Ion analysis method and ion analysis device

Abstract

An ion analysis method according to one embodiment of the present invention comprises: an ionization step for ionizing an object to be measured; an ion input step for inputting ions obtained in the ionization step or objective ions that are ions derived from said ions to a moving space (30) having a predetermined gas pressure adjusted within a range that allows particles to perform Brownian motion, and allowing the gravity and an electric field to act; a measurement step for acquiring first information on the motion by the action of the gravity and the electric field with respect to the objective ions inputted to the moving space, and acquiring second information on the Brownian motion; and a computation step for determining the mass or electric charge of the objective ions on the basis of the first and second information acquired in the measurement step. As a result, it is possible to accurately and efficiently measure the mass and electric charge of ions such as macromolecules and viruses.

Description

Ion analysis method and ion analysis device

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

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

[0003] "9. Measurement of elementary charge - Explanation of Millikan's measurement principle of elementary charge -", [Online], [Retrieved November 28, 2023], Shiga Prefectural Education Center, Internet <URL: https: / / www.shiga-ec.ed.jp / www / contents / 1440578636412 / files / kiki_phys_09.pdf> N.C. Contino and 1 other person, "Charge detection mass spectrometry for single ions with a limit of detection", International Journal of Mass Spectrometry, Vol. 345-347, 2013, pp. 153-159 "Charge Detection", [Online], [Retrieved November 28, 2023], Megadalton Solutions, Internet <URL: https: / / megadaltonsolutions.com / charge-detection / > Kenichiro Aoki, "For those who want to learn modern physics - from atoms to the universe," Keio University Press, May 20, 2011, pp. 30-32; Osamu Furuhashi and five others, "Development of a digital ion trap mass spectrometer," Shimadzu Review, Vol. 62, Nos. 3 and 4, 2005, "Dynamic Light Scattering (DLS) Measurement Principles," [Online], [Retrieved November 28, 2023], Consortium for Measurement Solutions (COMS), supporting the manufacturing industry, Internet: <https: / / unit.aist.go.jp / mcml / coms / nano-dls.html>

[0004] In Millikan's oil drop experiment, the target particle was a particle of known density, and the mass or charge of the charged particle was calculated using that known density. Therefore, if the density of the particle is unknown, the mass or charge of the charged particle cannot be determined.

[0005] On the other hand, there is an analytical method called CDMS (Charge Detection Mass Spectrometry) for measuring the mass of molecular / particle ions (Non-Patent Documents 2 and 3). In CDMS, molecular ions to be observed are introduced into a reciprocating reflector ion trap and repeatedly reciprocate within the ion trap. The ion trap is equipped with a detector that detects moving ions using electrostatic induction, and the induced charge signal obtained by the detector is Fourier transformed in a data processing unit. This Fourier transform process allows the mass-to-charge ratio (m / z; strictly speaking, this is the italicized "m / z," but here we will use the unitalicized "m / z") of the molecular ion to be determined from the frequency of the induced charge signal. At the same time, the ion's charge value can be determined from the amplitude of the signal, and the ion's mass can be calculated from the m / z and charge.

[0006] Thus, while CDMS can determine the mass or charge of ions of molecules / particles with unknown densities, it can give erroneous results unless the ion concentration is at the single-particle level. For example, if two ions with the same m / z pass near the detection area simultaneously or very close in time, they will have a larger induced charge, or a larger amplitude, than a single particle. If this is interpreted as a single particle, it will give an incorrect charge z and, as a result, an incorrect mass m. To avoid erroneous interpretations, it is necessary to lower the ion concentration to the single-particle level, which reduces measurement throughput, especially when measuring ions derived from various components contained in a sample.

[0007] The present invention has been made to solve these problems, and aims to provide a novel ion analysis method and ion analysis device that can measure the mass and charge of molecular ions using a technique that is completely different from CDMS.

[0008] A first aspect of the ion analysis method according to the present invention includes: an ionization step of ionizing a measurement target; an ion injection step of injecting target ions, which are ions obtained in the ionization step or ions derived from the obtained ions, into a movement space adjusted to a predetermined gas pressure within a range in which particles can undergo Brownian motion and in which gravity and an electric field can act; a measurement step of acquiring first information regarding the movement of the target ions introduced into the movement space due to the action of gravity or the action of gravity and an electric field, and acquiring second information regarding Brownian motion; and a calculation step of determining the mass or charge of the target ions based on the first and second information obtained in the measurement step.

[0009] A first aspect of the ion analysis apparatus according to the present invention is an apparatus for carrying out the ion analysis method of the first aspect, comprising: an ionization unit that ionizes a measurement target; an analysis chamber having an internal movement space adjusted to a predetermined gas pressure within a range in which particles can undergo Brownian motion and on which gravity acts; an electric field forming unit that selectively forms an electric field in the movement space; an ion injection unit that injects target ions, which are ions generated in the ionization unit or ions derived from the generated ions, into the movement space; a measurement execution unit that acquires, for the target ions injected into the movement space, first information regarding the movement due to the effects of gravity and the electric field and second information regarding Brownian motion; and an arithmetic processing unit that calculates the mass or charge of the target ions based on the first information and the second information.

[0010] A second aspect of the ion analysis method according to the present invention includes: an ionization step of ionizing a measurement object; an ion injection step of dropping target ions, which are ions obtained in the ionization step or ions derived from the ions obtained in the ionization step, into a movement space that is adjusted to a predetermined gas pressure, is subjected to gravity, and in which an electric field is formed that acts on the ions in a direction perpendicular or oblique to the direction of gravity; a separation step of spatially separating the target ions injected into the movement space from other ions by utilizing the movement of ions in the movement space that reflects differences in size and mass of ions due to the effect of gravity and the movement that reflects differences in charge of ions due to the effect of the electric field; and a detection step of detecting at least a portion of the target ions separated in the separation step.

[0011] A second aspect of the ion analysis apparatus according to the present invention is an apparatus for carrying out the ion analysis method of the second aspect, comprising: an ionization unit that ionizes a measurement target; an analysis chamber having an internal movement space adjusted to a predetermined gas pressure and on which gravity acts; an electric field forming unit that selectively forms, within the movement space, an electric field that acts on ions in a direction perpendicular or oblique to the direction of gravity; an ion injection unit that injects target ions, which are ions generated in the ionization unit or ions derived from the generated ions, into the movement space; and a detection unit that detects at least a portion of the target ions that are spatially separated from other ions in the movement space by the movement of the ions reflecting the difference in size and mass of the ions due to the effect of gravity and the movement reflecting the difference in charge of the ions due to the effect of the electric field.

[0012] The ion analysis method and apparatus of the first aspect observes the Brownian motion of an ion and the motion of an ion due to the action of gravity or the action of both gravity and an electric field, and calculates the mass or charge of the ion based on information about these motions. As a result, the ion analysis method and apparatus of the first aspect can determine the mass and / or charge of an ion of a large molecule without performing any prior operation to substantially isolate the ion.

[0013] On the other hand, in the ion analysis method and apparatus of the second aspect, both the action of gravity and the action of an electric field acting in a direction different from that of gravity make it possible to separate and detect ions of even large molecules according to their mass and charge. As a result, similar to the ion analysis method and apparatus of the first aspect, the ion analysis method and apparatus of the second aspect also makes it possible to determine either or both of the mass and charge of ions of large molecules without performing any prior operation to substantially isolate the ions.

[0014] FIG. 1 is a schematic configuration diagram of an ion analysis apparatus according to one embodiment of the present invention. FIG. 2 is a schematic configuration diagram of an ion analysis apparatus according to another embodiment of the present invention. FIG. 3 is a schematic configuration diagram of a modified example of the ion analysis apparatus shown in FIG. 2. FIG. 4 is a schematic configuration diagram of a modified example of the ion analysis apparatus shown in FIG. 2. FIG. 5 is a schematic configuration diagram of a modified example of the ion analysis apparatus shown in FIG. 1. FIG. 6 is an explanatory diagram of the principle of an ion analysis method according to the present invention. FIG. 7 is an explanatory diagram of a method for calculating the movement speed of ions when the effects of gravity and an electric field and the effect of diffusion exist simultaneously. FIG. 8 is an explanatory diagram of an example of a method for calculating the valence of ions. FIG. 9 is a graph showing the relationship between the valence of ions and the horizontal movement distance in FIG. 9.

[0015] [1] Principle of the ion analysis method of the present invention [1-1] Conventional reference methods and their problems When identifying a substance, the important thing is the molecular weight of the components that make up that substance, and what is desired to know with an ion analyzer including a mass analyzer is the mass of the ions derived from those components. However, ions of large molecules such as viruses and nanomedicines (nanotechnology-applied preparations), typically with a mass of MDa (= 10 6One of the problems when attempting to measure molecular ions at the (Da) level (hereinafter, ions of viruses and these giant molecules will be collectively referred to as "giant molecular ions." Here, "giant molecular ions" includes adduct ions and fragment ions generated during ionization, as well as product ions by CID, etc.) using existing general mass spectrometry techniques is that the distribution of ion valence is so wide that it is difficult to determine the valence, and in some cases, simply knowing the ion valence, or charge, is sufficient as a measurement result. Therefore, here we consider obtaining either the mass or charge of the giant molecular ions, or both.

[0016] Because ions are a type of particle and ions of large molecules can be considered particles, the inventors of the present invention initially conceived the idea of ​​applying Millikan's oil drop experiment to measuring the charge of ions of large molecules. However, for the following reasons, the measurement of the charge of charged oil drops in Millikan's oil drop experiment cannot be directly used to measure the charge of ions.

[0017] In Millikan's oil drop experiment, measurements reveal the relationship between the falling velocity v of the charged oil drop and the electric field strength E. This relationship is expressed by the linear equation v = αE - v0. In other words, even if the number of measurement data points is increased, only two pieces of information are obtained: the slope α of the linear equation and the Y-intercept, -v0. Meanwhile, the motion of the microparticles observed in Millikan's oil drop experiment involves the friction coefficient k of air resistance, so there are three unknowns to be determined (charge q, mass m, friction coefficient k). In other words, the amount of information obtained through measurement is one less than the number of unknowns. In fact, in Millikan's oil drop experiment, the material of the microparticle is known, so its density ρ is also known. The radius r of the microparticle can be obtained from the mass m, and the friction coefficient k can be calculated from this radius r according to Stokes' law of fluid mechanics. In this way, in Millikan's oil drop experiment, mass m and charge q are determined separately by eliminating one unknown. In other words, Millikan's oil drop experiment cannot determine the mass of an object to be measured whose material is unknown (i.e., whose coefficient of friction k is unknown).

[0018] [1-2] Diffusion Motion of Ions In contrast, the present inventors have noticed that when ions of large molecules are allowed to fall under the action of gravity, they are particles in a gas, and so in addition to the motion due to the action of gravity, they also undergo diffusive motion. Specifically, in this case, Brownian motion, which occurs as a result of a gas of known temperature colliding with the particles, is utilized. By observing the Brownian motion of the ions as they fall through a space in which a gravitational field or both a gravitational field and an electric field act and in which a gas exists, the diffusion coefficient can be calculated, and the mass of the ions can be determined from the result. In actual measurements, the diffusion motion and the drift motion due to the action of a gravitational field and / or an electric field occur simultaneously, so the motion of the ions must be treated as a diffusion drift, which is a combination of these motions.

[0019] It is generally known that the diffusion coefficient D of the Brownian motion of particles in a gas is expressed by Einstein's relational equation, which is the first historical example of the Fluctuation-Dissipation Theorem (Non-Patent Document 4). This equation is shown in (1). Here, k B is the Boltzmann constant, and T is the absolute temperature of the gas. μ is called the mobility, and corresponds to the speed that occurs when a force of unit magnitude is applied. Therefore, μ and the coefficient of friction k due to air resistance have the relationship μ = 1 / k. D = μk B T = k B T / k ... (1) Transforming this equation (1) gives the following equation (1A): kD = k B T = [constant] …(1A)

[0020] Equation (1A) implies that the larger the friction coefficient k, the smaller the diffusion coefficient D, and that to increase diffusion, the friction coefficient k must be reduced, which is consistent with the physical image. Furthermore, equation (1A) does not include information about the atoms and molecules that make up the gas. This is presumably because the fluctuation-dissipation theorem is a law that holds true as an average over a time scale that is sufficiently long compared to the time it takes for the gas to collide with the particle. In this case, it can be presumed that this holds true more easily for lighter gases such as helium (He) and hydrogen (H2).

[0021] Here, we are also dealing with the diffusion drift of microparticles under reduced pressure (but in the presence of gas), so it is necessary to understand the change in the friction coefficient k when the pressure p changes (the dependence of the friction coefficient k on the pressure p). Although this relationship is not directly proportional, it can be inferred that it is a monotonically increasing relationship in which the friction coefficient increases as the pressure increases. However, as the pressure approaches 1 atmosphere, it is thought that the friction coefficient saturates and changes very little. In other words, when the pressure p becomes so large that the mean free path of the gas becomes smaller than the particle size and it can be considered a fluid, Maxwell's kinetic theory of gases predicts that the friction coefficient will become constant. On the other hand, it is also obvious that k = 0 when p = 0.

[0022] Although it is desirable to investigate the pressure dependence of the friction coefficient k strictly through experiments, it can be estimated based on the results of particle trajectory simulations. In this simulation, the diffusion coefficient D can be determined from equation (1) by assuming a value for the friction coefficient k, and the particle trajectory can be predicted by providing an appropriate initial value and solving the equation of motion with random walk. In other words, by adjusting the friction coefficient k as a parameter in the simulation so as to reproduce the experimental results, it is possible to estimate the value of the friction coefficient k, which is thought to depend on pressure and gas type.

[0023] Next, we will discuss the feasibility of measuring the diffusion coefficient D based on the results of observations using a microscope such as that used in the Millikan oil drop experiment. In Perrin's experimental verification of Brownian motion, the movement of fine particles (radius: 213 nm) in a solvent such as water was observed using a microscope. The observed diffusion distance in this case was approximately 9 μm over 30 seconds. Based on this result, we will estimate how far a particle of similar size would diffuse in a gas over a measurement time of 0.3 seconds. Theoretically, the diffusion distance x at elapsed time t is x = √(2Dt), and the diffusion coefficient in a liquid is 10 times that of the diffusion coefficient in a gas. -4 Since the diffusion distance is twice as long, it should be approximately 90 μm, which is large enough to be optically distinguishable. For even smaller particles, such as viruses, the diffusion distance should be longer.

[0024] As an example, let us consider microscopic observation. If we assume that only diffusion movement occurs in the horizontal direction (the direction perpendicular to gravity), the average distance x that a particle moves horizontally over a time period Δt can be expressed by the following formula (2): x = √(2DΔt) ... (2) Therefore, by examining the distance that the particle moves horizontally at regular time intervals Δt, we can obtain x1, x2, x3, ..., x n If the measurement result is obtained as follows, the square mean value x av 2 is expressed by equation (3). av 2 = (x1 2 +x2 2 +x3 2 +...+x n 2 ) / n → 2DΔt(n → ∞) (3) That is, by repeatedly measuring the moving distance of the particle, the measurement accuracy can be improved.

[0025] In conclusion, it is quite possible to determine the diffusion coefficient D based on information obtained by observing the Brownian motion of ions of large molecules. In particular, it can be said that the accuracy of the diffusion coefficient D can be improved by repeatedly measuring the migration distance of ions over a certain period of time.

[0026] [1-3] Mass measurement using diffusion and drift motion Next, we will discuss a method for measuring the mass m of an ion when the above-mentioned diffusion motion and drift motion due to the effects of gravity and an electric field are present. First, consider the case where the direction of the electric field is opposite to the direction of gravity.

[0027] First, let us consider the drift motion of ions due to a gravitational field. The vertical terminal velocity v0 of a particle in zero electric field is expressed by the following equation (4), since [air resistance kv0] = [gravity mg]. v0 = mg / k ... (4) Combining this with the fluctuation-dissipation theorem, we obtain equation (5): m = v0k BT / (Dg) ... (5) Equation (5) means that the mass m can be determined from the measurable quantities v0 and D. As mentioned above, the diffusion coefficient D can be determined based on the observation of the ion's horizontal movement (= diffusion movement only), and the terminal velocity v0 can be determined based on the simultaneous vertical movement of the ion (= diffusion movement and movement due to the action of a gravitational field), so the ion's mass m can be calculated from these.

[0028] To accurately calculate the mass m from equation (5), the gravitational acceleration g must also be accurate. As is well known, the gravitational acceleration g varies considerably depending on the latitude and altitude of the measurement location, but an accurate value can be used depending on the measurement location. Furthermore, if it is possible to determine the m / z value of the target ion (however, the valence z is unknown) using a different mass spectrometer, even if the mass m is not accurate, it is possible to estimate the z value from the low-accuracy mass m and the accurate m / z value and calculate the accurate mass m.

[0029] The above-mentioned method for measuring the mass m of an ion requires that the diffusion coefficient D or friction coefficient k of the ion be measured in addition to the terminal velocity v of the ion. To achieve this, the following conditions must be met: - The temperature in the space in which the ions move must be constant and in thermal equilibrium. - The velocity of the particles in the space in which they move must be sufficiently slow so that a proportional relationship exists between frictional force and velocity. As long as the above conditions are met, there are essentially no limitations or restrictions imposed by the device, such as the limitations on charge resolution due to ion trajectories in CDMS.

[0030] Here, we will discuss a method for improving measurement accuracy when performing microscopic observation as an example. To improve measurement accuracy in this measurement technique, it is desirable for the particles to remain within the field of view of the microscope 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 at zero electric field. The second reason is that if the residence time is short, the number of data points required to calculate the diffusion coefficient D is small, making it difficult to improve its accuracy. That is, in terms of measurement accuracy, in this measurement technique, it is desirable for the fall velocity of the particles to be as small as possible. Of course, in practice, a low fall velocity reduces measurement throughput, so it is necessary to consider the balance between accuracy and throughput. To achieve a low fall velocity, it is advisable to adjust the strength of the electric field that attracts ions vertically upward so as to nearly cancel out the effect of gravity.

[0031] In a state where the effect of gravity is completely cancelled out by the electric field, in other words, in a pseudo-weightless (floating) state, the following equation (6) holds: 0 = mg + qE0 → m / q = -E0 / g ... (6) In other words, at this time, the specific charge m / q can be determined.

[0032] When an electric field of strength E (≒E0) is formed to reduce the falling speed of ions, equation (7) holds true: kv = mg + zE → m = vk B T / (g a D) ...(7) where g a = g (1 - E / E0) is the apparent effective gravitational acceleration. Incidentally, when the electric field strength E = 0, equation (7) becomes kv0 = mg → m = v0k B T / (gD), which coincides with the above equation (5). By making v<<v0, that is, by taking time for measurement, the measurement accuracy can be improved. This is the same as CDMS.

[0033] In a gravity-free (floating) state as shown in Equation (6), ion movement is caused solely by diffusion, making it possible to apply dynamic light scattering (DLS) as described in Non-Patent Document 6. DLS is a commonly used method for measuring the particle size of microparticles. It involves irradiating microparticles suspended in a solution with a laser beam to measure their particle size. Specifically, laser light is incident at a predetermined angle relative to the space in which the microparticles are moving. The scattered light from the microparticles exiting the space at a fixed angle is detected using a photon counting method, and the temporal change in intensity is recorded. The autocorrelation function is then calculated from the temporal change in the scattered light intensity. The diffusion coefficient is calculated from the relaxation rate (the inverse of the relaxation time), and the hydrodynamic particle size is calculated from the Stokes-Einstein equation, which expresses the relationship between the diffusion coefficient and particle size. In principle, the diffusion distance required for measurement is approximately the wavelength of the laser beam. Therefore, compared to direct observation of Brownian motion, DLS requires a smaller moving space and shortens the measurement time.

[0034] The above explanation is for the case where the direction of action of the electric field is opposite to the direction of action of gravity, but in reality, the direction of action of the electric field is not limited to this. In other words, the angle θ between the direction of action of the electric field and the direction of action of gravity can be any angle between 0 and 180°. Let us explain this. Now, suppose that gravitational acceleration g↑ (where *↑ represents the vector *) acts in the direction of action of gravity, and an electric field E↑ acts in the direction of action of the electric field, and that g↑ and E↑ form any angle θ as vectors.

[0035] In the steady state where the gravitational and electric fields described above exist, ions have a constant velocity (vector) v↑. In this case, kv↑ = qE↑ + mg↑ ... (8) and therefore the following equation (9) is obtained: v↑ = (q / k)E↑ + (m / k)g↑ ... (9) As already mentioned, there are three unknowns of interest (q, m, k), whereas equation (9) contains only two parameters, (q / k) and (m / k), and as mentioned above, if the diffusion coefficient D is unknown, then neither q nor m can be determined.

[0036] Therefore, the ion velocity v↑ and diffusion coefficient D are calculated as follows. Assuming that the drift motion due to the effects of gravity and an electric field is uniform linear motion, the velocity vector v↑ that fits the measurement data is determined using the least squares method as follows. Figure 8 is a diagram showing an example of an ion's movement trajectory. As shown in Figure 8, it is assumed that the ion moves from [1] → [2] → [3] → [4] → ... at a constant time interval Δt. Assuming a velocity vector v↑, the residual vector at each time interval Δt is calculated as follows: Δr1↑ = r1↑ - v↑Δt Δr2↑ = r2↑ - v↑Δt Δr3↑ = r3↑ - v↑Δt ...

[0037] The sum of squares of the absolute values ​​of the residual vectors above, R 2 Calculate the sum of squares R 2 Search for the velocity vector v↑ that minimizes R 2 = |Δr1↑| 2 +│Δr2↑│ 2 +│Δr3↑│ 2 +... In this case, the relationship between the velocity and the residual is linear, so that a unique solution can always be determined as the velocity vector v↑.

[0038] Once the velocity vector v↑ is determined, the diffusion coefficient D is then calculated. The residual vectors Δr1↑, Δr2↑, Δr3↑, ... obtained as a result of the least squares method contain only accidental components, which can be considered to be random walks. Therefore, the diffusion coefficient D can be calculated by analyzing the residual vectors. Specifically, Δr i ↑=(Δx i , Δy i ), its probability density function is a two-dimensional normal distribution {exp[-(Δx i ) 2 / (4DΔt)]}×{exp[−(Δy i ) 2 / (4DΔt)]}. Therefore, the simplest way is to 2The average value of these can be taken as 4DΔt, from which the diffusion coefficient D can be obtained. In this way, regardless of the direction of the electric field, the velocity and diffusion coefficient of the ion can be obtained from the measurement data obtained by observing the ion movement trajectory, and the mass m can be calculated.

[0039] As described above, the terminal velocity v of an ion can be determined by two methods: (a1) direct observation of the ion with a microscope and (a2) position measurement with a laser, and the diffusion coefficient D can be determined by two methods: (b1) direct observation with a microscope and (b2) DLS. Of course, the terminal velocity v and diffusion coefficient D of an ion may be determined by methods other than those described above, and the mass m of an ion may be calculated using the terminal velocity v and diffusion coefficient D of an ion determined by any combination of these methods.

[0040] [1-4] Measuring Charge Using a Horizontal Electric Field While the above measurement method uses an electric field to adjust the ion fall velocity, it is also possible to more actively use the electric field to separate ions according to their charge or valence. This is the second ion analysis method. In this method, the m / z value of the target ion is determined using a front-stage mass analysis unit, such as a digital ion trap (described below). However, if the target ion is a multivalent ion, the valence z is unknown, and therefore the mass m cannot be determined. If the valence z or charge q of the target ion can be determined using a method other than mass analysis, the accurate mass m can be determined. Therefore, as a method derived from the first ion analysis method, a two-dimensional extension of the method used in Millikan's oil drop experiment is used.

[0041] Typically, in this second analysis method, ions to be observed are allowed to fall vertically downward at a uniform velocity (terminal velocity) due to gravity in a gas atmosphere, and an electric field is formed that attracts the ions horizontally, causing them to move at a uniform velocity in the horizontal direction as well. He is the preferred gas used here for the following reasons.

[0042] Since the two electrons of He are strongly bound by the attractive force of the nucleus, the change in the wave function due to the influence of the charge of the approaching ion is small. Therefore, it is reasonable to think that the friction coefficient k resulting from the collision phenomenon between He and an ion is unlikely to change depending on the valence z of the ion (in fact, Millikan himself calculated the elementary charge on the assumption that the friction coefficient k does not depend on the valence z of the ion). If the friction coefficient k is constant regardless of the valence z, then the horizontal velocity v of the ion h is expressed as the following equation (10): kv h =zeE → v h = zeE / k ... (10) That is, the velocity v h is guaranteed to take equally spaced discrete values.

[0043] FIG. 7 is a diagram schematically illustrating the behavior of ions in this second ion analysis method. As described above, ions move at a constant velocity in the vertical direction (Z-axis direction) due solely to the action of gravity, i.e., fall. Because the velocity at this time depends on the size and weight of the particle, ions can be separated based solely on particle size and weight. Specifically, ions with a heavier mass (i.e., a stronger gravitational force) and a larger mass (i.e., relatively smaller friction) fall at a faster velocity. On the other hand, ions move at a constant velocity in the horizontal direction (here, Y-axis direction) according to their valence z or charge q. Therefore, they are discretely separated according to their valence z. That is, as shown in FIG. 7 , if falling ions have the same mass, they will reach different horizontal positions depending on their valence z. Specifically, the larger the valence z, the farther the ions will reach horizontally. This allows target ions with various valences z to be separated by their valences according to their fall position or falling trajectory. If the application simply involves separating ions by valence or determining whether ions of different valences exist, it is not necessary to determine the valences of the individual separated ions.

[0044] One method for determining the valence of each separated ion is to investigate in advance the relationship between the mass m and valence z of the ion, the electric field strength, and the fall position of the ion through a preliminary experiment, and then use this information to determine the valence z of the target ion from the fall position of the ion. Furthermore, different fall positions mean that the angle formed by the line of the ion's movement trajectory with respect to the Z axis changes depending on the valence z or the charge q. Therefore, for example, the valence z of the target ion can be estimated by the following calculation based on the fall position of the ion. The condition for this calculation to be possible is that multiple ions derived from the same component and differing in valence by one are observed.

[0045] As a simple example, consider the case where observations are made that show that ions originating from the same component, i.e., ions with the same mass m, fall at two adjacent positions, as shown in Figure 9. In this case, it is reasonable to consider that the valences of the ions falling at different positions differ by 1, so the valence of one ion (the one closer to the vertical fall position) is estimated to be z, and the valence of the other ion is estimated to be z+1.

[0046] As mentioned above, based on the premise that the coefficient of friction k is unlikely to change depending on the ion valence z (even if the coefficient of friction k does depend on the ion valence z, the change is so gradual that it can be considered practically nonexistent), for the same fine particle, even if the valence z is different, the gravity and frictional force acting in the vertical direction Z are the same, so that the particle should reach the same vertical coordinate Z in the same falling time. On the other hand, for the horizontal coordinate Y, if the coefficient of friction is the same based on the above premise, it should reach a horizontal position proportional to the valence z.

[0047] From this, the relationship between the ion valence (charge) z and the horizontal movement distance y can be graphed as shown in Figure 10. As can be seen from Figure 10, the quadratic function y = az is calculated using three data points P1 and P2 corresponding to z1 and z+1, plus point P0 where z = 0. 2+bz+c (however, since it passes through the origin, c = 0 automatically) should be uniquely determined. Under the above assumptions, the valence z is determined so that this quadratic function is closest to a straight line, and the valence z1 that minimizes the absolute value |a| of the coefficient of the quadratic term of the quadratic function is considered to be the most appropriate estimate. Since z1 is naturally a natural number, it is clear that its search is extremely easy. In this way, the valence z of an ion can be estimated from the fall positions of two ions whose valences differ by one.

[0048] The above concept can be extended to perform calculations even when three or more ions with different valences (z1) are observed, i.e., when there are three or more impact positions. One method that is considered to be the most versatile is to use a spline curve instead of a quadratic function. In this case, the condition of the smoothest curve passing through two or more data points is guaranteed by minimizing the elastic energy. Therefore, it is believed that using a spline curve makes it easy to search for the most appropriate valence z1.

[0049] However, when there are multiple fall locations and multiple types of particles, the spacing between the bright spots indicating the particle locations may appear irregular, making it difficult to determine the charge using a spline curve.However, even in such complex situations, the Fourier transform method can be used as a powerful and fast analysis method.

[0050] Using Figure 9, for the same particle with different valences z on a horizontal line where Y = constant, bright spots should be spaced at approximately equal intervals (ΔZ) in the Z direction. Then, if the light intensity distribution of the bright spots is Fourier transformed along the horizontal line where Y = constant, peaks will appear at discrete frequency positions such as 2π / ΔZ, 4π / ΔZ, 6π / ΔZ, etc. This allows us to identify not only the ΔZ value but also the bright spots that significantly contribute to the Fourier transform peak, making it possible to use spline curves based on these bright spots. Furthermore, if the position of the horizontal line where Y = constant is shifted vertically (up and down), the ΔZ value should also change linearly. Therefore, it is possible to determine the charge of the target particle (ion) by making maximum use of the two-dimensional information in the image obtained by observing the bright spots.

[0051] [1-5] Consideration of appropriate analytical conditions We will consider analytical conditions that are advantageous for improving measurement accuracy in each of the first and second ion analysis methods described above. Now, the distance that a target ion moves at drift velocity v during time t is vt, and the distance that the ion moves due to diffusion motion during the same time t is √(2Dt). Therefore, the ratio of the two, R (= [drift distance] / [diffusion distance]), is given by the following equation (11): R = vt / √(2Dt) = (v√t) / √(2D) = (mg a √t) / (k√(2D))=(mg a √t) / √(2kk B T) ... (11) Therefore, in the first ion analysis method, the condition advantageous for improving accuracy is to increase the diffusion distance, and R → 0, so that the apparent effective gravitational acceleration g a It is important to bring R close to 0 (to bring it close to a weightless, floating state). On the other hand, in the second ion analysis method, the condition that is advantageous for improving accuracy is to increase the drift distance, that is, R → ∞, so it is effective to reduce the friction coefficient k, that is, to lower the gas pressure (to increase the degree of vacuum).

[0052] Next, the configuration and operation of an embodiment of an ion analysis apparatus that utilizes the first ion analysis method and the second ion analysis method will be described.

[0053] [2] Ion Analysis Apparatus of the First Embodiment [2-1] Configuration FIG. 1 is a schematic diagram of an embodiment of an ion analysis apparatus utilizing the first ion analysis method. This ion analysis apparatus includes an ion source 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 in a vacuum chamber, and the interior of the vacuum chamber is evacuated to a predetermined gas pressure by a vacuum pump. The ion trap 2 and the ion drift unit 3 may be disposed in separate vacuum chambers with different degrees of vacuum. For convenience of explanation, FIG. 1 defines three mutually orthogonal axes, X, Y, and Z, in space. The Z-axis direction corresponds to the direction of gravity, i.e., the vertical direction.

[0054] The ion supply unit 1 generates ions to be measured and typically includes an ion source that ionizes components in a sample. The ionization method used in the ion source is not limited to a specific one. For example, if the sample is a liquid sample, a method for ionizing sample components under atmospheric pressure, such as electrospray ionization, atmospheric pressure chemical ionization, or probe electrospray ionization, can be used. In addition to the ion source, the ion supply unit 1 can also include an ion dissociation unit, such as a collision cell, that dissociates ions generated in the ion source using an appropriate method, such as collision-induced dissociation.

[0055] The ion trap 2 has the function of temporarily trapping the ions to be observed and defining the initial position at which they are dropped. Furthermore, although not essential, the ion trap 2 can have the function of restricting the m / z or m / z range of the ions to be dropped by discriminating the ions according to their m / z, as needed. This function of discriminating ions according to m / z is important when measuring complex molecules with various ion masses m and charges q.

[0056] The ion trap 2 may be any type capable of achieving the above-described functions. Here, the ion trap 2 is a three-dimensional quadrupole ion trap (also called a Paul trap) including a ring electrode 22 having a hyperboloid of revolution whose axis of symmetry extends in the Z-axis direction, and a pair of end cap electrodes 20, 21 arranged above and below the ring electrode 22 so as to face each other. An ion entrance aperture 20a is formed in the upper entrance end cap electrode 20, and an ion exit aperture 21a is formed in the lower exit end cap electrode 21. However, it is known that in such a three-dimensional quadrupole structure, the ring electrode 22 can be replaced with a cylindrical electrode and the end cap electrodes 20, 21 with planar electrodes, and an ion trap with such a simpler structure may also be employed.

[0057] Furthermore, the type and format of the ion trap 2 are not limited to a specific one, and may be a three-dimensional quadrupole ion trap or a two-dimensional ion trap. Here, a digital ion trap (see, for example, Non-Patent Document 5) is used, which uses a square-wave RF voltage as the RF voltage for forming the trapping electric field. However, an analog ion trap using a sinusoidal RF voltage may also be used. However, to efficiently trap ions of large molecules, it is desirable to use a digital ion trap and lower the RF voltage frequency as much as possible. Digital ion traps also have the advantage of being able to select ions in a specific m / z range with high resolution in a short time by using rough isolation and forward / reverse scanning. Furthermore, as will be described later, in order to limit the initial position of ions before they fall, it is desirable to use a three-dimensional ion trap, which can confine ions to a narrow region.

[0058] The ion drift section 3 provides a space in which ions fall due to the action of gravity and move due to Brownian motion. The gas pressure (or degree of vacuum) and temperature of this space are controlled with high precision. Here, the ion drift section 3 includes a pair of flat plate electrodes 31, 32 installed above and below at a predetermined distance, and an ion movement space 30 in which ions move is formed between these flat plate electrodes 31, 32. An ion passage opening 31a is formed in the upper flat plate electrode 31 at a position corresponding to the ion exit opening 21a of the ion trap 2.

[0059] The detection unit 4 is an optical detection means capable of detecting ions moving through the ion migration space 30. When observing viruses or large molecular ions, a method can be used in which light of a predetermined wavelength is irradiated onto the object to be measured and the scattered light is detected. Furthermore, since the size of detectable particles depends on the wavelength of the light used, a light source that emits light with a short wavelength (e.g., a 265 nm LED used exclusively for disinfection) can be used to detect smaller particles. Furthermore, when simultaneously observing many ions over a wide area, a sheet laser, which is used for visualizing fluids, can be used.

[0060] The voltage generator 5 includes a trap voltage generator 50 that applies a predetermined voltage to the ring electrode 22 of the ion trap 2, an end cap voltage generator 51 that applies predetermined voltages to the end cap electrodes 20 and 21, respectively, and a drift section voltage generator 52 that applies a predetermined voltage to the planar electrode 32 of the ion drift section 3. In Fig. 1, the planar electrode 31 is grounded and has zero potential, but it is also possible to apply a predetermined voltage other than zero to this planar electrode 31.

[0061] The data processing unit 10 is configured mainly with a computer including, for example, a CPU, and processes the detection results (observation image data and measurement data) obtained by the detection unit 4 .

[0062] [2-2] Analysis Operation An example of the analysis operation using this ion analyzer will now be described. The ion supply unit 1 ionizes component molecules contained in a given sample. Here, the component molecules are assumed to be, for example, viruses or large molecules. The ions generated by the ion supply unit 1 are introduced into the internal space of the ion trap 2 through the ion entrance opening 20a via an ion guide (not shown). During this ion introduction, a DC voltage of the same polarity as the ions is applied to the exit endcap electrode 21. This creates an electric field in the internal space of the ion trap 2, which applies a force to the ions introduced into the internal space generally in the negative direction of the Z axis (i.e., upward), preventing them from being ejected from the ion exit opening 21a. Simultaneously with or after a time delay after the ion introduction, a rectangular-wave RF voltage of a predetermined frequency is applied from the trap voltage generator 50 to the ring electrode 22. This creates an ion-trapping electric field in the internal space, trapping the ions in the internal space.

[0063] After ions are introduced into the internal space of the ion trap 2 and trapped by the electric field, a gas supply unit (not shown) introduces a predetermined inert gas as a cooling gas into the internal space. The trapped ions lose kinetic energy upon contact with the cooling gas and gather near the center of the ion trap 2. After a predetermined cooling period, the trapping voltage generator 50 stops applying the square-wave RF voltage to the ring electrode 22. This eliminates the electric field acting on the ions that had gathered near the center in the internal space of the ion trap 2, causing them to fall by gravity, passing through the ion exit aperture 21a and the ion passage aperture 31a and then into the ion movement space 30. At this time, a DC voltage may be applied to the two end cap electrodes to form a DC electric field between them, thereby accelerating the ions. In a digital ion trap, the initial positions of the ions within the ion trap 2 can be aligned or optimized by appropriately controlling the phase of the voltage when the application of the trapping voltage is stopped.

[0064] A specific high-purity gas is introduced into the ion migration space 30, and the gas pressure, i.e., the density of the gas molecules, is controlled with high precision. The temperature of the ion migration space 30 is also controlled with high precision. The ions introduced into the ion migration space 30 repeatedly come into contact with gas molecules whose density and temperature are controlled with high precision. Therefore, each introduced ion diffuses while undergoing Brownian motion. The detection unit 4 acquires data containing information indicating the position of each ion, for example, at regular time intervals. The data processing unit 10 calculates the horizontal migration distance per predetermined time for each ion based on this data. This determines the root mean square of the horizontal migration distance x due to Brownian motion, as described above, and the diffusion coefficient D of the ion is calculated from this information.

[0065] Gravity acts on the ions in the ion migration space 30, causing each ion to move (fall) in the Z-axis direction. That is, the ions undergo drift motion due to the gravitational field. When the voltage applied to the planar electrode 32 from the drift section voltage generator 52 is set to zero, the electric field acting on the ions in the ion migration space 30 becomes zero. The vertical terminal velocity v of the ions at this time can be calculated based on, for example, information indicating the positions of individual ions at regular time intervals obtained by the detector 4, as described above. In this way, the data processor 10 calculates the terminal velocity v and diffusion coefficient D of each ion based on the measurement data obtained by the detector 4, and then calculates the mass m of the ion from these values ​​using the above equation (6).

[0066] The value of gravitational acceleration g used in calculating mass can be, for example, a preset default value, a value selected based on gravity-related information such as the longitude and altitude of the measurement point entered by the user, or a value entered by the user.

[0067] The ion mass m of the ion analyzer of this embodiment can be measured as described above without forming an electric field in the ion migration space 30. Furthermore, as described above, by applying an appropriate voltage from the drift section voltage generator 52 to the planar electrode 32 to form an electric field in the ion migration space 30 that substantially cancels out the effect of gravity, the falling speed of the ions can be reduced and measurement accuracy can be improved.

[0068] Furthermore, by applying an appropriate voltage to the planar electrode 32 from the drift section voltage generator 52 to form a predetermined electric field in the ion movement space 30, the charge q (or valence z) of the ion being observed can be determined. That is, when there is no 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. For example, if the vertical velocity of the ion when there is no electric field is v0 and the same velocity when there is an electric field is v, the following equation (12) holds true: mg = kv0 = kv - qE → q = k(v - v0) / E = (v - v0) k B T / (DE) (12) The charge q can be obtained using equation (12). In other words, with this ion analyzer, it is possible to determine the mass m of the ion to be observed without forming an electric field in the ion movement space 30, and it is also possible to determine the charge q (or valence z) of the ion by also using the results of observation of the ion when an appropriate electric field is formed in the ion movement space 30.

[0069] Furthermore, as described above, the velocity and diffusion coefficient of the ion can be calculated from the results of observing the ion movement trajectory when an electric field is formed in the ion movement space 30, and the mass m of the ion can also be calculated.

[0070] Furthermore, by combining the above-described ion analyzer with other existing mass analyzers, a more accurate mass m can be determined. Specifically, the m / z of ions derived from the target large component molecule is determined using the other mass analyzer. However, since the valence z is unknown at this time, m cannot be determined from the m / z. On the other hand, when ions derived from the same component molecule are measured using the above-described ion analyzer as described above, the mass (approximate value) m can be determined, even if it is not an accurate value (at least, the accuracy is lower than that of the other mass analyzers described above). From this mass (approximate value) m, an approximate value of m / z can be determined assuming a value of z. Therefore, the value of valence z can be determined from the approximate value of m / z and the accurate value of m / z described above for which the valence z is unknown. This allows the accurate mass m to be determined from the accurate value of m / z.

[0071] In the ion analyzer of this embodiment, the detection unit 4 uses multiple cameras arranged to capture images of a wide range within the ion migration space 30 from various angles, and by examining the correlation between the multiple observation images obtained by these multiple cameras, information regarding the migration status and position changes of each ion can be obtained. This makes it possible to accurately track the trajectories of multiple ions falling simultaneously, facilitating comprehensive measurement of a large number of ions. Furthermore, by also capturing observation images of ions in the internal space of the ion trap 2, it is possible to correct for differences in the initial positions during the fall and improve measurement accuracy.

[0072] [3] Second Embodiment of Ion Analysis Apparatus Fig. 2 is a schematic diagram of an embodiment of an ion analysis apparatus utilizing the second ion analysis method. In the ion analysis apparatus of the first embodiment, in order to determine the diffusion coefficient D of an ion, it is necessary to observe the Brownian motion of ions falling due to the action of gravity or the action of gravity and an electric field. In contrast, the ion analysis apparatus of the second embodiment uses an electric field to spatially separate ions according to their charge or valence.

[0073] 2, components that are the same or substantially the same as those in the ion analyzer of the first embodiment are denoted by the same reference numerals, and detailed description thereof will be omitted. In the ion analyzer of this second embodiment, the ion drift section 6 includes a pair of plate-shaped electrodes 61, 62 spaced a predetermined distance apart in the Y-axis direction (more precisely, in the direction lying on the X-Y plane), and an ion migration space 60 is formed between these plate-shaped electrodes 61, 62. One plate-shaped electrode 61 is grounded, and when a predetermined voltage is applied from the drift section voltage generator 53 to the other plate-shaped electrode 62, an electric field that attracts ions in the horizontal direction (in this example, the Y-axis direction) is formed in the ion migration space 60.

[0074] A plurality of auxiliary electrodes 63 are disposed between the pair of plate-like electrodes 61, 62. By applying to each of these auxiliary electrodes 63 a stepped voltage that is lower (smaller in absolute value) than the voltage applied to the plate-like electrode 62, it becomes easier to make the horizontal strength of the electric field formed in the ion movement space 60 approximately constant. However, the auxiliary electrodes 63 are not essential components.

[0075] In this ion analyzer, similar to the ion analyzer of the first embodiment, once ions are sufficiently cooled in the ion trap 2 and the voltage application to the ring electrode 22 of the ion trap 2 is stopped, the ions fall from the ion trap 2 into the ion migration space 60. Each ion introduced into the ion migration space 60 is subjected to both vertically downward gravity and horizontal electric field forces. Because the ion migration space 60 is depressurized but contains gas molecules at a moderate density, ions fall at a speed according to their mass and size. At the same time, each ion is subjected to a horizontal electric field force of a certain strength, meaning that ions with larger charges move longer horizontal distances. Therefore, for example, ions with the same mass m derived from the same component molecule will fall along different trajectories and reach different horizontal positions if their charge q, i.e., valence z, differs.

[0076] The detection unit 4X is a detector capable of detecting the arrival of ions. The detection unit 4X can be a detector using the same method as the detection unit 4 in the ion analyzer of the first embodiment, i.e., an optical detection method, and the sheet laser detector described above is particularly useful for detecting ions over a wide area. However, since it is not necessary to observe the precise movement of ions, such as Brownian motion, a simpler detector may be used.

[0077] When the horizontal electric field is weak, the horizontal migration distance of ions is short, so the separation ability of ions by valence is low, but the detection results from the detection unit 4X make it possible to grasp the overall picture of a wide valence z distribution. From these results, the dependence of the friction coefficient k on the valence z can also be analyzed. On the other hand, when the horizontal electric field is strong, it is difficult to grasp the overall picture of the valence distribution, but the separation ability according to valence is high. In other words, it is possible to separate ions by valence. Therefore, by providing an aperture or slit at the bottom of the ion migration space 60, that is, at a specific position where the falling ions reach, it is possible to select only ions with a specific valence.

[0078] [4] Modification of the ion analyzer of the second embodiment Fig. 3 is a schematic diagram of a modification of the ion analyzer described above. In Fig. 3, components that are the same as or substantially the same as those in the ion analyzer of the second embodiment are designated by the same reference numerals, and detailed description thereof will be omitted.

[0079] As shown in Figure 3, this ion analyzer includes an ion drift section 7 with the same configuration, located downstream of an ion drift section 6 that generates an electric field that attracts ions in the horizontal direction. In other words, the two ion drift sections 6, 7 are cascade-connected. In the upper ion drift section 6, a weak horizontal electric field is used to roughly separate ions according to their valence. Because the separation power here is relatively low, ions are not completely separated according to their valence. Instead, ions within a specific valence range pass through an aperture opening 64a formed in an aperture plate 64 provided between the upper ion drift section 6 and the lower ion drift section 7 and are introduced into the lower ion drift section 7.

[0080] In the lower ion drift section 7, the introduced ions are separated according to their charge states with high resolution due to a strong horizontal electric field, making it possible to selectively detect only ions with specific charge states, even among multiply charged ions with large charge states.

[0081] 4 is a schematic diagram of a further modified example of the ion analyzer shown in FIG. 3 . In this modification, another ion trap 8 having a configuration similar to that of the ion trap 2 is placed between two cascade-connected ion drift sections 6 and 7. Ions within a specific charge range that have been separated and selected in the upper ion drift section 6 are temporarily trapped in the internal space of the ion trap 8 and cooled. This resets the spread of ions due to diffusion while they drift in the ion movement space 60 of the ion drift section 6, allowing for more accurate separation of ions according to their charge states in the lower ion drift section 7. This improves the selectivity of ions with the desired charge states.

[0082] 3 and 4, the electric field acts in the Y-axis direction in both of the two ion drift sections 6 and 7, but this does not have to be the same. It is also possible to use a configuration in which three or more ion drift sections are cascade-connected.

[0083] FIG. 5 is a schematic diagram of another modified example of the ion analyzer shown in FIG. 3 . In this modified example, only ions of a specific charge state selected in the ion drift section 6 are introduced into the quadrupole mass separator 9. The quadrupole voltage generator 58 applies a voltage to the quadrupole mass separator 9 such that only ions having a specific m / z value pass through the quadrupole mass separator 9, while other ions diverge along the way. As described above, for ions of the same size and weight, only ions having a specific charge state 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 having the target mass m and charge state z may also pass through the aperture opening 64a. In contrast, in the modified example shown in FIG. 5 , only ions having a specific mass m and a specific charge state z from among the ions that pass through the aperture opening 64a can be selected and detected by the quadrupole mass separator 9.

[0084] The ions selected by the quadrupole mass separator 9 can be detected using a detection unit 4 that is arranged to optically detect ions passing through a space near the exit of the quadrupole mass separator 9. In this case, a relatively long space in which ions can be detected can be secured in the direction of ion passage, thereby enabling more reliable ion detection.

[0085] [5] Ion analyzer of the third embodiment In the ion analyzer 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 separate entities, but if they are integrated, the configuration of the device can be further simplified. Figure 6 is a schematic diagram of an ion analyzer of the third embodiment that employs such a configuration.

[0086] In this ion analyzer, an observation opening 22a is provided in a portion of the ring electrode 22, allowing observation of the internal space through the observation opening 22a from a detector 4 located outside the ring electrode. By trapping and cooling ions to be observed in the internal space of the ion trap 2, the ions are collected near the center of the internal space, and then the application of the trapping voltage is stopped. As a result, the ions diffuse due to Brownian motion in the internal space and fall due to gravity. By appropriately adjusting the voltage applied to the end cap electrodes 20 and 21, an electric field can be created that approximately balances gravity. The ion's diffusion motion and drift motion due to the effects of 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, and the mass m is then determined. Ions that have fallen a predetermined distance in the internal space of the ion trap 2 can be pulled back to their original position (near the center of the internal space) by adjusting the voltage applied to the end cap electrodes 20 and 21.

[0087] In principle, this ion analyzer can repeat the cycle of ion capture → cooling → observation of Brownian motion and drift motion in a floating state → cooling → ... multiple times, and increasing the number of repetitions can improve measurement accuracy. When a large number of ions are simultaneously trapped inside the ion trap 2, Coulomb repulsion also occurs between ions, and this effect must be taken into consideration. However, numerical simulations that include this repulsion are possible. Therefore, by comparing the simulation results with experimental results in advance and calculating corrections for the Coulomb repulsion, it is possible to calculate the mass and / or charge with sufficient accuracy from the measurement results.

[0088] [6] Other Modifications The above-described ion analysis method and ion analysis device can be further modified as follows.

[0089] In the above-described ion analysis method, a gas at a pressure lower than normal atmospheric pressure is introduced into the ion migration space, and collisions between the target ions and this gas are utilized for measurement. The falling trajectory of ions in a gas varies depending on the geometric shape of the falling particles, so this can be considered a form of chromatography using gas. If the type of gas used is different, for example, if the gas is N or He, the interaction upon collision with the falling particles will vary. Furthermore, if the molecular structure of the target is different, the interaction with different types of gas will also differ, resulting in differences in terminal velocity v0. Therefore, by comparing the results of measuring target ions using different gases, it is possible to distinguish between ions with the same mass but different molecular structures.

[0090] Furthermore, although the above-described ion analysis method uses an optical technique for detecting scattered light or the like to observe or detect ions, a fluorescent substance may be added to the particles to be measured in advance in order to detect ions more accurately and efficiently. This allows the detection unit to selectively detect light of a wavelength corresponding to the fluorescent substance using, for example, a bandpass filter, thereby improving the signal-to-noise ratio of the detection signal and increasing the accuracy and efficiency of the measurement.

[0091] Furthermore, the above-described embodiments and modifications are merely examples of the present invention, and it is natural that modifications, additions, and deletions made as appropriate within the spirit of the present invention will also be encompassed within the scope of the claims of the present application.

[0092] Various Aspects It will be appreciated by those skilled in the art that the exemplary embodiments described above are examples of the following aspects.

[0093] (Item 1) One aspect of the ion analysis method according to the present invention includes an ionization step of ionizing a measurement target; an ion injection step of injecting target ions, which are ions obtained in the ionization step or ions derived from the ions obtained in the ionization step, into a movement space where the gas pressure is adjusted to a predetermined range within which particles can undergo Brownian motion and where gravity and an electric field can act; a measurement step of acquiring first information regarding the movement of the target ions introduced into the movement space due to the action of gravity or the action of gravity and an electric field, and acquiring second information regarding Brownian motion; and a calculation step of determining the mass or charge of the target ions based on the first and second information obtained in the measurement step.

[0094] (2) In the ion analysis method described in the above paragraph 1, the measuring step may acquire second information regarding Brownian motion of the target ions in a direction different from the direction in which gravity acts.

[0095] (Clause 3) In the ion analysis method described in the above paragraph 1, the calculation step may determine the velocity of the target ion from the first information, determine the diffusion coefficient of the target ion from the second information, and calculate the mass or charge of the target ion based on the velocity and diffusion coefficient.

[0096] (Item 20) One aspect of the ion analysis device according to the present invention is an ion analysis device for carrying out the ion analysis method described in Item 1, comprising: an ionization unit that ionizes a measurement target; an analysis chamber having an internal movement space on which gravity acts and which is adjusted to a predetermined gas pressure such that particles can undergo Brownian motion; an electric field forming unit that selectively forms an electric field in the movement space; an ion injection unit that injects target ions, which are ions generated in the ionization unit or ions derived from the generated ions, into the movement space; a measurement execution unit that acquires, for the target ions injected into the movement space, first information regarding the movement due to the effects of gravity and the electric field and second information regarding Brownian motion; and an arithmetic processing unit that calculates the mass or charge of the target ions based on the first information and the second information.

[0097] The ion analysis method described in paragraph 1 and the ion analyzer described in paragraph 20 observe the Brownian motion of ions and the motion of ions due to the action of gravity or the action of both gravity and an electric field, typically falling motion, and calculate the mass or charge of the ions based on information about these multiple motions. As described above, in CDMS, in order to detect the induced charge when the ions to be observed pass through, it is necessary to prevent multiple ions from passing near the detection unit simultaneously or in close proximity in time. However, the ion analysis method described in paragraph 1 and the ion analyzer described in paragraph 20 can effectively obtain information about the motion of individual ions unless the ion density is extremely high. This makes it possible to determine the mass and / or charge of ions of large molecules without performing prior operations that substantially isolate the ions. Furthermore, the ion analysis method described in paragraph 1 and the ion analyzer described in paragraph 20 perform measurements in an analysis chamber containing a gas of a certain density that generates Brownian motion of particles, thereby eliminating the need for an extremely high degree of vacuum and advantageously reducing analysis costs.

[0098] (4) The ion analysis method described in paragraph 1 may further include an m / z measurement step for measuring an m / z value for the target ion whose charge number z is not yet determined, and a second calculation step for determining a mass value more accurate 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.

[0099] Here, the "m / z measurement step" does not necessarily have to involve calculating the m / z value itself, but also includes selecting ions having a specific m / z value. Specifically, for example, it can include an operation of selecting only ions having a specific m / z value in the ion trap and leaving them inside, or conversely, selecting only ions having a specific m / z value and ejecting them from the ion trap. According to the ion analysis method described in Section 4, accurate mass can be determined even if the accuracy of the mass or charge calculated in the calculation step is not sufficiently high.

[0100] (Item 5) In the ion analysis method described in item 1, the measuring step may acquire the first information in a state where the electric field is zero.

[0101] When an electric field is not used, it is not possible to obtain information about the charge of the ion, but it is possible to calculate the mass of the ion using the first information and the second information.

[0102] (Item 6) In the ion analysis method described in Item 1, the measurement step may be to obtain first information regarding the motion of the target ion under an effective gravitational acceleration apparently adjusted by the action of an electric field in a state where the electric field is substantially present.

[0103] (Item 7) In the ion analysis method described in item 6, the measuring step may acquire the first information in the presence of an electric field that cancels the action of gravity.

[0104] In the ion analysis methods described in paragraphs 6 and 7, the falling speed of ions can be reduced by, for example, reducing the apparent effective gravitational acceleration, which increases the residence time of ions in the moving space, i.e., the time for observing the motion of target ions, thereby improving the accuracy of observing the motion and, in turn, improving the accuracy of the mass and charge calculated based on the observation results.

[0105] (Item 8) In the ion analysis method described in Item 1, the measurement step can use an optical technique to obtain first information regarding the movement of the target ions introduced into the movement space due to the action of gravity or the action of gravity and an electric field, and second information regarding Brownian motion.

[0106] Here, the optical technique for obtaining information on the movement of the target ions is, for example, the acquisition of a microscopic image of a predetermined area where one or more ions are present using one or more cameras. Specifically, particularly useful techniques include detecting scattered light by ions in response to irradiated light, detecting fluorescence emitted by ions in response to irradiation with excitation light, etc. Alternatively, it is also possible to use a dynamic light scattering method, which is used to measure the particle size of fine particles in liquid.

[0107] (Item 9) In the ion analysis method described in Item 8, the measurement step can be configured to irradiate light onto ions in the movement space and detect light scattered by or emitted from the ions, thereby determining the movement of the ions.

[0108] Here, it goes without saying that the technique for detecting scattered light also includes dynamic light scattering (DLS). According to the ion analysis method described in paragraph 9, the movement of individual ions in the movement space can be grasped with high accuracy. In particular, by irradiating ions with light of a short wavelength and detecting the scattered light, it is possible to detect not only large ions but also relatively small ions, and the range of m / z values ​​of the ions to be measured can be widened.

[0109] (Item 10) In the ion analysis method described in Item 1, the ion injection step can be configured to inject the target ions into the movement space by temporarily holding the target ions in an ion holding section provided outside the movement space and then releasing the ions at a predetermined timing.

[0110] (Item 11) In the ion analysis method described in Item 10, the ion holding unit may be an ion trap that is composed of a plurality of electrodes and can hold ions in a space surrounded by the plurality of electrodes by applying a rectangular wave voltage to at least one of the plurality of electrodes.

[0111] According to the ion analysis methods described in paragraphs 10 and 11, multiple ions can be injected into the movement space from approximately the same position at approximately the same time. This allows for efficient measurement. Furthermore, since measurements can be performed repeatedly at regular time intervals, this is convenient for measuring ions derived from a continuously supplied sample. Furthermore, according to the ion analysis methods described in paragraphs 9 and 10, ions can be caused to fall from a state in which the initial energy of the ions in the direction of gravity is approximately zero, allowing for highly accurate measurement.

[0112] (Item 12) In the ion analysis method described in Item 10, the ion injection step can include discriminating the ions held in the ion trap according to their m / z values, and then injecting the ions remaining as a result of the discrimination into the migration space.

[0113] According to the ion analysis method described in paragraph 12, even if ions derived from various components are generated in the ionization step, it is possible to narrow down the ions derived from a specific component and measure the mass and charge of the ions, thereby improving the accuracy of measuring the mass and charge.

[0114] (Item 13) Another aspect of the ion analysis method according to the present invention includes: an ionization step of ionizing a measurement object; an ion injection step of dropping target ions, which are ions obtained in the ionization step or ions derived from the ions obtained in the ionization step, into a movement space that is adjusted to a predetermined gas pressure, is subjected to gravity, and in which an electric field is formed that acts on the ions in a direction perpendicular or oblique to the direction of gravity; a separation step of spatially separating the target ions injected into the movement space from other ions by utilizing the movement of ions in the movement space that reflects differences in size and mass of ions due to the action of gravity and the movement that reflects differences in charge of ions due to the action of the electric field; and a detection step of detecting at least a portion of the target ions separated in the separation step.

[0115] (Item 21) Another aspect of the ion analysis apparatus according to the present invention is an apparatus for carrying out the ion analysis method described in Item 13, comprising: an ionization unit that ionizes a measurement target; an analysis chamber having an internal movement space adjusted to a predetermined gas pressure and subjected to gravity; an electric field forming unit that selectively forms an electric field inside the movement space that acts on ions in a direction perpendicular or oblique to the direction of gravity; an ion injection unit that injects target ions, which are ions generated in the ionization unit or ions derived from the generated ions, into the movement space; and a detection unit that detects at least a portion of the target ions spatially separated from other ions, based on the movement of the ions in the movement space that reflects the difference in size and mass of the ions due to the effect of gravity and the movement that reflects the difference in charge of the ions due to the effect of the electric field.

[0116] According to the ion analysis method described in paragraph 13 and the ion analysis apparatus described in paragraph 21, even ions of large molecules can be separated and detected according to their mass and charge by both the action of gravity and the action of an electric field acting in a direction different from the direction of gravity. This ion analysis method and apparatus also make it possible to determine either or both of the mass and charge of ions of large molecules without performing any prior operation to substantially isolate the ions. Furthermore, in particular, this ion analysis method and apparatus separates ions spatially, i.e., physically, according to their mass and charge, making it easy to detect ions and also enabling operations such as selectively extracting some of the separated ions (e.g., ions in a specific valence range) for more detailed measurements.

[0117] (Item 14) In the ion analysis method described in item 13, the electric field formed in the movement space can be a DC electric field that acts on the ions in a direction perpendicular to the direction in which gravity acts.

[0118] According to the ion analysis method described in paragraph 14, ions having the same mass will fall to different positions in the movement space depending on their charge states, which makes it easy to identify the charge states and simplifies the process of calculating the charge states.

[0119] (Item 15) The ion analysis method described in Item 13 may further include an m / z measurement step of measuring the m / z value of the target ion whose valence z is undetermined; and an arithmetic step of 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 the result and the m / z value obtained in the m / z measurement step.

[0120] According to the ion analysis method described in item 15, the accurate mass of a target ion can be easily determined.

[0121] (Item 16) In the ion analysis method described in Item 13, the detection step can obtain information related to the slope of the fall trajectory of the target ion, and the valence number z of the target ion can be obtained based on the information.

[0122] According to the ion analysis method described in paragraph 16, the charge number can be easily calculated by, for example, analyzing a photographed microscopic observation image.

[0123] (Item 17) In the ion analysis method described in Item 13, the ion injection step can be configured to inject the target ions into the movement space by temporarily holding the target ions in an ion holding section provided outside the movement space and then releasing the ions at a predetermined timing.

[0124] (Item 18) In the ion analysis method described in Item 17, the ion holding unit may be an ion trap that is composed of a plurality of electrodes and can hold ions in a space surrounded by the plurality of electrodes by applying a rectangular wave voltage to at least one of the plurality of electrodes.

[0125] According to the ion analysis methods described in paragraphs 17 and 18, multiple ions can be injected into the movement space from approximately the same position at approximately the same time. This allows for efficient measurement. Furthermore, since measurements can be repeated at regular time intervals, it is convenient for measuring ions derived from a continuously supplied sample. Furthermore, according to the ion analysis methods described in paragraphs 16 and 17, ions can be caused to fall from a state in which the initial energy of the ions in the direction of gravity is approximately zero, allowing for highly accurate measurement.

[0126] (Item 19) In the ion analysis method described in Item 18, the ion injection step can include discriminating the ions held in the ion trap according to their m / z values, and then injecting the ions remaining as a result of the discrimination into the migration space.

[0127] According to the ion analysis method described in paragraph 19, even if ions derived from various components are generated in the ionization step, it is possible to narrow down the ions derived from a specific component, measure the valence of the ions, and examine the valence distribution.

[0128] DESCRIPTION OF SYMBOLS 1...Ion supply unit 2, 8...Ion trap 20...Inlet end cap electrode 20a...Ion entrance aperture 21...Exit end cap electrode 21a...Ion exit aperture 22...Ring electrode 22a...Observation aperture 3, 6, 7...Ion drift unit 30, 60...Ion movement space 60...Ion movement space 31, 32, 61, 62...Plate-shaped electrodes 31a...Ion passage aperture 4, 4X...Detection unit 5...Voltage generation unit 50...Trap voltage generation unit 51...End cap voltage generation unit 52...Drift unit voltage generation unit 53...Drift unit voltage generation unit 58...Quadrupole voltage generation unit 63...Auxiliary electrode 64...Aperture plate 64a...Aperture opening 9...Quadrupole mass separator 10...Data processing unit

Claims

1. An ion analysis method comprising: an ionization step of ionizing an object to be measured; an ion injection step of injecting target ions, which are ions obtained in the ionization step or ions derived from the obtained ions, into a movement space in which the gas pressure is adjusted to a predetermined range in which the particles can undergo Brownian motion and in which gravity and an electric field can act; a measurement step of acquiring first information regarding the movement of the target ions introduced into the movement space due to the action of gravity or the action of gravity and an electric field, and acquiring second information regarding the Brownian motion; and a calculation step of calculating the mass or charge of the target ions based on the first and second information obtained in the measurement step.

2. The ion analysis method according to claim 1, wherein said measuring step obtains second information regarding Brownian motion of the target ion in a direction different from the direction in which gravity acts.

3. The ion analysis method according to claim 1, wherein in the calculation step, the velocity of the target ion is calculated from the first information, the diffusion coefficient of the target ion is calculated from the second information, and the mass or charge of the target ion is calculated based on the velocity and diffusion coefficient.

4. The ion analysis method according to claim 1, further comprising: an m / z measurement step for measuring an m / z value for the target ion whose charge z is undetermined; and a second calculation step for determining a mass value more accurate than the mass value obtained in the calculation step, or for 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 according to claim 1, wherein the first information is acquired in the measuring step in a state where the electric field is zero.

6. The ion analysis method according to claim 1, wherein the measuring step obtains first information regarding the motion of the target ion under an effective gravitational acceleration apparently adjusted by the action of a substantial electric field in the presence of the electric field.

7. The ion analysis method according to claim 6, wherein in said measuring step, the first information is obtained in the presence of an electric field that cancels the effect of gravity.

8. The ion analysis method according to claim 1, wherein the measurement step uses an optical technique to obtain first information regarding the motion of the target ions introduced into the movement space due to the action of gravity or the action of gravity and an electric field, and to obtain second information regarding Brownian motion.

9. The ion analysis method according to claim 8, wherein in the measurement step, the movement of the ions is grasped by irradiating the ions in the movement space with light and detecting light scattered by the ions or light emitted from the ions.

10. The ion analysis method according to claim 1, wherein in the ion injection step, the target ions are injected into the movement space by temporarily holding the target ions in an ion holding section provided outside the movement space and then releasing the ions at a predetermined timing.

11. The ion analysis method according to claim 10, wherein the ion holding section is an ion trap composed of a plurality of electrodes and capable of holding ions in a space surrounded by the plurality of electrodes by applying a rectangular wave voltage to at least one of the plurality of electrodes.

12. The ion analysis method according to claim 10, wherein in the ion injection step, the ions held in the ion trap are discriminated according to their m / z values, and ions remaining as a result of the discrimination are then injected into the migration space.

13. An ion analysis method comprising: an ionization step of ionizing a measurement target; an ion injection step of dropping target ions, which are ions obtained in the ionization step or ions derived from the ions, into a movement space that is adjusted to a predetermined gas pressure, is subjected to gravity, and in which an electric field is formed that acts on the ions in a direction perpendicular or oblique to the direction of gravity; a separation step of spatially separating the target ions injected into the movement space from other ions by utilizing ion movement in the movement space that reflects differences in ion size and mass due to the effect of gravity and movement that reflects differences in ion charge due to the effect of the electric field; and a detection step of detecting at least a portion of the target ions separated in the separation step.

14. The ion analysis method according to claim 13, wherein the electric field formed in the movement space is a DC electric field acting on the ions in a direction perpendicular to the direction of gravity.

15. The ion analysis method according to claim 13, further comprising: an m / z measurement step of measuring an m / z value for the target ion where the valence z is undetermined; and a calculation step of 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 the result and the m / z value obtained in the m / z measurement step.

16. The ion analysis method according to claim 13, further comprising the steps of: determining information relating to the gradient of the fall trajectory of the target ion by said detection step; and determining the charge number z of the target ion based on said information.

17. The ion analysis method according to claim 13, wherein in the ion injection step, the target ion is injected into the movement space by temporarily holding the target ion in an ion holding section provided outside the movement space and then releasing the ion at a predetermined timing.

18. The ion analysis method according to claim 17, wherein the ion holding section is an ion trap composed of a plurality of electrodes and capable of holding ions in a space surrounded by the plurality of electrodes by applying a rectangular wave voltage to at least one of the plurality of electrodes.

19. The ion analysis method according to claim 18, wherein in the ion injection step, the ions held in the ion trap are discriminated according to their m / z values, and ions remaining as a result of the discrimination are then injected into the migration space.

20. An ion analysis apparatus comprising: an ionization unit that ionizes a measurement target; an analysis chamber having an internal movement space in which gravity acts and which is adjusted to a predetermined gas pressure such that particles can undergo Brownian motion; an electric field forming unit that selectively forms an electric field in the movement space; an ion injection unit that injects target ions, which are ions generated in the ionization unit or ions derived from the generated ions, into the movement space; a measurement execution unit that obtains first information regarding the movement of the target ions due to the effects of gravity and the electric field and second information regarding the Brownian motion of the target ions injected into the movement space; and an arithmetic processing unit that calculates the mass or charge of the target ions based on the first information and the second information.

21. An ion analysis apparatus comprising: an ionization unit that ionizes a measurement target; an analysis chamber having an internal movement space adjusted to a predetermined gas pressure and in which gravity acts; an electric field forming unit that selectively forms, inside the movement space, an electric field that acts on ions in a direction perpendicular or oblique to the direction of gravity; an ion input unit that inputs target ions, which are ions generated in the ionization unit or ions derived from the generated ions, into the movement space; and a detection unit that detects at least a portion of the target ions spatially separated from other ions by the movement of ions in the movement space that reflects differences in size and mass of ions due to the effect of gravity and the movement that reflects differences in charge of ions due to the effect of the electric field.

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