JP2018163006A - Spectrophotofluorometer - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a spectrophotofluorometer with which it is possible to detect the fluorescence of a sample in a sample container at an optimum position free of obstacles on the surface of the sample container in a passage path of light (optical path).SOLUTION: A spectrophotofluorometer 1, designed to measure a sample stored in a sample container 100, comprises: a position adjusting mechanism 20 for adjusting the irradiation position of excitation light from an excitation-side spectroscope 12 and the sample container 100 to appropriate positions; a data processing unit 30 for controlling a photometer unit 10 and analyzing a sample; and an operation unit 40 for performing input/output. The position adjusting mechanism 20 includes a sample installation unit 21 for placing the sample container 100 and holding it in place, and a drive unit 22 for driving the sample installation unit 21, and has an in-plane adjustment mechanism 23 for adjusting the in-plane position of the sample container 100, a height adjustment mechanism 24 for adjusting the height position, and a circumferential position adjustment mechanism 25 for adjusting the circumferential position. The maximum output of a detector 16, reached while performing position adjustment, is determined as an optimum position for measurement.SELECTED DRAWING: Figure 1

Description

  The present invention relates to a spectrofluorometer that measures a sample in a sample container in a nondestructive manner.

  A method for analyzing a sealing material or liquid sealed (contained) in a sample container is known (Patent Documents 1 to 3). Also, a spectrofluorometer using fluorescence is known from the viewpoint of high sensitivity and information content (Non-Patent Document 1).

  Patent Document 1 discloses that a specially shaped container is used by a method of nondestructively measuring the gas composition in a sealed container. Patent Document 2 discloses that a liquid sample is put in a test tube and the liquid sample is measured with an absorptiometer. Patent Document 3 discloses an apparatus for inspecting a liquid-filled product filled with liquids for defects in containers, contents, and accessories.

JP-A-9-127001 JP 2001-33387 A JP 2003-130805 A

Spectroscopical Society of Japan Series 3 “Fluorescence Measurement-Application of Biological Science”, pages 45-77, January 20, 1983

  The measurement and inspection methods shown in Patent Documents 1 to 3 are applied only to a predetermined container. In Patent Document 2, it is necessary to open the sample every time the measurement is performed, and the analysis accuracy may be lowered. . In addition, there is a problem that measurement obstacles such as labels, bar codes, and stamps attached to the sample containers cannot be avoided, which cannot be applied to sample containers having different shapes.

  An object of the present invention is to provide a spectrofluorometer capable of detecting fluorescence of a sample in a sample container at an optimal position without an obstacle on the surface of the sample container in a light passage (optical path).

  The spectrofluorophotometer of the present invention holds a light source, an excitation-side spectroscope that generates excitation light by splitting light from the light from the light source, and a sample container containing a sample to be measured, and irradiation of the excitation light. A position adjusting mechanism capable of adjusting the position of the sample container so that the position and the sample container are relatively in a predetermined positional relationship; and a detector for detecting fluorescence emitted from the sample.

  As one aspect of the spectrofluorometer of the present invention, for example, the position adjustment mechanism is an in-plane adjustment mechanism capable of adjusting the in-plane position of the sample container, and a height adjustment capable of adjusting the height position of the sample container. It includes at least one of a mechanism and a circumferential adjustment mechanism capable of adjusting the circumferential position by rotating the sample container.

  As one aspect of the spectrofluorometer of the present invention, for example, the position adjusting mechanism includes a rotary height adjusting mechanism that can adjust the height while simultaneously adjusting the circumferential position by rotating the sample container.

  As one aspect of the spectrofluorometer of the present invention, for example, the position adjusting mechanism can adjust the position of the sample container so that the excitation light and the center of the cross section of the sample container coincide.

  As one aspect of the spectrofluorometer of the present invention, for example, it has a control unit that calculates the intensity of fluorescence detected by the detector, and the control unit controls the position adjustment mechanism based on the intensity of fluorescence. Then, the sample container is positioned.

  The spectrofluorometer of the present invention detects scattered light or fluorescence at each installation position with a position adjustment mechanism against measurement disturbance factors such as different outlines of sample containers and markings and labels on the outer periphery of the sample containers. Thus, the sample container can be set at an optimum position for evaluating the fluorescence characteristics.

The block diagram which shows an example of the spectrofluorometer which concerns on this invention. An example of 1st Embodiment of the position adjustment mechanism of the spectrofluorometer which concerns on this invention is shown, (a) Front view, (b) Side view. An example of 2nd Embodiment of the position adjustment mechanism of the spectrofluorimeter which concerns on this invention is shown, (a) Front view, (b) Side view. The graph which shows the intensity | strength and time change by the difference in the height position of a sample container using 1st Embodiment of a position adjustment mechanism. The graph which shows the intensity | strength and time change by the difference in the height position of a sample container using 2nd Embodiment of a position adjustment mechanism. The graph which shows the intensity | strength of the fluorescence with respect to an excitation wavelength in the measurement of the spectrofluorometer which concerns on this invention. The graph which shows the intensity | strength of a specific fluorescence wavelength in the measurement of the spectrofluorometer which concerns on this invention. In the measurement of the spectrofluorometer according to the present invention, the specific corresponding to the excitation light of a specific wavelength is a graph showing the intensity of long fluorescence.

  Hereinafter, a preferred embodiment of a spectrofluorometer according to the present invention will be described in detail with reference to FIGS.

  FIG. 1 is a configuration block diagram showing an example of a spectrofluorometer according to the present invention. The configuration of the spectrofluorometer of this embodiment will be described in detail with reference to FIG.

  The spectrofluorometer 1 of the present embodiment is a device that measures a sample contained in a sample container 100. The spectrofluorometer 1 is disposed in the photometer unit 10 and the photometer unit 10, and measures a sample in the sample container 100. Are provided with a position adjusting mechanism 20 for adjusting the sample container 100 to an appropriate position, a data processing unit 30 for controlling the photometer unit 10 and analyzing the sample, and an operation unit 40 for inputting and outputting.

  The photometer unit 10 includes a light source 11, an excitation-side spectroscope 12 that splits light from the light source 11 to generate excitation light, a beam splitter 13 that splits light from the excitation-side spectrometer 12, and a beam splitter 13. A monitor detector 14 that measures the intensity of a part of the dispersed light, a fluorescence-side spectrometer 15 that separates fluorescence emitted from the sample into monochromatic light, and a detector that detects an electrical signal of monochromatic fluorescence (fluorescence) Detector 16), an excitation-side pulse motor 17 that drives the diffraction grating of the excitation-side spectrometer 12, and a fluorescence-side pulse motor 18 that drives the diffraction grating of the fluorescence-side spectrometer 15.

  The position adjustment mechanism 20 includes a sample placement unit 21 that places and holds a sample container 100 that contains a sample to be measured, and a drive unit 22 that drives the sample placement unit 21.

  The data processing unit 30 includes a computer 31, a control unit 32 arranged in the computer 31, and an A / D converter 33 that digitally converts fluorescence from the sample. The operation unit 40 includes an operation panel 41 for inputting an input signal necessary for processing of the computer 31, a display unit 42 for displaying various analysis results processed by the computer 31, an operation panel 41, the display unit 42, and the computer. And an interface 43 for connecting the terminal 31 to the terminal 31.

  2A and 2B show an example of the first embodiment of the position adjusting mechanism 20, wherein FIG. 2A is a front view and FIG. 2B is a side view. FIGS. 3A and 3B show an example of the second embodiment of the position adjusting mechanism 20, wherein FIG. 3A is a front view and FIG. 3B is a side view. The position adjustment mechanism 20 will be described in detail with reference to FIGS.

  In the first embodiment shown in FIG. 2, a sample placement unit 21 for placing and holding the sample container 100 is provided on the top of the position adjustment mechanism 20, and the sample placement unit 21 is for holding the bottom of the sample container 100. Has a substantially circular recess 21a and a holding portion 21b surrounding the recess 21a. In the present embodiment, the position adjustment mechanism 20 includes an in-plane adjustment mechanism 23, a height adjustment mechanism 24, and a circumferential direction adjustment mechanism 25, and the sample placement unit 21 can adjust the in-plane position of the sample container 100. It is fixed to the in-plane adjustment mechanism 23 (see the XY direction in FIG. 2A).

  In addition, the drive unit 22 of the position adjustment mechanism 20 is electrically connected to the control unit 32 of the computer 31 and has a height pulse motor 22a and a circumferential pulse motor 22b in the first embodiment. The in-plane adjustment mechanism 23 is fixed on the height adjustment mechanism 24, and the height adjustment mechanism 24 moves in the height direction (see Z direction in FIG. 2A) by driving the height pulse motor 22a. Thus, the height position of the sample container 100 can be adjusted. Further, the circumferential direction adjustment mechanism 25 is fixed on the height adjustment mechanism 24, and the circumferential direction adjustment mechanism 25 is rotated in the circumferential direction (see the θ direction in FIG. 2A) by driving the circumferential pulse motor 22b. The circumferential position of the sample container 100 can be adjusted.

  In the second embodiment shown in FIG. 3, the sample placement unit 21 and the in-plane adjustment mechanism 23 are the same as those in the first embodiment, but the position adjustment mechanism 20 is a height adjustment mechanism 24 and a circumferential direction adjustment mechanism 25. And a rotary height adjusting mechanism 26 combined with the above. Moreover, the drive part 22 is a pulse motor similarly to 1st Embodiment. The rotary height adjusting mechanism 26 is a mechanism that can simultaneously adjust the height while rotating the sample container 100 to adjust the circumferential position. For example, the sample container 100 can be extended and contracted by a combination of screws, and the sample container 100 can be rotated in a spiral shape. The drive mechanism is screwed in a spiral shape that moves up and down, but there are also pneumatic and hydraulic mechanisms, and there is no particular limitation.

  The position adjustment mechanism 20 described above is a mechanism that sets an optimal measurement site for the installation of the sample container 100. The sample container 100 is, for example, a vial bottle or an ampoule bottle, and a label or inscription indicating the sample content such as a solution contained in the sample container 100 is applied to the surface of the sample container 100. The position adjustment mechanism 20 of the present embodiment is an effective means for nondestructive inspection in order to accurately acquire the fluorescence specification of the sample with the sample container 100 unopened in various shapes. That is, the position adjustment mechanism 20 minimizes the influence of measurement errors in different shapes of the sample container 100, the loss of light amount due to labels and markings, and the influence of reproducibility degradation.

  Using the position adjustment mechanism 20, the irradiation position of the excitation light generated by the excitation-side spectroscope 12 and the sample container 100 are relatively in a predetermined positional relationship (for example, the position where the detection of the detector 16 is maximized). An example of the procedure for installing and adjusting the sample container 100 will be described.

  (1) The sealed sample container 100 is placed on the sample setting part 21 of the position adjusting mechanism 20. At that time, the holding part 21 b of the sample setting part 21 is in contact with the outer periphery of the sample container 100, and the sample container 100 is placed on the sample setting part 21 in a stable state. The sample container 100 is an ampoule bottle, a vial bottle, or the like that encloses (accommodates) a medicine or sample, and is basically a transparent sample container 100 so as to transmit excitation light and fluorescence. Not intended for.

  (2) Next, the sample container 100 is adjusted in the in-plane direction (XY direction) by the in-plane adjustment mechanism 23 so that the center of the bottom surface of the sample container 100 and the center of the optical axis coincide.

  (3) Then, after the excitation light mask 50 is inserted between the beam splitter 13 and the position adjusting mechanism 20, the sample container 100 is irradiated with white light of zero-order light (transmitted light). The zero-order light is irradiated, and position detection is performed using the scattered light. The 0th order light may be laser light or the like.

  (4) The sample placement unit 21 is moved to the Z-axis origin in the height direction (Z-axis direction) by the height adjustment mechanism 24.

  (5) The sample setting portion 21 is rotated in the circumferential direction (θ direction) by the circumferential direction adjustment mechanism 25 at the Z-axis origin, and the output of the detector 16 in the 0th-order light is measured.

  (6) The Z-axis is changed by the height adjusting mechanism 24, and the sample setting section 21 is rotated in the circumferential direction by the circumferential direction adjusting mechanism 25 to measure the output of the detector 16. In the second embodiment, the adjustments (4) to (6) are performed by simultaneously moving (for example, spiraling) the height position and the circumferential direction with the rotary height adjusting mechanism 26. For example, the number of times of Z-axis measurement is calculated based on the height (input value) of the sample container 100 and the movement distance (input value) in the Z-axis direction, and the number of times is implemented.

  The position where the maximum output value of the detector 16 becomes the optimum position for measuring the sample in the sample container 100 at the position in the height direction and the position in the circumferential direction, and the fluorescence generated from the 90-degree direction of the excitation light at that position. Can be measured in an optimum state. The position (optimum position) through which the excitation light passes through the sample most reliably is free of obstructions existing on the surface of the sample container 100, such as a label or an inscription, and the center of the optical axis of the excitation light and the center of the cross section of the sample container 100, In particular, this is the position where the bottom center part matches.

  In the above-described embodiment, the zero-order light has been described. However, when the excitation wavelength and fluorescence wavelength of the sample to be measured are known in advance, the position may be detected with the optimum excitation wavelength and fluorescence wavelength for the sample. When the excitation-side spectroscope 12 that cannot use the 0th-order light is used, monochromatic light with little sample absorption may be irradiated as excitation light, and the detector 16 may detect the wavelength of the same monochromatic light. And when the sample container 100 of the same shape can be prepared, you may narrow an optimal position by carrying out rough adjustment using a pure water or a fluorescent reagent. Furthermore, when using pure water sealed in the sample container 100, Raman scattering may be used for position detection.

  Although the sample installation part 21 of this embodiment has explained the example which has the substantially circular recessed part 21a and the holding | maintenance part 21b surrounding the recessed part 21a, what is necessary is just to be able to mount the sample container 100 stably, and substantially. The holding portion 21b is not limited to a circular shape, and may be a substantially elliptical shape or a substantially rectangular shape, and may be any holding portion 21b that can hold the outer periphery of the sample container 100. Holding, diaphragm shape holding, slide bar type holding, etc., and are not limited to this embodiment.

  As described above, the position adjustment mechanism 20 is driven to obtain the optimum position. However, the position adjustment may be automatic or manual. In the case of the automatic type, it is possible to control the position adjustment mechanism 20 based on the intensity of the fluorescence using the control unit 32 that calculates the intensity of the fluorescence detected by the detector 16.

  The height adjusting mechanism 24 has a mechanism such as a screw feed type, a rack and pinion type, and a jack type. In the case of an automatic type, a stepping motor can be used. The circumferential direction adjusting mechanism 25 includes a mechanism such as a screw feed type. In the case of an automatic type, a stepping motor can be used. There are various height adjusting mechanisms 24, circumferential direction adjusting mechanisms 25, and rotary height adjusting mechanisms 26, and the mechanism is not limited to the mechanism.

  The optimum position is detected by the detector 16 installed in the 90-degree direction of the excitation light. When measuring the transmittance, a transmission detector is installed in the direction of the excitation light and 180 degrees. The optimum position may be detected by the flow. In addition, a diffraction grating is mainly used for the excitation-side spectroscope 12 and the fluorescence-side spectroscope 15, and a photomultiplier is used for the detector 16. However, a silicon photodiode or photomultiplier is used for the transmission detector, and the fluorescence-side spectroscope 15 is used. It does not have to be installed.

  The excitation light mask 50 inserted between the beam splitter 13 and the position adjusting mechanism 20 is used for the purpose of reducing the excitation light flux in order to increase the position confirmation accuracy. Normal excitation light is about 10 mm in the horizontal direction and about 5 mm in the vertical direction so as to fit the size of the 10 mm square cell. The excitation light beam can be reduced by inserting the excitation light mask 50 between the excitation light emitting lens and the sample container 100. The size is desirably 1/5 or less of the diameter of the sample container 100 (for example, about 4 mm when the outer diameter of the sample container 100 is 20 mm). However, if the excitation light beam is too small, noise increases, so it may be better to set a lower limit (for example, about 2 mm).

  4 and 5 are graphs showing the results of examining the output of the detector 16 at each height in order to determine the optimum position for measuring the sample accommodated in the sample container 100. FIG. The vertical axis represents intensity (I), the horizontal axis represents time (s), and six height positions from Z1 to Z6 were selected.

  The sample container 100 was placed on the position adjustment mechanism 20 and rotated in the circumferential direction at each height position (Zn: n = 1 to 6), and the change in strength was graphed. FIG. 4 is a measurement by the position adjustment mechanism 20 in the first embodiment, and becomes a discontinuous graph placed at the change position of each height position Zn. FIG. 5 shows the position adjusting mechanism 20 in the second embodiment, and a continuous graph is obtained even at the change position of each height position Zn. Based on the results of the graphs shown in FIG. 4 and FIG. 5, the sample container 100 to be measured is at the height position of Z2 having the highest intensity at the peak circumferential position θ (in the example, around 180 degrees), It is understood that it is optimal that the measurement is made.

  With the above method, the sample container 100 performs the continuous stability evaluation in the nondestructive inspection such as the quality check of the sample and the discrimination of the pseudo sample at the optimum position in the measurement by the position adjusting mechanism 20 of the present embodiment. Is possible. It is also possible to perform additional studies or other tests using the same sample.

  Analysis is performed by the spectrofluorometer 1 of the present embodiment using the sample container 100 set at the optimum position by the position adjusting mechanism 20. An example of the analysis method will be described with reference to FIGS. 1 and 6 to 8.

  Continuous light emitted from a light source 11 such as a xenon lamp of the spectrofluorometer 1 is split as excitation light by an excitation-side spectroscope 12 and passes through a beam splitter 13 to a sample in a sample container 100 installed in a position adjusting mechanism 20. Irradiated. At this time, the excitation light partially divided by the beam splitter 13 is measured by the monitor detector 14 for the amount of light (light intensity), and the fluctuation of the light source 11 is corrected.

  The fluorescence emitted from the sample is split into monochromatic light by the fluorescence side spectroscope 15 and detected as an electrical signal corresponding to the intensity of the fluorescence by the detector 16. The fluorescence intensity signal is converted into digital data via the A / D converter 33, and is taken in as signal intensity by the computer 31, and the measurement result is displayed on the display unit 42.

  The spectrofluorometer 1 of this embodiment is a wavelength drive system, and the excitation side spectroscope 12 is placed at a target wavelength position by driving the excitation side pulse motor 17 via the control unit 32 according to a command from the computer 31. Set. Further, the fluorescence side spectroscope 15 is also set to the target wavelength position by driving the fluorescence side pulse motor 18 through the control unit 32 in accordance with an instruction from the computer 31.

  The excitation-side spectroscope 12 and the fluorescence-side spectroscope 15 use optical elements such as diffraction gratings and prisms, and are powered by the excitation-side pulse motor 17 and the fluorescence-side pulse motor 18, for example, by a mechanism such as a gear and a cam. Spectral scanning can be performed by rotating a diffraction grating or a prism.

  In general, in the excitation spectrum for measuring the fluorescence intensity when the excitation wavelength of the excitation light is changed with respect to the measurement sample in the sample container 100, the excitation wavelength is changed from the measurement start wavelength to the measurement end wavelength by the excitation side spectroscope 12. Then, the excitation light of each wavelength is irradiated to the measurement sample, the change in the fluorescence of the specific wavelength is detected by the detector 16 through the fluorescence side spectroscope 15 set to the fixed wavelength at that time, and the A / D converter 33 is Via the computer 31 as signal strength.

  On the display unit 42, a two-dimensional spectrum as shown in FIG. 6 of the excitation wavelength and the fluorescence intensity is displayed as a measurement result. The graph of FIG. 6 shows the fluorescence intensity when the excitation wavelength is changed at a specific fluorescence wavelength.

  In addition, the fluorescence spectrum for measuring the fluorescence intensity for each wavelength when the measurement sample in the sample container 100 is irradiated with excitation light having a fixed wavelength and the fluorescence wavelength is changed is the excitation-side spectrum set to the fixed wavelength. Excitation light from the detector 12 is irradiated onto the measurement sample, and the fluorescence at that time is changed from the measurement start wavelength to the measurement end wavelength by the fluorescence side spectroscope 15, and the change in fluorescence for each wavelength is detected by the detector 16. The signal intensity is taken into the computer 31 via the / D converter 33.

  On the display unit 42, a two-dimensional spectrum as shown in FIG. 7 of the excitation wavelength and the fluorescence intensity is displayed as a measurement result. The graph of FIG. 7 shows the result of detecting the intensity of fluorescence in the broad wavelength band where the excitation light has a specific wavelength.

  On the other hand, the time-varying spectrum in which the measurement sample in the sample container 100 is irradiated with excitation light having a fixed wavelength and the intensity of fluorescence having a fixed wavelength is measured every unit time is an excitation-side spectroscope 12 set to a fixed wavelength. The measurement sample is irradiated with the excitation light from the light, the wavelength of the fluorescence-side spectroscope 15 is fixed for the fluorescence generated at that time, the intensity change of the fluorescence for each time is detected by the detector 16, and the A / D converter 33 is passed through. Is taken into the computer 31 as signal intensity.

  On the display unit 42, a two-dimensional spectrum as shown in FIG. 8 of the excitation wavelength and the fluorescence intensity is displayed as a measurement result. The graph of FIG. 8 shows the result of detecting the intensity of fluorescence of a specific wavelength corresponding to excitation light of a specific wavelength. If there is no change in the positional relationship of the sample container 100 or decomposition of the sample, the pattern is substantially constant.

  In addition, this invention is not limited to embodiment mentioned above, A deformation | transformation, improvement, etc. are possible suitably. In addition, the material, shape, dimension, numerical value, form, number, arrangement location, and the like of each component in the above-described embodiment are arbitrary and are not limited as long as the present invention can be achieved.

  The spectrofluorometer according to the present invention can be applied to the field in which a sample container is measured at an optimal position when a sample in a sample container having a different shape and labeled or engraved is subjected to nondestructive inspection.

DESCRIPTION OF SYMBOLS 1 Spectrofluorometer 10 Photometer part 11 Light source 12 Excitation side spectroscope 13 Beam splitter 14 Monitor detector 15 Fluorescence side spectroscope 16 Detector 20 Position adjustment mechanism 21 Sample installation part 22 Drive part 23 In-plane adjustment mechanism 24 Height Adjustment mechanism 25 Circumferential adjustment mechanism 26 Rotary height adjustment mechanism 30 Data processing unit 31 Computer 32 Control unit 40 Operation unit 100 Sample container

Claims (5)

A light source;
An excitation-side spectroscope that generates excitation light by splitting light from the light source;
A position adjusting mechanism capable of holding a sample container containing a sample to be measured and adjusting the position of the sample container so that the irradiation position of the excitation light and the sample container are relatively in a predetermined positional relationship; ,
A detector for detecting fluorescence emitted from the sample;
A spectrofluorometer comprising: The spectrofluorometer according to claim 1, wherein
The position adjustment mechanism includes an in-plane adjustment mechanism capable of adjusting an in-plane position of the sample container, a height adjustment mechanism capable of adjusting a height position of the sample container, and a circumferential position by rotating the sample container. A spectrofluorometer including at least one of a circumferential direction adjustment mechanism capable of adjusting the angle. The spectrofluorometer according to claim 1, wherein
The spectrofluorometer, wherein the position adjustment mechanism includes a rotary height adjustment mechanism that can simultaneously adjust the height while rotating the sample container to adjust the circumferential position. The spectrofluorometer according to claim 1, wherein
The spectrofluorometer, wherein the position adjusting mechanism is capable of adjusting the position of the sample container so that the center of the optical axis of the excitation light coincides with the center of the cross section of the sample container. The spectrofluorometer according to claim 1, wherein
Having a controller that calculates the intensity of the fluorescence detected by the detector;
The said control part is a spectrofluorometer which controls the said position adjustment mechanism based on the said fluorescence intensity, and positions the said sample container.

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