JP2001135269A - Two-phase micro-flow electrospray mass spectrometry - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a new ionization unit, for electro-spray ionization method(ESI) in mass spectrometer, that can apply nonaqueous solution as is and analyze solutions of inert solvent used in solvent extraction and substances produced at interface between a water and organic phases. SOLUTION: This method relates to an electrospray ionization unit, having a feeding path that can concurrently supply aqueous and organic phases to the ionizing portion, an aqueous-phase ionizing portion where the aqueous phase can fully be ionized, and a mixing portion where the organic phase is supplied into the aqueous-phase ionizing portion to mix the both, the electrospray ionization method using the electro-spray ionization unit, and a mass spectrometrometer by means of it. A capillary 2, that can be charged integrates a nonconductive tube 6 for feeding the organic phase 4, the tip end of the nonconductive tube has been shorter than the tip part of the chargeable capillary, and a mixing portion 3, that mixes the aqueous phase 5 and the organic phase 4, is provided between the tip ends of the nonconductive tube and the chargeable capillary.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to an electrospray ionizer which is an ion source in a mass spectrometer. The present invention provides a novel electro-electrode characterized by providing a supply path capable of simultaneously supplying an aqueous phase and an organic phase to an ionization section in order to ionize a hydrophobic substance into ions in an amount sufficient to obtain a mass spectrum. The present invention relates to a spray ionizer, an ionization method using the same, and a mass spectrometer using the same.

[0002]

2. Description of the Related Art Mass spectrometers (MS) are useful for identifying samples of biological substances such as proteins and environmental pollutants such as dioxins and elucidating the material structure. MS in combination with chromatography such as liquid chromatography and gas chromatography is rapidly developing. MS
Is to ionize a sample and analyze the ionized molecular species in an electric field. The MS apparatus mainly comprises a sample supply unit, an ion source, a mass analysis unit, and a detection unit. As the mass analysis unit, a quadrupole type, a magnetic field type, a time-of-flight type (TOF), and the like are used. I have. In order to introduce a sample into the mass spectrometer, it is necessary to ionize the molecules of the sample, and the sample is ionized by an ion source. Examples of ionization methods in an ion source include an electron impact method (EI),
There are various ionization methods such as chemical ionization (CI), electrospray ionization (ESI), and atmospheric pressure chemical ionization (APCI).

Among them, atmospheric pressure ionization methods such as electrospray ionization method (ESI) and atmospheric pressure chemical ionization method (APCI) can softly ionize a sample solution under atmospheric pressure and perform various liquid phase separations such as chromatography. It has the advantage that it can be easily connected to devices and is widely used.
Both ESI and APCI are ionized at a stage before entering the vacuum system of the mass spectrometer to form monovalent or polyvalent ions.
At this time, a protonated molecule is generated in the positive ion mode, and a deprotonated molecule is generated in the negative ion mode, and the ionized molecular species is subjected to measurement.

Electrospray, especially electrospray ion (ESI) using auxiliary spray gas
In the above, a stainless steel capillary (with an inner diameter of 70 to 100) having a flow path of a spray gas around the sample solution is used.
μm) and introduced into the ion source at a flow rate of 1 μL / min to 1 mL / min. When a high voltage of 3 to 4 kV is applied to the tip of the capillary, the sample coming out of the capillary becomes a charged mist and is discharged into the ion source. A high-speed atomization gas is emitted from the periphery of the capillary, and charged atomized substances are released straight and widely over the atomization gas. The ionized molecular species is introduced into an intermediate vacuum region through a pinhole called an orifice further from the tip of the capillary, and further introduced into the mass spectrometer.

[0005] ESI ionizes from a solution state. As a solvent at that time, a polar solvent capable of promoting ionization or a polar solvent of a water-containing system, for example, water / acetonitrile or water / methanol 1: 1 ( v / v) solutions are most often used. Therefore, when combined with liquid chromatography using such a polar solvent, MS can be measured in a solution state, and ESI can be measured.
Is widely used as a method for determining the structure of trace dissolved species.

However, the ESI method cannot use a solution of a nonpolar solvent, and is not suitable for the ESI method because many organic compounds such as proteins and dioxins are lipophilic and water-insoluble. is the current situation. Also,
The organic phase of the solvent extraction system cannot be directly used as a sample for the ESI method. Further, at present, non-ionized substances, that is, non-conductive inert organic solutions cannot be used by the ESI method. The ESI method is a convenient measurement method capable of performing mass spectrometry as it is by ionizing softly in a solution state under atmospheric pressure, and it has been demanded to expand the applicable range.

[0007]

SUMMARY OF THE INVENTION The present invention provides a novel ionization apparatus which can directly apply a non-aqueous solution to electrospray ionization (ESI) in a mass spectrometer. The present invention also provides a novel ionization apparatus capable of analyzing a solution of an inert solvent used in solvent extraction and a substance generated at an interface between an aqueous phase and an organic phase.

[0008]

In the electrospray ionization method (ESI), the excess charge necessary for ionizing a target substance is mainly due to electrolysis of a solvent, and a solvent which can be electrolyzed is used. I had to. Therefore, when only a non-conductive inert organic solvent is used, a charge required for ionizing the target substance cannot be obtained. In addition, it is possible to use a non-conductive inert organic solvent and a solvent that can be electrolyzed as a solvent, but if both are not uniformly mixed, that is, if they cannot be dissolved with each other, the tip of the spray can be used. Since the part of the capillary was extremely thin, usually 70 to 100 μm, both solvents could not be sprayed uniformly.

[0009] It is known that a relatively stable dispersion can be prepared if the volume ratio is about 1 to the aqueous phase per 1000 of the aqueous phase. The organic droplets may become large in the capillary before reaching the ionization part due to the difference in the stability of the water, or the droplets may move up and down due to the density difference with water and adsorb on the inner wall of the capillary. Further, since the size and time of the organic droplets cannot be controlled, there is no versatility. Further, for the same reason, it was impossible to ionize chemical species generated at the liquid-liquid interface.

The present inventors have further studied to solve this problem, and as a result, studied a method in which the aqueous phase and the organic phase are separately supplied to the ionization section, and the two are simultaneously mixed in the ionization section. did. As a result, it was found that when the solvent capable of being electrolyzed in the aqueous phase portion was introduced into the ionization portion in a state where it was sufficiently electrolyzed, a mass analysis spectrum was obtained. Furthermore, it has been surprisingly found that a mass spectrometric spectrum of the chemical species generated at the liquid-liquid interface between the aqueous phase and the organic phase can be obtained.

Therefore, the present invention provides a supply path capable of simultaneously supplying an aqueous phase and an organic phase to an ionization section, an aqueous phase ionization section in which the aqueous phase can be sufficiently ionized, and an organic phase in the aqueous phase ionization section. The present invention relates to an electrospray ionization apparatus having a mixing section supplied and mixed, an electrospray ionization method using the apparatus, and a mass spectrum apparatus using the same. The electrospray ionization device of the present invention further includes various devices used in the conventional electrospray ionization device, such as a spray gas supply path for supplying a spray gas and a dry gas supply device for drying ionized materials. You can also.

More specifically, the present invention incorporates a non-conductive tube for supplying an organic phase in a chargeable capillary, and the tip of the non-conductive tube has a chargeable tip portion. Electrospray ionization apparatus, wherein a mixing section in which an aqueous phase and an organic phase are mixed between the tip of the non-conductive tube and the tip of the capillary is provided,
The present invention relates to an electrospray ionization method using the device and a mass spectrum device using the same.

According to the electrospray ionization apparatus of the present invention, the organic phase supply path, that is, the fine droplets of the organic phase sprayed from the tip of the non-conductive tube are almost simultaneously sprayed in the mixing section. The droplets are covered with water supplied from a supply path of an aqueous phase, ie, a chargeable capillary, and the droplets are charged and ionized by electrolysis of water. Further, when the organic phase is covered with water, a liquid-liquid interface is generated, and the chemical species generated at this interface are directly ionized.

FIG. 1 shows an example of an electrospray ionization apparatus (hereinafter, referred to as an ESI apparatus) of the present invention. FIG.
The ESI apparatus 1 of the present invention shown in FIG. 1 is of a two-phase direct introduction type in which a non-conductive tube 6 serving as an organic phase supply path is contained inside a capillary 2 serving as an aqueous phase supply path. Things. The organic phase 4 is introduced from a non-conductive tube 6 and the aqueous phase 5 is introduced from a capillary 2.
The capillary 2 can be charged, and a high voltage is applied. The tip of the non-conductive tube 6 is designed to be slightly shorter than the tip of the capillary 2, and the organic phase 4 sprayed from the tip of the non-conductive tube 6 becomes water in the mixing section 3 inside the capillary 2. It is mixed with phase 5. At this time, the aqueous phase 5 is electrolyzed or charged by the high voltage applied to the capillary 2, and the charged aqueous phase is wrapped around the microdroplets sprayed from the organic phase 4 and discharged from the tip of the capillary 2. Is done. The microdroplets covered with the aqueous phase are dried like ordinary ESI, gradually release the solvent, and become ionized chemical species.

FIG. 2 shows the ESI apparatus of the present invention shown in FIG. 1 from the supply ports of the aqueous phase and the organic phase. The ESI device 1 shown in FIG. 1 is connected to a Teflon (registered trademark) tea connector 7 via a support member,
The non-conductive tube 6 inside the capillary 2 penetrates the Teflon tea connector 7 and reaches the supply port 9 of the organic phase 4. The other one port of the Teflon tea connector 7 is connected to a Teflon tube 8 which is a supply port of the aqueous phase 5. The aqueous phase 5 is supplied from a Teflon tube 8, its path is bent at a right angle by a Teflon tea connector 7, and is supplied to the portion of the ESI device 1 through the outside of the non-conductive tube 6 inside the capillary 2. On the other hand, the organic phase 4 is supplied from the supply port 9 and the non-conductive tube 6
Is supplied to the ESI device 1 through the inside. The chemical species ionized by the ESI device 1 passes through an interface or orifice 10 and is supplied to a mass spectrometer 11.

In order to send the organic phase into the aqueous phase with the smallest possible droplets, it is necessary to make the tip of the non-conductive tube 6 (hereinafter also simply referred to as a capillary) thin. For the processing, a silica capillary tip processing apparatus system as shown in FIG. 3 was constructed. The laser beam emitted from the carbon dioxide gas laser 21 is collected by the condenser lens 22 and applied to the capillary 23. A weight 24 is attached to the capillary 23, and a heat-resistant brick 25 is arranged behind the weight 24 for safety. The power supply and control of the carbon dioxide laser were performed using a stabilized DC power supply 26 and a function generator 27. The capillary should be 7 mm away from the focal length of the condenser lens so that the side you want to keep is down.
The position was determined from a burn mark where the laser was directly applied to the heat-resistant brick 25 at a distance of about 10 to 10 mm, and the position was adjusted until it was cut off as appropriate.

FIGS. 4 and 5 show the results of observing a silica capillary whose tip was thinned using this apparatus system with an optical microscope BX-60 (Olympus Optical Industry Co., Ltd.). FIG. 4 shows a silica capillary (GL Science) having an outer diameter of 150 μm and an inner diameter of 75 μm, and FIG.
The microscope image which processed the silica capillary (the same front) of 50 micrometers in inside diameter with this apparatus is shown. FIG. 4 shows a 10 × image of the portion where the thinning starts, and FIG. 5 shows a 10 × image of the tip portion. 4 and 5 are micrographs replacing the drawings. The tip diameter is about 12 μm outside diameter, about 8 μm inside diameter, and 50 μm inside diameter for a 75 μm inside diameter silica capillary.
For silica capillary, outer diameter is about 13μm, inner diameter is about 7μ
m, and the length from the portion where thinning started to the tip was about 2 mm for both capillaries.

A two-phase direct introduction electrospray ionization apparatus shown in FIG. 2 was assembled using the silica capillary thus prepared as a non-conductive tube 6. Using Teflon T-connector 7, stainless steel capillary 2 (200 μm inner diameter)
m, outer diameter 400 μm). The organic phase introduction side is
It was made to be in line with this stainless capillary, and the other was made into the aqueous phase introduction side. In order to mix the organic phase and the aqueous phase in this stainless capillary 2, a silica capillary of about 30 cm (outside diameter 150 μm, inside diameter 50 μm, tip Outer diameter 13μm,
(Inner diameter: 7 μm) was inserted, and was projected to the organic phase introduction side through the connector. The silica capillary of this diameter was used because it was desired to reduce the size of the organic phase droplets as much as possible and that the strength of the tip was stronger when the glass thickness of the original capillary was somewhat large. The silica capillary was introduced from the tip of the stainless capillary and fixed so that the tip of the capillary was narrowed inward by about 1 mm. At this portion, droplets of the organic phase solution are released into the aqueous phase solution flowing outside the silica capillary 6. The organic phase solution was introduced directly through this silica capillary. On the other hand, a Teflon tube 8 (outer diameter 1.58 mm, inner diameter 0.25 mm) of about 30 cm was attached to the aqueous phase introduction side to introduce the aqueous phase solution.

As the mass spectrometer, a time-of-flight mass spectrometer Jaguar (ESI-TOF-MS, Sens) used in an electrospray ionization unit equipped with the two-phase direct introduction electrospray ionizer of the present invention shown in FIG.
ar Larson-Davis). Figure 6 shows J
1 shows an aguar body. TOF-MS has a characteristic feature in that the measured mass range is wide, and the mass range is theoretically unlimited. However, conventionally, MALDI which has a low time resolution and is most suitable for the ionization section has been used. But,
Jaguar has improved 5000 spectra per second and low time resolution without deteriorating the merits of TOF-MS, and uses ESI for the ionization section. In addition, this E
The SI device has a simple structure and facilitates joint with other separation devices such as CE-MS and LC-MS. This was also a major advantage in incorporating the newly developed two-phase direct introduction device. FIG. 6 shows the prepared two-phase direct introduction device set in an electrospray ionization unit.

A Teflon tube (outer diameter 1.58 mm, inner diameter 0.25 mm) was attached to the aqueous phase introduction side via a deactivated silica capillary for the introduction of the organic phase and a gas tight syringe and a single microprocessor syringe, respectively. I connected the pump. Ordinary measurement conditions were as follows: measurement time: 6 minutes, integration: 51200, electrospray ionization section voltage: 4.4 kV, interface voltage: 600 V, skimmer voltage: 70 V, nozzle voltage: 100 to 500 V. The flow rate of the sample was 0.1 ml / hr for the aqueous phase,
The organic phase was changed from 0.001 ml / hr to 0.02 ml / hr. Note that a polyethylene glycol (PEG) solution was used for calibration.

In the two-phase direct introduction device prepared here, FIG.
Since the diameter of the stainless capillary is large as described above, there is a possibility that the ESI is insufficient at the normally applied ESI voltage (2 to 3 kV). Therefore, PE which is relatively easy to desolvent first
It was examined whether a spectrum could be obtained using the G solution.
For the measurement, a water-methanol 1: 1 solution was used for the aqueous phase, and
G solution was performed. FIG. 7 shows the spectrum.
In this spectrum measurement, the ESI voltage was 3.0 kV, the integration time was 3 minutes, the aqueous phase flow rate was 0.1 ml / hr, and the organic phase flow rate was 0.0.
The test was performed at 5 ml / hr. That the ESI is normally detected by the two-phase direct introduction device produced from the results and ions can be detected;
It was also confirmed that the capillary flowing in the organic phase was functioning normally.

Next, the possibility of mass spectrum measurement was examined using the sample solution. This means that in the mass spectrum measurement using a PEG solution, the solvent is water-methanol and the organic phase is water-methanol.
While the 1: 1 solution is a very suitable solvent for ESI-MS measurement, the solvent of the sample solution is an aqueous acetic acid solution saturated with toluene in the aqueous phase, and the organic phase is toluene. This is because it is expected that desolvation hardly occurs. As countermeasures, it has been considered to increase the ESI voltage to promote the generation of charged droplets and to reduce the flow rate of the organic phase as much as possible to increase the volume ratio with the aqueous phase. Therefore, the mass spectrum was measured using the ESI device of the present invention while changing the ESI voltage. 5.0 × 10 for the aqueous phase
⁻⁶ mol dm ⁻³ toluene-saturated aqueous solution of Cu (II) acetic acid, and the organic phase of 1.0 × 10 ⁻³ mol dm ⁻³ of the following formula (I)

[0023]

Embedded image

2- (5-bromo-2-pyridylazo) -5-diethylaminophenol (hereinafter referred to as 5-
It is called Br-PADAP. ) A toluene solution was used. 5-Br-PADAP used in this experiment is one type of chelating reagent, and is a red-brown crystal that is almost insoluble in water and soluble in alcohol and chloroform. Its acid dissociation mechanism is

[0025]

Embedded image

And the respective pK
The value of a is pKa₁Is 0.1, pKa ₂Is 2.02, p
Ka₃Is 11.3 (all in 50% ethanol)
You. This compound coordinates to the metal ion with N-N-O
It acts as a tridentate ligand and its visible absorption spectrum is complex.
It changes sharply by generation and the shift is large, so various
Excellent spectroscopy due to its excellent sensitivity as a highly sensitive colorimetric reagent for metals
The direct approach from the method is easy. Furthermore, large
Porphy known as a colorimetric reagent with a high molar extinction coefficient
The complexation reaction is faster than phosphorus. Copper is another metal
The complexation reaction is faster than this.
This compound was used in experiments on complexation reactions during two-phase direct introduction.
And Cu (II) ions as metal ions.

Hereinafter, Cu (II) -5-Br-PADAP
In the measurement of ESI-TOF-MS by two-phase direct introduction of the complex, this concentration was used unless otherwise specified. The flow rates were 0.1 ml / hr and 0.001 ml / hr, respectively. The obtained mass vectors when the voltage of the ESI device is 3.8 kV, 4.0 kV and 4.4 kV are shown in FIGS. 8, 9 and 10, respectively.

Droplets are not sprayed up to 3.5 kV,
Further, up to 3.8 kV, the generation of charged droplets was insufficient and the spectrum could not be obtained well, and 4.4 kV near the setting limit of the apparatus was not obtained.
, It was found that a sufficient mass spectrum could be obtained. From this result, the measurement was performed with the ESI voltage set to 4.4 kV. The mass spectra of FIGS. 8, 9 and 10 are obtained by measuring the Cu (II) -5-Br-PADAP complex, and it was found that mass spectra of organic compounds and the like can be obtained by the apparatus of the present invention. No specific peak derived from the complex or ligand could be confirmed from this spectrum. Therefore, it was considered that desolvation was promoted by increasing the nozzle voltage, clusters derived from the solvent were decomposed, and a complex peak was obtained as much as possible. In addition, the flow rate of the organic phase was increased to cope with the shortage of the amount of the organic phase.

By improving these points, a mass spectrum in which complex peaks clearly appeared could be obtained. This is shown in FIG. Main Cu (II) -5-Br-PAD
1: 2 complex [CuL (HL)] ⁺ and 1:
1 complex [CuL] ⁺ was confirmed, and 5-Br-PAD
A protonated product of AP [H ₂ L] ⁺ was also confirmed. In the spectrum, a 2: 3 complex [Cu ₂ L ₃ ] ⁺ ,
A peak indicating the presence of the 2: 2 complex [Cu (I) Cu (II) L ₂ ] ⁺ was also observed.

FIG. 12 shows that the Cu (II) concentration is 1.0 × 1.
0^-5mol dm^-3Figure on condition except that
11 shows the same mass spectrum as in FIG. Like this
Decompose and desolvate peaks that appear to be medium clusters.
A mass spectrum that cannot be obtained was obtained, and the 2: 3 complex [C
u2L₃]⁺2: 2 complex [Cu (I) Cu (II)
L ₂]⁺Could not be confirmed. However, the 1: 2 complex [C
uL (HL)]⁺And 1: 1 complex [CuL]⁺And 5-Br
-A protonated product of PADAP [H₂L]⁺Is catching
Therefore, as a result, this two-phase direct introduction ESI method
From: A complex reaction occurs via the mixed two phases,
Being able to capture that information. 2. Conventionally, a ligand peak was detected in the organic phase
Mass specs of samples in organic solvents, which have been considered difficult to use
Is possible. Was clearly shown.

To solve the problem pointed out above,
Since the nozzle voltage has been increased and the organic phase flow rate has a large effect on the spectrum, the change in the nozzle voltage, that is, the peak [CuL (HL)] ⁺ , [ The effect on CuL] ⁺ and [H ₂ L] ⁺ was examined. 30 to 30
This was performed using the ratio of the count number of the peak to the total ions counted at 1500 (m / z), or the count number. Nozzle voltage 100V, 200V, 300
FIG. 13 shows an example of a mass spectrum obtained when V, 400 V, and 500 V are changed. FIG. 13 shows the case where the nozzle voltage is set to 400V. The flow rate of the organic phase at this time is 0.02 ml / hr. FIG. 14 is a graph showing the relationship between the intensity of the [CuL (HL)] ⁺ , [CuL] ⁺ and [H ₂ L] ⁺ peaks and the number of counts, and the relationship between the peak voltage and the nozzle voltage. FIG. Further, as a comparison, a relationship diagram when the flow rate of the organic phase is 0.005 ml / hr is shown in FIG.
And FIG. From these figures, there is a tendency that the total count number is maximum when the nozzle voltage is 200 V, whereas [CuL (HL)] ⁺ , [CuL] ⁺ and [H ₂ L] ⁺ peak intensity generally increase with the nozzle voltage. Has been increased or maintained as it is. This means that [CuL (H
L)] ⁺ , [CuL] ⁺ and [H ₂ L] ⁺ are relatively stable against an increase in the collision activity due to an increase in the nozzle voltage, whereas the total count number probably includes peaks such as a solvent. It was shown that declustering and desolvation were caused by an increase in collision activity with an increase in nozzle voltage.

Here, the change in the flow rate of the organic phase [C
uL (HL)] ⁺ , [CuL] ⁺ and [H ₂ L] ⁺ . The flow rate of the organic phase is 0.00
1 ml / hr, 0.005 ml / hr, and 0.02 m
FIG. 18 shows an example of a mass spectrum obtained by changing l / hr. FIG. 18 shows the case where the flow rate is 0.02 ml / hr. At this time, the Cu (II) ion concentration was 5.0 × 1.
A ^{3 -} 0 ^-6 mol dm. [CuL (HL)] ⁺ , [CuL] ⁺
And [H ₂ L] ⁺ increased. This means that an increase in the amount of reagent due to an increase in the flow rate of the organic phase increases the chance of a complex reaction, and results in [CuL (HL)] ⁺ , [CuL
L] ⁺ is considered to have increased.

[CuL (HL)] ⁺ ,
It is considered that the increase in the count of [CuL] ⁺ , that is, the increase in the chance of the complex reaction, depends on the amount of the organic phase released into the aqueous phase solution per unit time. This means that by taking the time resolution of the complex count number for the measurement time of 6 minutes, the flow rate of the organic phase is slow, that is, when the amount of the organic phase per unit time is small, the count number of the complex is small, and conversely, the organic phase flow rate is low. When it is fast, that is, when the amount of organic phase per unit time is large, the number of complex counts should be large. Then, the 1: 2 complex [CuL (HL)] from which the peak was most remarkably obtained was obtained.
^With respect to ⁺ , the number of counts during the 6-minute measurement was examined by time resolution. FIG. 19 shows the time-resolved result of the count number of [CuL (HL)] ⁺ in the mass spectrum at the flow rate shown in FIG.

In normal ESI measurement, continuous [CuL]
The count of ⁺ was obtained as shown in FIG. 20, but in this two-phase direct introduction ESI-TOF-MS, the complex was obtained intermittently at any flow rate of the organic phase, and the complex was sprayed continuously. Turned out not to be. The appearance interval of the peak at this time was 0.001 ml of the organic phase flow rate.
/ Hr for about 100 seconds, 0.005 ml / hr for about 30 seconds
It could be estimated at 15 seconds at 0.02 ml / hr in seconds, and similar results were obtained with [CuL] ⁺ and [H ₂ L] ⁺ . From these results: The organic phase is not sprayed until it reaches a certain size in the aqueous phase solution. 2. While not sprayed, complex products in the two phases and materials in the organic phase do not diffuse into the aqueous phase solution. It has been suggested.

Here, based on the results so far, the reaction at the two-phase introduction part and the mass spectrum [CuL
(HL)] ⁺ and the origin of [CuL] ⁺ were examined.
In the study, solvent extraction was performed by adjusting the concentration and volume conditions as much as possible with the two-phase direct introduction ESI method, and the absorption spectra of both the aqueous phase and the organic phase were measured. In this experiment, a test tube with a lid was set to 1.0 × 10 ^{−3 to} 1.0 × 10 ⁻¹⁵ m.
oldm- ³ 5-Br-PADAP toluene solution 5m
and 25 ml of 5.0 × 10 ⁻⁶ moldm ⁻³ copper acetate aqueous solution saturated with toluene. This is because Cu (II) -5-Br-PAD of two-phase direct introduction ESI-TOF-MS
This is because, in the measurement of the AP complex, the volume ratio of the flow rate of the organic phase (0.02 ml / hr) and the flow rate of the aqueous phase (0.1 ml / hr) when the mass spectrum was best obtained was considered.
This mixed solution was stirred for 1 hour, separated into an organic phase and an aqueous phase by a centrifugal separator, and the respective absorption spectra were measured. In order to obtain only the absorption of the complex, a difference spectrum was obtained for each of the organic phase and the aqueous phase.

The results are shown in FIG. 21 (organic phase) and FIG.
(Aqueous phase). The organic phase having the same concentration as that in the two-phase direct introduction ESI-TOF-MS measurement is 1.0 × 10 ⁻³ mol d
Under the conditions of m ^{-3 5-Br-PADAP,} the organic phase, the absorption of the complex was observed in the aqueous phase both complex was found to be extracted into the organic phase. On the other hand, when the concentration of 5-Br-PADAP is 1.0 × 10 ⁻⁴ mol dm ⁻³ or 1.0 × 10 ⁻⁴ mol dm ^{−3.
At −5} mol dm ⁻³ , the complex was absorbed in the aqueous phase, but hardly observed in the organic phase. The composition of the Cu (II) -Br-PADAP complex in a toluene solution is reported as a 1: 2 complex [CuL] ⁺ . The Cu (II) -Br-PADAP complex present in the aqueous solution is a 1: 1 complex. Therefore, it was found that the concentration condition at this time by the two-phase direct introduction ESI method is a condition under which a complex reaction occurs through the two phases, and the complex is extracted into the organic phase.

In consideration of the above, the reaction in the two-phase introduction part was considered as shown in FIG. The organic phase droplets released into the aqueous phase solution were present in the stainless capillary until a certain size, suggesting that a complex reaction would occur. At this time, 1: 1 complex [CuL] ⁺ and 1: 2 complex [Cu
L ₂ ] is generated, [CuL] ⁺ is adsorbed on the interface on the aqueous phase side, and [CuL ₂ ] is adsorbed on the interface on the organic phase side or is extracted in the organic phase. From the above results, almost no diffusion of these products into the aqueous phase solution has occurred, and when the organic phase droplets have a certain size, a part or all of them are sprayed together with the aqueous phase solution,
It was considered that after desolvation of the charged droplets, they were detected as gas phase ions.

From the above, according to the ESI apparatus of the present invention, the substance in the non-polar organic solvent is also ionized and a mass spectrum is obtained, and if necessary, the mass spectrum of the chemical species generated at the liquid-liquid interface is obtained. I found that I could get it. The ESI device of the present invention mixes an organic phase and an aqueous phase in a mixing section, and preferably mixes an aqueous phase so as to enclose the organic phase. At that time, a part or all of the aqueous phase can be electrolyzed or charged. In this case, a high voltage is applied to the mixing section.

The mixing section of the ESI device of the present invention is made of a chargeable material so that the aqueous phase can be electrolyzed or charged. The chargeable material may be iron such as stainless steel, copper, nickel, or various alloys.
In the above example, stainless steel was used. The size of the mixing portion can be of various sizes, but preferably a medium size of a stainless steel capillary used for normal ESI. The mixing section can be separately provided as a mixing section, but mixing in a normal capillary is also preferable in terms of equipment. When the mixing section is provided inside the chargeable capillary, the tip of the non-conductive tube serving as the supply path of the organic phase is slightly shorter than the tip of the chargeable capillary, so that the non-conductive section is provided. Between the tip of the tube and the tip of the chargeable capillary can be a mixed portion of an aqueous phase and an organic phase. The distance between both tips is
The residence time in the mixing section is 0.1 to 3 seconds, preferably 0.5
It may be about 1.5 seconds. For example, when the residence time in the mixing section is 1 second and the flow rate of the organic phase or the aqueous phase, whichever is earlier, is 1 mm / sec, the distance between both ends may be 1 mm.

The capillary serving as the supply passage for the aqueous phase is chargeable, and its material is preferably the one used in the mixing section described above. The material of the non-conductive tube serving as the supply path of the organic phase is not particularly limited as long as it is non-conductive, but may be a silica tube, a glass tube, a plastic tube, or the like. The inner diameter is not particularly limited, but when the supply path for the organic phase is provided inside the supply path for the aqueous phase, the internal diameter is large enough to enter the supply path for the aqueous phase, and the flow of the aqueous phase can be sufficiently performed. Size is preferred. It is preferable that the tip of the non-conductive tube serving as the supply path of the organic phase is made thin enough to spray the organic phase. When the tube of the organic phase is provided in the above-mentioned capillary made of stainless steel, the inner diameter of the tip of the non-conductive tube is 1/5 of the inner diameter of the capillary.
It is about 0 to 1/2, preferably about 1/30 to 1/2, and more preferably about 1/30 to 1/10, but is not particularly limited.

The aqueous phase introduced into the ESI apparatus of the present invention may be water alone, but may be a mixed solvent of water and another solvent, for example, a mixed solvent such as water / methanol or water / acetonitrile. It may be. Further, as shown in the above-mentioned experimental example, a solution in which a chemical species is dissolved in addition to the solvent can be used. The aqueous phase used is preferably saturated in advance with the organic solvent used for the organic phase. The organic phase in the ESI apparatus of the present invention may be a non-polar solvent or a polar solvent, but preferably does not dissolve in water. Further, those which can easily remove the solvent are preferable. The organic phase is preferably purified, but may be unpurified. For example, an organic phase extracted with an organic solvent or an organic phase eluted by liquid chromatography can be used as it is.

In the method of the present invention, a chemical species that does not dissolve in water or a polar solvent can be used by dissolving it in an organic solvent, and a necessary mass spectrum can be obtained from that state.
In the method of the present invention, two or more chemical species are separated into an aqueous phase and an organic phase, and a mass spectrum of the chemical species generated by an interfacial reaction in the mixing section of the ESI device of the present invention is obtained. Can also be used for In this case, the water-soluble species are taken in the aqueous phase, the hydrophobic species are taken in the organic phase, the two are mixed in the mixing section, and the chemical species generated on the spot is directly converted into a mass spectrum according to the present invention. This is one of the major features.

The voltage applied to the chargeable capillary of the ESI device of the present invention may be the same level as that of ordinary ESI, or may be higher. The voltage can be appropriately set depending on the ionization situation. Also,
The nozzle voltage may be about the same as that of normal ESI, but may be higher. It can be set appropriately depending on the degree of ionization of the sample to be used.

In the ESI apparatus of the present invention, the injection gas used in the conventional ESI may be used if necessary. In this case, the propellant gas may be introduced from a propellant gas tube, but a propellant gas tube may be provided further outside the chargeable capillary of the present invention. The ESI apparatus of the present invention is to introduce a water phase and an organic phase separately, and to make various design changes within a range in accordance with the spirit of the present invention in which the aqueous phase and the organic phase are mixed and ionized by a charged mixing section. Can be.

The ESI device of the present invention can be attached to various mass analyzers in the same manner as the conventional ESI. Examples of the mass analyzer include a magnetic field type, a quadrupole type, and a time-of-flight type. The preferred mass analyzer is a time-of-flight (T
OF). As an attachment method, the ion source can be attached by a joint, and the E source of the present invention can be attached.
The SI device can also be integrated and incorporated. Therefore, the present invention also provides a mass spectrometer equipped with the above-described ESI device of the present invention.

[0046]

Next, the present invention will be described more specifically with reference to examples, but the present invention is not limited to these examples.

Example 1 (Production of a Coaxial Capillary) A silica capillary was heated and extended by a laser using a silica capillary tip processing apparatus shown in FIG. The carbon dioxide laser shown in FIG.
YNRAD)) 21 has a wavelength of 10.5 μm and an output of 12.
A 0-watt, 3 mm beam diameter, a plano-convex lens 22 (product name: Ge lens; manufactured by Japan Infrared Industries Co., Ltd.) having a focal length of 30 cm is arranged as a condensing lens,
The heat-resistant bricks 25 were arranged behind the set. When the laser was applied, a weight 24 was attached below the capillary 23 so that the capillary 23 was extended by gravity. The power supply and control of the carbon dioxide laser were performed using a stabilized DC power supply 26 (product name GP035-15R; manufactured by Takasago Seisakusho) and a function generator 27 (product name FG-273; manufactured by Kenwood). The position of the cavities was determined so as to be located at a distance of about 7 to 10 mm from the focal length of the plano-convex lens so that the side to be left was at a lower position, and from the burn mark of directly irradiating the heat-resistant brick 25 with the laser, and the position was adjusted until it was properly cut off. Outer diameter 150
A silica capillary having an inner diameter of 75 μm and an inner diameter of 75 μm (manufactured by GL Sciences Inc.), and then a silica capillary having an outer diameter of 150 μm and an inner diameter of 50 μm (manufactured by GL Sciences Inc.) are set in the capillary 23, and a laser is applied to the silica capillary to form a thin tip. Was manufactured.

The manufactured silica capillary whose tip was thinned was observed with an optical microscope BX-60 (manufactured by Olympus Optical Industries, Ltd.). Images were captured on a personal computer via a cooled CCD camera (product name Imagepoint; Photometrics). FIG. 4 shows a silica capillary (manufactured by GL Sciences) having an outer diameter of 150 μm and an inner diameter of 75 μm, and FIG.
1 shows a microscopic image of a silica capillary (manufactured by GL Sciences Inc.) having an inner diameter of 50 μm and processed by this apparatus.
FIG. 4 shows a 10 × image of the portion where the thinning starts, and FIG. 5 shows a 10 × image of the tip portion. 4 and 5 are micrographs replacing the drawings. The tip diameter is about 12 μm for the 75 μm inner diameter silica capillary, about 8 μm for the inner diameter, about 13 μm for the inner diameter about 50 μm for the silica capillary, and about 2 mm for the length from the part where the taper begins to taper to the tip for both capillaries. Was.

Next, a water flow test was conducted by flowing water through these capillaries. Water droplets were observed at the tip of both capillaries, and no significant damage was observed in the subsequent observation of the tip. The obtained silica capillary was physically usable. About 30 cm of the silica capillary (outside diameter 150 μm, inside diameter 50 μm, tip outside diameter 13 μm) formed in the stainless capillary 2 (inside diameter 200 μm, outside diameter 400 μm) in this manner.
The inner diameter of the silica capillary was 7 μm), and the silica capillary was introduced from the tip of the stainless steel capillary and fixed so that the thinned tip came about 1 mm inward to produce the desired coaxial capillary.

Example 2 (Manufacture of ESI device) The manufactured two-phase direct introduction coaxial capillary was incorporated into a T-shaped connector 7 made of Teflon so that the organic phase introduction side was aligned with this stainless capillary. The other side was the aqueous phase introduction side. The organic phase solution was introduced directly through this silica capillary. On the other hand, the Teflon tube 8 (outer diameter 1.58 m
m, inner diameter 0.25 mm) and the aqueous phase solution was introduced. An organic phase is introduced through a deactivated silica capillary, and a Teflon tube (outer diameter 1.58 m) is placed on the aqueous phase introduction side.
m, an inner diameter of 0.25 mm), and a gas tight syringe (Hamilton) and a single microprocessor syringe pump (Harvard 11; Harvard) were connected.
Thus, the two-phase direct introduction electrospray ionization apparatus shown in FIG. 2 was manufactured.

Example 3 (Mass Spectrometer and Measurement of Mass Spectrum) Time-of-flight mass spectrometer Jaguar (product name: ESI-
TOF-MS; sensor manufactured by Larson-Davis (Se
nsar Larson-Davis)), the two-phase direct introduction electrospray ionizer shown in FIG. 2 manufactured in Example 2 was attached to the electrospray ionizer, and a mass spectrometer was manufactured. FIG. 6 shows the Jaguar main body. The mass spectrum was measured using this mass spectrometer.
Ordinary measurement conditions were as follows: measurement time: 6 minutes, integration: 51200, electrospray ionization section voltage: 4.4 kV, interface voltage: 600 V, skimmer voltage: 70 V, nozzle voltage: 100 to 500 V. The sample solution was introduced into a gas tight syringe (Hamilton) and a single microprocessor syringe bomb, Harvard 11 (HARV (HARV)) for both the organic and aqueous phases.
ARD)), and the flow rate is 0.1 ml / h of the aqueous phase.
r, organic phase 0.001 ml / hr to 0.02 ml / hr
Was changed to. Note that a polyethylene glycol (PEG) solution was used for calibration.

Example 4 (Measurement of PEG solution spectrum) The spectrum of the PEG solution was measured using the mass spectrometer of Example 3. The measurement was performed with water-methanol 1: 1 in the aqueous phase.
A PEG solution was used for the solution and the organic phase. For the spectrum measurement, the voltage of the ESI section was 3.0 kV, the integration time was 3 minutes, and the aqueous phase flow rate was 0.
The reaction was performed at a flow rate of 1 ml / hr and an organic phase flow rate of 0.05 ml / hr. FIG. 7 shows the spectrum.

Example 5 (Measurement of Mass Spectrum of 5-Br-PADAP) A toluene solution of 2- (5-bromo-2-pyridylazo) -5-diethylaminophenol (5-Br-PADAP) was used as an organic phase, and acetic acid was used. Example 3 Using Copper Aqueous Solution as Aqueous Phase
The mass spectrum was measured using the mass spectrometer described in (1). The concentration of the organic phase is 1.0 × 10 ⁻³ mol dm
^-3 , and the aqueous phase was a 1.0 × 10 ⁻³ M aqueous acetic acid solution (pH = 4.0) containing 5.0 × 10 ⁻⁶ moldm ⁻³ (toluene saturated), which was an aqueous Cu (II) acetic acid solution. The flow rates were 0.001 ml / hr and 0.1 ml / hr, respectively. The mass spectra obtained when the voltage of the ESI device was 3.8 kV, 4.0 kV and 4.4 kV are shown in FIGS. 8, 9 and 10, respectively.

The nozzle voltage was set to 400 V and the flow rate of the organic phase was
Is a mass spectrum when is 0.02 ml / hr.
FIG. [H₂L]⁺(M / z = 349), [Cu
l] ⁺(M / z = 412), [Cul (HL)]⁺(M
/ Z = 760) was clearly observed. these
The peak is the organic phase droplet generated at the tip of the silica capillary.
This is measured when the person leaves intermittently after 10 to 30 seconds.
I understood. From this, the measured complex dissolves in the aqueous phase.
Not generated, generated in interface region and solvent evaporation process
It is thought that it was done. Further, the Cu (II) ion concentration is
5.0 × 10^-6mol dm^-3And the flow rate is 0.0
The mass spectrum at 2 ml / hr is shown in FIG.
You.

Example 6 (Measurement of Absorption Spectrum of 5-Br-PADAP) The absorption spectra of both the aqueous phase and the organic phase were measured. In a test tube with a lid, 1.0 × 10 ^{−3 to} 1.0 × 10 ⁻¹⁵ m
old dm- ³ 5-Br-PADAP toluene solution 5
5.0 × 10 ⁻⁶ mol dm with toluene and toluene saturation
25 ml of an aqueous solution of copper acetate- ³ was mixed. This mixed solution was stirred for 1 hour and separated into an organic phase and an aqueous phase by a centrifuge.
Each absorption spectrum was measured. In order to obtain only the absorption of the complex, a difference spectrum was obtained for each of the organic phase and the aqueous phase. The absorption spectrum was measured using a V-550 type UV-visible spectrophotometer (manufactured by JASCO Corporation). Measurement wavelength range is 2
00.0 nm to 900.0 nm, the data capture interval is 0.5 nm, the scan speed is 200 nm / min,
As a light source, a tungsten iodine lamp and a deuterium lamp were used. The cell for measuring the absorption spectrum has an optical path length of 10 m.
The experiment was performed using a quartz cell of m. All measurements are 25
The test was performed under the conditions of ± 1.5 ° C. The results are shown in FIG. 21 (organic phase) and FIG. 22 (aqueous phase).

[0056]

According to the present invention, a water-insoluble organic substance can be applied to a mass spectrometer very easily by using a two-phase flow structure at the sample introduction part in the electrospray ion method. In addition, mass spectrometry of a large number of chemical species such as biopolymers such as proteins and environmental hormones, environmental fine particles, and metal complexes can be performed very easily in the form thereof. As described above, by using the ESI device of the present invention, it is possible to directly perform mass spectrometry of organic or inorganic chemical species existing in a non-aqueous system. Furthermore, according to the ESI device of the present invention, chemical species generated at the liquid-liquid interface can be easily and accurately analyzed for mass.

[Brief description of the drawings]

FIG. 1 illustrates an outline of an electrospray ionization apparatus of the present invention.

FIG. 2 shows the assembled electrospray ionization device of the present invention shown in FIG.

FIG. 3 shows a silica capillary tipping machine system for manufacturing the tip of a non-conductive tube.

FIG. 4 is a micrograph instead of a drawing showing a microscope image of a 10 × image of a portion of a silica capillary having an outer diameter of 150 μm and an inner diameter of 75 μm processed by the apparatus shown in FIG.

5 is a micrograph instead of a drawing, showing a microscope image of a 10-times objective image of a tip portion obtained by processing a silica capillary having an outer diameter of 150 μm and an inner diameter of 50 μm by the apparatus shown in FIG. 3;

FIG. 6 shows a two-phase direct introduction device of the present invention using ESI-
1 shows a mass spectrometer of the present invention set in an electrospray ionization section of TOF-MS.

FIG. 7 is a mass spectrum of a polyethylene glycol (PEG) solution by ESI-TOF-MS in which the two-phase direct introduction device of the present invention is set.

FIG. 8 shows that the voltage of the ESI device of the present invention is 3.8 k.
It is an obtained mass spectrum when V is set.

FIG. 9 is a graph showing the voltage of the ESI device of the present invention set at 4.0 k;
It is an obtained mass spectrum when V is set.

FIG. 10 is a graph showing the voltage of the ESI device according to the present invention as 4.
It is the obtained mass spectrum at the time of 4 kV.

FIG. 11 shows Cu (II) -5-B by ESI-TOF-MS set with the two-phase direct introduction device of the present invention.
It is a mass spectrum of a r-PADAP complex.

FIG. 12 is a graph showing the case where the Cu (II) concentration by ESI-TOF-MS set with the two-phase direct introduction device of the present invention is 1.0 × 10 ⁻⁵ mol dm ^−3.
It is a mass spectrum of (II) -5-Br-PADAP complex.

FIG. 13 is a diagram showing a state in which the nozzle voltage is set to 400 by ESI-TOF-MS in which the two-phase direct introduction device of the present invention is set.
5 is a mass spectrum of a Cu (II) -5-Br-PADAP complex when V is V. FIG.

FIG. 14 shows [CuL (HL)] ⁺ and [CuL
L] ⁺ shows a comparison of the relationship between the peak intensity, the count number, and the nozzle voltage.

FIG. 15 shows a comparison of the relationship between the peak intensity of [H ₂ L] ⁺ , the count number, and the nozzle voltage.

FIG. 16 shows that the flow rate of the organic phase was 0.005 ml /
[CuL (HL)] ⁺ and [CuL] ⁺
3 shows a comparison of the relationship between the peak intensity, the count number, and the nozzle voltage.

FIG. 17 shows that the flow rate of the organic phase was 0.005 ml /
This shows a comparison of the relationship between the peak intensity of [H ₂ L] ⁺ , the count number, and the nozzle voltage at the time of hr.

FIG. 18 shows a flow rate of 0.02 ml by ESI-TOF-MS set with the two-phase direct introduction device of the present invention.
4 is a mass spectrum of the Cu (II) -5-Br-PADAP complex at the time of / hr.

FIG. 19 shows the results of time-resolved counts of [CuL (HL)] ⁺ in a mass spectrum by ESI-TOF-MS in which the two-phase direct introduction device of the present invention is set.

FIG. 20 shows the time-resolved result of the count number of [CuL] ⁺ in the mass spectrum by the conventional ESI-TOF-MS.

FIG. 21 shows Cu (II) -5-Br-PADA.
It is a difference spectrum of the toluene phase (organic phase) of the absorption spectrum of the P complex solution.

FIG. 22 shows Cu (II) -5-Br-PADA.
It is a difference spectrum of the water phase of the absorption spectrum of P complex solution.

FIG. 23 schematically shows an example of a reaction in a mixing section in the two-phase direct introduction method of the present invention. FIG.
HL in it indicates a ligand in the organic phase.

[Explanation of symbols]

 DESCRIPTION OF SYMBOLS 1 ESI apparatus 2 Capillary which is supply path of water phase 3 Mixing part 4 Organic phase 5 Water phase 6 Non-conductive tube which is supply path of organic phase 21 Carbon dioxide gas laser 22 Condensing lens 23 Capillary 24 Weight 25 Heat-resistant brick 26 Stabilized DC power supply 27 Function generator

Claims (8)

[Claims] 1. A supply path capable of simultaneously supplying an aqueous phase and an organic phase to an ionization section, an aqueous phase ionization section capable of sufficiently ionizing the aqueous phase, and an organic phase being supplied to the aqueous phase ionization section so that the two can be combined. An electrospray ionization device having a mixing section for mixing. 2. A supply path which can simultaneously supply an aqueous phase and an organic phase to an ionization section is a coaxial two-phase tube.
An electrospray ionization apparatus according to claim 1. 3. A supply path that can simultaneously supply an aqueous phase and an organic phase to an ionization section is a coaxial two-phase supply path that is provided inside a supply path that supplies an aqueous phase. An electrospray ionization apparatus according to claim 1 or 2. 4. The electrospray ionization apparatus according to claim 1, wherein the mixing section is inside the supply path of the aqueous phase. 5. The supply path for supplying an organic phase has a tip that is shorter than the tip of a supply path for supplying an aqueous phase, and a gap is formed between the two tips. Electrospray ionizer. 6. A method for ionizing a chemical species using the electrospray ionization apparatus according to claim 1. 7. A mass spectrum apparatus using the electrospray ionization apparatus according to claim 1 as an ion source. 8. The mass spectrum apparatus according to claim 7, wherein the mass spectrum is a time-of-flight type.