KR100944047B1 - Conductive Coupling Device for ESD Interface - Google Patents

Abstract

The present invention is a technique used to combine capillary electrophoresis (CE) separation technology and mass spectrometry (MS) detection technology [Reference 1: Withehiuse, CM, Dreyer, R. N, Yamashita, M. , Feen, JB, Anal . Chem . 1985 , 57 , 675-679.] Relates to a conductive coupling device for an Electrospray Ionization (ESI) interface.

Electrophoresis, Mass Spectrometry, Electrospray Ionization (ESI) Interface, Conductive Polymer

Description

Electro-conductive coupling device for a CE-MS ESI interface

The present invention relates to ESI (Electrospray Ionization) interface technology used to combine capillary electrophoresis (CE) separation technology and mass spectrometry (MS) detection technology [Reference 1: Withehiuse, CM, Dreyer, R. N, Yamashita, M., Feen, JB, Anal . Chem . 1985 , 57 , 675-679.] Relates to a conductive coupling device for an Electrospray Ionization (ESI) interface.

Capillary Electrophoresis-Mass Spectrometry (CE-MS) with capillary electrophoresis separation device with HPLC-MS (High Performance Liquid Chromatography-Mass Spectrometry) is one of the latest biochemical analysis techniques. However, unlike HPLC, CE is separated by applying an electric field, so the harmonious combination of the electric field of CE and the electric field applied for electro-spray ionization (ESI) of MS [Ref. 1] becomes a technical key of CE-MS. .

There are two types of CE-MS ESI interface technology using sheath flow and direct ESI. The method of using sheath flow is easy to apply electric field, but the sensitivity is reduced by dilution of sheath flow. Reference 2: Cao, P., Moini, M., J. Am . Soc . Mass Spectrom . 1977 , 8 , 561-564.]

The direct ESI method, which does not use the sheath flow, does not have the problem of desensitization caused by the sheath flow, but addresses various technical problems related to the application of the electric field. When the capillary tip of the electrophoretic capillary end is used as an ESI electrode, it is not easy to maintain stable electrical contact when attempting electrical contact by plating a microtip. An electrode is also inserted at the connection point between the electrophoretic capillary and the ESI capillary, which causes the fluid flow to become very unstable due to the electrolysis of water at the contact surface of the electrode with the solution, resulting in bubbles. In 2003, Whitt and Moini reported that CE-MS ESI was possible by making a conductive path directly on the capillary [Ref. 3: Whitt, JT, Moini, M., Anal . Chem . 2003 , 75 , 2188-2191.] This method is an ideal way to flow only the electricity to the external electrode in the state of blocking the flow of the fluid, but the manufacturing is very difficult and inconvenient to use. Drilling on glass tubes within a few hundred μm in diameter is not easy, and the additional required hydrofluoric acid etching is also highly technical and dangerous because of the toxicity of hydrofluoric acid. In particular, even if a successful passage has been successfully made, the conductive passage can easily become useless due to capillary tips that are easily blocked or damaged. Therefore, all the previous technologies regarding the interface of CE-MS are exposed to technical disadvantages or defects.

The present invention is to provide a high-sensitivity, high stability CE-MS ESI interface device to solve the instability problem when applying the electric field in the direct ESI method.

In addition, the present invention is to provide a CE-MS ESI interface device that can be made as convenient and easy to use the device.

The present invention is a capillary insertion hole and the lower end of the body is a conductive portion insertion hole formed in the inner peripheral surface of the capillary insertion hole is perforated; An electrophoretic capillary tube inserted into the capillary insertion hole such that a rear end thereof is exposed to the outside and a front end is located below the lower end of the conductive insertion hole; A capillary for ESI inserted into the capillary insertion hole such that a front end is exposed to the outside and a rear end is positioned below the lower end of the conductive insertion hole; And a conductive part inserted into the conductive insertion hole to apply a voltage to the electrolyte solution passing through the electrophoretic capillary and the ESI capillary through a gap formed between the electrophoretic capillary front end and the ESI capillary end. A conductive coupling device for an ESI interface.

In the present invention, the conductive portion may be a conductive polymer film, the conductive polymer film may be formed by injecting a liquid conductive polymer into the conductive portion insertion hole formed by high heat, the liquid conductive polymer is a liquid Nafion (Nafion May be).

The present invention is an electrode provided in the body; And an electrolyte solution preservation member covering an upper portion of the conductive part insertion hole and a predetermined portion of the electrode so that the conductive part and the electrode are energized. The body has a seating groove in which the electrolyte solution preservation member is seated. The electrode may be a conductor installed in the body by being wound into at least one through hole penetrating the bottom surface of the seating groove.

The present invention is provided with a capillary fixing means for electrophoresis and a capillary fixing means for ESI having a flange formed radially on the outer circumferential surface of the guide tube, wherein each capillary fixing means is the respective guide tubes for the electrophoresis Capillary tube and ESI capillary tube may be fixedly installed in each of the fixing means insertion hole formed at both ends of the body to guide the capillary insertion hole, the guide tube of each capillary fixing means is the deflection of each capillary One side end may be exposed to the outside of the body to prevent each other.

According to the present invention, a gas is generated at an electrode installed at the connection point by placing an electrode that applies a voltage to the connection point between the electrophoresis capillary and the ESI capillary, outside the connection point and the electrophoresis capillary tube and the ESI capillary tube. And it eliminates the conventional problem that causes instability in ionization, there is an advantage that can operate the CE-MS for a long time stable.

In addition, in the case of creating a passage of electric current with an externally installed electrode by using a minute gap present at the connection point between the electrophoretic capillary and the ESI capillary, the pressure in the electrophoretic capillary and the ESI capillary tube Solution may leak, but the present invention has the advantage that the high resolution of the CE can be transferred to the MS as it is, because the conductive membrane that allows the flow of current but blocks the flow of fluid at the source as a conductive part.

In addition, the present invention has a structure that can be detachably attached to the electrophoretic capillary and the ESI capillary through the guide tube of the electrophoretic capillary fixing means and the guide tube of the ESI capillary fixing means, these capillary tube as necessary Easily replaceable for maximum ease of use.

As such, it is determined that the biggest technical difficulty in CE-MS has been solved by the present invention, and thus, more active use of CE-MS technology is expected.

Hereinafter, an embodiment of the present invention will be described in detail with reference to the accompanying drawings.

1 is a block diagram of an embodiment of the present invention, Figure 2 is a cross-sectional view of the main part of the body of Figure 1, Figure 3 is a plan view of the main part of the body.

Referring to Figure 1 one embodiment of the present invention is a body 10, electrophoretic capillary 20, ESI capillary 30, conductive portion 40, electrode 50, electrophoretic capillary fixing means ( 60) and capillary fixing means 70 for electrophoresis.

Referring to FIG. 2, a capillary insertion hole 12 and a conductive portion insertion hole 14 are drilled in the body 10. The conductive insertion hole 14 is drilled to communicate with the capillary insertion hole 12, and the lower end is drilled to be formed on the inner circumferential surface of the capillary insertion hole 12. Capillary insertion hole 12 is formed in a straight line, the conductive portion insertion hole 14 is preferably formed to form a 90 ° with the capillary insertion hole (12). On the other hand, the body 10 may be made of a transparent plastic material.

1 and 2, the electrophoretic capillary 20 is inserted into the capillary insertion hole 12, the rear end of which is exposed to the outside and the front end is inserted so that the lower end of the conductive insertion hole (14).

1 and 2, the ESI capillary 30 is inserted into the capillary insertion hole 12, the front end is exposed to the outside and the rear end is inserted so that the lower end of the conductive insertion hole (14).

Referring to Figure 1, the electrophoretic capillary 20 and the ESI capillary 30 is inserted so that the ends thereof almost contact with each other. The electrophoretic capillary 20 and the ESI capillary 30 are conventional fused silica capillaries with an outer diameter of about 370 μm. Referring to FIG. 2, the diameter of the capillary insertion hole 12 was 400 μm so that the capillary tubes 20 and 30 could barely pass through the capillary insertion hole 12. On the other hand, the conductive portion insertion hole 14 had a diameter of 700 µm and a length of 1 mm.

1 and 2, the conductive portion 40 is inserted into the conductive portion insertion hole 14. The conductive part 40 applies a voltage to the electrolyte solution passing through the electrophoretic capillary 20 and the ESI capillary 30 through a gap formed between the electrophoretic capillary 20 and the ESI capillary 30. It is inserted into the conductive portion insertion hole 14 in order to. The conductive portion 40 is in close contact with the inner circumferential surface of the conductive inserting hole 14 so that the electrolyte solution passing through the electrophoretic capillary 20 and the ESI capillary 30 is not ejected to the upper portion of the conductive inserting hole 14. Is inserted.

The conductive portion 40 is a conductive polymer membrane. Thus, the charged medium or ions can move freely through the conductive portion 40, but the solution cannot pass through the conductive portion 40. The conductive polymer film may be formed by injecting a liquid conductive polymer into the conductive portion insertion hole 14 and molding the same at high heat. The liquid conductive polymer film may be a liquid Nafion. The formation process of the conductive portion 40 is as follows. The conductive insertion hole 14 is filled with a predetermined amount of a conductive polymer (eg, Nafion) in a solution state and molded at a high temperature (eg, 180 ^° C.). By repeating this, when the molding amount reaches an appropriate level, the conductive polymer membrane is moved from the lower end of the conductive insertion hole 14 toward the upper end by forcibly pushing the formed conductive polymer using a needle having a diameter of 700 μm. Make it hard. If the conductive polymer film (membrane) is formed to the upper end of the conductive insertion hole 14 is repeated, and stops, the electrophoretic capillary 20 and the ESI capillary 30 are inserted into the capillary insertion hole 12 and then Pass the fluid through the capillary tubes 20 and 30 to check whether the fluid leaks through the conductive insertion hole 14. In case of leakage, the conductive polymer film is stacked thicker and compressed so that the gap between the conductive polymer film and the conductive part insertion hole 14 is eliminated.

By using the conductive portion 40 as a conductive polymer membrane, electrolysis does not occur in the electrolyte solution flowing through the capillary tubes 20 and 30 even when a voltage is applied to the conductive portion 40.

Referring to FIG. 1, the body 10 is provided with an electrode 50 for applying a voltage to the conductive portion 14. The electrode 50 may be a platinum wire. The electrode 50 is connected to a power supply so that a voltage is applied. Although not shown in the figure, the body 10 is provided with an electrolyte solution preservation member (not shown). The electrolyte solution preservation member (not shown) uses a member having a high moisture content such as a sponge. The electrolyte solution preservation member (not shown) is provided with the conductive portion insertion hole 14 and the electrode (not shown) so that the conductive part 40 and the electrode 50 are energized when the electrolyte solution is preserved in the electrolyte solution preservation member (not shown). 50) is installed covering a predetermined portion.

1 to 3, the body 10 is provided with a mounting groove 15 on which an electrolyte solution preservation member (not shown) is seated. The conductive part insertion hole 14 is formed in the bottom surface of the seating groove 15. In particular, referring to FIG. 3, two through holes 19 may be formed on the bottom surface of the mounting groove 15 spaced apart from the conductive insertion hole 14 by a predetermined distance. Two through holes 19 are formed through the body 10 from the bottom surface of the mounting groove 15. In this case, the electrode 50 is fixed to the body 10 by being wound into two through holes 19. The two through holes 19 were drilled to a diameter of 700 μm at a position about 5 mm away from the conductive insertion hole 14, and the electrode 50 used a platinum wire having a diameter of 300 μm.

1 and 2, the electrophoretic capillary fixing means 60 and the ESI capillary fixing means 70 are radially formed on the outer circumferential surfaces of the guide tubes 62 and 72 and the guide tubes 62 and 72. It comprises a flange portion 64, 74.

Referring to FIGS. 1 and 2, the capillary fixing means 60 and 70 may include guide tubes 62 and 72 to guide the capillary tubes 20 and 30 to the capillary insertion holes 12, both sides of the body 10. Fixing means insertion holes 16 and 17 formed at the ends are respectively fixedly installed. Therefore, the electrophoretic capillary 20 and the ESI capillary 30 are formed through the guide tube 62 of the electrophoretic capillary fixing means 60 and the guide tube 72 of the ESI capillary fixing means 70. It can be detachably attached.

1 and 2, the guide tubes 62 and 72 of the capillary fixing means 60 and 70 have one end of the outside of the body 10 to prevent sagging of the capillary tubes 20 and 30, respectively. Extended exposure.

Plastic fittings (F-124S; manufactured by Upchurch, USA) were used as the flange portions 64 and 74, and tubing sleeves (F-185; manufactured by Upchurch, USA) were used as guide tubes 62 and 72. Used.

Hereinafter, the operation of the above-described embodiment will be described.

4 is an electrical connection diagram of a CE-MS interface using the conductive coupling device for the ESI interface of FIG.

Mass spectrometry and electrophoresis of creatinine and uric acid selected as representative of cations and anions, respectively, using the conductive coupling device for the ESI interface of FIG. The results were successfully obtained as follows.

Creatinine CE - MS  analysis

1. Composition of CE-MS system

CE-MS analysis of creatinine and D ₃ -isosubstituted creatinine was performed using the CE-MS system shown in FIG. 4. The CE device used was manufactured by the applicant. The voltage application device for the ESI is Bertan's Model 230. The MS device is Quatro Ultima from Waters.

FIG. 5 shows a spray image of an ESI formed by a CE-MS interface assembled using the conductive coupling device for the ESI interface of FIG. 1. Referring to FIG. 5, the solution pushed out by the electroosmotic flow (EOF) in the CE-MS system was confirmed that the spray was kept stable.

2. Creatinine Mass Spectrometry

FIG. 6 shows mass spectrometry spectra of creatinine (molecular weight 113) and D ₃ -isotopically substituted creatinine (molecular weight 116) obtained using the CE-MS interface assembled using the conductive coupling device for the ESI interface of FIG. 1.

Mass spectra of creatinine (molecular weight 113) and D ₃ -isotopically substituted creatinine under the following experimental conditions were obtained as shown in FIG. 6.

Experimental condition:

(1) Sample: creatinine (molecular weight 113) and D ₃ -isotopically substituted creatinine (molecular weight 116)

(2) Buffer: 20 mM ammonium acetate buffer (pH 4.0)

(3) ESI voltage: (+) 1.84 kV

(4) MS cone voltage: 170 V

(5) Electrophoretic voltage: 23 kV

(6) Electrophoretic capillary: inner diameter 50 µm, outer diameter 370 µm, length 60 cm

(7) Capillary for ESI: inner diameter 50 µm, outer diameter 370 µm, length 5 cm

3. CE-MS results of creatinine:

FIG. 7 shows electrophoretograms of creatinine and D ₃ -isotopically substituted creatinine obtained using a CE-MS interface assembled using the conductive coupling device for the ESI interface of FIG. 1.

CE-MS electrophoretograms of creatinine and isotope substituted creatinine under the following experimental conditions were obtained as shown in FIG. 7.

Experimental conditions (change the following items in the experimental conditions of paragraph 2 above)

(3) ESI voltage: (+) 2.0 kV

(5) electrophoretic voltage: 30 kV; Sample injection at 30 kV for 3 seconds.

Uric acid CE - MS  analysis

1. Composition of CE-MS system

The CE-MS system was constructed using the CE-MS system shown in FIG. 4 and CE-MS analysis of uric acid and isotope substituted uric acid was performed. The CE device used was manufactured directly by the applicant. The voltage application device for the ESI is Bertan's Model 230. The MS device is Quatro Ultima from Waters.

2. Uric Acid Mass Spectrometry

8 shows mass spectrometry spectra of uric acid (molecular weight 118) and isotopically substituted uric acid (molecular weight 170) obtained using a CE-MS interface assembled using the conductive coupling device for the ESI interface of FIG.

Mass spectra of uric acid and isotope substituted uric acid were obtained under the experimental conditions as shown in FIG. 8.

Experimental condition:

(1) Sample: uric acid (molecular weight 118) and ¹³ C ₂ -isotopically substituted uric acid (molecular weight 170)

(2) Buffer: 20 mM ammonium acetate buffer (pH 6.8)

(3) ESI voltage: (+) 2.10 kV

(4) MS cone voltage: 170 V

(5) Electrophoretic voltage: 27 kV

(6) Electrophoretic capillary: inner diameter 50 µm, outer diameter 370 µm, length 60 cm

(7) Capillary for ESI: inner diameter 50 µm, outer diameter 370 µm, length 5 cm

3. CE-MS Results of Uric Acid

9 shows electrophoretograms of uric acid and ¹⁵ N ₂ -isotopically substituted uric acid obtained using a CE-MS interface assembled using the conductive coupling device for the ESI interface of FIG. 1.

CE-MS electrophoretograms of uric acid and isotopically substituted uric acid were obtained in the following experimental conditions as shown in FIG. 9.

Experimental conditions (change the following items in the experimental conditions of paragraph 2 above)

(5) electrophoretic voltage: 27 kV; Sample injection for 2 seconds at 27 kV;

1 is a block diagram of an embodiment of the present invention.

Fig. 2 is a sectional view of an essential part of the body of Fig. 1;

3 is a plan view of the main part of the body;

4 is an electrical connection diagram of a CE-MS interface using the conductive coupling device for the ESI interface of FIG.

5 is a spray representation of an ESI formed by a CE-MS interface assembled using the conductive coupling device for the ESI interface of FIG.

FIG. 6 is a mass spectrometry spectrum of creatinine (molecular weight 113) and D3-isotopically substituted creatinine (molecular weight 116) obtained using a CE-MS interface assembled using the conductive coupling device for the ESI interface of FIG.

FIG. 7 is an electrophoretogram of creatinine and D3-isotopically substituted creatinine obtained using a CE-MS interface assembled using the conductive coupling device for the ESI interface of FIG.

8 is a mass spectrometry spectrum of uric acid (molecular weight 118) and isotopically substituted uric acid (molecular weight 170) obtained using a CE-MS interface assembled using the conductive coupling device for the ESI interface of FIG.

Figure 9 is an electrophoretogram of uric acid and 15N2-isotopically substituted uric acid obtained using a CE-MS interface assembled using the conductive coupling device for the ESI interface of Figure 1;

<Description of the symbols for the main parts of the drawings>

10: body

12: capillary insertion hole 14: conductive part insertion hole

15: Seating groove 16, 17: Fastening means insertion hole

19: through hole

20: capillary for electrophoresis 30: capillary for ESI

40: conductive part 50: electrode

60: capillary fixing means for electrophoresis 70: capillary fixing means for electrophoresis

Claims (8)

A body in which the capillary insertion hole and the lower end of the capillary insertion hole are formed in the inner circumferential surface of the capillary insertion hole; An electrophoretic capillary tube inserted into the capillary insertion hole such that a rear end thereof is exposed to the outside and a front end is located below the lower end of the conductive insertion hole; A capillary for ESI inserted into the capillary insertion hole such that a front end is exposed to the outside and a rear end is positioned below the lower end of the conductive insertion hole; A conductive part inserted into the conductive part insertion hole to apply a voltage to the electrolyte solution passing through the electrophoretic capillary and the ESI capillary through a gap formed between the electrophoretic capillary front end and the ESI capillary end; Conductive coupling device for an ESI interface comprising a. The method of claim 1, And said conductive portion is a conductive polymer film. The method of claim 2, And the conductive polymer film is formed by injecting a liquid conductive polymer into the conductive part insertion hole and forming the film at a high temperature. The method of claim 3, The conductive conductive polymer is an ESI interface, characterized in that the liquid Nafion (Nafion). The method according to any one of claims 1 to 4, An electrode provided in the body; An electrolyte solution storage member covering an upper portion of the conductive portion insertion hole and a predetermined portion of the electrode so that the conductive portion and the electrode are energized; Conductive coupling device for an ESI interface comprising a. The method of claim 5, The body is provided with a seating groove in which the electrolyte solution preservation member is seated, The electrode is a conductive coupling device for the ESI interface, characterized in that the conductor is installed in the body by being wound into at least one through-hole penetrating the bottom surface of the seating groove. The method according to any one of claims 1 to 6, Capillary fixing means for electrophoresis and capillary fixing means for ESI having a flange formed radially on the outer peripheral surface of the guide tube, Each of the capillary fixing means is fixed to each of the fixing means insertion holes formed at both ends of the body so that each guide tube guides the electrophoretic capillary and the ESI capillary to the capillary insertion hole. Conductive coupling device for ESI interface. The method of claim 7, wherein The guide tube of each capillary fixing means is a conductive coupling device for the ESI interface, characterized in that one side end is extended to the outside of the body to prevent the deflection of each capillary.

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