JP2020020623A - Cell holder for light scattering detector and light scattering detector - Google Patents

Abstract

To provide a cell holder capable of cleaning the inner surface of a flow path of a sample cell without removing the sample cell and also to provide a light scattering detector having the same.SOLUTION: In a cell holder 100, at least one holder 110 of a pair of holders 110, 210 holding both ends of a sample cell 10 is formed in a double flange structure 140 which includes: a first flange part 120 holding the sample cell 10; and a second flange part 130 holding a tube T connected to the sample cell 10.SELECTED DRAWING: Figure 2

Description

  INDUSTRIAL APPLICABILITY The present invention is used for a cell holder for holding a sample cell through which a liquid sample flows, and a particle detecting device for measuring the molecular weight, the radius of gyration (size), and the like of the fine particles dispersed in the liquid sample. The present invention relates to a light scattering detection device.

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

  FIG. 5 shows a coordinate system in the scattered light emission direction when a scattered light generation light source is arranged at the origin. As shown in FIG. 5, light is incident on the XY plane in the positive direction of the X direction, and the scattering angle from the traveling direction of the light on the XY plane is defined as θ, and the angle from the XY plane is defined as φ.

  Next, FIG. 6 is a plan view of a basic configuration example of the MALS detection device, and FIG. 7 is a side view. 6 and 7, 310 is a sample cell, 311 is a liquid sample, 320 is a light source, 321 is a condenser lens, 340 is a slit plate, 350 is an imaging lens, 360 is an aperture plate, and 370 is a detector.

  As shown in FIGS. 6 and 7, a liquid sample 311 is passed through a cylindrical sample cell 310, and light is emitted from a light source 320 so as to pass through the sample cell 310 and the center of the flow channel. Generally, a visible laser beam is used as a light source. The angle θ from the light traveling direction is defined as the scattering angle on the horizontal plane (on the XY plane), and the detector is located on the horizontal plane (on the XY plane) passing through the sample cell 310 and the center of the flow path so as to detect different scattering angles. A plurality of 370s are arranged. FIG. 6 shows an example in which two detectors 370 are arranged at arrangement angles of θ1 and θ2.

  FIG. 8 is a schematic view of a cell holder having a conventional structure. As shown in FIG. 8, the sample cell 310 is fixed by a lower holder 420 and an upper holder 430. A flow path 470 is formed inside the lower holder 420 and the upper holder 430, and connects the tube 440 and the flow path 312 of the sample cell 310. Tube 440 is fixed to lower holder 420 and upper holder 430 by fitting 450. The lower holder 420 and the holder are used to make the center axis of the flow path 312 of the sample cell 310 coincide with the center of the optical axis of the incident light on the XY plane, and also make the center axis of the tube 440 coincide with the center axis of the flow path 312. An O-ring 460 is attached to each of the holding portions 430.

JP-A-07-72068, JP 2015-11163 A "Analysis of Absolute Molecular Weight and Complex Formation of Proteins by Light Scattering Method", Masafumi Odaka, Biotechnology 89

  In the MALS detector, the sample adheres to the inner surface of the flow channel of the sample cell every time the measurement is repeated, and the background signal increases. The amount of increase in the background signal varies depending on the detector arrangement angle, and is more remarkable for a detector arranged at a lower scattering angle. The increase in the background signal reduces the measurement accuracy. It is effective to remove the sample adhered to the inner surface of the flow channel by brushing. Since the inner diameter of the flow path in the cell holder is as small as φ1.0 mm to φ1.5 mm, it is impossible to brush the cell flow path with the inner diameter of φ1.5 mm to φ2.0 mm through the cell holder. Therefore, when performing brushing cleaning, it was necessary to remove the sample cell from the cell holder. However, once the sample cell is removed, it is necessary to adjust the optical system again, and there is a problem in that the adjustment of the optical system is very troublesome.

  Then, an object of the present invention is to provide a cell holder which can clean the inside of a flow path of a sample cell satisfactorily and efficiently without removing a sample cell, and a light scattering detection device provided with the same.

  The cell holder of the light scattering detection device according to one embodiment of the present invention is a cell holder for holding a sample cell of the light scattering detection device, and at least one of a pair of holders holding both end portions of the sample cell. The holder is characterized by being formed in a double flange structure including a first flange portion holding the sample cell and a second flange portion holding a tube connected to the sample cell.

  In the configuration of the cell holder of the light scattering detection device, it is preferable that the sample cell is arranged along a vertical direction, and an upper holder of the pair of holders is formed in the double flange structure.

  Further, the first flange portion has a boss portion for holding the sample cell via a packing at a lower portion thereof, and a tube housing recess for housing the tube held by the second flange portion at an upper portion thereof. Preferably, the second flange portion is detachably connected to the first flange portion.

  Further, it is preferable that the second flange portion has a tube housing recess for housing the probe portion of the tube.

  A light scattering detection device according to one embodiment of the present invention is a light scattering detection device for detecting fine particles in a liquid sample, and irradiates the sample cell with a transparent sample cell holding the liquid sample and coherent light. A light source, an imaging optical system that collects light scattered from the sample cell to the surroundings with different scattering angles, and a detector that receives light collected from the imaging optical system, Is held by the cell holder according to any of the above.

  ADVANTAGE OF THE INVENTION According to this invention, the cell holder which can wash the inside of the flow path of a sample cell satisfactorily and efficiently without removing a sample cell, and a light-scattering detection apparatus provided with the same can be provided.

It is a side view of the light scattering detector concerning this embodiment. It is a schematic diagram of the assembled state of the cell holder concerning this embodiment. It is a schematic diagram of the disassembled state of the cell holder which concerns on this embodiment. FIG. 4 is an explanatory diagram of a state before and after washing an inner surface of a flow channel of a sample cell in the present embodiment. This is a coordinate system in the scattered light emission direction when a scattered light generation light source is arranged at the origin. FIG. 3 is a plan view of a basic configuration example of the MALS detection device. FIG. 3 is a side view of a basic configuration example of the MALS detection device. It is a schematic diagram of the cell holder of a conventional structure.

  Hereinafter, an embodiment of a cell holder and a light scattering detection device according to the present invention will be described with reference to the drawings. In each of the drawings, the components denoted by the same reference numerals have the same or similar configurations.

(Configuration of light scattering detection device)
First, a light scattering detection device incorporating a cell holder according to the present embodiment will be described with reference to FIG. FIG. 1 is a side view of the light scattering detection device according to the present embodiment. As shown in FIG. 1, a 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 plate 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 sample cell 10 is held by a cell holder 100 of the present embodiment described later.

  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 a light beam is invariant in time and is kept constant. After splitting the light beam by an arbitrary method, a large optical path difference is given and the light beam is superimposed again. Light that shows perfect coherence even when combined. As the light source 20, for example, a laser light source for irradiating a visible light laser is employed. There is no perfect coherent light in the natural world, and laser light oscillating in a single mode is light close to a coherent state.

  A condensing optical system 21 is disposed on an optical path L1 of incident light from the light source 20 to the sample cell 10. As the condenser optical system 21, for example, a single condenser lens is employed. This condensing lens is a plano-convex lens, and the incident side of the light from the light source 20 is formed as a convex surface, and the outgoing side is formed as a flat surface. In the present embodiment, a single condenser lens is used as the condenser optical system 21, but the condenser optical system 21 may be configured by combining a plurality of compound lenses or condenser mirrors.

  The light source 20 and the condensing optical system 21 are arranged such that 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 a plane (XY plane) including the sample cell 10 and the detector 70. It is arranged to be inclined. Specifically, the light source 20 and the condensing optical system 21 are arranged so that the incident light enters the sample cell 10 from obliquely above. By tilting the incident light with respect to the sample cell 10 (angle α), the interface between the glass and the air and the interface between the glass and the flow path of the sample cell 10 (hereinafter collectively referred to as “cell interface”). Stray light due to reflected light can be reduced. The laser light emitted from the light source 20 passes through the focusing optical system 21 and is focused near the central axis of the sample cell 10.

  On the optical path L2 of the light emitted from the sample cell 10, a detection optical system 30 is arranged. The detection optical system 30 of the present embodiment includes 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 at different scattering angles. As the imaging optical system 50, for example, a single imaging lens is employed. This imaging lens is a plano-convex lens, and the incident side of the scattered light from the sample cell 10 is formed as a plane, and the emission side is formed as a convex surface. In the present embodiment, a single imaging lens is employed as the imaging optical system 50, but the imaging optical system 50 may be configured by combining a plurality of compound lenses or imaging mirrors.

  The slit plate 40 is disposed 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 limits the scattering angle in the horizontal direction, and in order to take in a large amount of light flux in the vertical direction, the slit 41 is vertically long in the vertical direction, and at least the side along the vertical direction is formed. It is straight. Specifically, the slit 41 has a vertically long rectangular shape or a long hole shape in the vertical direction.

  The aperture plate 60 is disposed closer to the imaging optical system than the detector 70 on the optical path L2 of the light emitted from the sample cell 10. The aperture plate 60 has a function of limiting stray light by the opening width of the opening 61. The opening 61 of the aperture plate 60 opens in front of the light receiving surface of the detector 70.

  The detector 70 receives the light collected from the imaging 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 the present embodiment, for example, a photodiode (PD) is employed, but an array detector such as a two-dimensional CMOS may be employed.

[Configuration of cell holder]
Next, the configuration of the cell holder 100 according to the present embodiment will be described with reference to FIG. FIG. 2 is a schematic diagram of an assembled state of the cell holder according to the present embodiment.

  As shown in FIG. 1, a cell holder 100 according to the present embodiment is a device for holding a sample cell 10 of a light scattering detection device 1. The cell holder 100 according to the present embodiment includes a pair of holders 110 and 210, a first flange 120, a second flange 130, and a third flange 220. Hereinafter, each component will be described.

  The pair of holders 110 and 210 hold both ends of the sample cell 10. The sample cell 10 of the present embodiment is a transparent cylindrical cell that holds a liquid sample in the internal flow path 12. This sample cell 10 is arranged along the vertical direction. Therefore, the cell holder 1 according to the present embodiment includes a pair of the lower holder 210 holding the lower end of the sample cell 10 and the upper holder 110 holding the upper end of the sample cell 10.

  The lower holder 210 is formed of, for example, a metal such as stainless steel. The third flange portion 220 is a ring-shaped protrusion that protrudes radially outward, and the lower boss portion 230 and the upper boss portion 240 are integrally formed. A plurality of bolt insertion holes 221 are formed in the third flange portion 220 at equal intervals along the circumferential direction.

  In the lower boss 230, a tube housing recess 231 and a fitting mounting recess 232 are formed vertically. The fitting mounting recess 232 has an inner diameter larger than that of the tube housing recess 231. The tube housing concave part 231 and the fitting mounting concave part 232 have the same vertical center axis and communicate with each other. The probe portion P of the tube T is mounted in the tube storage recess 231 via a fitting F. The fitting F is mounted in the fitting mounting recess 232 in a state where the probe portion P of the tube T is mounted in the tube storage recess 231.

  A cell holding portion 241 for accommodating the lower end of the sample cell 10 is formed in the upper boss portion 240. The cell holding part 241 is formed as a columnar concave part. The inner diameter of the cell holder 241 is formed slightly larger than the outer diameter of the sample cell 10. A ring groove 242 for mounting the O-ring OR is formed on the inner peripheral wall of the cell holding portion 241. The O-ring OR aligns the center axis of the flow channel 12 of the sample cell 10 with the center of the optical axis of the incident light, and moves the sample cell 10 from the surroundings so that the tube T matches the center axis of the flow channel 12. Energize. On the cell contact surface of the cell holding portion 241, a flow path 243 communicating with the tube housing recess 231 is formed. A packing PK is mounted between the cell contact surface of the cell holding unit 241 and the lower end surface of the sample cell 10.

  The third flange 220 is detachably fixed to the lower gantry 250. A cylindrical through-hole 251 is opened in the lower frame 250. A female screw portion 253 is formed around the through hole 251 at a position corresponding to the bolt insertion hole 221 of the third flange portion 220. The lower boss 230 is dropped into the through hole 251, and the third flange 220 is arranged on the lower gantry 250. By aligning the bolt insertion hole 221 of the third flange portion 220 with the female screw portion 253 of the lower frame 250 and fastening the bolt B, the third flange portion 220 is fixed on the lower frame 250.

  On the other hand, the upper holder 110 is formed in a double flange structure 140 of a first flange portion 120 holding the sample cell 10 and a second flange portion 130 holding a tube T connected to the sample cell 10. . The upper holder 110 is formed of, for example, a metal such as stainless steel, similarly to the lower holder 210.

  The first flange portion 120 has a first boss portion 121 that holds the sample cell 10 protruding at the lower center thereof. A cell holding part 122 for holding the upper end of the sample cell 10 is formed below the first boss part 121. The cell holding part 122 is a cylindrical concave part, and has an inner diameter slightly larger than the outer diameter of the sample cell 10. A ring groove 123 for mounting the O-ring OR is formed on the inner peripheral wall of the cell holding part 122. The O-ring OR aligns the center axis of the flow path 12 of the sample cell 10 with the center of the optical axis of the incident light, and moves the sample cell 10 from the periphery to match the center axis of the tube T with the center axis of the flow path 12. Energize. A through hole (not shown) is formed in the cell contact surface of the cell holding part 122. A packing PK is mounted between the cell contact surface of the cell holding unit 122 and the upper end surface of the sample cell 10.

  In the upper part of the first boss part 121, a concave part 125 for accommodating the second boss part 131 protruding below the second flange part 130 is formed. The lower part of the concave portion 125 is reduced in diameter, and the concave portion 125 and the cell holding portion 122 communicate with each other.

  As described above, the second boss portion 131 is formed to protrude below the center of the second flange portion 130. Inside the second boss portion 131, a tube housing recess 132 for housing the probe portion P of the tube T is formed. A channel 133 for inserting the tube T is opened in the bottom surface of the tube storage recess 132. The fitting F is arranged on the probe part P of the tube T.

  The outer diameter of the first flange 120 is set to be larger than the outer diameter of the second flange 130. The first flange portion 120 is provided with two rows of bolt insertion holes 127 and 128 at equal intervals along the circumferential direction. The outer bolt insertion hole 128 is a hole for detachably fixing the first flange portion 120 to the upper base 150. The inner bolt insertion hole 127 is a hole for detachably connecting the second flange portion 130 to the first flange portion 120.

  The first flange portion 120 is detachably fixed to the lower frame 150. A cylindrical through-hole 151 is opened in the lower gantry 150. Around the through hole 151, female screw portions 153 and 154 are formed at portions corresponding to the bolt insertion holes 127 and 128 of the first flange portion 120. The first boss 121 of the first flange 120 is dropped into the through hole 151, and the first flange 120 is disposed on the lower gantry 150. By aligning the bolt insertion hole 128 of the first flange 120 with the female screw 154 of the lower frame 150 and fastening the bolt B, the first flange 120 is fixed on the lower frame 150.

  On the other hand, a plurality of bolt insertion holes 135 are formed in the second flange portion 130 at equal intervals along the circumferential direction. The bolt insertion hole 135 of the second flange portion 130 is formed at a position corresponding to the bolt insertion hole 127 inside the first flange portion 120. The bolt B is fastened by aligning the bolt insertion hole 135 of the second flange portion 130, the bolt insertion hole 127 inside the first flange portion 120, and the female screw portion 153 of the lower base 150, and fastening the bolt B. The flange 130 is fixed on the first flange 120 and the lower gantry 150.

  When assembling the lower holder 210, the sample cell 10, the upper holder 110, the tube T, and the fitting F, the central axis of the channel 12 of the sample cell 10 and the optical axis center of the incident light coincide with each other. The center of the sample cell 10 and the optical system are adjusted so that the center axis of the sample cell 12 coincides with the center axis of the sample cell 12.

[Operation of light scattering detector and cell holder]
Next, the operation of the light scattering detection device 1 and the cell holder 100 according to the present embodiment will be described with reference to FIGS. As shown in FIG. 1, a liquid sample is passed through a channel 12 of a cylindrical sample cell 10. When the passage of the liquid sample is completed, visible light, which is coherent light, is emitted 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 in the flow path of the sample cell 10. When laser light is incident on a liquid sample, the light impinges on fine particles contained in the liquid sample and is scattered at 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 is generated as stray light at the cell interface of the sample cell 10. Since the slit plate 40 is provided on the incident side of the imaging optical system 50, reflected light (stray light) can be restricted by the plate portion of the slit plate 40. Further, since the aperture plate 60 further restricts stray light, scattered light required for analysis is received on the light receiving surface of the detector 70.

  In the light scattering detection device 1, the sample adheres to the inner surface of the channel 12 of the sample cell 10 every time the measurement is repeated, and the background signal increases. As described above, brushing cleaning is effective for removing the sample adhering to the inner surface of the flow channel 12. Since the inner diameter of the flow channel 133 in the cell holder 100 is as small as φ1.0 mm to φ1.5 mm, the flow channel 12 having the internal diameter of φ1.5 mm to φ2.0 mm cannot be brushed through the cell holder 100.

  As shown in FIG. 2, the cell holder 100 according to the present embodiment includes a first flange portion 120 in which the upper holder 110 holds the sample cell 10 and a second flange portion 130 in which the tube T connected to the sample cell 10 is held. , And a double flange structure 140. Therefore, when brushing and cleaning the inner surface of the flow channel 12 of the sample cell 10, the bolt B fastened to the second flange portion 130 is removed, and the second flange portion 130 is detached from the first flange portion 120. The second boss 131 of the second flange 130 is accommodated in the recess 125 of the first flange 120. A tube housing 135 is formed in the second boss 131 of the second flange 130, and the probe P of the tube T is housed in the tube housing 135.

  Therefore, as shown in FIGS. 2 and 3, when the second flange portion 130 is removed from the first flange portion 120, the probe portion P and the fitting F of the tube T can be removed together with the second flange portion 130.

  FIG. 3 is a schematic view of the disassembled state of the cell holder according to the present embodiment. As shown in FIG. 3, the sample cell 10 is held between the cell holder 241 of the lower holder 210 and the cell holder 122 of the first flange 120 of the upper holder 110. As described above, the inner diameter of the flow path 133 formed in the second boss 131 of the second flange 130 is φ1.0 mm to φ1.5 mm, but the second boss 131 is integrated with the second flange 130. Since it is formed, it does not exist in the disassembled state of FIG. 3 (see FIG. 2).

  Accordingly, the inner surface of the flow channel 12 of the sample cell 10 can be brushed and cleaned through the concave portion 125 of the first flange portion 120. Since the inner surface of the flow channel 12 can be brushed and washed without removing the sample cell 10 from the cell holder 100, it is not necessary to readjust the optical system again.

(Washing experiment)
A cleaning experiment was performed to confirm the operation and effect of the present embodiment. FIG. 4 is an explanatory diagram of a state before and after cleaning the inner surface of the flow channel of the sample cell in the present embodiment. As shown in FIG. 4, with the sample cell held in the cell holder according to the present embodiment, the detector output (mV) before and after cleaning was measured for each detector arrangement angle (θ) in the state of FIG. . At a low arrangement angle of less than 30 degrees, a slight decrease in the output of the detector was observed, but when the arrangement angle exceeded 30 degrees, the output of the detector coincided before and after cleaning, and a high cleaning effect could be confirmed. Was. In this manner, an experiment for confirming the cleaning state can be performed with the sample cell held in the cell holder.

  As described above, according to the cell holder 100 according to the present embodiment, the inner surface of the flow path of the sample cell can be cleaned well and efficiently without removing the sample cell.

  Further, according to the light scattering detection device 1 provided with the cell holder 100, when a deposit is generated in the flow path 12 of the sample cell 10 and the background signal increases, the second holding of the tube T in the holder 110 is performed. By simply removing the flange portion 130, the inner surface of the flow channel 12 can be brushed and cleaned. The inside of the sample cell 10 is kept airtight. Therefore, it is possible to perform the brushing cleaning in a state in which the solvent is enclosed, and it is possible to remove the deposit and reduce the background signal.

  At the time of cleaning, the cell holder 100 holding the sample cell 10 is fixed to the gantry 150, 250, so that the position of the sample cell 10 does not change, and it is unnecessary to readjust the optical system. Further, since the adjustment of the optical system is unnecessary, the cleaning effect can be confirmed by confirming the background light. Further, at the time of washing, a solution such as a surfactant that enhances the washing effect is sealed in the flow channel 12 of the sample cell 10, so that washing can be performed efficiently.

  The above embodiments are intended to facilitate understanding of the present invention, and are not intended to limit the present invention. Each element included in the embodiment and the arrangement, material, condition, shape, size, and the like of the embodiment are not limited to those illustrated but can be appropriately changed. It is also possible to partially replace or combine the configurations shown in different embodiments.

DESCRIPTION OF SYMBOLS 1 ... Light scattering detector 10 ... Sample cell 12 ... Flow path 20 ... Light source 50 ... Imaging optical system 70 ... Detector 100 ... Cell holder 110 ... Upper holder 120 ... First flange part 121 ... First boss part 130 ... Second Flange part 135 Tube recessed part 140 Double flange structure 210 Lower holder T Tube P Probe part PK Packing

Claims (5)

A cell holder for holding a sample cell of the light scattering detection device,
At least one of a pair of holders holding both end portions of the sample cell,
A first flange portion for holding the sample cell;
A second flange portion for holding a tube connected to the sample cell;
A cell holder for a light scattering detection device, wherein the cell holder is formed in a double flange structure.   The cell holder of the light scattering detection device according to claim 1, wherein the sample cell is arranged along a vertical direction, and an upper holder of the pair of holders is formed in the double flange structure. The first flange portion has a boss portion that holds the sample cell via a packing at a lower portion thereof, and has a tube housing concave portion that houses the tube held by the second flange portion at an upper portion thereof. ,
The cell holder of the light scattering detection device according to claim 1 or 2, wherein the second flange portion is detachably connected to the first flange portion.   The cell holder of the light scattering detection device according to claim 3, wherein the second flange portion has a tube housing recess for housing a probe portion of the tube. A light scattering detection device for detecting fine particles in a liquid sample,
A transparent sample cell for holding a liquid sample,
A light source for irradiating the sample cell with coherent light,
An imaging optical system that collects light scattered with different scattering angles from the sample cell to the surroundings,
A detector for receiving light collected from the imaging optical system,
At least,
A light scattering detection device, wherein the sample cell is held by the cell holder according to any one of claims 1 to 4.

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