injection side end cap electrode

[0261] 21 a ion emission opening

[0262] 22 Ring Electrode

[0263] 22a Observation opening

[0264] 3, 6, and 7 Ion drift sections

[0265] 30, 60 Ion Movement Space

[0266] 60 Ion Movement Space

[0267] 31, 32, 61, 62 flat plate electrodes

[0268] 31a ions through the opening

[0269] 4. 4X Testing Department

[0270] 5 Voltage Generating Unit

[0271] 50-well voltage generation unit

[0272] 51 End Cap Voltage Generator

[0273] 52 Drift Section Voltage Generation Section

[0274] 53 Drift section voltage generation section

[0275] 58 Quadrupole Voltage Generator

[0276] 63 auxiliary electrode

[0277] 64-hole plate

[0278] 64a hole opening

[0279] 9. Four-pole mass separator

[0280] 10. Data Processing Department.

Claims

1. An ion analysis method, characterized in that, have: The ionization step ionizes the object being measured; The ion introduction step involves introducing the ions obtained in the ionization step or the ions derived from those ions, i.e., the target ions, into a moving space. This moving space is adjusted to a specified pressure within the range where the particles can undergo Brownian motion and where gravity and an electric field can act. The measurement steps involve acquiring first information related to the motion caused by gravity or the action of gravity and electric field for the target ion introduced into the moving space, and acquiring second information related to Brownian motion. The calculation step involves determining the mass or charge of the target ion based on the first and second information obtained in the measurement step.

2. The ion analysis method as described in claim 1, characterized in that, In the measurement step, second information related to the Brownian motion of the target ion in a direction different from the direction of gravity is obtained.

3. The ion analysis method as described in claim 1, characterized in that, In the calculation step, the velocity of the target ion is determined based on the first information, the diffusion coefficient of the target ion is determined based on the second information, and the mass or charge of the target ion is calculated based on the velocity and the diffusion coefficient.

4. The ion analysis method as described in claim 1, characterized in that, It also has: The m / z measurement step involves measuring the m / z value of the target ion when the valence z is undetermined. The second calculation step involves determining a more accurate mass value than the mass value obtained in the calculation step, or narrowing down the m / z value, based on the mass or charge value obtained in the calculation step and the m / z value obtained in the m / z measurement step.

5. The ion analysis method as described in claim 1, characterized in that, In the measurement step, the first information is acquired while the electric field is zero.

6. The ion analysis method as described in claim 1, characterized in that, In the measurement step, under the condition that an electric field is actually present, first information related to the motion of the target ion under the effective gravitational acceleration that is apparent to be adjusted by the action of the electric field is obtained.

7. The ion analysis method as described in claim 6, characterized in that, In the measurement step, the first information is acquired under the condition that an electric field exists that counteracts the effect of gravity.

8. The ion analysis method as described in claim 1, characterized in that, In the measurement step, optical methods are used to acquire first information related to the motion caused by gravity or the action of gravity and electric field for the target ion introduced into the moving space, and second information related to Brownian motion is acquired.

9. The ion analysis method as described in claim 8, characterized in that, In the measurement step, light is irradiated onto the ions in the moving space, and the light scattered in the ions or emitted from the ions is detected, thereby determining the movement of the ions.

10. The ion analysis method according to claim 1, characterized in that, In the ion introduction step, after the target ion is temporarily held in an ion holding part located outside the moving space, the ion is released at a predetermined time, thereby introducing the target ion into the moving space.

11. The ion analysis method as described in claim 10, characterized in that, The ion holding part is an ion trap, which is composed of multiple electrodes. By applying a rectangular wave voltage to at least one of the multiple electrodes, ions can be held in the space surrounded by the multiple electrodes.

12. The ion analysis method as described in claim 10, characterized in that, In the ion introduction step, after identifying the ions held in the ion trap based on the m / z value, the ions remaining after the identification are introduced into the moving space.

13. An ion analysis method, characterized in that, have: The ionization step ionizes the object being measured; In the ion introduction step, the ions obtained in the ionization step or the ions derived from the ions, i.e. the target ions, are introduced into a moving space. The moving space is adjusted to a specified gas pressure and gravity acts therein, and an electric field is formed that acts on the ions in a direction orthogonal or oblique to the direction of gravity. The separation step utilizes the movement of ions in the moving space, which reflects the differences in size and mass of ions due to gravity and the movement of ions, which reflects the differences in charge of ions due to the electric field, to spatially separate the target ions introduced into the moving space from other ions. The detection step detects at least a portion of the target ions separated in the separation step.

14. The ion analysis method as described in claim 13, characterized in that, The electric field formed in the moving space is a DC electric field acting on the ions in a direction orthogonal to the direction of gravity.

15. The ion analysis method as described in claim 13, characterized in that, It also has: The m / z measurement step involves measuring the m / z value of the target ion when the valence z is undetermined. The calculation step involves determining the charge q or valence z of the target ion based on the detection result in the detection step, and calculating the mass of the target ion based on this result and the m / z value obtained in the m / z measurement step.

16. The ion analysis method as described in claim 13, characterized in that, The detection step is used to obtain information related to the slope of the target ion's trajectory, and the valence z of the target ion is determined based on this information.

17. The ion analysis method as described in claim 13, characterized in that, In the ion introduction step, after the target ion is temporarily held in an ion holding part located outside the moving space, the ion is released at a predetermined time, thereby introducing the target ion into the moving space.

18. The ion analysis method as described in claim 17, characterized in that, The ion holding part is an ion trap, which is composed of multiple electrodes. By applying a rectangular wave voltage to at least one of the multiple electrodes, ions can be held in the space surrounded by the multiple electrodes.

19. The ion analysis method as described in claim 18, characterized in that, In the ion introduction step, after identifying the ions held in the ion trap based on the m / z value, the ions remaining after the identification are introduced into the moving space.

20. An ion analysis device, characterized in that, have: The ionization section ionizes the object being measured. The analysis chamber has an internal movement space that is adjusted to a prescribed air pressure where particles can undergo Brownian motion and where gravity acts. An electric field forming unit selectively forms an electric field in the moving space; An ion input unit inputs a target ion into the moving space. The target ion is an ion generated by the ionization unit or an ion originating from the ion. The measurement execution unit acquires first information related to the motion caused by gravity and electric field and second information related to Brownian motion for the target ion that has been introduced into the moving space. The processing unit calculates the mass or charge of the target ion based on the first information and the second information.

21. An ion analysis device, characterized in that, have: The ionization section ionizes the object being measured. The analysis chamber has a movable space inside that is adjusted to a specified air pressure and subjected to gravity; An electric field forming unit selectively forms an electric field within the moving space that acts on ions in a direction orthogonal or oblique to the direction of gravity. An ion input unit inputs a target ion into the moving space. The target ion is an ion generated by the ionization unit or an ion originating from the ion. The detection unit detects at least a portion of the target ions that are spatially separated from other ions by the movement of ions in the moving space due to the movement of ions reflecting differences in size and mass caused by gravity and the movement of ions reflecting differences in charge caused by the electric field.

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