JPH0886772A - Capillary electrophoresis device - Google Patents

Abstract

(57) [Summary] [Structure] The light emitted from the UV light source 1 is imaged at the slit position by the condenser lens 7, and the pinhole 2 is formed at the light source imaging position.
And set the light source size to the capillary inner diameter or less. The light from the light source is collimated by the lens 8 and the half mirror 10
After passing through, the image is formed in the capillary 4 by the condenser lens 9. The branched light from the half mirror 10 is for reference.
The branched light and the light that has passed through the capillary 4 are respectively received by the light receiver 5, processed by the signal processor 6, and the processing result is output. The condenser lens 9 is combined with the diaphragm 3 that controls the solid angle of the light incident on the capillary. The incident light on the capillary 4 passes through only the space where the sample in the center of the capillary passes, and blocks the light received by the light receiver without passing through the space through which the sample passes. [Effect] Trace components can be detected without treatment such as fluorescent labeling and derivatization.

Description

Detailed Description of the Invention

[0001]

The present invention relates to capillary electrophoresis (C
E) Regarding the device.

[0002]

2. Description of the Related Art Conventionally, as a method for detecting a capillary electrophoresis device, an ultraviolet absorption detection method, a laser excitation fluorescence detection method, an electrochemical detection method, a mass spectrometry method and the like have been described as "Capillary Electrophoresis", pp55.
-154, Elsevier, 1992 ". Among them, the ultraviolet absorption detection method involves injection, separation, and separation of the sample without performing derivatization of the sample.
Since detection can be performed on one column, it has a compact device configuration and has elements as a general-purpose physicochemical instrument. A liquid chromatography (hereinafter abbreviated as LC) detection unit, which has already been completed as a product, is diverted and used as the ultraviolet absorption detection unit.

[0003]

Since the optical path length of the ultraviolet (UV) detection system of the general-purpose capillary electrophoresis device depends on the inner diameter of the capillary (20-100 μm), it is about two orders of magnitude shorter than the optical path length of LC. As a result, the detection sensitivity also decreases. In addition, the presence of background light resulting from the light received by the light receiver without passing through the center of the capillary among the light transmitted through the capillary reduces the S / N in photodetection and at the same time destabilizes the baseline. There was a problem of becoming a factor of.

In summary, the conventional device has the following two problems. (1) Since the UV light source typified by deuterium discharge tubes, mercury lamps, xenon lamps, cadmium lamps, zinc lamps, etc. is not a point light source, the detection system condensing unit has a multifocal size larger than the inner diameter of the capillary, and thus the capillary is used. The light emitted from the focus off the center becomes the background noise. (2) Ordinarily, the light-transmitting window of the detection portion is used by closely contacting the slit with the capillary from which the polyimide coating on the surface is peeled off.
This slit / capillary was used in a state where it could not completely block the light received by passing through the fused silica portion out of the central portion of the capillary through a narrow gap or an unnecessarily wide slit width.

An object of the present invention is to solve the above problems of the prior art and to provide a capillary electrophoresis apparatus capable of detecting a trace amount.

[0006]

To improve the detection sensitivity of a general-purpose CE device, it is considered that improvement of the light source brightness, blocking of background noise light, and amplification of the optical path length contribute. The present invention has the following configurations to achieve the above object.
(1) Since the radiance cannot be increased by using any condensing system, use the place with the highest brightness of the light source to make it an appropriate size. (2) UV of CE device
In the optical system of the detector, in order to control the size of the light source focused inside the capillary tube, a pinhole with a size close to the inner diameter of the capillary should be placed at the light source imaging position of the focusing system.
(3) In order to block the light that does not pass through the inside of the capillary tube out of the light incident on the capillary of the light source of a minute size adjusted by the pinhole, calculate the angle (exit pupil) that looks into the image plane from the capillary, and calculate this. Use an appropriate size diaphragm in combination with a lens close to the capillary. The same applies when selecting the light emitted from the capillary. (4) Similar to the above, a light transmission window is provided by providing minute holes on the capillary coating layer so that incident light or outgoing light to the capillary can be efficiently selected. The size of the capillary-integrated light transmission window should be calculated based on the numerical value calculated from the exit pupil or the entrance pupil.

[0007] That is, the present invention is a capillary electrophoresis having a light source, a capillary for sample migration, a condensing means for condensing the light from the light source to the capillary, and a light detecting means for detecting the light transmitted through the capillary. In the device, the condensing means has means for selecting an image from a light source having a high brightness at a predetermined image forming position and forming an image, and a pinhole arranged at the image forming position. It is characterized in that the image formation size of the light source formed in the above is made substantially equal to the inner diameter of the capillary, and the condensing means further has a diaphragm means for controlling the solid angle of the light incident on the capillary. Are arranged on the light incident side of the capillary or on the light emitting side of the capillary.

The capillary used in the present invention has a covering material, and a light entrance window and a light exit window are formed in the covering material at predetermined positions facing each other, and the area of the light exit window is the light entrance window. The feature is that it is larger than the area of.

[0009]

The signal intensity (S) is increased by selecting a high-luminance portion in advance from the non-uniform luminance distribution of the UV light source. The pinhole provided at the image forming position of the light source of the condensing system adjusts the UV light source size having a spatial spread to the inner diameter of the capillary, while the diaphragm inserted in the condensing system is the solid angle of incidence into the capillary. To contribute to the reduction of background noise (N) received by the light receiver without passing through the space where the sample in the capillary exists. The light transmission window formed of a pinhole provided on the fused silica capillary coating film controls the solid angle of the light incident on the capillary to achieve the same effect. Even when reducing the background noise light on the light emission side from the capillary, a light transmission window formed of a pinhole is provided on the light emission side.

[0010]

EXAMPLE In the example of the present invention, light source imaging of the photodetection system of the capillary electrophoresis apparatus is performed by selecting a portion having high light source brightness, and at the same time, inserting a pinhole at the image forming position,
Adjust the light source size to a size close to the inner diameter of the capillary.
In the photodetection system of the capillary electrophoresis device, a lens,
By inserting a diaphragm that controls the solid angle of the incident light in the capillary into the condensing unit that uses a zone plate such as a mirror, the central portion of the capillary transmitted light, that is, the space where the sample in the capillary passes, is passed. Instead, the light received by the light receiver is blocked to reduce background noise.

The diaphragm is provided in front of the capillary to adjust the size of the exit pupil, or in the rear of the capillary to adjust the size of the entrance pupil.
Background light is reduced by blocking the light of the light transmitted through the capillary that is received by the light receiver without passing through the space through which the sample in the capillary passes.

In a photodetection system of a capillary electrophoresis apparatus, micropores of which size is adjusted are provided on a capillary coating film to serve as a light transmission window, and a capillary in which the light transmission window and the capillary are integrated is used. . The micropores of which the size is adjusted on the capillary coating film are provided on the light incident side or the light emitting side, or both, and the size of the micropores provided on the coating film is the space through which the sample in the capillary passes in the light transmitted through the capillary. The size is adjusted so that the light received by the light receiver does not pass through.

(First Embodiment) FIG. 1 shows the configuration of the first embodiment. The light emitted from the UV light source 1 is a condenser lens 7
The image is formed at the slit position. The pinhole 2 is placed at this light source imaging position so that the light source size is at least equal to or smaller than the inner diameter of the capillary. The light from the light source whose size has been adjusted by the pinhole 2 is collimated by the lens 8 and the half mirror 10
After passing through, the image is formed in the capillary 4 by the condenser lens 9. The branched light from the half mirror 10 is for reference. The branched light and the light that has passed through the capillary 4 are
The light is received by the light receiver 5, processed by the signal processor 6, and the processed result is output. In FIG. 1, the optical path is shown by a solid line and a dotted line.

The condenser lens 9 is combined with a diaphragm 3 (for example, a diameter of 20 mm) for controlling the solid angle of light incident on the capillary. In the fused silica capillary 4 fixed to the support, the light transmitting portion of the capillary was formed by peeling off a part of the polyimide resin coating.

FIG. 2 is a diagram showing a part of the optical path of the first embodiment. The incident light to the capillary 11 passes only through the space at the center of the capillary where the sample passes, and this sample passes. The light received by the photodetector is blocked without passing through the space to reduce background noise and improve the detection sensitivity.

Specimen (Neuromedin, 0.25 μg / μ
l), sodium phosphate buffer (pH 7), UV214
In the measurement system using nm, the noise is 0.3 nA and the signal strength is 9.2 nA in the conventional pinhole size (diameter of 0.6 mm), whereas the noise is 0.3 nA and the signal strength is 80.6 nA in this embodiment. The performance was improved about 9 times in terms of S / N ratio.

(Second Embodiment) FIG. 3 is a sectional view showing the structure of a capillary of the second embodiment. FIG. 4 is a diagram showing a part of the optical path of the second embodiment. In the capillary electrophoresis apparatus, a circular pinhole 13 or 14 is formed by using an excimer laser on the polyimide coating film 12 in the light transmitting portion (light incident and light emitting positions) of the capillary 11.
Is provided, and the light transmission window and the capillary are integrated. When selecting the light irradiating the capillary on the incident side, the size of the pinhole 13 is adjusted (for example, a diameter of 100 μm).
m), the background noise resulting from the light received by the light receiver without passing through the space where the sample in the center of the capillary passes is reduced, and at the same time, the device configuration is simplified. Further, when the light is selected on the side of exiting from the capillary, the same effect is brought about by adjusting the size of the pinhole 14.

[0018]

EFFECTS OF THE INVENTION Since the UV detection sensitivity is improved, it becomes possible to detect a trace amount in a living body, such as a trace component or a component change, without performing a treatment such as fluorescent labeling or derivatization. Since the pre-column or post-column for derivatization is not necessary, the device configuration is simple, and the problems resulting from derivatization efficiency or decomposition by light are eliminated and the analysis of the results becomes easy.

[Brief description of drawings]

FIG. 1 is a block diagram showing an outline of a device configuration of a first embodiment.

FIG. 2 is an explanatory view showing a part of an optical path of the first embodiment.

FIG. 3 is a cross-sectional view showing the structure of a capillary having a light transmission window according to a second embodiment.

FIG. 4 is an explanatory view showing a part of an optical path of a second embodiment.

[Explanation of symbols]

1 ... Light source, 2 ... Pinhole, 3 ... Aperture, 4 ... Capillary, 5 ... Photoreceiver, 6 ... Signal processor, 7,8, 9 ... Lens, 10 ... Half mirror, 11 ... Fused silica capillary, 12 ... Polyimide coating film, 13, 14 ... Pinhole.

Claims (5)

[Claims] 1. A capillary electrophoresis apparatus having a light source, a capillary for causing a sample to migrate, a condenser for condensing light from the light source on the capillary, and a photodetector for detecting light transmitted through the capillary. In the above, the light condensing means has means for selecting an image from a portion of the light source having a high brightness at a predetermined image forming position from the light source and forming an image, and a pinhole arranged at the image forming position, A capillary electrophoresis apparatus, wherein an image forming size of the light source formed at a pinhole position is substantially equal to an inner diameter of the capillary. 2. The capillary electrophoresis apparatus according to claim 1, wherein the light condensing unit further includes a diaphragm unit for controlling a solid angle of light incident on the capillary. 3. The capillary electrophoresis apparatus according to claim 2, wherein the diaphragm means is arranged on a light incident side of the capillary or a light emitting side of the capillary. 4. The capillary electrophoresis apparatus according to claim 1, wherein the capillaries have a coating material, and a light entrance window and a light exit window are respectively formed at positions of the capillaries facing the coating material. 5. The capillary electrophoresis device according to claim 4, wherein the area of the light exit window is larger than the area of the light entrance window.