JPH05164737A - Capillary electrophoretic method and apparatus capable of performing on-line pretreatment - Google Patents

Abstract

PURPOSE:To concentrate an objective component by directly injecting an actual specimen containing protein by capillary electrophoresis to analyze the same. CONSTITUTION:A microporous column 8 packed with a functional packing agent 10 is provided on the injection side of a three-way branched capillary. High voltage is applied to the capillary on its drain side while the injection end of the capillary is immersed in a specimen solution to generate electroosmotic flow from the column 8 to a drain and an unnecessary component is discharged to the drain and a component to be analyzed is distributed to the column 8. Next, the injection end is immersed in a buffer solution and high voltage is applied to a separation capillary 2 to execute capillary electrophoretic separation and the component to be analyzed is detected by a detector 22.

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention relates to a capillary electrophoresis method used for rapid and high resolution analysis of biopolymers such as proteins and nucleic acids, drugs, optical isomers and other ionic low molecular weight compounds, and its capillary electrophoresis. The present invention relates to an electrophoretic device for carrying out a migration method.

[0002]

2. Description of the Related Art Capillary electrophoresis, which is a high-performance electrophoretic technology, has the advantages of high resolution, rapid analysis, and extremely low sample consumption. , Is attracting attention in fields such as medicine and food. The capillary used usually has an inner diameter of 50 to 100 μ.
It is made of fused silica and has a length of 20 to 100 cm and a polyimide coating on its outer surface. The capillary is filled with a migration buffer or a polymerized gel.
After the nl-order sample is injected into one end of the capillary, a high voltage of about 30 kV is applied between both ends of the capillary to migrate the sample components, and the sample components are separated and detected near the other end of the capillary.

Capillary electrophoresis is a widely known method for separation and analysis, but so far, the majority of high-purity preparations have been analyzed. Particularly, it is difficult to separate and analyze a specific component existing in a complicated matrix such as a biological sample. Direct injection analysis of biological samples such as plasma, serum and urine not only interferes with the detection of the target component by the protein peak but also has a fatal effect on the subsequent analysis when adsorption on the fused silica surface of the capillary occurs. give. To avoid such problems, most biological samples require rigorous multi-step sample cleanup procedures, such as solvent extraction.

An example of application of capillary electrophoresis to drug analysis in plasma is reported by Nakagawa et al. (Chem.
Pharm. Bull., 36 , 1622 (1988); ibid, 37 , 707 (1988)
See). According to the report, the addition of SDS micelles slows the migration of proteins and allows the separation of drug peaks. Further, after the peak of the target drug is detected, the capillary can be washed and the next analysis can be performed without waiting for the detection of other unnecessary components.

On the other hand, a spectroscopic detector (Spectrop) in which a part of a capillary having an inner diameter of 100 μm or less is used as a detection cell as it is
Although the hotometric detector has a fate that the concentration sensitivity is poor, one of the attempts to improve this problem is the sample zone online concentration. There are the following two types of online concentration methods: a and b. (A) Isotachophoretic concentration method: For example, it is well known that a sufficiently dilute sample can be injected in a large amount as compared with a background migration buffer to expect a concentration effect by isotachophoresis. (B) A method of trapping or delaying the target component in the filler; a section in which the filler such as polymer beads or CPG (control pore glass) beads is sandwiched by a porous frit is provided in a part of the capillary to provide a multi-component. It is a concentration method in which only a specific target component is trapped or delayed when a system sample passes through. For example, using antibody-immobilized beads
J. Liq. Chromatogr., 14 (5), 997-1015 (1991).

[0006]

SUMMARY OF THE INVENTION It is not preferable to introduce unnecessary components, particularly high-molecular-weight impurities that tend to cause problems, to separation capillaries, and capillary electrophoresis is used for clinical applications such as monitoring of drug amount in biological samples. In order to establish it as a usage method, it is necessary to avoid introducing unnecessary components. For on-line concentration, isotachophoretic concentration methods interfere with the solvent peak of the sample depending on the pH of the buffer used. In the method in which a packing material that traps or delays the target component is sandwiched in a part of the capillary, unnecessary components are eventually introduced into the separation column, and thus sufficient washing is required. An object of the present invention is to provide a capillary electrophoresis method which directly injects and analyzes an actual sample and also has an effect of concentrating a target component, and an electrophoresis apparatus used for the method.

[0007]

In the capillary electrophoresis method of the present invention, the capillaries are of a three-way branch type in which the capillaries for injection, the separation capillaries and the drain capillaries are branched, and unnecessary components are eluted and analyzed in the injection capillaries. After filling the pretreatment filler that holds the target component, immersing the injection capillary end in the sample solution, applying a high voltage between the injection capillary end and the drain capillary end, and discarding unnecessary components to the drain The end of the injection capillary is immersed in a buffer solution, and a high voltage is applied between the end of the injection capillary and the end of the separation capillary to separate and analyze the components to be analyzed held in the pretreatment filler.

In order to realize this capillary electrophoresis method, the capillary electrophoresis apparatus of the present invention is branched into injection capillaries, separation capillaries and drain capillaries, and unnecessary components are eluted in the injection capillaries to be analyzed. A three-way branched capillary filled with a pretreatment filler that retains the components, a high voltage is independently applied between the injection capillary end and the drain capillary end, and between the injection capillary end and the separation capillary end. And a power supply device that can be applied.

Examples of the functional fillers used as the pretreatment fillers include the following a to d. (A) Protein-coated ODS;
This is a protein (for example, BSA) irreversibly adsorbed on the surface of ODS silica. The surface of the hole is normal O
DS (Chromatographia, 19 , 466 (1984); Anal.
Biochem., 151 , 358 (1985)). (B) ISRP (Internal Surfice Reversed Phase) (c) SHP (Shielded Hydrophobic Phase) All of these packing materials are columns for direct injection, which are designed to prevent proteins from contacting the partitioning and binding phase by a size exclusion mechanism. It is a packing material and is eluted as in reverse phase liquid chromatography. (D) Antibody-immobilized column This specifically binds only the antigen by the immune reaction. On the contrary, there is also one in which an antigen is immobilized and an antibody is specifically bound. The bound antigen or antibody is eluted by changing pH (J. Chromatogr., 544 , 13 (1991), Chromatogra.
phia, 27 , 574 (1989)). In addition, by attempting to create a capillary with a dialysis membrane at the injection end, it is possible to introduce only low-molecular substances as a sample from a multi-component system sample in which high-molecular substances and low-molecular substances coexist, and deproteinize simultaneously with injection. It is possible to have a function of performing preprocessing such as.

[0010]

With respect to the injection capillary end of the three-way branch type capillary, the drain capillary end and the separation capillary end are respectively immersed in different migration buffers, and two blocks of circuits are formed between the injection end and the injection end via the electrodes. Switching of two flow paths, that is, a flow path from the injection end to the drain capillary end and a flow path from the injection end to the separation capillary end is performed by turning on and off the voltage application of each block. First, when the injection end is immersed in the sample solution, a high voltage is applied to the drain capillary side, and the separation capillary side is electrically floated, an electroosmotic flow occurs from the injection end to the drain direction, and the pretreatment is performed. The sample passes through the filler portion. The substances that are not retained by the pretreatment filler and the solvent required between them do not enter the separation capillary and flow to the drain side and are discarded. After this pretreatment, the sample solution is changed to a buffer solution by immersing the injection end, a high voltage is applied to the separation capillary side, and the drain capillary side is electrically floated. As a result, the components retained in the pretreatment filler are detected by performing electrophoresis on the separation capillary side.

As the pretreatment filler, for example, one which distributes a low molecular weight compound such as protein coat ODS, ISRP or SHP and elutes the protein at a high recovery rate is used.
A sample containing a drug, a metabolite, a peptide, etc. can be directly injected into a biological fluid for analysis. That is, unnecessary substances and solvents that have passed through the pretreatment filler in the electroosmotic flow due to migration to the drain side move to the drain side, and are then retained in the pretreatment filler by electrophoresis on the separation capillary side. Only the target substance can be eluted and separated by electrophoresis. At this time, a concentration effect can be expected at the same time.

[0012]

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS FIG. 1 schematically shows the apparatus configuration of one embodiment. The capillary is a three-way branch type capillary in which the separation capillary 2, the drain capillary 4 and the injection capillary 6 are branched at the branch position A. A part of the injection capillary 6 is a microbore column 8 packed with a packing material. The microbore column 8 is filled with a functional filler such as a protein coat ODS according to the purpose.

The end of the injection capillary 6 is immersed in the sample solution or buffer solution. Reference numeral 16 represents a buffer solution containing a buffer solution. The end of the separation capillary 2 is dipped in the buffer solution in the detection side buffer container 18, and the end of the drain capillary 4 is dipped in the buffer solution in the drain side buffer container 20. The electrode 26 is immersed in the sample solution and the buffer solution at the end of the injection capillary, the electrode 28 is immersed in the buffer solution at the end of the separation capillary, and the electrode 32 is immersed in the buffer solution at the end of the injection capillary. Analytical detector 22
Is provided at the end of the separation capillary 2, and the signal of the detector 22 is output to the recorder 24 and recorded.

A power supply 30 is provided between the electrode 26 at the end of the injection capillary and the electrode 28 at the end of the separation capillary (A block).
A high voltage is applied by the power supply 34 between the electrode 26 at the end of the injection capillary and the electrode 32 at the end of the drain capillary (B block). Each of the power supplies 30 and 34 is provided with a switch so that a high voltage can be independently applied to the A block and the B block. Reference numerals 31 and 35 are ammeters of the respective blocks.

FIG. 2 shows an example of the microbore column 8. The microbore column 8 has a porous silica gel base or CPG in a fused silica capillary with an inner diameter of about 100 μm.
A filler 10 such as glass beads is enclosed by two porous glass frits 12 and 14. Such a microbore column is manufactured by dry-packing borosilicate glass beads on the end of the capillary and sintering, then dry-packing or slurry-packing the functional filler therein, and finally plugging with sintered glass. can do.

The operation of the embodiment shown in FIG. 1 will be described. The end of the injection capillary 6 is dipped in the sample solution, the end of the separation capillary 2 is dipped in the detection side buffer solution, and the end of the drain capillary 4 is dipped in the drain side buffer solution. B
A high voltage is applied to the drain side of the block power supply 34 (for example, 10 kV or less for several minutes), and the A block side is not turned on. At this time, when the ground for the high voltage of the B block and the potential of the end of the separation capillary 2 are both ground, an electric circuit is generated in the separation capillary 2 as well, so that the switch of the power supply 30 is switched. To disconnect the detection side buffer solution so that it floats electrically. As a result, the electroosmotic flow flows from the microbore column 8 toward the drain, and liquid chromatography mode separation according to the packing material is performed. For example,
When a protein-coated ODS column is used as the microbore column 8, a serum sample is introduced into the drain at a high recovery rate by the introduction of the serum sample, and the drug in the serum is distributed to the ODS silica in the pores.

Next, the sample container at the end of the injection capillary is replaced with an elution buffer container containing an organic solvent, for example, about 70% acetonitrile, and a high voltage is applied by the power source 30 of the A block, contrary to the case of sample injection. The target component is eluted toward the separation capillary 2 by turning off the power supply 34 of the B block (for example, for 10 seconds at 10 kV or less).
Next, a high voltage is applied (for example, 20 kV) from the power source 30 of the A block, and capillary electrophoresis separation is performed. Even when the power supply on the B block side is turned off, if the end of the drain capillary is dropped to the ground, the eluted component may flow to the drain side, so it is electrically floated. As a result, the total amount of the drug in serum is introduced into the separation capillary 2, and the concentrated sample with respect to the injected sample is separated by capillary electrophoresis and detected by the detector 22.

The concentration effect of the capillary column containing the pretreatment filler is, for example, 50 mM phosphate buffer (pH 7.
When a capillary containing protein-coated ODS is used as the migration buffer (0), the drug (eg, propranolol) can be concentrated at least 100 times, and the peak area (height) is proportional to the injection time / concentration, It showed linearity. Further, the decrease in the resolution due to the application of the concentrated gel was 10% or less. Direct injection of serum sample added with propranolol using a three-way branched capillary,
When UV detection (280 nm) is applied to each of the capillaries (drain and separation) in the washing and separating processes, the impurities and unnecessary components (mainly serum proteins) are eluted only in the drain capillaries during the washing process by electroosmotic flow, and the separating process Then, the target drug propranolol was quantitatively detected only in the separation capillary.

In FIG. 1, in order to apply a high voltage independently to the A block and the B block, power supplies 30 and 34 are provided in each block, but one power supply is used and a high voltage relay is used. The voltage may be applied to any one of the blocks.

[0020]

INDUSTRIAL APPLICABILITY In the present invention, since the liquid chromatography pretreatment column and the capillary electrophoresis are brought online by electroosmotic flow, different separation modes can be combined without lowering the resolution. Moreover, by directly introducing and removing unnecessary components from the system before entering the separation capillary, analysis can be performed without overlapping the peak of the target component and leaving no adverse effect on the separation capillary. Since the target component is trapped in the pretreatment column, the target component is also concentrated from the injected sample. The range of application is expanded by properly using various functional fillers according to the purpose of separation. The present invention can be applied to direct injection high-performance liquid chromatographic analysis of a sample to speed up the analysis, and can also be applied to the fractional analysis of the amount of the free form or the bound form of a protein-bound drug.

[Brief description of drawings]

FIG. 1 is a schematic configuration diagram showing an apparatus of an embodiment.

FIG. 2 is an enlarged sectional view showing a pretreatment microbore column in the same Example.

[Explanation of symbols]

 2 Capillary for separation 4 Capillary for drain 8 Microbore column for pretreatment 10 Functional packing material 16 Buffer container 18 Detection side buffer container 20 Drain side buffer container 22 Detector 26, 28, 32 Electrode 30, 34 Power supply

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁵ Identification number Office reference number FI technical display location G01N 30/14 A 8506-2J

Claims (2)

[Claims] 1. A three-way branch type in which a capillary is branched into an injection capillary, a separation capillary, and a drain capillary, and the injection capillary is filled with a pretreatment filler that elutes unnecessary components and holds components to be analyzed. , The end of the injection capillary is immersed in the sample solution, a high voltage is applied between the end of the injection capillary and the end of the drain capillary to discard unnecessary components to the drain, and then the end of the injection capillary is immersed in a buffer solution for injection. A capillary electrophoresis method in which a high voltage is applied between a capillary end and a separation capillary end to separate and analyze a component to be analyzed held in the pretreatment filler. 2. A three-way branched capillary which is branched into an injection capillary, a separation capillary and a drain capillary, and the injection capillary is filled with a pretreatment filler that elutes unnecessary components and retains components to be analyzed. A capillary electrophoresis apparatus comprising: a power supply device capable of independently applying a high voltage between an end of an injection capillary and an end of a drain capillary and between an end of an injection capillary and an end of a separation capillary.