JPH0641916B2 - Spectrofluorometer - Google Patents

Description

Description: FIELD OF THE INVENTION The present invention relates to a spectrofluorometer, and more particularly, to obtain accurate measurement data by obtaining a device function that corrects measurement spectrum data over the entire measurement wavelength range. Spectrofluorometer capable of

[Background of the Invention]

An excitation light source for analyzing the sample, an excitation side optical system including an excitation side spectroscope for irradiating the sample with excitation light from this light source, and a fluorescence side for analyzing the fluorescence emitted from this sample In a spectrofluorometer equipped with a fluorescence-side optical system consisting of a spectroscope and a fluorescence detector, a conventional spectrum correction method is described in the literature "Fluorescence analysis" -Kodansha Scientific (P81-87). As shown, first, the device function of the optical system on the excitation side is obtained by measuring the measured excitation light amount of the light source using a photon meter and a standard sample (normally, high concentration Rhodamine B), and then There is an optical quantum meter method in which the device function (correction function) of the fluorescence side optical system is obtained by using the device function of the excitation side optical system based on the measured excitation light amount previously obtained using the optical quantum meter. Further, as shown in the above-mentioned document, using a tungsten lamp whose output is known as another method for obtaining the device function of the fluorescence side optical system, by measuring the light from this lamp with the fluorescence side optical system, There is a tungsten lamp method for obtaining the device function of the fluorescence side optical system by comparing the measured value with a theoretically obtained theoretical value.

However, according to the former method among the methods for obtaining the device function of these fluorescence side optical systems, the device function is 200 nm to
Only the function in the wavelength region of 600 nm was obtained, and the latter method could only obtain the device function in the wavelength region of 330 nm or more.

Therefore, in order to obtain the device function of the fluorescence side optical system over the entire measurement wavelength, it is necessary to obtain each device function by two methods. At this time, the measurement results of the two methods should be the same. As a light source used for the tungsten lamp method, a high-performance tungsten lamp that can obtain the same measurement result as that of the photon meter method in the wavelength range to be measured must be used.

[Object of the Invention]

An object of the present invention is to provide a continuous fluorescence-side optical system in the entire measurement wavelength without using a high-performance tungsten lamp as a light source of a tungsten lamp method for obtaining a device function of a fluorescence-side optical system of a spectrofluorometer. Find the device function,
Another object of the present invention is to provide a spectrofluorometer capable of correcting measured spectrum data.

[Outline of Invention]

The spectrofluorometer of the present invention irradiates a standard sample with excitation light from a light source prior to sample measurement, measures the amount of excitation light of the light source by a photon meter, and then excites the light source obtained using this photon meter. Based on the device function of the excitation side optical system from the amount of light, obtain the device function of the fluorescence side optical system from short wavelength to medium wavelength, also, by measuring the light from the light source of known output with the fluorescence side optical system , By obtaining the device function of the fluorescence side optical system from the middle wavelength to the long wavelength from the difference between the measured value and the theoretical value, these methods uniquely find the same device function for the same target optical system device. Regarding the medium wavelength region, with respect to the specific wavelength in the medium wavelength region, the device function of the fluorescence side optical system with a wavelength longer than the specific wavelength and from the medium wavelength to the long wavelength, and the short wavelength shorter than the specific wavelength. From wavelength to medium wavelength Using the device function of the fluorescence side optical system, so as to match these device functions at a specific wavelength, one or both of these device functions are corrected and calculated, and continuous from a short wavelength to a long wavelength. By obtaining the device function of the fluorescence side optical system, the measured spectrum data is corrected using this device function to obtain an accurate analysis result.

Example of Invention

 An embodiment of the present invention will be described with reference to the drawings.

FIG. 1 shows a principle diagram of an embodiment of the present invention. First, using a photon meter and a standard sample, obtain the measured excitation light intensity of the excitation light source,
Using the device function of the excitation-side optical system based on this, then by the photon meter method to obtain the device function of the fluorescence-side optical system from the device function of the excitation-side optical system based on the measured excitation light quantity, further by the tungsten lamp method A device function is obtained from the difference between the measured value of the light source and the theoretical value, and these two device functions are matched at 500 nm to obtain a continuous device function in the entire wavelength range.

As a method of making these device functions continuous, first, the wavelength characteristic of the device in the wavelength range of 200 nm to 600 nm is measured by the photon measurement method, and the device function of the fluorescence side optical system which is the reciprocal thereof is stored in the storage device. Next, when reading the device function of the fluorescence side optical system in the wavelength range of 500 nm or more by the tungsten lamp method into the storage device, make sure that the device function value of 500 mm in the photonmeter method and the device function value of 500 mm in the tungsten lamp method match. Then, the whole device function of the tungsten lamp method is multiplied by a coefficient and stored in the storage device. The device function thus obtained gives 2
Continuous spectrum correction is possible in the entire measurement wavelength range from 00 mm to 800 mm.

FIG. 2 shows the configuration of an embodiment of the spectrofluorimeter according to the present invention. In FIG. 2, the light emitted from the light source 1 is guided to the sample chamber 3 via the excitation side spectroscope 2 and the sample Is irradiated. The fluorescence emitted from the sample enters the detector 7 via the fluorescence side spectroscope 6. What is currently targeted is correction of the device function of the fluorescence-side optical system including the fluorescence-side spectroscope 6 and the detector 7. The sample chamber 3 has an optical quantum meter installation place 4, but a tungsten lamp installation place 5 exists separately from this. The wavelength characteristic data of the device and the actual measurement data are both R from the detector 7 via the A / D converter 9 and the bus line 12.
It is read into AM16.

The RAM 16 is independently provided with a device function storage unit 17 for the excitation side optical system, a device function storage unit 18 for the fluorescence side optical system by the photon meter method, and a device function storage unit 19 for the fluorescence side optical system by the tungsten lamp method. There is. The microprocessor unit (MPU) 14 corrects and calculates one or both of the device functions of these two fluorescence side optical systems based on a program stored in the ROM 15 to obtain a corrected device function. The function data is stored in the RAM 16, the measurement data of the sample is corrected based on the device function data of the excitation side optical system stored in the RAM 16, and the corrected spectrum is stored in the storage system 11 via the D / A converter 10. Record.

FIG. 3 is an example of the correction spectrum actually taken according to the above-mentioned embodiment. The corrected spectrum for a wide wavelength range is actually recorded. This data is a corrected spectrum taken for the purpose of calculating the quantum yield, and it is necessary to correct the bottom of the spectrum correctly. This is one of the examples in which the present invention is most effectively used.

〔The invention's effect〕

According to the spectrofluorimeter of the present invention, the tungsten lamp used in the tungsten lamp method has a long wavelength range of 3 to 3.
It is not necessary to use a high-performance one that can be used up to a short wavelength of around 30 nm, it is sufficient that it can output its output in the medium wavelength range or more, and the device function obtained from this tungsten lamp and an optical quantum meter Even if the device function obtained by the method does not match, there is an effect that a continuous device function can be obtained in the entire measurement wavelength range.

[Brief description of drawings]

FIG. 1 is a diagram for explaining the principle of the present invention, FIG. 2 is a configuration diagram of an embodiment of a spectrofluorophotometer according to the present invention, and FIG. 3 is obtained by the spectrofluorophotometer according to the present invention. It is a figure which shows an example of the corrected spectrum. 1 ... Light source, 2 ... Excitation-side spectrometer, 3 ... Sample chamber, 6 ...
... Fluorescence side spectroscope, 7 ... Detector, 8 ... Mechanical system control circuit, 9 ... A / D converter, 10 ... D / A converter, 14
... MPU, 15 ... ROM, 16 ... RAM.

Claims (2)

[Claims] 1. An excitation-side optical system having an excitation-side light source, an excitation-side spectroscope for irradiating a sample placed in a sample chamber with the excitation light from the excitation-light source, and analyzing fluorescence emitted from the sample. In the spectrofluorometer equipped with a fluorescence-side optical system having a fluorescence-side spectroscope and a fluorescence detector, a photon meter and a light source whose output is known are provided in the sample chamber, and the excitation light source obtained by the photon meter is provided. Based on the pumping light quantity, a device function of the pumping side optical system is obtained, the calculating means for storing in the first storage unit, the calculating means uses the optical quantum meter from the pumping light quantity of the pumping light source to the pumping side optical Based on the system device function,
Obtaining the device function of the fluorescence side optical system from the short wavelength to the medium wavelength, it is stored in the second storage unit, and the amount of light emitted from the light source of the known output is measured by the fluorescence side optical system, From the difference between the measured value and the theoretical value, to obtain the device function of the fluorescence side optical system from the medium wavelength to the long wavelength, is to be stored in the third storage unit, the arithmetic means is a specific wavelength in the intermediate wavelength. With respect to the device function of the fluorescence side optical system from the medium wavelength to the long wavelength having a wavelength longer than the specific wavelength, and the fluorescence side optical system from the short wavelength to the medium wavelength having a wavelength shorter than the specific wavelength. And the device function of
Either or both of these device functions are corrected so that the device function of the fluorescence side optical system from the short wavelength to the medium wavelength and the device function of the fluorescence side optical system from the long wavelength to the medium wavelength match. The device function of the fluorescence-side optical system that was calculated to obtain a continuous fluorescence-side optical system from a short wavelength to a long wavelength is stored in the fourth storage unit, and the device function of the fluorescence-side optical system stored in the fourth storage unit at the time of sample measurement. A spectrofluorometer, which corrects measured spectrum data based on 2. The spectrofluorophotometer according to claim 1, wherein
A spectrofluorometer, characterized in that a tungsten lamp is used as the light source whose output is known.