JPH0219897B2 - - Google Patents

Description

[Detailed Description of the Invention] The present invention measures fluorescence and phosphorescence generated when sample components developed by electrophoresis or chromatography are excited with monochromatic excitation light. This invention relates to a photometer for measuring secondary light of developed components for analysis.

FIG. 1 is a perspective view of a conventional photometer for measuring secondary light. The sample in this case is a cylindrical transparent gel housed in a test tube 9, in which sample components are developed into a disk shape by electrophoresis, and this gel-like object to be measured is generally called a disk gel. I'm here. Light source 1 that generates high-intensity continuous wavelength light such as a xeno lamp
The light is transmitted through a commonly used excitation side spectrometer 2.
The light enters the spectrometer through the entrance slit 3. This incident light is reflected by a concave mirror (not shown), becomes a convergent light beam, enters the plane diffraction grating 13, and generates a spectrum in the horizontal direction including the output slit 4. Therefore, by rotating the plane diffraction grating 13 about the axis in the direction of the grating lines, monochromatic light of any wavelength can be emitted from the output slit 4.

Since the entrance slit 3 and the exit slit 4 of the excitation side spectrometer 2 are vertically long as shown in the figure, the output light beam from the exit slit 4 has a vertically long rectangular cross section. However, the sample components developed on the disc gel in the test tube 9 are distributed in a disc-shaped manner in which they are stacked vertically. Therefore,
The vertically elongated emitted light beam must be condensed by a horizontally long rectangular hole opened in the slit plate 11, and the disk gel must be irradiated with the light beam parallel to the sample components. The amount of light used at this time is a portion of the amount of light emitted from the output slit 4, and most of the light is blocked.

The test tube 9 is guided by a test tube guide 10, moved up and down by a test tube moving mechanism 12, and scanned with monochromatic light of a wavelength selected by the excitation side spectrometer 2. At this time, a plurality of expanded components made of different substances generate fluorescence and phosphorescence of various wavelengths, and the excitation light side spectrometer 2
The light is introduced into the spectrometer through the entrance slit 6 of the light-emitting spectrometer 5, which is installed on the same plane in a direction 90 degrees different from that of the light source. In general, fluorescence emitted by a substance has a wavelength unique to the substance that is different from the wavelength of the excitation light, so when the plane diffraction grating 14 is rotated, the wavelength distribution of the secondary light is determined by the detector 8. detected and displayed or recorded. The excitation-side spectrometer 2 and the emission-side spectrometer 5 are the same spectrometers as described above, but are installed in 90-degree different directions to prevent the influence of excitation light. Slit the image of 4 on plate 1
A lens formed on the sample component layer 1 and a lens formed on the entrance slit 6 are installed between them to effectively condense light.

The thus configured photometer for measuring secondary light moves the test tube 9 up and down and measures the amount of fluorescence and phosphorescence generated by the developed sample component, that is, the concentration of the sample component. In particular, sample components that generate secondary light have higher sensitivity than other absorbance measurement methods, and trace analysis is also possible. Further, it has the advantage that it becomes possible to identify the sample component substances by measuring the wavelength distribution of the secondary light.

However, as mentioned above, the excitation light obtained from the excitation side spectrometer 2 is blocked by the slit plate 11, and only a portion of it is utilized. If the horizontally elongated hole of the slit plate 11 is enlarged in the vertical direction, the amount of light used will increase;
Since a wide beam of light scans the spread layer of sample components, the resolution of the components decreases and broad peaks are recorded, which is undesirable. In addition, in the case of a small amount such as a biological sample, the amount of developed components is also small, so it is necessary to irradiate the object with strong excitation light, so it has been desired to irradiate the object to be measured with a narrow and powerful excitation light.

Furthermore, improvements similar to those described above are desired in the light-emitting side spectrometer 5 as well. That is, the component layer that generates fluorescence in the test tube 9 is oriented horizontally, and the secondary light image focused by a lens (not shown) is oriented horizontally at right angles to the entrance slit 6. It is occurring in Therefore, the amount of secondary light introduced into the light-emitting spectrometer 5 and utilized here is extremely small, and there is a drawback that it is impossible to measure a small amount of secondary light.

FIG. 2 is a perspective view showing another example of a conventional photometer for measuring secondary light; in this case, the chromato plate 18
This is a device that measures the amount of sample components spread on the top. The same parts as in FIG. 1 are given the same reference numerals, but the difference from FIG. The point is that the image of the exit slit 4 is formed into a horizontally elongated image using a complicated reflection optical system. In this way, the horizontally elongated excitation light beam scans in alignment with the length direction of the developed sample component, so that the excitation effect is improved. Further, since the developed sample component images are formed so as to coincide with each other on the incident slit of the light-emitting side spectrometer 5, the secondary light emitted by the sample components can be efficiently introduced into the light-emitting side spectrometer 5.

The reflective optical system is composed of a plane mirror 15 and toroid mirrors 16 and 17. This toroid mirror 16 forms an image of the output slit 4 on the back surface of the microplate 18 as shown by the broken line, and the toroid mirror 17 focuses the secondary light generated by the developed component that matches the broken line of the chromato plate 18. An image is formed on the entrance slit 6. Note that when the exit slit 4 of the excitation side spectrometer 2 is installed so as to face the entrance slit 6 of the emission side spectrometer 5, the plane mirror 15 can be omitted.

The photometer for secondary light measurement using the reflective optical system configured in this way can rationally irradiate the developed sample components of the plate-shaped object 18 with excitation light and efficiently measure and detect the secondary light. However, since a plurality of reflecting mirrors must be installed, the configuration is complicated, and in particular, it is expensive because two toroid mirrors are used. Furthermore, since a plurality of surface reflecting mirrors are used, the reflectance is likely to decrease due to dust that falls when the chromato plate 18 is replaced, steam generated from the chromato plate 18, and the like. In this case, since three reflecting mirrors are used, there is a drawback that the effect appears as the cube of the reflectance.

That is, conventional photometers for measuring secondary light have low secondary light generation efficiency and detection efficiency, and attempts to avoid this drawback would require a complicated reflective optical system, which would be expensive and result in low reflectance. It has disadvantages such as being easy to change. In addition, in order to use it exclusively for phosphorescence measurement, the excitation light of the secondary light optical system is intermittent, and the phosphorescence is measured while the excitation light is blocked. The drawbacks are also common to phosphorescence measurements.

The purpose of the present invention is to provide a secondary light side photometer for developing components that can be easily used for measuring phosphorescence and that can be used to measure phosphorescence with high sensitivity over a long period of time with a simple configuration. , the object to be measured consisting of the chromatogram obtained by developing the sample components is irradiated with excitation light from the excitation side spectrometer, and the object to be measured is moved in the direction in which the sample components are developed, so that the sample components are generated. In a photometer for measuring secondary light of an expanded component, in which secondary light is spectrally detected by an emission side spectrometer, the spectral dispersion direction of the excitation side spectrometer and the emission side spectrometer is the same as the moving direction of the object to be measured. The excitation side optical axis and the longitudinal axis of the excitation beam cross section, the secondary light side optical axis and the secondary beam cross section, and the layer of the sample component exist in the same plane. and having a long entrance slit and an exit slit in a direction perpendicular to the spectral dispersion direction, and between the exit slit of the excitation side spectrometer and the object to be measured and between the entrance slit of the emission side spectrometer. a light condensing member installed between the object to be measured, a means for continuously moving each layer of the sample component of the object to be measured in a direction perpendicular to the plane, and the exit slit of the excitation side spectrometer. and a stopper made of a rotating body, which intermittents the excitation light and makes it possible to measure only the phosphorescence emitted by the sample component when the excitation light is interrupted.

FIG. 3 is a perspective explanatory view of one embodiment of the present invention, showing a sample developed on a chromato plate as a sample to be measured. In this figure, Figures 1 and 2
The same parts as in the figure are given the same reference numerals, and 19 is a plate-shaped holder that locks the chromato plate 18 at the four corners, and 20 is a plate-shaped holder that has a notch in a part and is attached to a rotation axis parallel to the excitation optical axis. Choppers 21a and 21b, which are attached and rotated so that excitation light passes through a notch and are blocked at other parts, are a slit plate 11, an excitation side spectrometer 2, and an emission side spectrometer 5, respectively. 22 is a slider to which a holder 19 is attached and which is guided by two guides 26a and 26b by an endless thread 25 wound around pulleys 23 and 24 and moves up and down; 27 indicates sample components on the chromato plate 18. Also, 2
8 and 29 are the entrance slit and exit slit of the excitation side spectrometer 2, respectively, 30 and 31 are the entrance slit and the exit slit of the emission side spectrometer 5, respectively, which are horizontally long;
These are the planar diffraction gratings of the excitation side spectrometer 2 and the emission side spectrometer, respectively, which have been rotated 90 degrees from those shown in FIG.

This photometer uses spectroscopes 2 and 5 of vertical dispersion type, and focuses the light so that an image of an output slit 29 is formed on a slit of a slit plate 11 with a lens 21a. The horizontally elongated linear monochromatic light beam passing through the slit illuminates the chromato plate 18 that moves up and down in the vicinity of the slit plate 11. Since the sample components 27 are spread vertically on the chromato plate 18, when the chromato plate 18 is moved up and down, the excitation light sequentially scans the sample components 27 to generate fluorescence and phosphorescence. The secondary light generated by the sample component 27 is focused by the lens 21b, is introduced into the spectrometer through the entrance slit 30 of the light-emitting spectrometer 5, is spectrally separated, and is detected by the detector 8.

Note that in this photometer, the entrance slits 28, 3
0 and the exit slits 29, 31 are all horizontally long, and the ruled lines of the plane diffraction gratings 32, 33 and their rotation axes are horizontal, so the spectral dispersion directions of both spectrometers are the same as each other. They are distributed in the vertical direction including the emission slits 29 and 31. Generally, it is reasonable to provide a spectrometer with an entrance slit and an exit slit that are horizontally elongated in the spectral dispersion direction in order to increase the amount of output light. The component 28 layer can be excited with a large amount of excitation light, and the secondary light generated by the sample component 28 layer can be introduced most efficiently. Therefore, the concentration of sample components can be detected with high sensitivity, and even trace components can be detected. In addition, when reducing the input and output slit widths of both spectrometers and the vertical direction of the hole in the slit plate 11, the wavelength purity of the excitation light and detection light is improved, and the vertical width of the light beam irradiating the sample component layer is reduced. Since it is thin, it is effective for improving measurement accuracy, such as being able to record a concentration curve with high resolution of sample components.

As described above, since the excitation side spectrometer and the emission side spectrometer are configured to be rotated by 90 degrees from the conventional configuration, measurement accuracy can be easily improved.

The above is a case where the excitation light simultaneously photometrically detects the secondary light generated by the sample components, but some sample components generate strong phosphorescence, and it is better to measure only the phosphorescence. may be effective in identifying The chopper 20 shown in FIG. 3 is used for phosphorescence measurement, and the chopper 20 has a notch formed in a part of a disc and is rotated by attaching it to a rotating shaft parallel to the excitation optical axis. Therefore, the excitation light passes through the notch and is blocked at other parts. In this way, it becomes possible to photometer only phosphorescence when excitation light is cut off. That is, by synchronizing the rotation of the chopper 20 with the gating for the output signal of the detector 8, and opening the gate when the chopper 20 blocks the excitation light, the phosphorescence emitted by the sample components can be separated from the fluorescent light and scattered light. It is measured by

Since the photometer constructed in this manner does not use a plurality of mirrors as shown in FIG. 2, the reflectance does not decrease during use and the sensitivity does not decrease. Further, the configuration of the chopper 20 is relatively easy to install, and phosphorescence measurement can be easily performed. The longitudinal direction of the exit slit 29 of the excitation spectrometer 2 and the input slit 30 of the emission spectrometer 5 coincide with the longitudinal direction of the expanded component of the object to be measured, so that the sample component is excited with the largest amount of excitation light, and its secondary It becomes possible to introduce light into the most effective emission spectrometer.

The photometer for measuring secondary light of this embodiment has the effect of being able to distinguish between fluorescence and phosphorescence and measure them with high sensitivity for a long period of time without using a complicated reflective optical system.

The photometer for secondary light measurement of developed components of the present invention has the advantage of being able to measure the concentration of sample components with high sensitivity and precision over a long period of time with a relatively simple configuration, and being easily usable for phosphorescence measurements. It has

[Brief explanation of drawings]

Figure 1 is a perspective view of a conventional photometer for measuring secondary light.
FIG. 2 is a perspective view of another conventional photometer for measuring secondary light, and FIG. 3 is an explanatory perspective view of a photometer for measuring secondary light, which is an embodiment of the present invention. 1...Light source, 2...Excitation side spectrometer, 5...Emission side spectrometer, 8...Detector, 11...Slit plate,
18...Chromato plate, 19...Holder,
20...Chopper, 21...Lens, 22...
Slider, 23, 24...Pulley, 25...
Thread, 26...Guide, 27...Sample component, 28,
30...Incidence slit, 29, 31...Output slit, 32, 33...Plane diffraction grating.

Claims (1)

[Claims] 1 Irradiate the object to be measured consisting of a chromatogram obtained by developing the sample component with excitation light from the excitation side spectrometer, and move the object to be measured in the direction in which the sample component is developed, so that the sample component is generated. In a photometer for measuring secondary light of an expanded component, in which secondary light is spectrally detected by an emission side spectrometer, the spectral dispersion direction of the excitation side spectrometer and the emission side spectrometer is the same as the moving direction of the object to be measured. The excitation side optical axis and the longitudinal axis of the excitation beam cross section, the secondary light side optical axis and the secondary beam cross section, and the layer of the sample component exist in the same plane. death,
a long entrance slit and an exit slit in a direction perpendicular to the spectral dispersion direction, and between the exit slit of the excitation side spectrometer and the object to be measured, and between the entrance slit of the emission side spectrometer and the a light condensing member installed between the object to be measured, a means for continuously moving each layer of the sample component of the object to be measured in a direction perpendicular to the plane, and the exit slit of the excitation side spectrometer; A development characterized in that a stopper is provided between the object to be measured and a rotating body that intermittents the excitation light and makes it possible to measure only the phosphorescence emitted by the sample component when the excitation light is interrupted. Photometer for secondary light measurement of components.