JP2012093092A - Planer column, and separation system and separation method using the same - Google Patents

Abstract

PROBLEM TO BE SOLVED: To perform chromatographic separation and electrophoretic separation.SOLUTION: A flow channel in which samples flow is formed between a pair of glass substrates. In the flow channel, a planer column with a structure having an organic monolith in the sample flow direction and having a pair of electrodes to apply voltage in the direction perpendicular to the sample flow is used to perform chromatographic separation in the flow direction and, by simultaneously applying voltage in the direction perpendicular to the flow, to perform electrophoretic separation. Consequently, sample components are two-dimensionally separated in the planer column.

Description

  The present invention relates to a flat column column for performing electrophoretic separation and chromatographic separation, a separation system using the same, and a separation method.

  Bioanalysis requires comprehensive separation and analysis of a wide variety of components. In order to separate a wide variety of components, a method using a plurality of methods (separation modes) such as two-dimensional electrophoresis and two-dimensional chromatography is widely used (see Patent Document 1, Non-Patent Documents 1 and 2). ).

JP 2005-312402 A

“Fast, comprehensive two-dimensional liquid chromatography.”, Stoll Dwight R; Li Xiaoping; Wang Xiaoli; Carr Peter W; Porter Sarah E G; Rutan Sarah C, J. Chromatogr. A (2007), 1168, 3-43. Nobuaki Okumura, Katsuya Nagai “Two-dimensional electrophoresis in proteomics” Molecular Medicine, separate volume, Cancer Genomics, 39: 158-165 (2002)

  In the conventional method, after the separation in one separation mode is completed, the entire separated sample or a part thereof is further separated in another mode. Therefore, there are problems such as (1) longer separation time, (2) complicated operation procedures, and (3) the necessity of a complicated flow path switching system.

  In view of the above points, an object of the present invention is to provide a flat column column that simultaneously performs chromatographic separation and electrophoretic separation, a separation system using the same, and a separation method.

  In order to achieve the above object, the present invention provides a flow path through which a sample flows between a pair of substrates, has a stationary phase for chromatography in the flow direction of the sample in the flow path, and A flat plate column having a pair of electrodes for applying a voltage in a direction crossing the sample flow is a first feature.

  The present invention includes the above-described flat plate column, first introducing means for continuously introducing a mobile phase into the flow path of the flat plate column, and second liquid that introduces a sample solution into the flow path of the flat plate column. A separation system having introduction means and power supply means for applying a voltage between the pair of electrodes is a second feature.

  In the separation system described above, the present invention has an interface attached to the plate-type column for carrying out liquid separation for chromatographic separation. The interface includes a mobile phase supply port and a mobile phase supply port. A sample solution introduction port located on the downstream side, the first introduction unit supplies the mobile phase from the mobile phase supply port, and the second introduction unit includes the sample solution introduction unit. A third feature is that the sample solution is supplied from the mouth.

  In the third aspect of the present invention, the interface has a triangular shape in which the mobile phase supply port is present at the apex, and the mobile phase supplied from the mobile phase supply port faces the flat column. The fourth feature is that it is widened.

  The present invention uses the above-described flat column, and the sample introduced into the flat column is chromatographically separated by the chromatographic stationary phase, and at the same time, a voltage is applied between the pair of electrodes. A separation method for electrophoretic separation of the sample is a fifth feature.

It is a figure which shows the whole structure of the flat column in 1st Embodiment of this invention. It is a figure which shows the whole structure of the separation apparatus using the flat column in 1st Embodiment of this invention.

(First embodiment)
Two electrodes (gold-plated copper foil, 10 x 50 mm, thickness 0.05 mm) are bonded to both ends of a glass plate (40 x 50 mm) whose surface is modified with 3-methacryloxypropyltrimethoxysilane. Further, a glass plate modified with 3-methacryloxypropyltrimethoxysilane was adhered onto the plate, and a flat glass empty column having a rectangular cross section (30 mm × 0.05 mm) having electrodes at both ends was prepared. In this flat glass empty column, butyl methacrylate (monomer, 24%), ethylene dimethacrylate (crosslinking agent, 16%), 1-decanol (pore forming agent, 34%), cyclohexanol (pore forming agent) , 26%), a polymerization reaction solution consisting of 2,2-dimethoxyphenyl-2-acetophenone (photoinitiator, 1% with respect to the total of monomer and crosslinking agent) was injected. An organic monolith was prepared by in situ photopolymerization by irradiating a flat glass empty column filled with the reaction solution with ultraviolet light (intensity of about 4 mW / cm2, wavelength of 254 nm) in a thermostat kept at 0 ° C. After photopolymerization, methanol was sucked and fed to remove unreacted substances, and a flat glass column having electrodes at both ends shown in FIG. 1 was prepared.

  The separation system shown in FIG. 2 was configured using the flat glass column shown in FIG. A triangular interface was attached to the prepared flat glass column in order to send liquid for chromatographic separation. The mobile phase supplied from the mobile phase supply port existing at the apex of the triangular interface using the mobile phase liquid feeding pump constituting the first introducing means flows through the flow path in the triangular interface, as shown in the figure. And continuously introduced into the plate column. A sample solution introduction port is separately provided in the interface, and the sample solution can be introduced into the interface (in the column) by using a sample solution introduction pump serving as a second introduction means. The introduced sample can be chromatographically separated by an organic monolith in a flat column. Moreover, the introduced sample can be electrophoresed by applying a voltage to the electrodes installed at both ends of the flat column using a power supply device that constitutes a power supply means.

  Using the apparatus shown in FIG. 2, methanol to which 0.1% trifluoroacetic acid was added was continuously supplied as a mobile phase to a flat column. The flat column column was irradiated with 365 nm ultraviolet light, and fluorescence was observed using a CCD camera.

  When pyrene and quinine are used as a sample without voltage application, the movement speed of the highly hydrophobic pyrene in the mobile phase flow direction is slow, and the interaction between the hydrophobic phase and the stationary phase is small. It was confirmed that chromatographic separation was possible because of the high movement speed of. It was also confirmed that when voltage was applied (150 V), quinine was electrophoresed in the direction across the flow. This cross flow (electrophoresis) was not observed in the neutral substance pyrene. That is, it was confirmed that electrophoretic separation was possible.

  According to the above-described embodiment, the flow path through which the sample flows is formed between the pair of rectangular glass substrates, and in the flow path, the organic monolith is provided in the flow direction of the sample, and the flow direction crosses the flow of the sample. By using a plate type column having a pair of electrodes for applying a voltage to the column, chromatographic separation and electrophoretic separation can be simultaneously performed. In other words, by using a flat column with electrodes on both ends that can orthogonally separate chromatographic separation and electrophoretic separation, chromatographic separation proceeds in the flow direction, and at the same time, voltage is applied across the flow. Perform electrophoretic separation. As a result, the sample components are two-dimensionally separated in the flat column.

(Other embodiments)
In the above-described embodiment, as a polymerization reaction solution for preparing an organic monolith, “butyl methacrylate (monomer, 24%), ethylene dimethacrylate (crosslinking agent, 16%), 1-decanol (pore-forming agent” , 34%), cyclohexanol (pore-forming agent, 26%), 2,2-dimethoxyphenyl-2-acetophenone (photoinitiator, 1% based on the sum of monomer and cross-linking agent) Other solutions (for example, a monomer for glycidyl methacrylate and a pore size preparation for water) can be substituted.

  In the above-described embodiment, a photopolymerization reaction is used when preparing an organic monolith, but other reactions (for example, a thermal polymerization reaction) can also be used.

  In the embodiment described above, a flat column is prepared using an organic monolith, but other than this (for example, an inorganic monolith, a filler, etc.) can be substituted.

  In the above-described embodiment, the case where an organic monolith is used as a stationary phase for chromatography has been described, but other than this (for example, an inorganic monolith, a filler, etc.) can be substituted as a stationary phase.

  In the above-described embodiment, glass is used as a component of the flat column, but other than that (for example, quartz, Teflon (registered trademark), PEEK, metal whose surface is subjected to insulation treatment, etc.) is substituted. You can also.

  In the above-described embodiment, a gold-plated copper foil is used as the electrode attached to the flat column, but other (for example, gold, platinum, graphite, etc.) can be substituted, and any other than 0.05 mm can be used. Can be used.

  In the above-described embodiment, a 40 × 50 mm glass plate and a 10 × 50 mm electrode are used, but any other size can be used instead.

Claims (5)

  A channel through which the sample flows is formed between a pair of substrates, has a chromatographic stationary phase in the flow direction of the sample in the flow channel, and applies a voltage in a direction crossing the sample flow in the flow channel A flat column having a pair of electrodes. A flat column according to claim 1;
First introduction means for continuously introducing a mobile phase into the flow path of the flat plate column;
A second introduction means for introducing a sample solution into the flow path of the flat column;
And a power supply means for applying a voltage between the pair of electrodes. Having an interface attached to the plate column for carrying out liquid separation for chromatography separation;
This interface is provided with a mobile phase supply port and a sample solution introduction port located downstream of the mobile phase supply port.
The first introduction means supplies the mobile phase from the mobile phase supply port,
The separation system according to claim 2, wherein the second introduction unit supplies the sample solution from the sample solution introduction port.   The interface has a triangular shape in which the mobile phase supply port is present at the apex, and the mobile phase supplied from the mobile phase supply port spreads toward the flat plate column. The separation system according to claim 3.   The flat plate column according to claim 1, wherein the sample introduced into the flat plate column is chromatographically separated by the stationary phase for chromatography and simultaneously introduced by applying a voltage between the pair of electrodes. A separation method comprising electrophoretic separation of the sample.