JP7078115B2 - Light scattering detector - Google Patents

Description

The present invention relates to a light scattering detection device used in a fine particle detection device for measuring the molecular weight, turning radius (size), etc. of fine particles dispersed in a liquid sample.

Size exclusion chromatography (SEC) is known as a method for separating fine particles such as proteins dispersed in a liquid sample. In recent years, as a chromatography detection device, a multi-angle light scattering (MALS) detection device has been used in addition to an ultraviolet (UV) absorbance detection device and a differential refractive index detection device. The MALS detector has a feature that the molecular weight and particle size of the measurement sample can be calculated (see Patent Documents 1 and 2).

FIG. 7 shows a coordinate system in the scattered light radiation direction when a scattered light generating light source is arranged at the origin. As shown in FIG. 7, light is incident on the XY plane in the positive direction in the X direction, and the scattering angle of the light on the XY plane from the traveling direction is defined as θ, and the angle from the XY plane is defined as φ.

Next, FIG. 8 shows a plan view of a basic configuration example of the MALS detection device, and FIG. 9 shows a side view. In FIGS. 8 and 9, 110 is a sample cell, 111 is a liquid sample, 120 is a light source, 121 is a condenser lens, 140 is a slit plate, 150 is an imaging lens, 160 is an aperture plate, and 170 is a detector. As shown in FIGS. 8 and 9, the liquid sample 111 is passed through the inside of the cylindrical sample cell 110, and light is irradiated from the light source 120 so as to pass through the sample cell 110 and the center of the flow path. Visible laser light is usually used as the light source. The angle θ from the traveling direction of light is defined as the scattering angle on the horizontal plane (on the XY plane), and the detector is placed on the horizontal plane (on the XY plane) passing through the sample cell 110 and the center of the flow path so as to detect different scattering angles. A plurality of 170s are arranged. FIG. 8 shows an example in which two detectors 170 are arranged at the arrangement angles of θ1 and θ2.

In the case of the cylindrical sample cell 110, there is a problem that the reflected light at the interface between the glass and the air and the interface between the glass and the flow path enters the detector 170 as stray light and deteriorates the detection accuracy of the detector 170. .. As a solution to this, the inventor has found a method for reducing the stray light by tilting (angle α) the incident light with respect to the sample cell 110 as shown in FIG. 9 (see Non-Patent Document 1). ..

Next, FIGS. 10 and 11 show the relationship between the scattered light intensity and the scattered angle. That is, FIGS. 10 and 11 are the results of calculating the scattering pattern of particles having a refractive index of 1.59 in a solvent having an incident wavelength of 660 nm and a refractive index of 1.33 according to the theoretical formula of Mie scattering. The particle size was 1 nm, 100 nm, and 500 nm. FIG. 10 is a scattering angle pattern in the horizontal direction (θ direction), and FIG. 11 is a scattering angle pattern in the vertical direction (φ direction). When the sample is sufficiently smaller than the wavelength of the incident light, the scattered light in the θ direction is isotropically generated, and the scattered light intensity does not depend on the scattering angle. As the particle size of the sample increases, the scattered light becomes more scattered forward (in the direction in which θ is smaller). In the φ direction, the scattered light intensity tends to decrease as φ increases.

Further, in the MALS detection device, a device capable of measuring a sample having a low concentration and having a high S / N ratio is desirable. For that purpose, an optical system that efficiently enters the scattered light generated from the sample into the detector is required. In other words, the solid angle of the scattered light entering each detector needs to be large. When increasing the solid angle of the detector, if the solid angle in the horizontal direction (on the optical axis plane) is increased, the angular resolution of each detector will deteriorate, so it is desirable to increase the solid angle in the vertical direction. .. However, increasing the detector size in order to increase the solid angle is not preferable because the dark current increases.

In order to increase the solid angle without increasing the detector size, a method of condensing scattered light with a lens or the like is adopted (Non-Patent Document 1). Specifically, the arrangement is such that the scattered light generated in the cell flow path is imaged by the detector via the imaging lens. Therefore, in order to reduce the solid angle in the horizontal direction and increase the solid angle in the vertical direction, a slit having a narrow opening width in the horizontal direction and a wide opening length in the vertical direction is provided on the incident side of the imaging lens. .. As a result, scattered light can be efficiently detected without deteriorating the angular resolution.

Japanese Unexamined Patent Publication No. 07-72068, Japanese Unexamined Patent Publication No. 2015-11163 "Analysis of Absolute Molecular Weight and Complex Formation of Proteins by Light Scattering Method", Masafumi Odaka, Biotechnology Vol. 89

In order to increase the amount of light entering the detector, it is necessary to set a large vertical aperture length of the slit arranged on the incident side of the imaging lens. However, if the vertical opening length of the slit is set large, the reflected light at the interface between the glass and air of the sample cell and the interface between the glass and the flow path is incident on the detector as stray light. Since the reflected light has a higher intensity than the scattered light, the dynamic range of the detector is lowered, and the detection accuracy of the detector is deteriorated due to the fluctuation of the intensity of the stray light. On the other hand, if the tilt angle α of the incident light is set large in order to remove this stray light, the scattering angle φ in the vertical direction becomes large as shown in FIG. 11, the scattered light intensity entering the slit decreases, and S / The N ratio decreases.

Therefore, according to the present invention, the solid angle in the vertical direction of the incident side slit of the imaging lens can be set large without setting the tilt angle α of the incident light to be large, and the measurement is performed with high S / N ratio and high detection accuracy. It is an object of the present invention to provide a light scattering detection device capable of performing.

The light scattering detection device according to one aspect of the present invention is a light scattering detection device for detecting fine particles in a liquid sample, and is a transparent sample cell holding the liquid sample and the liquid sample in the sample cell. A light source that irradiates coherent light from an angle inclined with respect to the central axis of the A slit plate having a slit for limiting an angular range and a detector that receives light from the imaging optical system are provided, and the central axis of the slit is the central axis of the imaging optical system. It is characterized in that it is arranged eccentrically in one direction from the vertical direction.

In the configuration of the light scattering detection device, it is preferable that the slit is formed vertically in the vertical direction.

Further, the slit has at least a straight side along the vertical direction and is arranged eccentrically upward in the vertical direction from the central axis of the imaging optical system. When the lower length in the vertical direction from the central axis is a and the upper length is b, it is preferable to have a relationship of a <b .

A plurality of detection optical systems from the sample cell to the detector are arranged around the sample cell at equal intervals from the cell center axis, and are the lengths of the lower length a and the upper length b of the slit. The ratio is preferably adjusted under the condition of a ≦ b according to the arrangement angle of each detector with respect to the sample cell.

According to the present invention, the solid angle in the vertical direction of the incident side slit of the imaging lens can be set large without setting the tilt angle α of the incident light to be large, and the measurement is performed with high S / N ratio and high detection accuracy. It is possible to provide a light scattering detection device that can be used.

It is a side view of one Embodiment of the light scattering detection apparatus which concerns on this invention. It is a partially enlarged side view of the light scattering detection apparatus which concerns on this embodiment. It is a top view of the light scattering detection apparatus which concerns on this embodiment. It is a side view for demonstrating arrangement and member size in a ray tracing simulation. It is a top view for demonstrating the ray tracing simulation result when the detector is arranged at θ = 15 degrees. It is a side view for demonstrating the ray tracing simulation result when the detector is arranged at θ = 15 degrees. This is a coordinate system in the scattered light radiation direction when a scattered light generation light source is placed at the origin. It is a top view of the basic configuration example of a MALS detection device. It is a side view of the basic configuration example of a MALS detection device. It is explanatory drawing of the relationship (horizontal scattering angle pattern) of the scattered light intensity and the scattering angle. It is explanatory drawing of the relationship (the scattering angle pattern in the vertical direction) of the scattered light intensity and the scattering angle.

Hereinafter, an embodiment of the light scattering detection device according to the present invention will be described with reference to the drawings. In each figure, those with the same reference numerals have the same or similar configurations.

[Structure of light scattering detector]
First, the configuration of one embodiment of the light scattering detection device according to the present invention will be described with reference to FIGS. 1 to 3. FIG. 1 is a side view of an embodiment of a light scattering detection device according to the present invention. FIG. 2 is a partially enlarged side view of the light scattering detection device according to the present embodiment. As shown in FIGS. 1 and 2, the light scattering detection device 1 according to the present embodiment is a device that detects the molecular weight and the radius of gyration (size) of fine particles such as proteins dispersed in a liquid sample. The light scattering detection device 1 includes a sample cell 10, a light source 20, a slit plate 40, an imaging optical system 50, an aperture 60, and a detector 70. Hereinafter, each component will be described.

The sample cell 10 is a transparent cylindrical cell that holds a liquid sample in an internal flow path. The sample cell 10 is formed of, for example, colorless and transparent quartz glass.

The light source 20 irradiates the sample cell 10 with coherent light. "Coherent light" means that the phase relationship of light waves at any two points in the luminous flux is constant over time and is kept constant. After dividing the luminous flux by an arbitrary method, a large optical path difference is given and superposition is performed again. Light that shows complete coherence even when combined. As the light source 20, for example, a laser light source for irradiating a visible light laser is adopted. There is no perfect coherent light in nature, and the laser light oscillated in single mode is close to the coherent state.

A condensing optical system 21 is arranged in the optical path L1 of the incident light from the light source 20 to the sample cell 10. As the condensing optical system 21, for example, a single condensing lens is adopted. This condenser lens is a plano-convex lens, and the incident side of the light from the light source 20 is a convex surface, and the emission side is a flat surface. In the present embodiment, a single condensing lens is adopted as the condensing optical system 21, but the condensing optical system 21 may be configured by combining a plurality of composite lenses and condensing mirrors.

In the light source 20 and the condensing optical system 21, the optical axis of the coherent light incident on the sample cell 10 from the light source 20 is at a predetermined angle (tilt angle α) from the plane (XY plane) including the sample cell 10 and the detector 70 . It is arranged so as to be inclined. Specifically, the light source 20 and the condensing optical system 21 are arranged so that the incident light is incident on the sample cell 10 from diagonally above. By tilting the incident light with respect to the sample cell 10 (angle α), the interface between the glass and air and the interface between the glass and the flow path of the sample cell 10 [hereinafter, collectively referred to as “cell interface”. ) Can reduce stray light due to reflected light. The laser light emitted from the light source 20 passes through the condensing optical system 21 and then condenses in the vicinity of the central axis of the sample cell 10.

The detection optical system 30 is arranged on the optical path L2 of the light emitted from the sample cell 10. The detection optical system 30 of this embodiment is composed of a slit plate 40, an imaging optical system 50, an aperture plate 60, and a detector 70.

The imaging optical system 50 collects light scattered from the sample cell 10 to the surroundings with different scattering angles. As the imaging optical system 50, for example, a single imaging lens is adopted. This imaging lens is a plano-convex lens, and the incident side of the scattered light from the sample cell 10 is formed on a flat surface and the emitted side is formed on a convex surface. In the present embodiment, a single imaging lens is adopted as the imaging optical system 50, but the imaging optical system 50 may be configured by combining a plurality of composite lenses and imaging mirrors.

The slit plate 40 is arranged between the sample cell 10 and the imaging optical system 50 on the optical path L2 of the light emitted from the sample cell 10. The slit plate 40 limits the scattering angle range incident on the imaging optical system 50. That is, the slit 41 opened in the slit plate 40 is vertically long in the vertical direction in order to limit the scattering angle in the horizontal direction and capture a large amount of light beam in the vertical direction, and at least the sides along the vertical direction are provided. It is a plumb bob. Specifically, the slit 41 has a vertically long rectangular shape or an elongated hole shape.

The central axis 41S of the slit 41 is arranged eccentrically in one direction in the vertical direction from the central axis 50S of the imaging optical system 50. Since the irradiation light of the light source 20 is incident from diagonally above the sample cell 10, the reflected light RL generated at the interface between the air and the glass and the interface between the glass and the liquid (hereinafter referred to as “cell interface”) of the sample cell 10. Is likely to be biased downward as stray light. Therefore, the central axis 41S of the slit 41 of the present embodiment is arranged eccentrically upward in the vertical direction from the central axis 50S of the imaging optical system 50 in order to limit the reflected light (stray light) RL.

Specifically, as shown in FIG. 2, when the downward length of the slit 41 in the vertical direction from the central axis 50S of the imaging optical system 50 is a and the upward length is b, the downward length of the slit 41 is defined. The relationship between the a and the upper length b is set so that a <b. The incident light may be incident from below to above, and in that case, the relationship between the lower length a and the upper length b of the slit 41 is set so that a> b.

The aperture plate 60 is arranged on the optical path L2 of the light emitted from the sample cell 10 on the imaging optical system side of the detector 70. The aperture plate 60 has a function of limiting stray light, and the aperture 61 is open in front of the light receiving surface of the detector 70.

The detector 70 receives light from the image pickup optical system 50. That is, the light receiving surface of the detector 70 is located at the focal point of the imaging optical system 50. As the detector 70 of this embodiment, for example, a photodiode (PD) is adopted, but an array detector such as a two-dimensional CMOS may be adopted.

FIG. 3 is a plan view of an embodiment of the light scattering detection device according to the present embodiment. As shown in FIG. 3, a plurality of detection optical systems 30 from the sample cell 10 to the detector 70 are arranged around the sample cell 10 at equal intervals d from the cell central axis S. The angle θ from the traveling direction of light is defined as the scattering angle on the horizontal plane (on the XY plane). A plurality of detectors 70 are arranged on a horizontal plane (on the XY plane) passing through the sample cell 10 and the cell central axis S so that different scattering angles can be detected. In the aspect of FIG. 3, two detection optical systems 30 and 31 are arranged at the arrangement angles of θ1 and θ2.

When a plurality of detectors 70 are provided around the sample cell 10 at equal intervals d, the lengths of the lower length a and the upper length b of the slit 41 are provided according to the arrangement angle of each detector 70 with respect to the sample cell 10. The ratio is adjusted under the condition of a ≦ b. That is, in FIG. 3, the detection optical system 31 arranged at the scattering angle θ2 is less affected by the reflected light at the cell interface than the detection optical system 30 arranged at the scattering angle θ1. Since the detection optical system 31 having a scattering angle θ2 = 90 degrees is less affected by the reflected light at the cell interface, it is possible to optimize the solid angle in the vertical direction by setting a large and bringing it closer to b. ..

[Action of light scattering detector]
Next, the operation of the light scattering detection device 1 according to the present embodiment will be described with reference to FIGS. 1 to 6.

As shown in FIGS. 1 and 2, the liquid sample 11 is passed through the flow path of the cylindrical sample cell 10. When the liquid sample 11 has been passed through the liquid sample 11, the visible laser light, which is coherent light, is irradiated from the light source 20 via the condensing optical system 21. The visible laser light travels along the optical path L1, so that the laser light is incident on the liquid sample 11 in the flow path of the sample cell 10. When the laser beam is incident on the liquid sample 11, the light hits the fine particles contained in the liquid sample 11 and is scattered with a predetermined scattering angle. Then, the scattered light emitted from the sample cell 10 passes through the slit 41 of the slit plate 40, passes through the imaging optical system 50 and the aperture plate 60, and is received on the light receiving surface of the detector 70.

When scattered light is emitted from the sample cell 10, reflected light RL is generated as stray light at the cell interface of the sample cell 10. Since the irradiation light of the light source 20 is incident from diagonally above the sample cell 10 at a tilt angle α, the reflected light (stray light) generated at the cell interface of the sample cell 10 tends to be biased downward. Since the slit 41 of the present embodiment is eccentrically arranged upward in the vertical direction from the central axis 50S of the imaging optical system 50, the reflected light (stray light) RL biased downward by the plate portion of the slit plate 40 is limited. be able to.

By eccentrically arranging the slit 41 of the slit plate 40 upward from the central axis 50S of the imaging optical system 50 in the vertical direction in this way, scattered light useful for analysis is positively incident on the imaging optical system 50 . Can be done. The imaging optical system 50 forms an image on the light receiving surface of the detector 70 , and the aperture plate 60 is arranged in front of the light receiving surface. Therefore, the aperture plate 60 further limits the stray light, and the scattered light required for the analysis can be received by the light receiving surface of the detector 70.

Specifically, when the lower length of the slit 41 in the vertical direction from the central axis 50S of the imaging optical system 50 is a and the upper length is b, the lower length a and the upper length b of the slit 41 are defined. The relationship of is set so that a <b. That is, by setting the downward length a of the slit 41 to be small, the reflected light RL at the cell interface can be blocked by the plate portion of the slit plate 40. By setting the length b in the upward direction to a large value with the effective diameter of the imaging optical system 50 as the upper limit, the solid angle in the vertical direction can be increased.

Further, as shown in FIG. 3, a plurality of detection optical systems 30 from the sample cell 10 to the detector 70 are arranged around the sample cell 10 at equal intervals d from the cell central axis S. When the light scattering detection device 1 is provided with a plurality of detection optical systems 30 at equal intervals d around the sample cell 10, the length ratio between the lower length a and the upper length b of the slit 41 is the length ratio with respect to the sample cell 10. It is adjusted under the condition of a ≦ b according to the arrangement angle of each detector 70. That is, in the detection optical system 31 arranged at a scattering angle θ2 = 90 degrees, which is less affected by the reflected light RL at the cell interface, a is set large and is brought closer to b to optimize the solid angle in the vertical direction. Can be done.

[Ray tracing simulation]
A ray tracing simulation was performed to confirm the action and effect of the above embodiment. FIG. 4 is a side view for explaining the arrangement and member dimensions in the ray tracing simulation. FIG. 5 is a plan view for explaining the result of the ray tracing simulation when the detector (PD) is arranged at θ = 15 degrees, and FIG. 6 is a side view thereof.

As shown in FIG. 4, the sample cell 10 is, for example, a transparent cylindrical cell having an inner diameter of 1.6 mm and an outer diameter of 8.0 mm. An imaging lens (plano-convex lens) is arranged at a position 47 mm from the central axis S of the sample cell 10. The imaging lens has, for example, a convex diameter of φ12.7 mm and a focal length of 38 mm. The aperture plate 60 and the PD of 2.4 mm are arranged at a position 140 mm from the central axis S of the sample cell 10. The slit 41 of the slit plate 40 is formed as an opening having a horizontal (XY direction) width of 3 mm and a vertical direction (Z direction) length of 8.5 mm, for example. Regarding the vertical opening of the slit 41, for example, the downward length a from the XY plane including the central axis 50S of the imaging optical system 50 is 2.5 mm in the −Z direction, and the upward length b is 6 in the + Z direction. It is set to 0.0 mm.

As shown in FIGS. 5 and 6, by setting the relationship between the lower length a and the upper length b to a <b, the reflected light RL generated at the cell interface of the sample cell 10 is the plate of the slit plate 40 . Since it is blocked by the portion and does not reach the detector 70, accurate measurement is possible.

Further, the stray light due to the reflected light RL at the cell interface depends on the arrangement angle θ of the detector 70. When θ is small, the influence of the reflected light RL at the cell interface is large, so a needs to be shortened, but when θ is close to 90 degrees, the reflected light RL at the cell interface hardly occurs. By optimizing the slit 41 under the condition of a ≦ b according to the arrangement angle of the detector 70, it is possible to reduce the stray light and improve the S / N ratio.

As described above, according to the light scattering detection device 1 of the present embodiment, the slit 41 of the slit plate 40 arranged on the incident side of the imaging optical system 50 without setting the tilt angle α of the incident light to be large. It is possible to provide a light scattering detection device capable of setting a large stereoscopic angle in the vertical direction and measuring with high detection accuracy at a high S / N ratio.

The above embodiment is for facilitating the understanding of the present invention, and is not for limiting the interpretation of the present invention. Each element included in the embodiment and its arrangement, material, condition, shape, size, and the like are not limited to those exemplified, and can be appropriately changed. Further, it is possible to partially replace or combine the configurations shown in different embodiments.

1 ... Light scattering detection device 10 ... Sample cell 20 ... Light source 30 ... Detection optical system 40 ... Slit plate 41 ... Slit 41S ... Slit central axis 50 ... Imaging optical system 51S ... Imaging optical system central axis 70 ... Detector

Claims (4)

A light scattering detector for detecting fine particles in a liquid sample.
A transparent sample cell that holds a liquid sample,
A light source that irradiates the sample cell with coherent light from an angle inclined with respect to the central axis of the liquid sample .
An imaging optical system that collects light scattered from the liquid sample to the surroundings with different scattering angles.
A slit plate in which a slit for limiting the scattering angle range incident on the imaging optical system is formed , and
A detector that receives light from the imaging optical system and
Equipped with
A light scattering detection device characterized in that the central axis of the slit is eccentrically arranged in one direction in the vertical direction from the central axis of the imaging optical system. The light scattering detection device according to claim 1, wherein the slit is vertically elongated in the vertical direction. The slit has at least a straight side along the vertical direction, and is arranged eccentrically upward in the vertical direction from the central axis of the imaging optical system .
The first or second aspect of the present invention, which has a relationship of a <b when the lower length of the slit in the vertical direction from the central axis of the imaging optical system is a and the upper length is b . Light scattering detector. A plurality of detection optical systems from the sample cell to the detector are arranged around the sample cell at equal intervals from the cell central axis.
The third aspect of claim 3 , wherein the length ratio between the lower length a and the upper length b of the slit is adjusted under the condition of a ≦ b according to the arrangement angle of each detector with respect to the sample cell. Light scattering detector.

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