JP3063344B2 - Capillary electrophoresis method and apparatus capable of online pretreatment - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. 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 the capillary electrophoresis method. The present invention relates to an electrophoresis apparatus for performing an electrophoresis method.

[0002]

2. Description of the Related Art Capillary electrophoresis, a high-performance technology for electrophoresis, has the advantages of being capable of high-resolution, rapid analysis, and requiring very little sample consumption. , Medicine, food and the like. The capillary used usually has an inner diameter of 50-100μ
m, made of fused quartz with a length of 20 to 100 cm, and a polyimide coating on the outer surface. The capillary is filled with an electrophoresis buffer or a polymerized gel.
After an 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, sample components are caused to migrate, separated, and detected near the other end of the capillary.

[0003] Capillary electrophoresis is a separation and analysis means that has come to be widely known, but so far, the analysis of high-purity samples has been the majority. In particular, it is difficult to separate and analyze a specific component present in a complex 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 due to protein peaks, but also has a fatal effect on subsequent analysis if adsorption occurs on the fused silica surface of the capillary. give. In order to avoid such a problem, most biological samples require a strict multi-step sample clean-up operation by, for example, solvent extraction.

As an example of application of capillary electrophoresis to the analysis of drugs in plasma, there is a report 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 down protein transfer and allows drug peaks to be separated. 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) using a part of a capillary having an inner diameter of 100 μm or less as a detection cell as it is.
Although the hotometric detector has the fate of poor concentration sensitivity, one attempt to remedy this problem is sample zone online enrichment. There are the following two types of online enrichment methods a and b. (A) Isokinetic electrophoretic enrichment method; for example, it is well known that a large amount of a sample that is sufficiently diluted as compared with a background electrophoresis buffer can be injected to achieve an isokinetic electrophoretic enrichment effect. (B) A method of trapping or delaying a target component in a filler; providing a section in which a filler such as a polymer bead or a CPG (control pore glass) bead is sandwiched by a porous frit in a part of a capillary to form a multi-component. This is a concentration method that traps or delays only a specific target component when a system sample passes. For example, using antibody-immobilized beads
J. Liq. Chromatogr., 14 (5), 997-1015 (1991).

[0006]

It is not preferable to introduce unnecessary components, particularly problematic polymer impurities, into the separation capillary. Capillary electrophoresis is not suitable for clinical applications such as monitoring the amount of drugs in biological samples. The introduction of unnecessary components must be avoided in order to establish the usage. For on-line concentration, isotachophoretic concentration methods interfere with the solvent peak of the sample, depending on the pH of the buffer used. In a method in which a filler that traps or delays a target component is sandwiched in a part of the capillary, unnecessary components are eventually introduced into the separation column, so that sufficient washing is required. An object of the present invention is to provide a capillary electrophoresis method that directly injects and analyzes an actual sample and also has an effect of concentrating a target component, and an electrophoresis apparatus used in the method.

[0007]

According to the capillary electrophoresis method of the present invention, the capillary is formed into a three-way branched type having a capillary for injection, a capillary for separation, and a capillary for drain, and unnecessary components are eluted and analyzed in the capillary for injection. After filling the pretreatment filler holding the target component, immersing the injection capillary end into the sample solution, and applying a high voltage between the injection capillary end and the drain capillary end to discard unnecessary components to the drain Then, the end of the injection capillary is immersed in the 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 analysis target component held in the pretreatment filler.

In order to realize this capillary electrophoresis method, the capillary electrophoresis apparatus of the present invention branches into an injection capillary, a separation capillary, and a drain capillary, and elutes unnecessary components into the injection capillary to analyze. A high voltage is independently applied between the three-way branched capillary filled with the pretreatment filler holding the components, 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 to which the voltage can be applied.

Examples of the functional filler used in the pretreatment filler include the following ad. (A) Protein-coated ODS;
In this method, a protein (for example, BSA) is irreversibly adsorbed on the ODS silica surface. The surface of the hole is a regular O
DS (Chromatographia, 19 , 466 (1984); Anal.
Biochem., 151 , 358 (1985)). (B) ISRP (Internal Surfice Reversed Phase) (c) SHP (Shielded Hydrophobic Phase) Each of these packing materials is a direct injection column designed to prevent proteins from coming into contact with the distributed binding phase by a size exclusion mechanism. It is a packing material and eluted in the same manner as in reversed-phase liquid chromatography. (D) Antibody-immobilized column This specifically binds only the antigen by an immune reaction. On the other hand, there is a method in which an antigen is immobilized to specifically bind an antibody. The bound antigen or antibody is eluted by changing the 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, only low-molecular substances can be introduced as a sample from a multi-component sample in which high-molecular substances and low-molecular substances coexist, and deproteinization occurs simultaneously with injection. Such a pre-processing function can be provided.

[0010]

The drain capillary end and the separation capillary end are respectively immersed in separate electrophoresis buffers with respect to the injection capillary end of the three-way branched capillary, and a two-block circuit is formed between the injection capillary end and the injection end via electrodes. Switching between the two flow paths, the flow path from the injection end to the drain capillary end and the flow path from the injection end to the separation capillary end, is performed by turning on / off the voltage application of each block. First, 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. The sample passes through the filler portion. Substances that are not retained in the pretreatment filler and the solvent required therebetween flow into the separation capillary without flowing into the separation capillary and are discarded. After this pretreatment, the injection end is immersed in the sample solution with a buffer solution, a high voltage is applied to the separation capillary side, and the drain capillary side is electrically floated. Thus, the components held in the pretreatment filler are detected by electrophoresis on the separation capillary side.

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

[0012]

FIG. 1 schematically shows the structure of an apparatus according to an embodiment. The capillary is a three-way branched capillary in which a separation capillary 2, a drain capillary 4, and an injection capillary 6 are branched at a branch position A. A part of the injection capillary 6 is a micro-bore column 8 packed with a filler. The microbore column 8 is filled with a functional filler such as protein-coated ODS according to the purpose.

The end of the injection capillary 6 is immersed in a sample solution or a buffer solution. Reference numeral 16 denotes a buffer solution containing a buffer solution. The end of the separation capillary 2 is immersed in the buffer solution in the detection-side buffer container 18, and the end of the drain capillary 4 is immersed in the buffer solution of the drain-side buffer container 20. The electrode 26 is immersed in the sample solution and buffer solution at the end of the capillary for injection, the electrode 28 is immersed in the buffer solution at the end of the capillary for separation, and the electrode 32 is immersed in the buffer solution at the end of the capillary for injection. 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 capillary for injection and the electrode 28 at the end of the capillary for separation (A block).
A high voltage is applied between the electrode 26 at the injection capillary end and the electrode 32 at the drain capillary end (B block) by the power supply 34. Each of the power supplies 30 and 34 is provided with a switch so that a high voltage can be applied to the A block and the B block independently of each other. 31 and 35 are ammeters of each block, respectively.

FIG. 2 shows an example of the microbore column 8. The microbore column 8 contains a porous silica 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 at the end of the capillary and sintering, then dry-packing or slurry-packing the functional filler, 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 immersed in the sample solution, the end of the separation capillary 2 is immersed in the detection buffer solution, and the end of the drain capillary 4 is immersed in the drain buffer solution. B
A high voltage is applied to the drain side by the power supply 34 of the block (for example, at 10 kV or less for several minutes), and the power is not turned on to the A block side. At this time, if the ground for the high voltage of the B block and the potential of the end of the separation capillary 2 are both at the ground, an electric circuit is also generated in the separation capillary 2, so the switch of the power supply 30 is switched. To make the detection-side buffer liquid electrically float. As a result, the electroosmotic flow flows from the microbore column 8 to the drain, and the separation in the liquid chromatography mode according to the packing material is performed. For example,
When a protein-coated ODS column is used as the microbore column 8, the serum protein is discarded to the drain at a high recovery rate by introducing a serum sample, and the drug and the like in the serum are distributed to the ODS silica in the pores.

Next, the sample container at the end of the capillary for injection 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 supply 30 of the A block in reverse to the time of sample injection. The target component is eluted in the direction of the separation capillary 2 by turning off the power supply of the B block (for example, 10 kV or less for about 10 seconds).
Next, a high voltage is applied by the power supply 30 of the A block (for example, 20 kV) to execute capillary electrophoresis separation. Even when the power supply on the B block side is turned off, if the end of the capillary for the drain falls to the ground, the eluted component may flow to the drain side, so that it is electrically floated. As a result, the total amount of the drug in the serum is introduced into the separation capillary 2, and the concentrate concentrated with respect to the injected sample is separated by capillary electrophoresis and detected by the detector 22.

The concentration effect of the packed capillary column for pretreatment is, for example, 50 mM phosphate buffer (pH 7.0).
When 0) is used as an electrophoresis buffer and a capillary containing protein-coated ODS is used, 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. In addition, the decrease in separation ability by attaching the concentrated gel was 10% or less. Using a three-way branched capillary to directly inject a serum sample to which propranolol was added,
When the capillaries (for drain and separation) detect ultraviolet light (280 nm) in the washing and separation processes, in the washing process by electroosmotic flow, contaminants and unnecessary components (mainly serum proteins) are eluted only in the drain capillaries. Detected the target drug propranolol quantitatively only in the separation capillary.

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

[0020]

According to 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 separation ability. Moreover, by directly introducing and removing unnecessary components before entering the separation capillary, the analysis can be performed without overlapping the peak of the target component and without leaving any adverse effect on the separation capillary. Since the target component is trapped in the pretreatment column, there is also an effect that the target component is concentrated from the injected sample. If various functional fillers are properly used according to the purpose of separation, the applicable range is expanded. The present invention is applied to high-performance liquid chromatography analysis by direct injection of a sample to speed up the analysis, and can be applied to, for example, differential analysis of free and bound amounts of a protein-bound drug.

[Brief description of the drawings]

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

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

[Explanation of symbols]

 2 Capillary for separation 4 Capillary for drain 8 Microbore column for pretreatment 10 Functional filler 16 Buffer container 18 Buffer container on detection side 20 Buffer container on drain side 22 Detector 26, 28, 32 Electrode 30, 34 Power supply

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ identification code FI G01N 27/26 325Z 331H (58) Investigated field (Int.Cl. ⁷ , DB name) G01N 27/447 B01D 35/06 G01N 1 / 10 G01N 30/14 G01N 30/16

Claims (2)

(57) [Claims] 1. A capillary having a three-way branching structure in which a capillary is divided 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 a component to be analyzed. After immersing the injection capillary end into the sample solution and applying a high voltage between the injection capillary end and the drain capillary end to discard unnecessary components to the drain, the injection capillary end is immersed in the buffer solution and injected. 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 a capillary for injection, a capillary for separation, and a capillary for drain, and which is filled with a pretreatment filler for eluting unnecessary components and retaining a component to be analyzed in the capillary for injection. A capillary electrophoresis apparatus comprising: a power supply device capable of independently applying a high voltage between an injection capillary end and a drain capillary end and between an injection capillary end and a separation capillary end.