JP2003130848A - Method and apparatus for mass spectrometry of solution - Google Patents

Abstract

PROBLEM TO BE SOLVED: To generate positive or negative ions with high efficiency by disposing an electrode in the neighborhood of a spray capillary to apply electric field to liquid and spraying liquid in a sonic spray ionizing method. SOLUTION: An ion source has a capillary for spraying liquid, and an orifice in which the tip site of the capillary is inserted. Gas flows in the neighborhood of the tip of the capillary so that the flow velocity of the gas is equal to about sonic velocity, and a voltage is applied between the liquid in the capillary and the orifice or the electrode in the neighborhood of the capillary. The polarity of ions generated can be controlled in accordance with the polarity of the voltage applied to the electrode. Multiply-charged ions of materials having large mass such as protein or the like can be generated efficiently with high reproducibility.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to an ion source and a mass spectrometer using the same, and particularly to an ion source suitable for ionizing a sample existing in a liquid and introducing the sample into a mass spectrometer, and a mass using the same. The present invention relates to an analyzer, a liquid chromatograph / mass spectrometer coupling device, and a Capillary electrophoresis / mass spectrometer coupling device.

[0002]

2. Description of the Related Art Analytical Chemistry 66
(1994) p. 4557 to p. 4559 (Ana
Lytical Chemistry, 66 (199
4) pp 4557-4559) has a description about the sonic spray ionization method. A gas is caused to flow coaxially from the outside of the capillary, and the liquid is sprayed at the end of the capillary to stably generate ions and charged droplets. When the gas flow velocity at the tip of the capillary is sonic, the amount of positive and negative ions generated by atomization becomes maximum.
In this case, neither electric field nor voltage is applied to the liquid. The generated atomizing gas is neutral as a whole, and the amount of positive ions and the amount of negative ions are equal.

Also, Journal of Physical Chemistry 88 (1984), pages 4451 to 4
Page 459 (Journal of Physical
Chemistry, 88 (1984) pp4451-4459) or US Pat. No. 5,130,538 describes electrospray ionization. A high voltage of 2.5 kV or more is applied between the metal capillary into which the liquid is introduced and the counter electrode (ion intake port of the mass spectrometer). That is, a high voltage is applied to the liquid. As a result, charged droplets are sprayed from the liquid toward the counter electrode in the space to which the electric field is applied (electrostatic spray phenomenon), and ions are generated from the charged droplets. The polarities of the charged droplets and ions match the polarities of the applied voltage. The electrospray ionization method can generate ions having a large charge number and is often used for analysis of proteins and the like. The liquid flow rate is usually used in the range of 0.001 to 0.01 ml / min.

Analytical chemistry 5
9 (1987) pages 2642 to 2646 (An.
analytical Chemistry, 59 (198)
7) pp2642-2646) or US Pat. No. 4861
No. 988 describes the ion spray ionization method. As with the electrospray ionization method, a high voltage of 2.5 kV or more is applied between the liquid at the end of the spray capillary and the counter electrode (ion intake port of the mass spectrometer). As a result, ions or charged droplets are sprayed from the liquid toward the counter electrode in the space to which the electric field is applied.
(Electrostatic atomization phenomenon) Unlike the electrospray ionization method, in the ion spray ionization method, a gas is caused to flow outside the atomization capillary in order to promote vaporization of charged droplets. Therefore, since the same electrostatic atomization phenomenon is used to generate ions, the generated ion species are exactly the same as in the electrospray ionization method. The polarities of the charged droplets and ions match the polarities of the applied voltage. But,
The liquid flow rate is used in the range of 0.01 to 1 ml / min, which is higher than that of the electrospray ionization method.
The maximum gas flow velocity is 216 m / s.

In the case of liquid gas spraying, a description is given in which an electrode is provided in the vicinity of the spraying part and charged droplets or ions are generated by applying a high voltage of 3.5 kV thereto. Journal of Chemical Physics 71 (1979) Fourth
Pages 451 to 4463 (Journal of C
chemical physics, 71 (1979) pp
4451-4463) or U.S. Pat. No. 4300044.
No. In this example, both the liquid and the gas are discharged from an injection needle having an inner diameter of 0.3 mm. However, unlike the above-described sonic spray ionization method and ion spray ionization method, the direction in which the liquid is discharged and the direction of the gas flow are orthogonal to each other. The velocity of the gas stream is unknown.

When a mixed solution containing a large number of substances mixed therein is introduced into a mass spectrometer, the obtained mass spectrum may be too complicated to identify the substances. Therefore, in the analysis of the mixed solution, a method is used in which the mixed solution is first separated using a means such as a liquid chromatograph or a capillary electrophoresis device, and the extract is introduced into the mass spectrometer. That is, there are a liquid chromatograph / mass spectrometer coupling device and a capillary electrophoresis / mass spectrometer coupling device that realize the separation and analysis of a mixed solution online. In a liquid chromatograph, a solution is separated by causing a solution to flow through a column filled with a packing material at a constant flow rate by a pump. Therefore, the potential of the extract is the ground potential. On the other hand, in the capillary electrophoresis apparatus, a high voltage of about 20 to 30 kV is applied to both ends of the capillary with respect to the solution introduced into the capillary to separate the solution by electrophoresis. Normally, the potential on the outlet side of the capillary is set to the ground potential, and the potential of the extract is set to the ground potential.

[0007]

In a mass spectrometer, ions are analyzed by m / z, which is a value obtained by dividing the mass m of an ion by its charge number z. A mass spectrometer with a very large upper limit of mass analysis range is not realistic because the entire apparatus is huge and expensive. Therefore, in a general mass spectrometer, the upper limit of the mass analysis range is about m / z = 1000 to 2000.

In the above-mentioned conventional technique, ions are mainly generated from minute charged droplets. In the prior art relating to the electrospray ionization method and the ion spray ionization method, charged droplets having a high charge density are generated,
It is possible to generate multiply-charged ions having a charge number z of 3 or more. Therefore, even a substance having a huge mass such as a protein having a mass m of about tens of thousands can be analyzed because the value of m / z falls within the mass analysis range. However, in the conventional technology relating to the sonic spray ionization method and the fourth conventional technology, charged droplets having a low charge density tend to be generated, and ions having a small charge number z tend to be generated. That is,
Generation and mass spectrometry of monovalent and divalent ions such as peptides are possible. However, in the analysis of proteins with a very large mass number m, it is not possible to generate multiply charged ions with a charge number z of 3 or more, so the m / z value deviates from the mass analysis range and the mass It has the drawback that it cannot be analyzed.

Further, in the prior art relating to the electrospray ionization method or the ion spray ionization method, a high voltage is applied to the liquid, and ions or charged droplets are generated by the electrostatic atomization phenomenon. The droplets generated by this phenomenon include minute charged droplets having a high charge density, but also include large charged droplets of a micron order having a low charge density. The large charged droplets have too large a mass as compared with the charge amount, and it is difficult to control the movement of the charged droplets by an electric field or a magnetic field. Therefore, when mass analysis is performed using a quadrupole mass spectrometer or a quadrupole ion trap mass spectrometer, large charged droplets reach the detection unit without being mass separated and are detected as random noise. In particular, in the related art related to the ion spray ionization method,
Since the liquid flow rate is high, the generation of large charged droplets is remarkable. A similar problem exists in the above-mentioned fourth prior art.
In the fourth prior art, the liquid is atomized by the gas flow. However, in the spraying section, the direction in which the liquid is discharged and the direction in which the gas flows are orthogonal to each other. Therefore, at the end of the injection needle from which the liquid is discharged, there is a difference in gas flow velocity between the upstream side and the downstream side of the gas flow, and the droplet size generated on the downstream side becomes larger in size. There's a problem. In addition, electrodes (US Pat. No. 430004)
The electrode 27) in FIG. 2 of No. 4 is a rod. Therefore, the charge densities of the liquid droplets generated in the region close to the electrode and the region far from the electrode in the liquid spray portion are different. Therefore, many large droplets having a low charge density are generated. In the fourth prior art described above, a counter gas flow is generated at the ion intake port of the mass spectrometer to promote vaporization of droplets, but sufficient vaporization is impossible. As described above, ions are mainly generated from minute charged droplets, and it is known that the ion generation efficiency is higher as the droplet size is smaller. The efficiency of ion generation from large charged droplets is so low that it can be ignored. Therefore, there is a problem that due to the generation of large charged droplets, the ion detection sensitivity is significantly lowered due to the reduction of signal intensity and the increase of noise level.

Further, in the prior art relating to the electrospray ionization method and the ion spray ionization method, it is necessary to adjust the position of the ion source every time the spraying is started to optimize the ion intensity, and the operation is complicated. There are inherent problems with the electrostatic spray phenomenon. This is because the electrostatic atomization phenomenon has a property that the shape of the atomized gas generated is significantly different due to the contamination or wetting of the atomizing capillary or the counter electrode (mass spectrometer ion intake port). Therefore, the conventional techniques relating to the electrospray ionization method and the ion spray ionization method have problems that they require skill and have extremely low operability.

Further, in the prior art relating to the electrospray ionization method and the ion spray ionization method, a liquid chromatograph / mass spectrometer coupling device in which a solution separating means such as a liquid chromatograph or a capillary electrophoresis device is coupled on the upstream side, When used in a capillary electrophoresis / mass spectrometer combination device, a high voltage is applied to the entire solution separation means, so there is a risk of electric shock during handling. The potential of the entire solution separation means may be set to the ground potential for use, but in this case, since a kind of electrophoresis is realized in the capillary, reproducibility of ion generation and ion analysis is remarkably reduced. Therefore, there is a problem that the liquid chromatograph / mass spectrometer coupling device and the capillary electrophoresis / mass spectrometer coupling device do not exhibit sufficient functions in terms of operation and function.

[0012]

In order to achieve the above object, a flow passage for flowing a liquid to a predetermined position, and at least a part of the flow passage for the liquid is an insulator, and the liquid passage is provided through the insulator. There is provided an ion source having an electric field applying means for applying an electric field and a spraying means arranged downstream of the liquid to which the electric field is applied and spraying the liquid to generate a charged gas.

In order to achieve the above object, a flow passage for flowing a liquid to a predetermined position, and at least a part of the flow passage for the liquid is an insulator, and an electric field is applied through the insulator. Means for applying electric field, a gas supply unit for supplying gas, a supply port for supplying gas from the gas supply unit, an injection port for discharging gas, and a holding unit for holding the flow passage, An ion source having is provided.

Further, in order to achieve the above object, a separation means for separating the sample liquid containing the mixture, a flow passage for flowing the separated sample liquid to a predetermined position, and the flow of the sample liquid. At least a part of the passage is an insulator, and an electric field applying unit that applies an electric field through the insulator, a gas supply unit that supplies gas, a supply port that supplies gas from the gas supply unit, and discharge the gas. There is provided a mass spectroscope including a spraying unit that is a chamber including an injection port and a holding unit that holds the flow passage, and an analyzing unit that performs mass analysis and analysis of the sprayed gas.

In order to achieve the above object, a separation means for separating the sample liquid containing the mixture, a flow passage for flowing the separated sample liquid to a predetermined position, and the flow of the sample liquid At least a part of the passage is an insulator, and an electric field applying unit that applies an electric field through the insulator, a gas supply unit that supplies gas, a supply port that supplies gas from the gas supply unit, and discharge the gas. There is provided a mass spectrometer including a spraying unit, which is a chamber including an injection port and a holding unit that holds the flow passage, and a quadrupole mass spectrometer that performs mass analysis and analysis of the sprayed gas.

In order to achieve the above object, a separation means for separating the sample liquid containing the mixture, a flow passage for flowing the separated sample liquid to a predetermined position, and the flow of the sample liquid. At least a part of the passage is an insulator, and an electric field applying unit that applies an electric field through the insulator, a gas supply unit that supplies gas, a supply port that supplies gas from the gas supply unit, and discharge the gas. There is provided a mass spectrometer including a spraying unit, which is a chamber including an injection port and a holding unit that holds the flow passage, and a quadrupole trap mass spectrometer that performs mass analysis and analysis of the sprayed gas.

In order to achieve the above object, a separating means for separating the sample liquid containing the mixture, a detecting means for detecting the characteristics of the separated sample liquid, and a predetermined sample liquid for the separated sample liquid. A flow passage for flowing to the position, an electric field applying means for applying at least a part of the flow passage of the sample liquid to the sample liquid through the insulator, a gas supply unit for supplying a gas, and the gas A spraying means which is a room consisting of a supply port for supplying gas from a supply part, an injection port for discharging gas, and a holding part for holding the flow passage, and an analysis means for mass-analyzing and analyzing the sprayed gas, There is provided a mass spectrometer having a control unit for controlling the electric field applying unit and the analyzing unit by a signal from the detecting unit.

Further, in order to achieve the above object, a part or all of an injection port for spraying a liquid to generate polyvalent ions having an electric charge of at least trivalent, and an injection port of the injection port. There is provided a mass spectrometer having a first electrode arranged around the second electrode and a second electrode arranged to face the first electrode and generate a uniform electric field between the first electrode and the first electrode. It

In order to achieve the above object, a liquid separating means for separating a liquid, a metal for defining a potential of the liquid separated by the liquid separating means, a flow passage to which the liquid is supplied, and the flow passage. An electric field applying means electrically isolated from the flow passage for applying an electric field to the liquid flowing in the passage,
A part that is disposed downstream of the liquid to which the electric field is applied and that has a spraying means that sprays the liquid to generate charged droplets and that sprays an ion source to generate multivalent ions having an electric charge of at least three Alternatively, a jet port formed entirely of an insulator, a first electrode arranged around the jet port, and a first electrode arranged so as to face the first electrode are uniformly provided between the jet electrode and the first electrode. Provided is a mass spectrometer coupled with a liquid separating means, which comprises a mass spectrometer having a second electrode for generating a different electric field.

Further, in order to achieve the above object, between a first electrode arranged around an injection port for spraying a liquid and a second electrode arranged so as to face the first electrode. Provided is a mass spectrometry method in which multiply charged ions having an electric charge of at least trivalent are generated in a uniform electric field generated in and are analyzed by a mass spectrometer.

In order to achieve the above object, a separation step of separating the liquid, a step of applying an electric field to the separated liquid having a fixed potential, and a first step arranged around the injection port. Mass to be mass-analyzed by multiply-charged ions having at least trivalent charges passing through a uniform electric field generated between an electrode and a second electrode arranged to face the first electrode. A mass spectrometric method comprising the steps of supplying to an analyzer, mass spectrometric analysis, and analysis.

Further, in order to achieve the above-mentioned object, between a first electrode arranged around an injection port for spraying a liquid and a second electrode arranged so as to face the first electrode. At least 3 charges are generated in the uniform electric field generated in
An electric field application step of changing the polarity of an electric field applied to the liquid according to the polarity of multivalent ions having a valence is provided.

In order to achieve the above object, an electric field applying step of changing the polarity of an electric field applied to a liquid, a first electrode arranged around an injection port for spraying the liquid, and the first electrode A step of generating multiply-charged ions having a polarity corresponding to the polarity of the electric field in which a charge is at least trivalent in a uniform electric field generated between the second electrode arranged to face the electrode; A mass spectrometry method is provided including.

The object of the present invention is to analyze a substance having a large mass number, so that a highly efficient ion source, a highly sensitive mass spectrometer or a high sensitivity ion source capable of producing and analyzing multiply charged ions having a charge number z of 3 or more can be realized. It is to provide an efficient ionization method and a highly sensitive mass spectrometry method.

Another object of the present invention is to provide a high-sensitivity mass spectrometer and a high-sensitivity mass spectrometric analysis method in which large charged droplets that cause a reduction in ion detection sensitivity are not generated so much.

Still another object of the present invention is to provide a highly efficient ion source, a highly sensitive mass spectrometer or a highly efficient ionization method, and a highly sensitive mass spectrometry which have high operability.

Still another object of the present invention is that the potential of the liquid can be set to the ground potential, is safe, has high reproducibility of ion generation and ion analysis, and is a highly efficient ion source and highly sensitive mass spectrometer. Alternatively, it is to provide a highly efficient ionization method and a highly sensitive mass spectrometry method.

In the conventional sonic spray ionization method,
The atomization initially produces electrically neutral droplets. Although the droplet contains the same number of positive and negative ions, the density distribution of the positive and negative ions in the vicinity of the surface is significantly different from that in the central portion due to the surface potential determined by the combination of the droplet and the surrounding gas. Further, the sonic gas flow separates fine droplets from the droplet surface. Since the number of positive and negative ions is different in these fine droplets, the charge density is low, but the entire droplet is charged. Ions are generated from fine charged droplets by the ion evaporation process or complete vaporization of charged droplets.

In the ionization method and ion source of the present invention, an electric field is applied to the liquid introduced into the capillary in the sonic spray ionization method from an electrode installed outside. For example, when a negative voltage is applied, the positive ion density in the vicinity of the liquid surface is significantly higher than the negative ion density. Therefore, when the liquid is sprayed by the gas flow, positively charged droplets are preferentially generated, and the spray gas is positively charged. In the droplet, Coulomb repulsive force causes positive ions to concentrate near the surface. The charge density of these charged droplets is high as compared with the charge density of the charged droplets generated in the conventional sonic spray ionization method. Furthermore, when the sonic gas flow separates the fine droplets from the surface of the droplets, fine droplets with extremely high charge density are generated. It is known that ions are generated from fine droplets by the ion evaporation process or complete evaporation of charged droplets. In the former case, the ion generation efficiency of multiply charged ions is generally much lower than that of monovalent ions and is ignored, but when the charge density of the droplets is high, the Coulomb repulsion in the droplets is significantly large. Therefore, the ion generation efficiency of multiply charged ions also becomes high. Therefore, multiply charged ions can also be generated. In the ionization method of the present invention, the density of positive or negative ions near the surface of the liquid can be made extremely high before being sprayed. Therefore, it is meaningless to apply an electric field to the droplets generated by the spray. It is necessary to apply an electric field to the liquid before it is sprayed.

Further, in the mass spectrometer and the mass spectrometric method of the present invention, the electrostatic atomization phenomenon is not used for the ion generation, and therefore the mass spectrometer ion intake port and the atomization capillary are prevented from becoming dirty.
Ion formation is not affected by wetting. Therefore, the device is easy to handle and the reproducibility of ion generation is high.

Further, in the mass spectrometer and the mass spectrometry method of the present invention, the potential of the liquid introduced into the ion source can be set to the ground potential. This makes it easy to connect a capillary electrophoresis device, a liquid chromatograph device, or the like upstream of the ion source. Further, the bond does not impair the reproducibility of ion generation.

The method of applying an electric field from the outside to a liquid includes
There is a method in which an insulator such as quartz or resin is used as the material of the capillaries and a voltage is applied to a metal orifice into which the ends of the capillaries are inserted. Yet another way is
There is a method in which an insulator is used as the material of the capillary and the outer surface near the end of the capillary is metal-coated and a voltage is applied to the outer surface of the capillary. Furthermore, there is also a method of inserting an insulator capillary into a metal tube to which a voltage is applied. In either method, it is necessary to keep the distance from the liquid surface to the electrode constant for highly efficient ion generation. When a conductor such as a metal is used as the material of the capillaries, it is necessary to apply a high voltage to the electrodes because the metal capillaries have an electric shield effect.

[0033]

1 is a block diagram of an ion source according to an embodiment of the present invention. The liquid is introduced into the capillary 2 from the flow passage 1. The tip of the capillary 2 is coaxially inserted into the orifice 3. The gas supplied from the gas supply unit 4 is introduced into the ion source main body 6 through the gas introduction pipe 5, and is discharged to the outside through the orifice 3. Orifice 3
Is made of metal, and a voltage is applied from the power source 7 between the orifice 3 and the metal tube 8. Since there is no conduction between the orifice 3 and the liquid, no current flows, but an electric field is applied to the liquid. The liquid reaching the end of the capillary 2 is atomized by gas. Ions and fine charged droplets are generated in the spray gas.

FIG. 2 shows a sectional view of an ion source according to an embodiment of the present invention. The liquid is introduced at a flow rate of 5 to 100 microliters per minute into the metal tube 8 whose potential is set to the ground potential, and then the quartz capillary 2 (inner diameter 0.1 mm, outer diameter 0.2
mm). Orifice 3 (0.4 mm inner diameter, 1 m thickness)
The orifice plate 9 with m) is made of duralumin, and a voltage is applied from the power source 7. The capillary 2 is inserted into a Teflon (registered trademark) tube 10 and a stainless tube 11, and is fixed to the stainless tube 11 with an adhesive. Also,
The space between the two stainless steel tubes 11 electrically insulates the liquid from the ion source body 6. The central axes of the capillary 2 and the orifice 3 coincide with each other, and the end of the capillary 2 is exposed from the orifice plate 9 by about 0.2 mm. The stainless tube 11 is also used to fix the capillary 2 to the ion source body 6. When the orifice plate 9 is cooled by adiabatic expansion of gas, the generated droplets may condense and may not be atomized. In this case, large charged droplets are generated as a result, and the amount of ions is reduced. This problem can be avoided by installing a heater in the orifice plate 9 or the ion source body 6 to heat it, or by heating the gas introduced into the ion source with the heater in advance. The gas is introduced into the gas introduction pipe 5 by a gas cylinder or a gas compressor. A gas flow rate control device such as a mass flow controller or a purge meter is installed in the middle of the process to control the flow rate of gas used for atomization. Just the gas flow rate is 3 per minute
In the case of liter, the gas flow velocity near the end of the capillary 2 becomes the speed of sound.

It is known that ions are produced from minute charged droplets having a diameter of 10 nanometers or less. In this embodiment, fine charged droplets are generated by gas atomization as in the sonic spray ionization method. The average droplet size generated by gas atomization decreases as the gas flow velocity increases, but the average droplet size increases as the gas flow velocity becomes supersonic. This is because a shock wave is generated. (See FIG. 9) Therefore, when the gas flow velocity is sonic velocity, the average droplet size is minimized, and the most efficient ion generation is performed. The smaller the wall pressure of the capillary 2, the higher the generation efficiency of fine droplets, and consequently the higher the ion generation efficiency.

To generate the sonic flow, assuming that the pressure around the ion source is 1 atm and the thickness of the orifice 3 is ignored, a gas pressure of about 2 atm is required on the upstream side of the orifice 3. However, depending on the thickness of the orifice plate 9, the reduced pressure in the orifice 3 may not be negligible. It is convenient to provide a pressure gauge on the ion source body 6 and adjust the gas pressure to generate a sonic gas flow. In this case, the orifice 3
It is safe to adjust the pressure so that a gas pressure of about 0.5 to 5 atm can be applied upstream. It is also realistic to generate a sonic flow by a gas flow rate adjusting means such as a purge meter or a mass flow meter. If the thickness of the orifice 3 is too thick, the pressure reduction inside the orifice 3 becomes large and it becomes necessary to pressurize unnecessarily. Ion source body 6
It does not need to be thicker than the wall thickness of. Conversely, orifice 3
If the thickness is too thin, the pressure difference between the inside and the outside of the ion source body 6 cannot be sufficiently endured. Therefore, it is realistic that the diameter is 0.5 to 1 mm, which is thicker than the diameter of the capillary 2. Ion source body 6 upstream of the orifice 3
Needs a space to be a gas reservoir so that the gas pressure is not reduced. The size of the space needs to be 5 times or more the diameter of the capillary 2.

FIG. 3 shows a sketch of the ion source according to the embodiment of the present invention shown in FIG. The atomized gas is discharged in the front direction. The cross section of the ion source body 6 in the direction perpendicular to the central axis of the capillary 2 is quadrangular, but it may be circular. The orifice plate 9 has four holes in addition to the orifice 3.
Four screws are used to fix the. The orifice plate 9 is fixed with screws by adjusting the center of the orifice 3 and the center of the capillary 2 under the microscope. In this way, a uniform gas flow is formed at the end of the capillary 2. The dimensions in the figure show that the ion source of the present invention is extremely compact.

FIG. 4 shows the dependency of the total ion current generated by the spray on the voltage applied to the orifice 3. This is the result of an experiment when a solution containing 47.5% methanol / 47.5% water / 5% acetic acid was used as a liquid. It is shown that negative ions and charged droplets are generated when a positive voltage is applied, and vice versa when a negative voltage is applied. That is, it is shown that when a voltage having a polarity opposite to that of ions to be generated is applied, ions having a desired polarity are generated. Also, when the absolute value of the applied voltage is 1 kV or less, the ion current decreases with the increase of the applied voltage, but when the absolute value of the applied voltage is about 1 kV or more, the ion current does not change much and saturates. Is shown. This indicates that the ion density of the same sign near the surface of the liquid just before spraying reached the limit. Therefore, it is practical to set the absolute value of the applied voltage in the range of 1 kV to 2 kV based on the experimental result.

Assuming that the electric conductivity of the liquid is sufficiently high, the potential of the central axis of the capillary 2 can be regarded as 0 V, and the electric field strength is 5000 kV / m (= 1000 V / 0.2 m).
m). Therefore, when the inner diameter of the orifice 3 is larger, or when the electrodes are installed at positions farther from each other, it is necessary to apply a higher voltage.

FIG. 5 shows the result of mass spectrometric analysis of the ions generated when the applied voltage was set to -1 kV. This is a cytoc with a concentration of 1 micromol / liter (mmol / L)
home-C (a kind of protein, molecular weight about 1250
0) A solution (a solvent is 48% methanol water added with 5% acetic acid) was introduced into the capillary at a flow rate of 30 microliter / min, and ions produced by atomization using a sonic gas flow were analyzed by a quadrupole mass spectrometer. It is the result of analysis (mass spectrum). A series of multivalent ions from 13 to 20 valences centering on 16 valent ions (m / z = 766) in which 16 protons are added to a cytochrome-C molecule are clearly shown in the region below m / z = 1000 To be detected.

Up to now, the ion spray ion method and the electrospray ionization method (the ion generation principle is almost the same) have been used for the generation of multiply charged ions. Therefore, ions were generated from the same cytochrome-C solution using the ion spray ion method, and the ions were detected with the same quadrupole mass spectrometer. The result is shown in FIG.
The vertical axis is the same as in the case of FIG. Regarding the multiply charged ions, the same spectrum turn of the mass spectrum was obtained, but the ionic strength was 2.6 compared with the result of Fig. 5.
About twice as low. This corresponds to the ion detection sensitivity obtained by setting the applied voltage to about 700 V in the ion source of the present invention. Further, it is shown in FIG. 6 that the noise level is significantly higher than that in FIG. This noise is because a large amount of large charged droplets are generated in the ion spray ion method as compared with the ionization method of the present invention, and they are detected as random noise. The S / N ratio is about 9 times smaller than that in the case of FIG. Therefore, it is shown that more highly sensitive ion detection can be performed by using the ionization method of the present invention shown in FIG. Further, in FIG. 6, m / z
Many ions originating from the solvent are detected in the region of 300 or less. Familiarity is required to analyze such complex mass spectra.

7 and 8 show myoglobin solution (concentration: 1 micromol / liter, solvent: 48% methanol water with 5% acetic acid added) and hemoglobin solution (concentration: 1 micromol / liter, solvent: 5% acetic acid added). 48% methanol water). myoglobin has a molecular weight of 17
It is 200, and a series of multiply-charged ions having a valence of 18 to 26 centering on a 22-valent ion (m / z = 772) having 22 protons added is detected. hemoglob
in is a protein having a molecular weight of 64500 in which two kinds of two kinds of subunits are bound to each other. In FIG. 8, the mass spectrum of the subunit is observed.

FIG. 9 shows the dependence of the spray gas flow rate F on the relative intensity of the 16-valent ions in FIG. The ion source used is that of the embodiment shown in FIG. The gas flow velocity has a relationship of increasing as the gas flow rate increases. In this experiment, it is already known that the gas flow velocity corresponds to the sound velocity (Mach 1) when the gas flow rate is about 3 liters / min, and the gas flow velocity is Mach 2 or less when it is about 7 liters / min. (Analytical Chemistry 66 (1994) No. 45)
57 to 4559 (Analytical Ch
estry, 66 (1994) pp 4557-45
59)) shows that no ions are generated when the gas flow rate is 1.4 liters / min or less, and the ionic strength increases with an increase in the gas flow rate when the gas flow rate is 1.4 liters / min or more. It is shown that the intensity becomes maximum at about 3 liters / minute, and at flow rates higher than that (supersonic region), the ionic strength decreases due to the generation of shock waves. From this, the dependence of the ion intensity on the gas flow rate is similar to that of the conventional sonic spray ionization method, but the atomized gas produced from the ion source of the present invention is characterized by being positively or negatively charged as a whole. .

As described above, it is also possible to control the gas flow rate by adjusting the gas flow rate F using the flow rate adjusting means. In the example of the ion source shown in FIG.
The area S of the cross section perpendicular to the capillary in the gap between the orifice and the capillary is calculated as 9.4 × 10 −8 m 2. If F is the standard state (20 ° C, 1 atm), F
/ S is estimated to be 250 m / s when F is 1.4 liters / minute, and about 1200 m / s when F is 7 liters / minute. It should be noted that the unit of F / S is velocity, but it is different from gas flow velocity.

It is also possible to control the gas flow rate by the gas pressure in the ion source. In FIG. 9, ion generation starts at about 1.2 atm. Assuming isentropic flow, Mach of about 0.5 at a gas pressure of 1.2 atm and Mach of 1 at about 2 atm are achieved when the thickness of the orifice is sufficiently thin. 7.8 bar is required to achieve Mach 2. Incidentally, Mach 3 requires about 40 atm, which is a problem in practical use. In order to control the gas flow velocity with the gas pressure, it is realistic to adjust the gas pressure within the range of 0.5 to 5 atm.

In this ion source, the orifice diameter is 0.4.
mm, and the outer diameter of the quartz capillary is 0.2 mm. Therefore, the cross-sectional area S between the orifice and the capillary is calculated to be 9.4 × 10 −8 m 3. Therefore, using the parameter F / S, when F is 1.4 liter / min, it is 250 m / s, and the state of sound velocity is 53
It becomes 0 m / s. (Note that the unit of parameter F / S is velocity, but it is not related to the actual gas flow velocity.) Even if F is increased to 7 (F / S = 1200 m / s) or more, The ion generation efficiency does not increase, only the consumption increases. Therefore, the F / S is actually 250-
Used in the range of 1200 m / s. Since S is determined by the structure of the ion source, it is convenient to adjust the gas flow rate F using a flow rate controller such as a mass flow meter or a purge meter.

The gas is used for atomizing the liquid. Therefore, the type of gas hardly contributes to ion generation. Actually, it is convenient to use nitrogen gas. This is because it is inexpensive and is effective for vaporizing droplets because it is dry. Similar effects can be obtained by using air, oxygen, carbon dioxide, a rare gas or the like in addition to nitrogen gas.

FIG. 10 shows a sectional view of an ion source according to another embodiment of the present invention. Quartz capillary 2 (inner diameter
Aluminum is vapor-deposited on the outer surface of a terminal portion 5 mm having a diameter of 0.01 mm and an outer diameter of 0.05 mm, and a voltage is applied from a power source 7. As described in the description of FIG. 4, in order to efficiently generate ions, it is necessary to generate an electric field strength above a certain level.
In this embodiment, the voltage applied to the electrodes can be set to a low voltage as compared with the embodiment of FIG. In this embodiment,
Since the ion source body 6 can be set to the ground potential, the safety during operation is extremely high.

FIG. 11 shows a sectional view of an ion source according to still another embodiment of the present invention. Resin capillary 2
Is inserted into the tubular electrode 12, and a voltage is applied to the electrode 12 from the power supply 7. The resin capillary 2 and the electrode 12 are fixed with an insulating adhesive. If a sufficiently high voltage is applied, the tubular electrode 12 is not always the capillary 2.
Need not be installed coaxially with. However, in the case of the coaxial shape, ion generation is performed with a low applied voltage. Similarly, if a sufficiently high voltage is applied to the electrode 12, the electrode 12 does not necessarily have to be tubular. The applied voltage is much lower when the electrode 12 is placed before the end of the capillary 2 than when it is not.

FIG. 12 shows a sectional view of an ion source according to still another embodiment of the present invention. Quartz capillary 2
Is heated by a heating block 14 which is heated by a heater 13. Therefore, the liquid introduced into the capillary 2 is sprayed by heating. Due to the electric field generated by the electrode 12, the droplets generated by spraying in the capillary 2 are charged, and charged droplets and ions are generated. An infrared laser or a lamp can be used to heat the liquid.

FIG. 13 shows a sectional view of an ion source according to still another embodiment of the present invention. Quartz capillary 2
The liquid introduced into the gas injection unit 1 from the lateral direction at the end thereof.
It is atomized by the gas stream generated from No. 5. in this case,
Capillary 2 produces relatively large charged droplets
The position adjustment of can be done very easily. In the case where it is not particularly necessary to generate ions and generation of charged droplets is sufficient, this embodiment provides an extremely simple charged droplet generation device.

FIG. 14 shows a sectional view of an ion source according to still another embodiment of the present invention. Quartz capillary 2
The liquid introduced into is converted into droplets by the ultrasonic transducer 16. The electric field generated by the electrode 12 causes the droplet to be charged when the droplet is generated. The charged atomizing gas diffuses widely in space. The embodiments shown in FIGS. 12 to 14 have an advantage that ions can be generated without using a gas cylinder, a compressor or the like.

FIG. 15 shows a block diagram of a mass spectrometer according to an embodiment of the present invention. The liquid is introduced into the capillary 2 from the flow passage 1 at a constant flow rate. The tip of the capillary 2 is coaxially inserted into the orifice 3. Nitrogen gas supplied from the gas supply unit 4 is introduced into the ion source body 6 through the gas introduction pipe 5 and is discharged to the outside through the orifice 3. A voltage is applied from the power source 7 to the metal orifice plate 9. As a result, an electric field is applied to the liquid that has reached the end of the capillary 2. The liquid to which the electric field is applied is sprayed under atmospheric pressure according to the gas flow from the orifice 3. Generally, a uniform electric field exists in the space between the orifice plate 9 where the atomized gas is generated and the pores 17 at the mass spectrometer inlet. Ions and charged droplets generated in the atomized gas are introduced into the vacuum through the pores 17 at the entrance of the mass spectrometer, and are mass separated by the electric field or magnetic field in the mass analysis section 18 installed in the high vacuum section. The mass-separated ions are detected by the ion detector 19. The output of the ion detector 19 is the computer 2
Sent to 0 and parsed. The power supply 7 and the mass spectrometer 18 can be controlled by the computer 20. That is, the positive / negative ions generated by the intermittent voltage signal generated by the power supply 7 can be mass-separated in synchronization with the ion generation and the ions can be detected. For example, when the potential of the metal tube 8 is set to the ground potential and a rectangular wave voltage with an amplitude of 1 kV is applied to the orifice plate 9 by the power supply 7 to generate positive and negative ions, the positive and negative ions are analyzed in synchronization with it. You can Whether the capillary 2 or the metal tube 8 and the pores 17 are set to the same potential so that the electric field does not exist in the space where the spray gas is generated, or the potential difference of about 200 V is generated, the ion detection is performed. Is no problem at all.

When the potential of the metal tube 8 is set to the ground potential, the potential of the liquid flowing in the capillary 2 can be set to the ground potential. Therefore, it is easy to connect a solution separation means such as a capillary electrophoresis device or a liquid chromatograph to the flow passage 1.

FIG. 16 shows a block diagram of a capillary electrophoresis / mass spectrometer coupling device according to an embodiment of the present invention. The mixed solution is introduced into the capillary electrophoresis apparatus 21, and is electrophoresed and separated by the high voltage applied to both ends of the capillary. The separated solution is detected by the detector 22 and then introduced into the capillary 2. The tip of the capillary 2 is coaxially inserted into the orifice 3. Nitrogen gas supplied from the gas supply unit 4 is introduced into the ion source body 6 through the gas introduction pipe 5 and is discharged to the outside through the orifice 3. The orifice plate 9 is made of metal, and a voltage is applied from the power source 7. The liquid reaching the end of the capillary 2 is atomized by gas under atmospheric pressure. Ions and charged droplets generated in the atomized gas are introduced into the vacuum through the pores 17 at the entrance of the mass spectrometer, and are mass separated by the electric field or magnetic field in the mass analysis section 18 installed in the high vacuum section. The mass-separated ions are detected by the ion detector 19. The output of the ion detector 19 is sent to the computer 20 and analyzed. The power supply 7 and the mass spectrometer 18 can be controlled by the computer 20.
That is, the positive / negative ions generated by the intermittent voltage signal generated by the power supply 7 can be mass-separated in synchronization with the ion generation and the ions can be detected. For example, when the potential of the metal tube 8 is set to the installation potential and the power source 7 applies a rectangular wave voltage with an amplitude of 1 kV to the orifice plate 9 to generate positive and negative ions, the positive and negative ions are analyzed in synchronization with it. You can Whether the capillary 2 or the metal tube 8 and the pores 17 are set to the same potential so that the electric field does not exist in the space where the spray gas is generated, or the potential difference of about 200 V is generated, the ion detection is performed. Is no problem at all. The mass spectrometer 18 includes any mass spectrometer such as a quadrupole mass spectrometer, a quadrupole trap mass spectrometer, a magnetic field mass spectrometer, a time-of-flight mass spectrometer, or a Fourier transform mass spectrometer. Can be used. By setting the potential of the metal tube 8 to the ground potential, the capillary electrophoresis device 21
The potential of the mixed solution of can also be set to the ground potential.
Therefore, the capillary electrophoresis device 21 can be operated without any trouble in the separation of the mixed solution,
Highly sensitive separation analysis of mixed solutions is realized online. Even if a solution separation means such as a liquid chromatograph device is used instead of the capillary electrophoresis device 21, the highly sensitive separation analysis of the mixed solution is realized in exactly the same manner online.

FIG. 17 shows a block diagram of a capillary electrophoresis / mass spectrometer coupling device according to an embodiment of the present invention. The mixed solution is introduced into the capillary electrophoresis apparatus 21, and is electrophoresed and separated by the high voltage applied to both ends of the capillary. The separated solution is detected by the detection unit 22 and then introduced into the capillary 2 through the metal tube 8 set to the ground potential. The tip of the capillary 2 is coaxially inserted into the orifice 3. Nitrogen gas supplied from the gas supply unit 4 is introduced into the ion source body 6 through the gas introduction pipe 5 and is discharged to the outside through the orifice 3. The orifice plate 9 is made of metal, and a voltage is applied from the power source 7. The liquid reaching the end of the capillary 2 is atomized by gas under atmospheric pressure. Ions and charged droplets generated in the atomized gas are introduced into the vacuum through the pores 17 at the entrance of the mass spectrometer, and are mass separated by the electric field or magnetic field in the mass analysis section 18 installed in the high vacuum section. The mass-separated ions are detected by the ion detector 19. The output of the detection unit 22 is sent to the computer 20. Based on this, a signal instructing the power supply 7 and the mass spectrometer 18 is sent from the computer 20. Then, the output of the ion detector 19 is sent to the computer 20 and analyzed.

The substances separated by the capillary electrophoresis apparatus 21 include those that become positive ions and those that become negative ions by spraying. Therefore, for example, even if a substance that becomes a negative ion is separated, in the state where only positive ions can be generated by spraying or only positive ions can be analyzed, the substance cannot be analyzed. Therefore, while the separated substance is sprayed from the capillary 2, the polarity of the voltage applied to the orifice plate 9 is reversed to perform both positive ion analysis and negative ion analysis. Since the potential of the metal tube 8 is set to the ground potential, performing positive and negative ion analysis does not affect the capillary electrophoresis device 21. Even if a solution separation means such as a liquid chromatograph device is used instead of the capillary electrophoresis device 21, the highly sensitive separation analysis of the mixed solution is realized in exactly the same manner online.

[0058]

INDUSTRIAL APPLICABILITY The ion source of the present invention and the mass spectrometer using the same have high stability and reproducibility of ion generation as compared with the electrospray ionization method and the ion spray ionization method using the electrostatic atomization phenomenon. Even if the spraying is interrupted and then restarted, the ion generation is reproduced, so that the operability of the ion source is extremely high. This is because only gas atomization of the solution is used in ion generation.

[Brief description of drawings]

FIG. 1 is a configuration diagram of an ion source according to an embodiment of the present invention.

FIG. 2 is a sectional view of an ion source according to an embodiment of the present invention.

FIG. 3 is a sketch of an ion source according to an embodiment of the present invention.

FIG. 4 shows the voltage dependence of the total ion current applied to the orifice 3.

FIG. 5: Mass spectrum obtained from a cytochrome-C solution.

FIG. 6 is a mass spectrum obtained from a cytochrome-C solution by an on-spray ionization method.

FIG. 7: Mass spectrum obtained from myoglobin solution.

FIG. 8: Mass spectrum obtained from a hemoglobin solution.

FIG. 9: Dependence of ionic strength on atomization gas flow rate.

FIG. 10 is a sectional view of an ion source according to another embodiment of the present invention.

FIG. 11 is a sectional view of an ion source according to still another embodiment of the present invention.

FIG. 12 is a sectional view of an ion source according to still another embodiment of the present invention.

FIG. 13 is a sectional view of an ion source according to still another embodiment of the present invention.

FIG. 14 is a sectional view of an ion source according to still another embodiment of the present invention.

FIG. 15 is a configuration diagram of a mass spectrometer according to an embodiment of the present invention.

FIG. 16: Capillary electrophoresis / in one embodiment of the present invention
The block diagram of a mass spectrometer coupling device.

FIG. 17 is a configuration diagram of a capillary electrophoresis / mass spectrometer coupling device according to another embodiment of the present invention.

[Explanation of symbols]

1: Flow path, 2: Capillary, 3: Orifice, 4:
Gas supply unit, 5: gas introduction pipe, 6: ion source body, 7:
Power supply, 8: metal tube, 9: orifice plate, 10: tube, 1
1: stainless steel tube, 12: tubular electrode, 13: heater,
14: heating block, 15: gas injection part, 16: supersonic oscillator, 17: pore, 18: mass spectrometric part, 19: ion detection part, 20: calculator, 21: capillary electrophoresis device, 22: detection part .

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁷ Identification code FI theme code (reference) H01J 49/10 H01J 49/10 (72) Inventor Minoru Sakairi 1-280 Higashi Koigokubo, Kokubunji, Tokyo Hitachi Ltd. Central Research Laboratory (72) Inventor Yasushi Takada 1-280 Higashi Koikekubo, Kokubunji, Tokyo Hitachi Central Research Laboratory (72) Inventor Takayuki Nabeshima 1-280 Higashi Koikeku, Kokubunji, Tokyo Hitachi Research Center Co., Ltd. In-house (72) Inventor Hideaki Koizumi 1-280, Higashi-Kengokubo, Kokubunji-shi, Tokyo F-term in Hitachi Central Research Laboratory (reference) 2G045 DA36 FA40 GC30 5C038 EE02 EF04 GG08 GH05 GH08 GH11

Claims (3)

[Claims] 1. A sample liquid containing a protein is circulated in a flow passage, a voltage with a reversed polarity is applied to the flow passage, and a gas is discharged from a gas supply device so that a gas flow is generated at the tip of the flow passage. By forming, spraying the sample liquid,
A method for mass spectrometric analysis of a protein, comprising ionizing the protein, and mass-analyzing the ionized ion in an analyzer. 2. The method for mass spectrometric analysis of protein according to claim 1, wherein the voltage application is performed by the first electrode, the second electrode and the voltage application means. 3. The flow passage has a capillary at the tip,
The gas flow has a gas flow rate of F at 20 ° C. and 1 atm.
When the cross-sectional area of the plane perpendicular to the capillary between the second electrode and the capillary is S, the value of F / S is in the range of 250 m / sec to 1200 m / sec. The method for mass spectrometric analysis of protein according to claim 2.