JPWO2009118775A1 - Electrophoresis / electrospray ionizer - Google Patents

Abstract

A control flow path (34), which is a capillary tube, connects between the sprayer (21) for performing electrospray and the control reservoir (30) storing the background electrolyte liquid (30a), and is immersed in the electrolyte liquid (30a). Thus, the control electrode (35) is arranged. The control electrode (35) is positioned higher than the inlet end (34a) of the control channel (34). Since the electrical resistance value between both ends of the control flow path (34) can be accurately obtained from the dimensions of the flow path and the electrical resistivity of the electrolyte solution, the electrical resistance value and the high voltage power supply (16, 36) are applied respectively. Therefore, the potential Vp of the electrolyte solution in the nebulizer (21) can be calculated with high accuracy, and the potential Vp can be stably controlled according to the output voltage of the high-voltage power source (36). Further, bubbles generated from the surface of the control electrode (35) due to the oxidation-reduction reaction do not enter the flow path (34), and reliable contact between the electrolyte solution (30a) and the control electrode (35) is ensured. Component separation in the flow path (14) and ionization in the sprayer (21) can be performed stably.

Description

  The present invention relates to an electrophoresis / electrospray ionization apparatus that ionizes a sample separated by capillary electrophoresis using an electrospray ionization method. The apparatus according to the present invention can typically be used in a capillary electrophoresis / mass spectrometer that combines a capillary electrophoresis apparatus and a mass spectrometer.

  The capillary electrophoresis (CE) method is suitable for separating components having similar structures, such as optical resolution and isotope separation, as well as analysis of biological components such as peptides, proteins, DNA, and sugars. Yes, it is widely used for applications such as clinical medicine, pharmaceuticals, and monitoring of environmental substances. Generally, in a capillary electrophoresis apparatus, an ultraviolet-visible spectrophotometer, a fluorescence spectrophotometer, or the like is often used as a detector. However, in order to perform high-precision and high-sensitivity analysis, the capillary electrophoresis apparatus Conventionally, a capillary electrophoresis / mass spectrometer (hereinafter abbreviated as “CE / MS”) using a mass spectrometer as a detector is known.

  In CE / MS, a so-called atmospheric pressure ionization interface is used to ionize sample components in a sample solution eluted from a capillary separation channel. As one of the typical atmospheric pressure ionization interfaces, the sample liquid that reaches the tip of the nozzle is charged, and the sample liquid is ionized mainly by Coulomb attractive force (or repulsive force) while spraying the sample liquid into the atmospheric pressure atmosphere. There is an electrospray ionization method (ESI). In an electrospray ionization interface used in a liquid chromatograph mass spectrometer (LC / MS), a high voltage is directly applied to an electrode provided in contact with the sample liquid in the vicinity of the tip of the nozzle of the nebulizer, so that the sample liquid In general, a structure in which an electric charge is imparted to is used. On the other hand, in the electrospray ionization interface of CE / MS, various configurations have been conventionally proposed in order to apply a potential suitable for electrospray to the sample liquid that has reached the vicinity of the nozzle tip of the sprayer.

  For example, in the devices described in Patent Documents 1, 2, 3, etc., a background electrolyte for separating and moving sample components by means of a wetted electrode provided in the capillary separation channel and in direct contact with the sample solution A predetermined high voltage is applied to the liquid (buffer). However, in such a configuration, bubbles generated from the surface of the wetted electrode due to the oxidation-reduction reaction are mixed with the background electrolyte solution in the separation flow path, and this causes a problem that ionization of the sample component in the sprayer becomes intermittent. In addition, since the electric resistance value between both ends of the separation channel fluctuates due to the influence of such bubbles, the separation of the sample components may become unstable and inaccurate.

  In addition, in the apparatus described in Patent Document 4, a predetermined high voltage is applied to the background electrolyte solution using the conductive material coated on the outer wall surface of the outlet end of the nozzle made of a non-conductive material as a wetted electrode. ing. However, since the outlet end of the nozzle is disposed in a gas phase at approximately atmospheric pressure, the background electrolyte solution is almost continuously lost from the nozzle outlet end by evaporation. For this reason, it is difficult to ensure stable contact between the liquid contact electrode and the background electrolyte solution, and it is difficult to stably apply a potential to the background electrolyte solution in the separation channel. As a result, it is difficult to draw the sample into the separation channel, and it is also difficult to perform accurate component separation in the separation channel and stable ionization in the sprayer.

  Further, in the apparatus described in Non-Patent Document 1, the outer wall surface of the flow path is contacted with the background electrolyte solution that oozes out from the flow path through the pores formed in the flow path wall of the separation flow path. A liquid electrode is installed, and a potential is applied to the background electrolyte solution by applying a voltage to the electrode. However, similarly to the above configuration, the outer wall surface of the separation channel is a gas phase at atmospheric pressure, so that the background electrolyte solution is almost continuously lost from the outer wall surface by evaporation. For this reason, it is still difficult to ensure stable contact between the wetted electrode and the background electrolyte solution. As a result, it is difficult to draw the sample into the separation channel, and it is also difficult to perform accurate component separation in the separation channel and stable ionization in the sprayer.

  In the devices described in Patent Documents 5 and 6 and Non-Patent Document 2, the outlet end of the separation channel, the inlet end of the sprayer, and the liquid contact electrode are arranged so as to be immersed in the solution stored in the same reservoir. A voltage is applied to the solution in the reservoir from the liquid contact electrode. In this case, the outlet end of the separation channel and the inlet end of the sprayer are provided in close proximity, but in order to control the potential in the gap as intended, the electrical resistance between the gap and the wetted electrode You need to know the value exactly. However, since it is difficult to accurately know the electrical resistance value in the solution in the reservoir, it is difficult to accurately control the potential in the gap as intended. Therefore, it is difficult to accurately control component separation and ionization. By making the distance between the gap and the wetted electrode as short as possible in the solution in the reservoir, it is possible to reduce the electrical resistance value between them to a negligible level. However, in that case, bubbles generated from the liquid contact electrode are liable to enter the separation flow path or the flow path in the sprayer, which may be a factor that makes component separation in the separation flow path and ionization in the sprayer unstable.

  In the devices described in Patent Documents 7 and 8 and Non-Patent Document 3, although the outlet end of the separation channel, the inlet end of the sprayer, and the wetted electrode are arranged so as to be immersed in the solution in the same reservoir, the separation The gap between the outlet end of the flow path and the inlet end of the sprayer is covered with a separation membrane such as an ion exchange membrane, a permeable membrane, or porous silica, and the background electrolyte solution passing through the gap and the solution in the reservoir The structure is such that direct contact is avoided. However, the electrical resistance value of the separation membrane is much larger than that of an aqueous buffer generally used as a background electrolyte solution, and it is difficult to accurately know the electrical resistance value of the separation membrane. Therefore, it is still difficult to accurately control the potential in the gap as intended, and therefore it is also difficult to accurately control component separation in the separation channel and ionization in the sprayer.

As described above, conventionally known various configurations are:
(1) Lack of stability of contact between the liquid contact electrode for applying voltage and the background electrolyte solution;
(2) Bubbles generated on the surface of the wetted electrode due to the oxidation-reduction reaction enter the separation channel or the channel of the sprayer.
(3) It is difficult to accurately obtain the electrical resistance value between the liquid contact electrode and the sprayer.
At least one of the problems. For this reason, it is difficult to accurately and stably control the potential of the sample liquid at the nozzle tip, more specifically, at the nozzle tip, which greatly affects the efficiency of ionization. As a result, it is difficult to perform ionization at or near the best, and the reliability of analysis may be impaired.

US Pat. No. 4,994,165 US Pat. No. 5,856,671 US Pat. No. 6,863,790 US Pat. No. 5,788,166 US Pat. No. 5,505,832 US Pat. No. 6,067,749 US Pat. No. 5,993,633 US Patent Application Publication No. 2006/0057556 Moini M, “Design and Performance of a Universal Sheathless Capillary Electrophoresis to Mass Spectrometer Interface Using a Split-Flow Technique universal sheathless capillary electrophoresis to mass spectrometry interface using a split-flow technique), Anal. Chem., 2001, 73, 3497-3501. Chen YR and three others, “A Low-Flow Cee / Electrospray Ionization MS Interface for Capillary Zone Electrophoresis Large-Volume Sample Stacking and Mysler Electrokinetic chromatography (A low-flow ce / electrospray ionization MS interface for capillary zone electrophoresis, large-volume sample stacking, and micellar electrokinetic chromatography), Analytical Chemistry (Anal. Chem.), 2003, 75th. Volume, pages 503-508 Whit JT and one other, "Capillary electrophoresis to mass spectrometry interface using a porous junction", Analytical chemistry ( Anal. Chem.), 2003, 75, 2188-2191.

  The present invention has been made in view of the above problems, and its main purpose is to solve the above-mentioned problems (1), (2), and (3), and the potential of the sample liquid that has reached the nozzle tip of the sprayer. An electrophoretic / electrospray ionization apparatus that can stably and satisfactorily perform component separation in the separation flow path and ionization in the sprayer by enabling accurate and stable control as intended. It is to provide.

In order to solve the above-mentioned problems, the present invention is an electrophoresis / electrophoresis method in which sample components are separated by electrophoresis, and ionized by being sprayed in a substantially atmospheric pressure atmosphere while applying charges to the separated sample components. In electrospray ionization equipment,
a) a separation channel for separating sample components;
b) an inlet reservoir in which a predetermined solution is stored and an inlet end of the separation channel is immersed;
c) an inlet electrode immersed in the solution in the inlet reservoir and disposed higher than the inlet end of the separation channel;
d) a control reservoir in which a predetermined solution is stored;
e) a control flow path arranged such that the inlet end is immersed in the solution in the control reservoir;
f) a control electrode immersed in the solution in the control reservoir and disposed at a position higher than the inlet end of the control flow path;
g) The outlet end of the separation channel and the outlet end of the control channel are connected so that the solution in both channels can be contacted, and the solution sent from the separation channel can A sprayer that can be sprayed into,
h) voltage applying means for applying a voltage to each of the inlet electrode and the control electrode;
In the state where the inlet electrode and the control electrode are electrically connected via the solution in the separation channel, the sprayer, and the control channel, a voltage is applied to both electrodes by the voltage applying means. The sample component is electrophoresed and separated in the separation channel by application, and the solution is sprayed by applying a charge to the solution by the potential generated in the solution in the sprayer.

  In the electrophoresis / electrospray ionization apparatus according to the present invention, typically, the separation channel and the control channel can be configured to be non-conductive capillary tubes. Moreover, the solution stored in each reservoir can be an electrolyte solution having appropriate conductivity.

  In the electrophoresis / electrospray ionization apparatus according to the present invention, the liquid contact so as to be immersed in a solution (for example, background electrolyte solution) stored in the inlet reservoir and the control reservoir, not in the separation channel or the control channel. Since the electrodes (inlet electrode and control electrode) are arranged, the contact between the liquid contact electrode and the solution is reliably and stably performed. Therefore, it is possible to reliably separate the components while applying a potential to the solution in the separation channel and moving the sample injected into the separation channel smoothly. Moreover, an electric potential can be reliably given to the solution in a nebulizer, and ionization by electrospray can be performed.

  Further, in each reservoir, since it is disposed at a position higher than the opening at the inlet end of the separation channel and the control channel, that is, a position close to the interface of the solution, bubbles are generated from the electrode surface by the oxidation-reduction reaction. Even when the bubbles are generated, the bubbles rise to the solution interface and diffuse into the gas phase, and the bubbles do not enter the separation channel or the control channel. Therefore, the flow of the solution and the flow of the sample liquid are not hindered by the bubbles. Further, the conductivity through the solution is not inhibited by bubbles.

  In addition, the control electrode and the sprayer are connected via a control flow path, and the potential applied to the solution in the sprayer is filled in the control flow path, more precisely, the electrical resistance value of the control flow path. It depends on the electric resistance value between both ends of the control channel by the solution. That is, if this electrical resistance value is obtained with high accuracy, the potential applied to the solution in the sprayer can be controlled with high accuracy. As described above, when the control flow path is a capillary tube, the cross-sectional area of the flow path is quite small, and thus the electric resistance value between both ends of the control flow path shows a somewhat large value. Therefore, the electrical resistance value of the gap between the control channel inlet end and the control electrode in the solution in the control reservoir is smaller than the electrical resistance value between the control channel ends if the gap is narrow. Small enough to be ignored.

  When the control channel is filled with the solution, the electrical resistance value between both ends of the control channel can be calculated from the cross-sectional area and length of the control channel and the electrical resistivity of the solution. Therefore, as one aspect of the electrophoresis / electrospray ionization apparatus according to the present invention, from the cross-sectional area and length of the control channel and the electrical resistivity of the solution filled in the channel, the distance between both ends of the control channel is determined. The electric resistance value can be calculated, and the electric resistance value can be used to calculate the potential generated in the solution in the sprayer.

  With this configuration, the potential generated in the solution in the nebulizer can be accurately calculated, so that the electrospray ionization can be appropriately performed, for example, so that the ionization efficiency is stably maintained at the best state or a state close thereto. It becomes easy to control the voltage applied to the control electrode.

In addition, the electric resistance value between both ends of the control flow path can be obtained by actual measurement without using the above calculation. That is, as another aspect of the electrophoresis / electrospray ionization apparatus according to the present invention,
A reference reservoir in which a predetermined solution is stored and an outlet end of the control channel is immersed;
A reference electrode immersed in the solution in the reference reservoir and maintained at ground potential;
When a voltage is applied to the control electrode by the voltage application means in a state where the control electrode and the reference electrode are electrically connected via the solution in the control flow path, the applied voltage and correspondingly Calculating an electric resistance value between both ends of the control flow path from the flowing current, and calculating an electric potential generated in the solution in the nebulizer using the electric resistance value;
It is good also as a structure further equipped with. According to this configuration, it is not necessary to measure the electrical resistivity of the solution and to collect accurate dimensional information of the control channel.

  Further, in the electrophoresis / electrospray ionization apparatus according to the present invention, the inlet reservoir and the control reservoir can be sealed, respectively, an inlet pressure adjusting means for adjusting the pressure applied to the solution in the inlet reservoir, and the control reservoir Control pressure adjusting means for adjusting the pressure applied to the solution in the inside, and by pressurizing the solution in each reservoir by the inlet pressure adjusting means and the control pressure adjusting means, the separation flow path and the control It can be set as the structure filled with the solution in the flow path.

  Since the outlet end of the separation channel and the outlet end of the control channel are opened to a substantially atmospheric pressure in the sprayer, if the solution in the inlet reservoir and the control reservoir is set to an appropriate pressure higher than the atmospheric pressure, the separation channel The solution is smoothly fed into the channel from the inlet end and the inlet end of the control channel, and the channel can be filled with the solution in a relatively short time. Further, the pressurization of the solution by the inlet pressure control means and the control pressure control means can be used supplementarily to move the sample when the sample is separated into components. Thereby, the time required for component separation can be shortened. Further, when the sample liquid is injected into the separation channel from the inlet end after the separation channel is filled with the solution, pressurization by the inlet pressure adjusting means can be used. At this time, a reservoir for storing the sample liquid is prepared instead of the inlet reservoir for storing the solution, the inlet end of the separation channel is immersed in the sample liquid in the reservoir, and the inside of the reservoir is pressurized. That's fine. Of course, the injection of the sample liquid into the separation channel can be performed by utilizing the potential difference between the voltages applied to the inlet electrode and the control electrode, regardless of pressurization.

  Moreover, as an aspect of the electrophoresis / electrospray ionization apparatus according to the present invention, the nebulizer has a confluence portion where the solution from the separation channel and the solution from the control channel merge inside the nebulizer, It may be a structure in which the merged solution flows out from the nozzle to the outside. As another aspect of the electrophoresis / electrospray ionization apparatus according to the present invention, the nebulizer has a double pipe in which a pipe through which the solution from the separation channel flows and a pipe through which the solution from the control channel flows. It can also be set as the structure where both solutions contact on the outer side of the open end of both pipe lines. In any embodiment, the solution supplied from the separation channel and the solution supplied from the control channel in the sprayer are in contact with each other, and electrical connection between the inlet electrode and the control electrode can be ensured through the solution.

  According to the electrophoresis / electrospray ionization apparatus according to the present invention, the contact between the liquid contact electrode and the solution for applying a voltage to the solution connected to the separation channel, the nebulizer, and the control channel is reliably and stably performed. In addition, air bubbles are not mixed in the flow path. Thereby, an electric potential can be reliably and stably applied to the solution in the nebulizer, and electrospray ionization in the nebulizer can be favorably performed. Moreover, component separation can also be performed satisfactorily by applying a stable potential to the solution in the separation channel.

  In addition, the electrical resistance value between both ends of the control flow path is accurately obtained, and by using this value, the potential applied to the solution in the sprayer can be controlled stably and accurately as the user intended. Therefore, it is possible to stably maintain a good ionization state as compared with the conventional case, and to improve the accuracy of component separation in the separation channel.

The schematic block diagram of one Example of CE / MS carrying the electrophoresis / electrospray ionization apparatus which concerns on this invention. The block diagram of a capillary electrophoresis part and an atmospheric pressure ionization interface part. The block diagram of a capillary electrophoresis part and an atmospheric pressure ionization interface part. The figure which shows an example of a sprayer. The enlarged view of a control reservoir. The figure which shows the other example of a sprayer.

Explanation of symbols

DESCRIPTION OF SYMBOLS 1 ... Capillary electrophoresis part 2 ... Atmospheric pressure ionization interface part 3 ... Mass analysis part 4 ... Mass separator 5 ... Detector 10 ... Inlet reservoir 10a, 30a, 37a ... Background electrolyte solution 12, 32 ... Gas supply pipe 13 , 33 ... Pressure regulating section 14 ... Separation channel 14a ... Inlet end 14b ... Outlet end 15 ... Liquid contact electrode (inlet electrode)
16, 36 ... High-voltage power supply 17 ... Sample inlet reservoir 17a ... Sample solution 20 ... Spray chamber 21 ... Sprayer 21a ... Nozzle 21b ... Separation inlet end 21c ... Control inlet end 21d ... Spray port 22 ... Counter electrode 23 ... Ion introduction port 24 ... Control part 30 ... Control reservoir 34 ... Control flow path 34a ... Inlet end 34b ... Outlet end 35 ... Wetted electrode (control electrode)
37 ... Reference reservoir 38 ... Reference electrode 40 ... Nebulizer 41 ... Electrolyte liquid line 42 ... Sheath liquid line 43 ... Sheath gas line

  Hereinafter, an embodiment of CE / MS equipped with an electrophoresis / electrospray ionization apparatus according to the present invention will be described with reference to the drawings. FIG. 1 is a schematic configuration diagram of the CE / MS of the present embodiment.

  In the capillary electrophoresis unit (CE) 1, a plurality of sample components in a given sample are separated and introduced into the atmospheric pressure ionization interface unit 2 with a temporal difference. The atmospheric pressure ionization interface unit 2 ionizes sample components by spraying a sample solution containing the separated sample components into the atmosphere by electrospray ionization as described later. The generated ions are separated for each mass (strictly m / z) by the mass separator 4 of the mass analyzer 3 and detected by the detector 5. As the mass analysis unit 3, various types such as a quadrupole, a flight time, and an ion trap can be used.

  2 and 3 are configuration diagrams of the main parts of the capillary electrophoresis unit 1 and the atmospheric pressure ionization interface unit 2.

  The atmospheric pressure ionization interface unit 2 has a spray chamber (ionization chamber) 20 that is maintained in a substantially atmospheric pressure atmosphere, and an ion introduction port 23 that sends ions to the mass analysis unit 3 is formed in the spray chamber 20. An electrode 22 is provided. The counter electrode 22 is grounded as shown in FIG. 2, or an appropriate DC bias voltage is applied by a power source (not shown). Opposite to the counter electrode 22, a sprayer 21 is provided that ionizes the sample component while spraying a micro droplet containing the sample component into the spray chamber 20. The sprayer 21 can be attached to and detached from the wall surface forming the spray chamber 20.

  FIG. 4 is a schematic sectional view showing an example of the sprayer 21. The sprayer 21 includes a tapered nozzle 21a having a spray port 21d for ejecting a solution at a tip thereof, a separation inlet end 21b to which an outlet end 14b of a separation channel 14 described later is connected, and a control channel 34 also described later. And a control inlet end 21c to which the outlet end 34b is connected. The flow paths of both the inlet ends 21b and 21c merge inside the sprayer 21 and are directed to the nozzle 21a.

  In the capillary electrophoresis unit 1, the inlet end 14 a of the separation channel 14, which is a capillary tube made of a non-conductive material (for example, glass) for separating sample components, is stored in the inlet reservoir 10. It is immersed in the solution 10a. The inlet reservoir 10 is a substantially hermetically sealed container, and gas (for example, nitrogen, synthetic air, etc.) adjusted to a predetermined gas pressure by the first pressure adjusting unit 13 is supplied through the gas supply pipe 12. . A liquid contact electrode (hereinafter referred to as “inlet electrode”) 15 is installed so as to be immersed in the solution 10 a stored in the inlet reservoir 10, and a predetermined high voltage is supplied from the inlet electrode 15 to the solution 10 a by the first high-voltage power supply 16. A voltage can be applied. In this example, the solution 10a is a background electrolyte solution for electrophoresis. In addition to the inlet reservoir 10, a sample inlet reservoir 17 in which a sample liquid 17a to be analyzed is stored is provided so as to be exchangeable by a reservoir automatic exchange mechanism (not shown).

  On the other hand, the inlet end 34 a of the control channel 34, which is a capillary tube different from the separation channel 14, is immersed in the solution 30 a stored in the control reservoir 30. The control reservoir 30 is a container that can be substantially sealed like the inlet reservoir 10, and a gas (for example, nitrogen, synthetic air, etc.) adjusted to a predetermined gas pressure by the second pressure adjusting unit 33 is supplied through the gas supply pipe 32. It has become so. In addition, a liquid contact electrode (hereinafter referred to as “control electrode”) 35 is installed so as to be immersed in the solution 30 a stored in the control reservoir 30, and a predetermined high voltage is supplied from the control electrode 15 to the solution 30 a by the second high voltage power source 36. A voltage can be applied. In this example, the solution 30a is also a background electrolyte solution.

  Further, as shown in FIG. 3, a reference reservoir 37 in which a solution 37 a is stored separately from the inlet reservoirs 10, 17 and the control reservoir 30 is additionally provided (for example, as an option). 34b can be removed from the control inlet end 21c of the sprayer 21 and immersed in the solution 37a. Such spatial movement of the outlet end 34b of the control flow path 34 may be performed manually by the person in charge of analysis, or a mechanism for automatically switching the connection destination may be provided. The solution 37a is the same as the solution 30a stored in the control reservoir 30, and is a background electrolyte solution in this example. In addition, a grounded reference electrode 38 is provided so as to be immersed in the solution 37a. As a result, the reference electrode 38 is stably fixed at zero potential.

  FIG. 5 is an enlarged view of the control reservoir 30. As shown in FIG. 5, the inlet end 34a of the control channel 34 is inserted deeply in the background electrolyte solution 30a until it reaches the vicinity of the inner bottom surface of the control reservoir 30, and the control electrode 35 is installed in close proximity thereto. Yes. Both are positioned so that the lower end of the control electrode 35 is slightly higher (by height d) than the inlet end 34 of the control flow path 34, that is, a position close to the interface of the background electrolyte solution 30a. Therefore, as will be described later, even if bubbles are generated on the surface of the electrode 35 due to the oxidation-reduction reaction when the electrodes 15 and 35 are energized, the bubbles rise as they are and diffuse into the upper space in the control reservoir 30, and the inlet end 34a. Does not enter the control flow path 34. Thus, in order to avoid intrusion of bubbles, the height d is preferably large, but the electrical resistance value of the gap between the control electrode 35 and the inlet end 34a increases as d increases. Since it is desirable that the electrical resistance value be as small as possible, it is preferable to determine an appropriate d in consideration of both.

  In the inlet reservoirs 10 and 17 as well, the positional relationship between the inlet end 14 a of the separation channel 14 and the inlet electrode 15 can be determined in the same manner as the control reservoir 30 described above. However, d may be set to zero or a negative value when no bubbles are generated during energization.

  The operations of the pressure adjusting units 13 and 33 and the high voltage power supplies 16 and 36 are all controlled by a control unit 24 including a CPU. The high-voltage power supplies 16 and 36 have a function of monitoring the output current flowing in response to outputting a DC voltage having a voltage value instructed by the control unit 24 and sending the current value to the control unit 24. ing. The control unit 24 may be integrated with the control unit that controls the operation of the mass analysis unit 3 or may be a separate unit.

  Next, electrophoresis / mass spectrometry in the CE / MS having the above configuration will be described in order.

(A) Filling the separation channel 14 and the control channel 34 with the background electrolyte solution First, as a preparatory stage for electrophoresis in the capillary electrophoresis section 1, the separation channel 14, the control flow separation 34, and the flow in the sprayer 21 Fill the road with background electrolyte solution. That is, as shown in FIG. 2, the outlet end 34 b of the control channel 34 is connected to the control inlet end 21 c of the sprayer 21, and the inlet end 34 a is immersed in the background electrolyte 30 a in the control reservoir 30. The outlet end 14 b of the separation channel 14 is connected to the separation inlet end 21 b of the sprayer 21, and the inlet end 14 a is immersed in the background electrolyte solution 10 a in the inlet reservoir 10. Under the control of the control unit 24, when the gas appropriately regulated by the first pressure regulating unit 13 is fed into the inlet reservoir 10, the outlet end 14b of the separation channel 14 is at substantially atmospheric pressure. The background electrolyte solution in the inlet reservoir 10 is sucked into the separation channel 14 by the differential pressure across the separation channel 14 and filled therein.

  In parallel with this, also in the second pressure adjusting unit 33 on the control reservoir 30 side, the gas whose pressure is adjusted appropriately is fed into the control reservoir 30. Since the outlet end 34b of the control flow path 34 is also at substantially atmospheric pressure, the background electrolyte solution 30a in the control reservoir 30 is sucked into the control flow path 34 by the differential pressure across the control flow path 34 and filled therein. Is done. The background electrolyte solution filled in the separation channel 14 and the background electrolyte solution filled in the control channel 34 are merged inside the sprayer 21 and filled from the spray port 21d after filling the internal channel of the nozzle 21a. leak. Thereafter, pressurization by the pressure adjusting units 13 and 33 is stopped, and the inside of the inlet reservoir 10 and the control reservoir 30 is returned to substantially atmospheric pressure. In this way, when the separation channel 14, the control channel 34, and the inner channel of the sprayer 21 are filled with the conductive background electrolyte solution, the inlet electrode 15 on the inlet reservoir 10 side and the control electrode on the control reservoir 30 side are filled. 35 is electrically connected via a background electrolyte solution (at least not in an insulating state).

(B) Sample Injection into Separation Channel 14 Next, a predetermined amount of sample solution is injected and held near the inlet in the separation channel 14. That is, the inlet reservoir 10 is replaced with the sample inlet reservoir 17 by the above-described reservoir automatic replacement mechanism (not shown), and the inlet end 14 a of the separation channel 14 is immersed in the sample liquid 17 a in the sample inlet reservoir 17. From this state, the sample liquid 17a is introduced into the separation channel 14, and either pressure injection or electrokinetic injection can be selected as the method.

(B-1) Pressurized injection As in the case of filling the background electrolyte solution described above, the gas appropriately adjusted by the first pressure adjusting unit 13 under the control of the control unit 24 is supplied to the sample inlet reservoir 17. The sample liquid 17 a is pressurized and sucked into the separation channel 14. When an appropriate amount of the sample liquid 17a is injected into the separation channel 14, the pressurization by the first pressure adjusting unit 13 is stopped, and the inside of the sample inlet reservoir 17 is returned to substantially atmospheric pressure. As a result, the sample liquid is held in the predetermined range from the inlet end 14a of the separation channel 14. In this case, since a part of the background electrolyte solution previously filled in the separation channel 14 is discharged to the outside from the ejection port 21d, there is no special consideration as in the case of electrokinetic injection described later. However, the spray port 21d at the tip of the nozzle 21a does not dry.

Under the control of (B-2) electrokinetic injection control unit 24, a predetermined voltage V ₁ is applied to the entrance electrode 15 immersed from the first high-voltage power source 16 in the sample solution 17a, while the second high-voltage power source 36 the predetermined voltage V ₂ is applied to the control electrode 35 which is immersed in a background electrolyte solution 30a from. Here, in order for the sample liquid 17a to be smoothly injected into the separation channel 14, it is necessary to appropriately set the potential difference between both ends of the separation channel 14, but this is present in the nozzle 21a together with it. It is necessary to keep the potential Vp of the background electrolyte solution in an appropriately low range so that electrospray does not occur. Considering this, the applied voltages V ₁ and V ₂ are determined, but in general, V ₂ has a polarity opposite to that of V ₁ . When the output voltage of the first high-voltage power source 16 is assumed to be fixed, the potential Vp of the background electrolyte solution in the nozzle 21a includes an output voltage V ₂ of the second high-voltage power supply 36, in a state where the background electrolyte solution filled was It is possible to calculate from the product of the electrical resistance value between both ends of a certain control flow path 34 and the output current value of the second high-voltage power supply 36 (that is, the potential difference between both ends of the control flow path 34). Therefore, the output voltage of the second high-voltage power supply 36 may be controlled so that the potential Vp becomes an appropriate value.

  The electrical resistance value between both ends of the control channel 34 can be obtained by calculation from the electrical resistivity of the background electrolyte solution, the cross-sectional area and the channel length of the control channel 34. Therefore, by storing the value obtained in this way in the control unit 24, it is possible to calculate the potential Vp at the time of energization and perform control so that this becomes an appropriate value. In addition, the electrical resistance value between both ends of the control flow path 34 can be measured by a method described later.

  When an appropriate amount of the sample solution 17a is injected into the separation channel 14, the voltage application by the first high-voltage power supply 16 and the second high-voltage power supply 36 is stopped, and both the inlet electrode 15 and the control electrode 35 are grounded. Returned to As a result, the sample solution 17a is held in the flow channel within a predetermined range from the inlet end 14a of the separation flow channel 14.

  In the case of this electrokinetic injection, even when the sample liquid 17a is injected into the separation channel 14, the background electrolyte solution filled in the separation channel 14 hardly flows out from the spray port 21d at the tip of the nozzle 21a. . Therefore, the background electrolyte solution is lost due to evaporation from the spray port 21d, and the spray port 21d may be dried to be clogged. Therefore, for example, in the configuration in which the sprayer 21 is detachable from the wall surface of the spray chamber 20 as described above, the sprayer 21 is removed and the tip of the nozzle 21a is placed in the background electrolyte solution 37a in the reference reservoir 37 during electrokinetic injection. It is good to immerse in. Thereby, drying of the spray nozzle 21d can be prevented.

(C) Separation and ionization of sample components After the sample liquid 17a is held near the inlet of the separation channel 14 by pressure injection or electrokinetic injection as described above, the sample inlet is provided by the above-described reservoir automatic exchange mechanism (not shown). The reservoir 17 is replaced with the inlet reservoir 10. Then, the inlet end 14 a of the separation channel 14 is immersed again in the background electrolyte solution 10 a in the inlet reservoir 10. Under the control of the control unit 24, a predetermined voltage V ₁ ′ is applied from the first high-voltage power source 16 to the inlet electrode 15 immersed in the background electrolyte solution 10a, and the background electrolyte solution 30a is supplied from the second high-voltage power source 36. A predetermined voltage V ₂ ′ is applied to the control electrode 35 immersed therein. Here, the output voltage of the second high-voltage power supply 36 is adjusted by a method similar to that at the time of electrokinetic injection so that the potential Vp of the background electrolyte solution in the nozzle 21a becomes a desired value at which electrospray occurs. .

  Due to the potential difference generated at both ends of the separation channel 14, the sample liquid 17a held near the inlet of the separation channel 14 moves as a whole toward the outlet end 14b, and the sample components are separated. In addition, since the background electrolyte solution in the nozzle 21a has a potential sufficient for electrospraying, the charged electrolyte solution is sprayed into the spray chamber 20 so as to be torn off from the spray port 21d by Coulomb attractive force or repulsive force. . When the sample component separated in the separation channel 14 reaches the nozzle 21a, the sample component is mixed in the fine droplets to be sprayed, and the component is ionized and directed to the counter electrode 22 through the ion introduction port 23. It is sent to the mass analyzer 3.

  At this time, the gas adjusted to a predetermined pressure through the gas supply pipe 12 is supplied into the inlet reservoir 10 to moderately pressurize the background electrolyte solution 10a, thereby assisting the movement of the sample solution 17a in the separation channel 14. You may make it do.

  After completion of mass analysis by performing sample component separation and ionization as described above for a predetermined time, voltage application by the first high-voltage power supply 16 and the first high-voltage power supply 36 is stopped, and both the inlet electrode 15 and the control electrode 35 are used. Returned to ground potential.

(D) Measurement of electric resistance value between both ends of control flow path 14 As described above, the electric resistance value between both ends of the control flow path 34 can be obtained by calculation. You can ask for it. That is, as shown in FIG. 3, the outlet end 34 b of the control flow path 34 is removed from the control inlet end 21 c of the sprayer 21 and immersed in the background electrolyte solution 37 a stored in the reference reservoir 37. The transfer of the outlet end 34b of the control flow path 34 is generally performed manually by the person in charge of analysis, but may be automatically performed by providing an appropriate transfer mechanism or the like. Then, the gas that has been moderately regulated by the second pressure regulating unit 33 is sent to the control reservoir 30 to pressurize the background electrolyte solution 30a, thereby filling the control electrolyte 34 with the background electrolyte solution 30a.

  Next, a predetermined voltage is applied from the second high voltage power source 36 to the control electrode 35. The distance between the control electrode 35 and the inlet end 34a of the control flow path 34 is very short, and the electrical resistance value between them is negligibly small. On the other hand, the distance between the reference electrode 38 and the outlet end 34b of the control channel 34 in the reference reservoir 37 is very short, and the electrical resistance value between the two is so small that it can be ignored. Accordingly, it can be considered that the potential at the inlet end 34a of the control flow path 34 is equal to the output voltage of the second high-voltage power supply 36, and the potential at the outlet end 34b of the control flow path 34 is equal to the ground potential. Therefore, the potential difference ΔVc between both ends of the control flow path 34 is a difference between the output voltage of the high-voltage power supply 36 and the ground potential (usually zero). Further, the current Ic flowing in the control flow path 34 in accordance with the potential difference ΔVc is equal to the output current in the second high voltage power source 36. Since the electrical resistance value Rc between both ends of the control flow path 34 is ΔVc / Ic, the electrical resistance value Rc can be calculated from the output voltage and output current of the high-voltage power supply 36. When the electrical resistance value Rc is obtained in this way, voltage application by the second high-voltage power supply 36 is stopped and the control electrode 35 is returned to the ground potential.

  As described above, according to the CE / MS of this embodiment, the bag stored in the inlet reservoir 10 or the control reservoir 30 is not in the separation channel 14, the control channel 34, or the sprayer 21. Since the inlet electrode 15 and the control electrode 35 are placed in the ground electrolyte solutions 10a and 30a, the contact between the electrodes 15 and 35 and the electrolyte solutions 10a and 30a is ensured and stable (for example, the influence of evaporation of the solution). Secured) Thereby, an electric potential can be reliably given to the background electrolyte solution in a flow path, and an electric current can be supplied. In addition, since the inlet electrode 15 and the control electrode 35 are installed at a position higher than the inlet ends 14a and 34a of the separation channel 14 and the control channel 34, bubbles are generated from the surfaces of the electrodes 15 and 35 by the oxidation-reduction reaction. Even if it occurs, the bubbles can be prevented from entering the flow paths 14 and 34. Thereby, it can be avoided that bubbles are accumulated in the flow paths 14 and 34 to impair the conductivity through which the electrolyte solution is passed and the smooth movement of the sample solution is prevented.

  Furthermore, the control electrode 35 and the sprayer 21 are connected by a control flow path 34 that is a capillary tube, and an electric resistance value between both ends of the control flow path 34 and an output voltage of the second high-voltage power supply 36 The electric potential applied to the electrolyte solution in the nozzle 21a of the sprayer 21 can be obtained with high accuracy. This makes it possible to easily and stably control the potential that has a great influence on the electrospray state, that is, the ionization state, with high accuracy. Thereby, the state of component separation in the separation channel 14 and the state of ionization in the sprayer 21 can be stably controlled.

  As another example of the atomizer of the atmospheric pressure ionization interface unit 2 in the CE / MS of the above embodiment, a configuration as shown in FIG. 6 may be used. The sprayer 40 has a triple pipe structure, and a background electrolyte solution supplied from the separation channel 14 flows through a central electrolyte solution channel 41. A sheath liquid containing a large amount of a polar organic solvent such as acetonitrile or methanol, which is supplied from the control flow path 34, flows through the outer sheath liquid pipe 42. Further, a sheath gas such as nitrogen is supplied to the outermost sheath gas conduit 43, and sheath gas flowing out from the end of the sheath fluid conduit 42 is surrounded by the sheath gas ejected from the end of the sheath gas conduit 43. It is suppressed to spread. As shown in FIG. 6, the background electrolyte liquid flowing out from the end of the electrolyte liquid conduit 41 and the sheath liquid flowing out from the end of the sheath liquid conduit 42 are connected by a liquid reservoir 44 formed outside the end. Thereby, the conductivity of the solution in both the pipes 41 and 42, and hence the solution in the separation channel 14 and the control channel 34, is ensured. When a sufficient potential is applied to the electrospray, spraying occurs outside this pool 44.

  In the case of the structure of the sprayer 40, the solution junction, that is, the liquid reservoir 44 faces the spray chamber 20 and is easily dried. Therefore, at the time of electrokinetic injection as described above, the sheath liquid may be supplied to the sprayer 40 through the control flow path 34 by pressurizing the inside of the control reservoir 30 by the second pressure adjusting unit 33. As a result, the sheath liquid oozes out almost continuously from the end of the sheath liquid duct 42, and the liquid pool 44 can be maintained. Similarly, when separating and ionizing sample components, the sheath liquid may be fed by pressurization.

  It should be noted that the above embodiment is merely an example of the present invention, and it is obvious that any appropriate changes, modifications, additions, etc. within the scope of the present invention are included in the scope of the claims of the present application.

In each reservoir, the electrode is disposed at a position higher than the opening at the inlet end of the separation channel and the control channel, that is, a position close to the interface of the solution. Even when bubbles are generated, the bubbles rise to the solution interface and diffuse into the gas phase, and the bubbles do not enter the separation channel or the control channel. Therefore, the flow of the solution and the flow of the sample liquid are not hindered by the bubbles. Further, the conductivity through the solution is not inhibited by bubbles.

On the other hand, the inlet end 34 a of the control channel 34, which is a capillary tube different from the separation channel 14, is immersed in the solution 30 a stored in the control reservoir 30. The control reservoir 30 is a container that can be substantially sealed like the inlet reservoir 10, and a gas (for example, nitrogen, synthetic air, etc.) adjusted to a predetermined gas pressure by the second pressure adjusting unit 33 is supplied through the gas supply pipe 32. It has become so. Further, a liquid contact electrode (hereinafter referred to as “control electrode”) 35 is installed so as to be immersed in the solution 30 a stored in the control reservoir 30, and a predetermined high voltage is supplied from the control electrode 35 to the solution 30 a by the second high voltage power source 36. A voltage can be applied. In this example, the solution 30a is also a background electrolyte solution.

FIG. 5 is an enlarged view of the control reservoir 30. As shown in FIG. 5, the inlet end 34a of the control channel 34 is inserted deeply in the background electrolyte solution 30a until it reaches the vicinity of the inner bottom surface of the control reservoir 30, and the control electrode 35 is installed in close proximity thereto. Yes. Control the lower end of the electrode 35 is slightly than the inlet end 34 a of the control passage 34 (height d only) a high position, i.e., to a position near the interface of the background electrolyte solution 30a, both are positioned respectively . Therefore, as will be described later, even if bubbles are generated on the surface of the electrode 35 due to the oxidation-reduction reaction when the electrodes 15 and 35 are energized, the bubbles rise as they are and diffuse into the upper space in the control reservoir 30, and the inlet end 34a. Does not enter the control flow path 34. Thus, in order to avoid intrusion of bubbles, the height d is preferably large, but the electrical resistance value of the gap between the control electrode 35 and the inlet end 34a increases as d increases. Since it is desirable that the electrical resistance value be as small as possible, it is preferable to determine an appropriate d in consideration of both.

(A) Filling the separation flow path 14 and the control flow path 34 with the background electrolyte solution First, as a preparatory stage for electrophoresis in the capillary electrophoresis section 1, the separation flow path 14, the control flow path 34, and the sprayer 21 Fill the channel with background electrolyte. That is, as shown in FIG. 2, the outlet end 34 b of the control channel 34 is connected to the control inlet end 21 c of the sprayer 21, and the inlet end 34 a is immersed in the background electrolyte solution 30 a in the control reservoir 30. The outlet end 14 b of the separation channel 14 is connected to the separation inlet end 21 b of the sprayer 21, and the inlet end 14 a is immersed in the background electrolyte solution 10 a in the inlet reservoir 10. Under the control of the control unit 24, when the gas appropriately regulated by the first pressure regulating unit 13 is fed into the inlet reservoir 10, the outlet end 14b of the separation channel 14 is at substantially atmospheric pressure. The background electrolyte solution in the inlet reservoir 10 is sucked into the separation channel 14 by the differential pressure across the separation channel 14 and filled therein.

(B-1) Pressurized injection As in the case of filling the background electrolyte solution described above, the gas appropriately adjusted by the first pressure adjusting unit 13 under the control of the control unit 24 is supplied to the sample inlet reservoir 17. The sample liquid 17 a is pressurized and sucked into the separation channel 14. When an appropriate amount of the sample liquid 17a is injected into the separation channel 14, the pressurization by the first pressure adjusting unit 13 is stopped, and the inside of the sample inlet reservoir 17 is returned to substantially atmospheric pressure. As a result, the sample liquid is held in the predetermined range from the inlet end 14a of the separation channel 14. In this case, earlier because some background electrolyte solution filled in the separation flow path 14 is discharged to the outside from the mists port 21d, perform special considerations, such as during electrokinetic injection, which will be described later Even if not, the spray port 21d at the tip of the nozzle 21a does not dry.

(D) Measurement of electric resistance value between both ends of control flow path 34 As described above, the electric resistance value between both ends of the control flow path 34 can be obtained by calculation. You can ask for it. That is, as shown in FIG. 3, the outlet end 34 b of the control flow path 34 is removed from the control inlet end 21 c of the sprayer 21 and immersed in the background electrolyte solution 37 a stored in the reference reservoir 37. The transfer of the outlet end 34b of the control flow path 34 is generally performed manually by the person in charge of analysis, but may be automatically performed by providing an appropriate transfer mechanism or the like. Then, the gas that has been moderately regulated by the second pressure regulating unit 33 is sent to the control reservoir 30 to pressurize the background electrolyte solution 30a, thereby filling the control electrolyte 34 with the background electrolyte solution 30a.

Claims (7)

In an electrophoresis / electrospray ionization apparatus that separates sample components by electrophoresis and ionizes them by spraying them in a substantially atmospheric pressure atmosphere while applying charge to the separated sample components.
a) a separation channel for separating sample components;
b) an inlet reservoir in which a predetermined solution is stored and an inlet end of the separation channel is immersed;
c) an inlet electrode immersed in the solution in the inlet reservoir and disposed higher than the inlet end of the separation channel;
d) a control reservoir in which a predetermined solution is stored;
e) a control flow path arranged such that the inlet end is immersed in the solution in the control reservoir;
f) a control electrode immersed in the solution in the control reservoir and disposed at a position higher than the inlet end of the control flow path;
g) The outlet end of the separation channel and the outlet end of the control channel are connected so that the solution in both channels can be contacted, and the solution sent from the separation channel can A sprayer that can be sprayed into,
h) voltage applying means for applying a voltage to each of the inlet electrode and the control electrode;
In the state where the inlet electrode and the control electrode are electrically connected via the solution in the separation channel, the sprayer, and the control channel, a voltage is applied to both electrodes by the voltage applying means. Electrophoresis characterized in that the sample components are electrophoresed and separated in the separation channel by application, and the solution is sprayed with an electric potential generated by the potential generated in the solution in the nebulizer. / Electrospray ionizer.   2. The electrophoresis / electrospray ionization apparatus according to claim 1, wherein the separation channel and the control channel are non-conductive capillary tubes.   2. The electrophoresis / electrospray ionization apparatus according to claim 1, wherein a cross-sectional area and a length of the control flow path and an electrical resistivity of a solution filled in the flow path are determined between both ends of the control flow path. An electrophoretic / electrospray ionization apparatus, further comprising arithmetic means for calculating an electric resistance value and calculating an electric potential generated in the solution in the nebulizer using the electric resistance value. The electrophoresis / electrospray ionization apparatus according to claim 1,
A reference reservoir in which a predetermined solution is stored and an outlet end of the control channel is immersed;
A reference electrode immersed in the solution in the reference reservoir and maintained at ground potential;
When a voltage is applied to the control electrode by the voltage application means in a state where the control electrode and the reference electrode are electrically connected via the solution in the control flow path, the applied voltage and correspondingly Calculating an electric resistance value between both ends of the control flow path from the flowing current, and calculating an electric potential generated in the solution in the nebulizer using the electric resistance value;
An electrophoretic / electrospray ionization apparatus, further comprising:   The electrophoresis / electrospray ionization apparatus according to any one of claims 1 to 3, wherein the inlet reservoir and the control reservoir are each sealable, and an inlet regulator that adjusts a pressure applied to a solution in the inlet reservoir. Pressure adjusting means, and control pressure adjusting means for adjusting the pressure applied to the solution in the control reservoir, and pressurizing the solution in each reservoir by the inlet pressure adjusting means and the control pressure adjusting means, An electrophoresis / electrospray ionization apparatus characterized in that a solution is filled in the separation channel and the control channel.   2. The electrophoresis / electrospray ionization apparatus according to claim 1, wherein the nebulizer has a confluence portion where a solution from the separation channel and a solution from the control channel merge inside the nebulizer, An electrophoresis / electrospray ionization apparatus characterized in that the merged solution flows out from a nozzle to the outside.   2. The electrophoresis / electrospray ionization apparatus according to claim 1, wherein the nebulizer has a double-pipe structure in which a pipe through which the solution from the separation channel flows and a pipe through which the solution from the control channel flows. An electrophoresis / electrospray ionization apparatus characterized in that both solutions are in contact with each other outside the open ends of both pipes.

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