JPS62235548A - Fluorescence absorption analysis method and device - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

[Detailed Description of the Invention] [Industrial Application Field] The present invention relates to a method and apparatus for fluorescence absorption analysis, particularly for rapidly measuring the fluorescence and absorption spectra of a sample rich in a single component or 4JI components. The present invention relates to a fluorescence absorption analysis method and apparatus suitable for.

[Conventional technology]

The spectrofluorometer needle commonly used for fluorescence measurement uses a white light source with a wide wavelength range, such as a 1,000 senon lamp, and uses a spectrometer to extract monochromatic light and apply it to the sample. It is irradiating. However, for this type of device,
Compared to laser light, the monochromaticity of the extracted light is inferior, and the light intensity is low, and when using optical fibers, the light collection efficiency is not good, so the sensitivity of fluorescence measurement is lower than that when using laser light. is also low.

As a result, fluorescence analysis using laser light is now becoming mainstream.

Among devices that use a laser as a light source, devices that can measure both fluorescence and absorption optical properties at the same time include JP-A-58-37545 and JP-A-58-37546.
As described in the publication, a dye laser is excited by an N 2 laser and the respective laser beams are used as excitation light for fluorescence measurement and irradiation light for absorption measurement using optical means. The feature is that two laser beams, the N2 laser and the dye V-laser, can be used. However, in this case, there are only two wavelengths that can be used at the same time, and in order to measure the absorption spectrum, it is necessary to convert the wavelength of one dye laser using a diffraction grating, or to replace the dye. The equipment is expensive. That is, in this Hinoki apparatus, no consideration was given to the quick measurement of absorption spectra.

[Problem that the invention seeks to solve]

The above-mentioned conventional technology has no problem in measuring fluorescence spectra, but in measuring absorption spectra, it does not take into account the need to quickly change the wavelength of the irradiated laser light, There was a problem in that it was not possible to measure absorption spectra that required rapid measurement, such as absorption spectra of substances in a certain state.

An object of the present invention is to provide an apparatus that can quickly and easily measure the fluorescence spectrum and absorption spectrum of a sample.
Ruru.

[Means for solving problems]', 7. The above purpose is to irradiate a fluorescent substance with excitation light to generate fluorescent 1,5' light, to irradiate this transmitted light to a sample liquid, and to detect the transmitted light that has passed through the sample liquid. This is achieved by calculating the absorbance of the substance to be measured from the spectrum and analyzing the concentration of the substance.

[Effect]

The light emitted from the laser is split into two directions by a rotating plate or beam splitter that has a reflective surface and a transmitting surface, and one is used as excitation light that is directly irradiated onto the sample and causes the sample to emit fluorescence. used. The nine fluorescence obtained as a result is guided to a spectrometer and a detector through a condensing lens and measured.

The other laser beam irradiates the dye, and the resulting light emission from the dye is irradiated onto the sample as light for measuring the absorption spectrum.The transmitted light is detected and the emission spectrum of the dye, which has been separately monitored, is measured. It is possible to obtain the absorption spectrum of a sample by performing calculation processing using this method.

〔Example〕

An embodiment of the present invention will be explained below with reference to FIG.

First, the overall configuration of the present invention will be described. The present invention is.

Excitation light source 1. Optical means for splitting the excitation light 20.

Colors with unique emission spectra! 10 and its container, an optically transparent dye cell 9; a sample 8 and its container, an optically transparent sample cell; A concave mirror 11, a mini 7-12 for condensing the emitted light 21 of the dye 10, a condenser lens 13 for converting the condensed light into parallel light, and a condenser for condensing the fluorescence 23 and transmitted light 22 from the sample 8. Concentrating lenses 14, 15. It consists of a spectroscope 17 that separates the fluorescence 23 and transmitted light 22 of the sample 8, a photodetector 18 that detects the separated light, and a data processing device 19 that processes measurement data.

Next, the characteristics and operations of individual components will be explained.

First, as the light source used for the excitation light source IK, a laser beam with high light intensity and excellent monochromaticity is suitable.

Strong light intensity increases fluorescence intensity, and excellent true color properties improve the discrimination between scattered light such as Raman light from water and fluorescence from the sample, allowing highly sensitive measurements. The laser used is N! It is possible to widen the selection range of excitation light wavelengths by selecting a wide variety of lasers depending on the sample to be measured, or by installing one dye laser at the same time.

Excitation light 20 from excitation light source 1 is transmitted to beam splitter 2
One part is sent to a spectrometer 171C to divide the intensity of the excitation light into 72 parts. The excitation light 2 transmitted through the beam splitter 2 is further divided into the sample self side and the dye cell 9 side by optical means for splitting the light. Fig. s1 shows a case where a rotary chopper 3, which rotates around the driving device #L4'lC and has a reflection plate and a gap through which light passes, is used as the optical means. In this case, the excitation light is sent alternately to the sample self side and the dye cell 9 side at a time interval of 1rlJmK, which is the shape and rotation speed of the chopper 3.
0 can be derived. Alternatively, if a beam splitter is used as this optical means, the excitation light 20 can be sent in both directions at the same time.

The nine excitation lights 20 are divided into two and used for fluorescence and absorption spectrum measurements, respectively. First, the absorption spectrum measurement system, which is a feature of the present invention, will be explained.

One of the divided excitation lights is sent to a dye cell 9 containing a dye 10.
is irradiated. The irradiated excitation light 20 is transmitted to the dye cell 11
The light travels back and forth between the concave reflecting mirrors 11 installed on both sides of the dye 10 until it is absorbed by the dye 10. In this way, the operation is
This can be achieved by irradiating the excitation light 20 from a hole slightly apart from the center of the concave reflecting mirror 11. This significantly improves the conversion efficiency. That is, the quantum yield of the dye 10 used is 90 or more.

It is possible to bring the conversion efficiency closer to that point. The light 21 emitted from the dye 10 by the excitation light 20 is emitted isotropically, so in order to efficiently collect the light, the paraboloid ξ2-12 and the condensing lens 13 are arranged as shown in the figure. The optical loss can be suppressed to as low as 1111υ. The sample 8 is irradiated with the light 21 having the wavelength range of the dye 101 obtained in this manner, and the light 22 transmitted through the sample 8 is sent to the spectroscope 17 and detected by the photodetector 18. On the other hand, the light 21 of 1 dye IO is parabolic, 9
A part of the light 4u) is taken out through the hole drilled in -12, guided by the reflecting mirror 5 to the spectrometer 17, and detected by the photodetector 18. By using a position-sensitive detector such as a two-dimensional photomultiplier tube as the photodetector 18 used here, the transmitted light 22 and the dye 1 are arranged so that the wavelength is on the X-axis side and the dye 1 is on the Y-axis side.
By separating the light emission 21 from 0 and 0, the spectra of each light can be measured simultaneously, and the information can be processed by the data processing device 19 to instantly obtain the absorption spectrum of the sample. As will be described later, in the present invention, a photodetector 1B using this position-sensitive detector detects excitation light 20, emission 21 of the dye 10, and transmitted light 22 of the sample.
, the fluorescence 23 of the sample can be measured spectroscopically at the same time, which makes the measurement bright.

The other excitation light 20 that is multi-divided by the chopper 3 is irradiated onto the sample 8 . At this time, the concave mirror 11 is arranged so as to sandwich the sample self, and as described above, the sample 8 is efficiently
excite. The fluorescence 23 of the sample 8 emitted at this time is #
& Optical lenses 14 and 15 collect the light at a solid angle as large as possible, the light is separated by a spectrometer 17, and a photodetector 18
As mentioned above, the spectrum is measured instantaneously. This measured spectrum is processed by the data processing device 19 together with the monitored excitation light 20, and the fluorescence relative intensity is determined.As described above, according to the present invention, the fluorescence and absorption spectra of the sample can be quickly and easily measured. It is possible to obtain

Next, Figure 2 shows the analytical targets that can be particularly effectively measured by the apparatus according to the present invention and the methods for measuring them. I will explain using

Analyzes that can be particularly effectively measured in the present invention include:
First, we measured the absorption spectrum of a sample of excited state J.
The other is simultaneous measurement of fluorescent and non-fluorescent substances.

First, the former will be explained.

Measuring the absorption spectrum of the cut substance in the excited state is effective for obtaining information about the electron orbit of the substance, but the usual method is to measure the absorption spectrum of the cut substance in the excited state. It takes time to measure the yield spectrum, and accurate data cannot always be obtained.

However, since the apparatus of the present invention can instantaneously measure absorption spectra, highly accurate measurements are possible.

The spectrum of the excitation light 20 has strong intensity and high monochromaticity, as shown in (a). The fluorescence spectrum obtained by irradiating the sample 8 with this light is Φ). On the other hand, (c) is the excitation light 2
The emission spectrum of dye 10 obtained by irradiating dye 10 with rhodamine, which is used in dye lasers, etc.
The spectrum of pigments such as coumarin is a broad spectrum as shown in (c). And (d) is the transmitted light obtained by irradiating the sample with the light of (c), and the P a7 isle changes due to absorption by the sample. This (c) and (d)
Ic(λ
>, Xa (λ), then according to the Lampert-Beer equation, the absorbance A(λ) of the sample is 1, λ = 10g(Ic(λ〆Ia(λ))
(1) λ: Determined by wavelength, as shown in (e).

Since the absorption spectrum from the excited state of the sample usually has a peak on the longer wavelength side than the fluorescence 22 of the sample, the dye 10 to be used may be selected to have an emission spectrum in that wavelength range.

On the other hand, in the case of the latter measurement of a sample containing a mixture of fluorescent and non-fluorescent substances, the method is the same as the former.

However, in this case, a dye 10 that can measure the absorption spectrum of a non-fluorescent substance may be selected.

As described above, according to the present invention, it is possible to measure an object to be analyzed, which has been difficult in the past.

Another embodiment of the invention is shown in FIG.

This example is an effective method for JIJ1'&, where the sample is shaken up so much that fluorescence cannot be detected from the side.

That is, the excitation light 20 split by the chopper 3 is irradiated onto the sample by a small excitation light reflection mirror 24 installed between the sample and the coming lens 15, and the irradiated surface is By measuring the fluorescence 23 of the sample emitted from the sample, the optical surface fluorescence of the sample can be measured, and therefore,
This method has the effect of being able to measure the fluorescence of a wood sample with an absorption amount.

Yet another embodiment of the invention is shown in FIG.

In this embodiment, excitation light 20 from an excitation light source 1 is transmitted through an optical fiber 24 to irradiate the dye 1O and the sample 8, and further,
By transmitting the transmitted light 22 and fluorescence 23 of the sample through the optical 7 eyeper 26 and the luminescence 21 of the dye 10 through the optical fiber 25 to the spectrometer 17, the analysis cell 6 can be installed at a location away from the light source and the measuring equipment. , has the effect that remote analysis is possible.

However, when an optical fiber is used as in this embodiment, it is necessary to take some measures to efficiently focus the fluorescence 23 of the sample onto the end face of the optical fiber 26. This is done by using multiple condensing lenses with different focal lengths in order to capture the fluorescence 23 of the sample at as large a solid angle as possible compared to the light intake angle (approximately 200 degrees) of the light 7 eyeper 26. be. In Fig. 4.

By installing another light-coming lens 15 on the optical fiber 26 side on the 14-ft sample self side of the condensing V-shaped lens with a short focal length, it is possible to reduce
This has the effect of making it possible to focus the fluorescence 23 with twice the sensitivity. “A further embodiment of the present invention is shown in FIG.

In this embodiment, the dye cell 9 has two layers.

By adding different dyes 10 and 10', the light emission 21 of two colors, 110 and 10', can be used for absorbance measurement at the same time. By multi-layering the dye cells 9 in this way, it is possible to obtain the light emission 21 of the dye 10 with a much wider band.

Yet another embodiment of the invention is shown in FIG.

This embodiment is characterized in that the sample cell and the dye cell 9 are in direct contact with each other. By doing so, it is possible to omit an optical system such as a condensing lens, resulting in a simpler device configuration.

Yet another embodiment of the invention is shown in FIG.

This embodiment has a distinctive feature in that an integrating sphere 27 is used in place of the reflecting mirror for cell recovery.

By doing so, there is an effect that the light collection efficiency can be improved to the maximum.

Colors used in this invention! In this method, a mixture of multiple dyes is used, or even a single component uses a dye that emits light in a very wide wavelength range (e.g., the entire visible light, or a wavelength range including the ultraviolet and infrared regions). This has the effect of making it possible to measure the absorption spectrum of any sample using only that dye.

〔Effect of the invention〕

According to the present invention, it is possible to perform absorption spectroscopy by irradiating a sample with emitted light having a unique spectrum obtained by irradiating a dye with excitation light from an excitation light source.
The effect is that absorption spectrum measurement and fluorescence spectrum measurement can be performed quickly and easily with one spectrometer and photodetector.

[Brief explanation of drawings]

Fig. 1 is a cross-sectional view of one embodiment of the present invention, Fig. 2 is a flowchart of the measured spectrum and data processing of the present invention, Figs. FIG. 5 is a sectional view of an embodiment using the optical fiber of the invention, and FIG. 5 is a longitudinal sectional view of an embodiment using a multilayer dye cell. FIG. 6 is a sectional view of a simpler embodiment of the present invention, and FIG. 7 is a sectional view of an embodiment using an integrating sphere in the present invention. 1... Excitation light source, 2... Beam splitter-13.
... Chopper, 4... Drive device, 5... Reflection mirror, 6... Analysis cell, 7... Sample cell, 8... Sample, 9... Pigment cell, 10... Pigment , 11... concave reflective mirror, 12°°. Parabolic reflecting mirror, 13, 14. 15... Condensing lens, 16-°° slit, 17... Spectrometer, 18...
- Photodetector, 19... Data processing device, 2o... Excitation light. 21... Luminescence of dye, 22... Light transmitted through sample, 23
°°. Fluorescence of sample, 24.258 26... light 7 Aipa. 27... Integrating sphere.

Claims (1)

[Claims] 1. From the absorption spectrum obtained by irradiating a fluorescent substance with excitation light to generate fluorescence, irradiating this fluorescence to a sample liquid, and detecting the transmitted light transmitted through the sample liquid. A patented fluorescence absorption analysis method that analyzes the concentration of a substance to be measured by determining the absorbance of the substance through arithmetic processing. 2. The fluorescence absorption analysis method according to claim 1, wherein the fluorescent substance is a fluorescent dye solution. 3. The fluorescence absorption analysis method according to claim 1 or 2, which uses fluorescence from a plurality of fluorescent substances. 4. The fluorescence absorption analysis method according to claim 1, 2 or 3, characterized in that a mixture of a plurality of fluorescent dye solutions is used. 5. (a) Excitation light source, (b) Dye cell containing a fluorescent substance that irradiates excitation light, (c) Sample cell that irradiates fluorescence, (d) Spectrometer for transmitted light, (e) Transmitted light spectrum and (f) a data processing device for calculating the absorption spectrum intensity of the sample from the spectrum intensity and the fluorescence spectrum intensity of the fluorescent substance. 6. The fluorescence absorption spectrometer according to claims 1 and 5, characterized in that excitation light from an excitation light source is transmitted to the dye via an optical fiber. 7. The fluorescence absorption spectrometer according to claim 5 or 6, characterized in that the fluorescence from the dye is transmitted to the sample through an optical fiber. 8. The fluorescence absorption spectrometer according to claim 5, 6, or 7, characterized in that a position-sensitive detector is used as the photodetector. 9. Claim 5, characterized in that means is provided for condensing fluorescence from the sample using a plurality of condensing lenses.
6. The fluorescence absorption spectrometer according to item 6, 7, or 8.