阅读说明：本技术 荧光分光光度计、分光测定方法以及荧光分光光度计用控制软件 (Fluorescence spectrophotometer, spectrometry method, and control software for fluorescence spectrophotometer ) 是由 渡边康之 于 2017-04-28 设计创作，主要内容包括：荧光分光光度计(1)具备：光源部(11),其能够向试样配置部(12)发出多个波长的光；检测部(13),其对来自试样配置部(12)的光中的规定波长范围内的光的强度进行测定；激发波长输入部(30),其用于受理多个激发波长的输入；测定波长范围决定部(221),其与所述多个激发波长对应地决定与各激发波长对应的透射光测定波长范围和发光测定波长范围；空白测定执行部(222),其接收空白测定的执行指示,来使光源部(11)按顺序发出所述多个激发波长的光,并测定穿过了试样配置部(12)的透射光测定波长范围内的光的强度；以及实际测定执行部(223),其接收实际测定的执行指示,来使光源部(11)按顺序发出多个激发波长的光,并测定穿过了试样配置部(12)的透射光测定波长范围内的光的强度和从所述试样配置部发出的发光测定波长范围内的光的强度。(a fluorescence spectrophotometer (1) is provided with: a light source unit (11) that can emit light of a plurality of wavelengths to the sample arrangement unit (12); a detection unit (13) that measures the intensity of light within a predetermined wavelength range from among the light from the sample arrangement unit (12); an excitation wavelength input unit (30) for receiving input of a plurality of excitation wavelengths; a measurement wavelength range determination unit (221) that determines a transmission light measurement wavelength range and a light emission measurement wavelength range corresponding to each excitation wavelength, in accordance with the plurality of excitation wavelengths; a blank measurement execution unit (222) that receives an instruction to execute blank measurement, causes the light source unit (11) to emit light of the plurality of excitation wavelengths in order, and measures the intensity of light within the measurement wavelength range of transmitted light that has passed through the sample arrangement unit (12); and an actual measurement execution unit (223) that receives an instruction to execute actual measurement, causes the light source unit (11) to emit light of a plurality of excitation wavelengths in order, and measures the intensity of light in the measurement wavelength range of transmitted light that has passed through the sample arrangement unit (12) and the intensity of light in the measurement wavelength range of emitted light from the sample arrangement unit.)

1. A fluorescence spectrophotometer is characterized by comprising:

a) A sample arrangement unit for arranging a sample;

b) A light source unit capable of emitting light of a plurality of wavelengths to the sample arrangement unit;

c) A detection unit that measures the intensity of light in a predetermined wavelength range among the light from the sample arrangement unit;

d) An excitation wavelength input unit for receiving input of a plurality of excitation wavelengths, which are a part or all of the plurality of wavelengths, by a user;

e) A measurement wavelength range determination unit that determines a transmission light measurement wavelength range and a light emission measurement wavelength range corresponding to each excitation wavelength according to a predetermined rule, in accordance with the input excitation wavelengths;

f) A blank measurement execution unit that receives a blank measurement execution instruction from a user, and controls the light source unit and the detection unit to execute the following blank measurement: causing the light source unit to emit light of the plurality of excitation wavelengths in order, and measuring the intensity of light in the wavelength range of the transmitted light passing through the sample arrangement unit for each light of the excitation wavelength; and

g) An actual measurement execution unit that receives an instruction to execute an actual measurement by a user, and controls the light source unit and the detection unit to execute the actual measurement as follows: the light source unit is caused to emit light of the plurality of excitation wavelengths in order, and the intensity of light in the transmitted light measurement wavelength range that has passed through the sample arrangement unit and the intensity of light in the emission measurement wavelength range emitted from the sample arrangement unit are measured for each excitation wavelength of light.

2. The spectrofluorometer of claim 1,

The transmitted light measurement wavelength range is determined with a wavelength shorter than each of the excitation wavelengths by a predetermined wavelength as a lower limit wavelength and a wavelength longer than each of the excitation wavelengths by a predetermined wavelength as an upper limit wavelength, and a range of a predetermined wavelength width with the upper limit wavelength of the transmitted light measurement wavelength range as a lower limit wavelength is determined as a light emission measurement wavelength range.

3. The spectrofluorometer of claim 1,

A first wavelength, a second wavelength and a third wavelength are inputted by a user, a transmitted light measurement wavelength range is determined with a wavelength shorter than each of the excitation wavelengths by the first wavelength as a lower limit wavelength and a wavelength longer than each of the excitation wavelengths by the first wavelength as an upper limit wavelength, and a light emission measurement wavelength range is determined with a wavelength longer than each of the excitation wavelengths by the second wavelength as a lower limit wavelength and a third wavelength as an upper limit wavelength.

4. The spectrofluorometer of claim 1,

the blank measurement execution section further measures the intensity of light within the emission measurement wavelength range emitted from the sample arrangement section.

5. The spectrofluorometer according to claim 1, further comprising:

h) And a quantum efficiency calculation unit that obtains an external quantum efficiency and/or an internal quantum efficiency based on the transmitted light intensity and the emission intensity obtained by the blank measurement and the actual measurement.

6. The spectrofluorometer according to claim 5, further comprising:

i) And an analysis result display unit that displays the external quantum efficiency and/or the internal quantum efficiency at the plurality of excitation wavelengths in one direction so that the values of the external quantum efficiency and/or the internal quantum efficiency at the plurality of excitation wavelengths can be visually recognized.

7. The spectrofluorometer according to claim 1, further comprising:

j) and a spectrum creation unit that creates and displays a spectrum based on the transmitted light intensity and the emission intensity obtained by the blank measurement and the actual measurement.

8. a spectroscopic measurement method using a fluorescence spectrophotometer having a light source unit, a sample arrangement unit, and a detection unit,

a) Receiving input of a plurality of excitation wavelengths by a user;

b) determining a transmitted light measurement wavelength range and a light emission measurement wavelength range corresponding to each excitation wavelength in accordance with a predetermined rule in correspondence with the plurality of excitation wavelengths;

c) The following blank measurements were performed: causing the light source unit to emit light of the plurality of excitation wavelengths in order, and measuring the intensity of light in the wavelength range of the transmitted light passing through the sample arrangement unit for each light of the excitation wavelength; and

d) The following actual measurements were performed: the light source unit is caused to emit light of the plurality of excitation wavelengths in order, and the intensity of light in the transmitted light measurement wavelength range that has passed through the sample arrangement unit and the intensity of light in the emission wavelength measurement range that has been emitted from the sample arrangement unit are measured for each excitation wavelength of light.

9. Control software for a spectrofluorometer, characterized in that a computer communicably connected to a spectrofluorometer having a light source unit, a sample arrangement unit, and a detection unit is operated as:

a) an excitation wavelength input unit for receiving input of a plurality of excitation wavelengths by a user;

b) A measurement wavelength range determination unit that determines a transmission light measurement wavelength range and a light emission measurement wavelength range corresponding to each excitation wavelength in accordance with a predetermined rule in accordance with the plurality of excitation wavelengths;

c) A blank measurement execution unit that receives a blank measurement execution instruction from a user, and controls the light source unit and the detection unit to execute the following blank measurement: causing the light source unit to emit light of the plurality of excitation wavelengths in order, and measuring the intensity of light in the wavelength range of the transmitted light passing through the sample arrangement unit for each light of the excitation wavelength; and

d) An actual measurement execution unit that receives an instruction to execute an actual measurement by a user, and controls the light source unit and the detection unit to execute the actual measurement as follows: the light source unit is caused to emit light of the plurality of excitation wavelengths in order, and the intensity of light in the transmitted light measurement wavelength range that has passed through the sample arrangement unit and the intensity of light in the emission measurement wavelength range emitted from the sample arrangement unit are measured for each excitation wavelength of light.

Technical Field

The present invention relates to a fluorescence spectrophotometer, a spectrometry method, and control software for a fluorescence spectrophotometer. In particular, the present invention relates to a fluorescence spectrophotometer, a spectroscopic measurement method, and control software for a fluorescence spectrophotometer that can be preferably used when measurement is performed using excitation light of different multiple wavelengths.

Background

A molecular structure is designed for a functional substance such as an organic EL light emitting device, a photocatalyst, a molecular sensor using photoreaction, and the like to absorb light of a specific wavelength to achieve a target function. Specifically, the molecular structure is designed to have an electronic state in which light of a specific wavelength is absorbed to transition to an excited state, and fluorescence or phosphorescence of a specific wavelength is released to return to a ground state. The ratio of the number of photons of light emitted from a sample as a functional substance to the number of photons of light irradiated to the sample is referred to as the external quantum efficiency. The ratio of the number of photons of light emitted from a sample to the number of photons of light absorbed by the sample is referred to as the internal quantum efficiency. The external quantum efficiency and the internal quantum efficiency are used as an index for evaluating a functional substance.

The external quantum efficiency and the internal quantum efficiency are obtained by measuring the intensity of fluorescence or phosphorescence emitted from a sample by irradiating excitation light of a specific wavelength using a fluorescence spectrophotometer. The fluorescence spectrophotometer includes an excitation light source unit having a light source and a spectroscopic unit, an integrating sphere for collecting fluorescence or phosphorescence emitted from a sample, and a detection unit having a spectroscopic unit and a detector. When the external quantum efficiency and the internal quantum efficiency are obtained by measurement using a fluorescence spectrophotometer, blank (blank) measurement is first performed. In the blank measurement, for example, a cuvette (cuvette cell) in which only a solvent (blank sample) for dissolving an actual sample is enclosed is irradiated with excitation light, and the intensity (intensity a) of light (transmitted light) passing through the cuvette is measured. Next, a cuvette in which a real sample solution obtained by dissolving a real sample in the solvent is enclosed is irradiated with excitation light, and the intensity (intensity B) of light (transmitted light) passing through the cuvette is measured. In addition, the intensity (intensity C) of fluorescence or phosphorescence emitted from the actual sample solution is also measured. After the measurement, the numbers of photons A to C were obtained from the intensities A to C, respectively. The external quantum efficiency is obtained from the ratio of the photon number C to the photon number a, and the internal quantum efficiency is obtained from the ratio of the photon number C to the photon number a-B obtained by subtracting the photon number B from the photon number a (for example, patent document 1).

The fluorescence spectrophotometer is operated in many cases by receiving an instruction from dedicated control software. When the user starts the control software and instructs to start measurement, a screen for inputting measurement conditions is displayed. When the user inputs measurement conditions such as the wavelength of the excitation light, the measurement wavelength range of the transmitted light, and the measurement wavelength range of the fluorescence (or phosphorescence), the user is then prompted to place the cuvette in which the blank sample is enclosed at a predetermined position in the spectrofluorometer. When the user has set the cuvette and instructs the start of blank measurement, the control software causes the excitation light source section of the spectrophotometer to irradiate the blank sample in the cuvette with excitation light of a wavelength specified by the user, and measures the intensity of transmitted light that has passed through the blank sample in the wavelength range input by the user in the detection section. When the measurement of the blank sample is finished, the user is urged to place the cuvette in which the actual sample solution is enclosed. When the user has set the cuvette and instructs the start of measurement, the control software irradiates the excitation light of a wavelength specified by the user to the actual sample solution in the cuvette, and measures the intensity of transmitted light passing through the actual sample solution and the intensity of fluorescence or phosphorescence emitted from the actual sample solution in the wavelength range input by the user, respectively.

Disclosure of Invention

problems to be solved by the invention

In recent years, research into functional substances having a plurality of functions has been carried out. That is, a substance that emits fluorescence or phosphorescence with respect to light of different wavelengths is being studied. In such studies, the above-described blank measurement and actual measurement were performed for excitation lights of different multiple wavelengths. In addition, there are the following cases: the above-described blank measurement and actual measurement were also performed at a plurality of wavelengths for a single-function functional substance to search for an optimum excitation light wavelength that maximizes the external quantum efficiency or internal quantum efficiency.

most of the cuvettes used in spectrofluorometers are made of glass or plastic, and the light absorption characteristics are slightly different among individuals. Therefore, if different cuvettes are used for the blank measurement and the actual measurement, a difference occurs in the amount of light absorption of the cuvettes between the blank measurement and the actual measurement, and the measurement accuracy is lowered. Therefore, in order to perform measurement with high accuracy, it is necessary to use the same cuvette for both blank measurement and actual measurement.

In the case of measuring excitation light of a plurality of wavelengths by the control software used in the related art, blank measurement and actual measurement are performed for light of a first wavelength, and then blank measurement and actual measurement are performed for light of a second wavelength. At this time, there are the following problems: it is necessary to repeat the processes of washing the cuvette after completion of blank measurement at the first wavelength and enclosing the actual sample solution to perform actual measurement, and washing the cuvette again after completion of actual measurement and enclosing the blank sample to perform blank measurement at the second wavelength, and it takes a lot of time and labor to wash the cuvette, enclose the blank sample or the sample solution, and place the cuvette. In addition, there are the following problems: since many control software perform measurement independently for excitation light of each wavelength, it is necessary to input the wavelength of excitation light, the measurement wavelength range of transmitted light, and the measurement wavelength range of fluorescence (or phosphorescence) every time measurement of excitation light of one wavelength is performed, which takes time and effort.

Here, the case where a blank sample and an actual sample, which are liquid, are measured is described as an example, but the same problem as described above is also present in the case where a gas sample or a solid sample is sealed in a sample container and measured. In addition, for example, in the case of a solid sample formed by depositing an optical functional substance on a substrate, measurement can be performed without enclosing the solid sample in a sample container, but in such a case, it is also necessary to repeatedly place a blank sample (substrate only) and an actual sample (substrate on which an optical functional substance is deposited) for each measurement, which takes a lot of time and effort.

The present invention addresses the problem of providing a fluorescence spectrophotometer capable of easily performing blank measurement and actual measurement for excitation light of a plurality of wavelengths, a measurement method using the fluorescence spectrophotometer, and fluorescence spectrophotometer control software.

Means for solving the problems

A fluorescence spectrophotometer according to a first aspect of the present invention, which has been made to solve the above problems, includes:

a) A sample arrangement unit for arranging a sample;

b) A light source unit capable of emitting light of a plurality of wavelengths to the sample arrangement unit;

c) A detection unit that measures the intensity of light in a predetermined wavelength range among the light from the sample arrangement unit;

d) An excitation wavelength input unit for receiving input of a plurality of excitation wavelengths, which are a part or all of the plurality of wavelengths, by a user;

e) A measurement wavelength range determination unit that determines a transmission light measurement wavelength range and a light emission measurement wavelength range corresponding to each excitation wavelength according to a predetermined rule, in accordance with the input excitation wavelengths;

f) A blank measurement execution unit that receives a blank measurement execution instruction from a user, and controls the light source unit and the detection unit to execute the following blank measurement: causing the light source unit to emit light of the plurality of excitation wavelengths in order, and measuring the intensity of light in the wavelength range of the transmitted light passing through the sample arrangement unit for each light of the excitation wavelength; and

g) An actual measurement execution unit that receives an instruction to execute an actual measurement by a user, and controls the light source unit and the detection unit to execute the actual measurement as follows: the light source unit is caused to emit light of the plurality of excitation wavelengths in order, and the intensity of light in the transmitted light measurement wavelength range that has passed through the sample arrangement unit and the intensity of light in the emission measurement wavelength range emitted from the sample arrangement unit are measured for each excitation wavelength of light.

the light source unit may be an appropriate light source unit such as the following light source unit: a light source unit configured to combine a continuous light source such as a lamp that emits continuous light in a wavelength range including the plurality of excitation wavelengths with a beam splitter; a light source unit having a plurality of monochromatic light sources each emitting light of one of the plurality of excitation wavelengths; and a light source unit in which a discrete light source that emits discrete light including light of the plurality of excitation wavelengths and a beam splitter are combined.

The detection unit that measures the intensity of light in the predetermined wavelength range measures the intensity of light in the specified wavelength range based on control from the outside.

the procedure of measurement by the spectrofluorometer according to the present invention will be described.

First, input of a plurality of excitation wavelengths by a user is received. This processing can be performed by various methods such as the following method, in addition to the user inputting the excitation wavelength itself: displaying a plurality of excitation wavelengths in a list and allowing a user to select a plurality of excitation wavelengths from the list; alternatively, a table or the like is prepared in advance in which the name or type of the sample to be measured is associated with a plurality of excitation wavelengths or transmitted light measurement wavelength ranges and light emission measurement wavelength ranges, and the name or type of the sample is input by the user to read out the plurality of excitation wavelengths or the like associated with the name or type.

Next, the measurement wavelength range determining unit determines, in accordance with a predetermined rule, measurement wavelength ranges of light (transmitted light) that has passed through the sample arrangement unit (blank sample and actual sample arranged in the sample arrangement unit by the user) and fluorescence or phosphorescence (light emission) that has been emitted from the sample arrangement unit (actual sample arranged in the sample arrangement unit by the user) corresponding to the respective excitation wavelengths, in accordance with a plurality of excitation wavelengths input by the user. The predetermined rule mentioned here may be, for example, a rule in which a transmitted light measurement wavelength range is determined with a wavelength shorter than each excitation wavelength by a predetermined wavelength as a lower limit wavelength and a wavelength longer than each excitation wavelength by a predetermined wavelength as an upper limit wavelength, and a range of a predetermined wavelength width with the upper limit wavelength of the transmitted light measurement wavelength range as a lower limit wavelength is determined as a measurement wavelength range of light emission. As described above, the user may input the name or type of the sample and then read the measurement wavelength range of the transmitted light and the emission light corresponding to the name or type. In addition, the user may input the upper limit wavelength and the lower limit wavelength of the measurement wavelength range of the emitted light.

Next, when the user places a blank sample in the sample arrangement portion and instructs to perform blank measurement, the blank measurement execution portion operates the light source portion and the detection portion to sequentially emit light of a plurality of excitation wavelengths from the light source portion, and measures the intensity of light in the measurement wavelength range of transmitted light that has passed through the sample arrangement portion (blank sample arranged in the sample arrangement portion) for each excitation wavelength of light.

When the user places the actual sample in the sample arrangement portion and instructs to perform the actual measurement after the blank measurement of all the excitation wavelengths is completed, the actual measurement execution portion operates the light source portion and the detection portion to sequentially emit light of a plurality of excitation wavelengths from the light source portion, and measures the intensity of light in the measurement wavelength range of transmitted light that has passed through the sample arrangement portion (the actual sample arranged in the sample arrangement portion) and the intensity of light in the measurement wavelength range of emitted light from the sample arrangement portion for light of each excitation wavelength.

In the spectrophotometer according to the present invention, since blank measurements are sequentially performed for a plurality of excitation wavelengths and then actual measurements are sequentially performed for a plurality of excitation wavelengths, the blank samples can be simply replaced with actual samples after the measurement of the blank samples for all the excitation wavelengths is completed, and the measurement can be easily performed. Further, since the analyst only needs to input a plurality of excitation wavelengths before the start of measurement, the labor and time of the analyst can be reduced as compared with the measurement using the conventional apparatus.

in addition, a second aspect of the present invention, which has been made to solve the above problems, is a spectrometry method using a fluorescence spectrophotometer having a light source unit, a sample arrangement unit, and a detection unit,

a) Receiving input of a plurality of excitation wavelengths by a user;

b) determining a transmitted light measurement wavelength range and a light emission measurement wavelength range corresponding to each excitation wavelength in accordance with a predetermined rule in correspondence with the plurality of excitation wavelengths;

c) The following blank measurements were performed: causing the light source unit to emit light of the plurality of excitation wavelengths in order, and measuring the intensity of light in the wavelength range of the transmitted light passing through the sample arrangement unit for each light of the excitation wavelength; and

d) The following actual measurements were performed: the light source unit is caused to emit light of the plurality of excitation wavelengths in order, and the intensity of light in the transmitted light measurement wavelength range that has passed through the sample arrangement unit and the intensity of light in the emission wavelength measurement range that has been emitted from the sample arrangement unit are measured for each excitation wavelength of light.

A third aspect of the present invention, which has been made to solve the above problems, is a control software for a fluorescence spectrophotometer having a light source unit, a sample arrangement unit, and a detection unit, the control software causing a computer communicably connected to the fluorescence spectrophotometer to operate as:

a) an excitation wavelength input unit for receiving input of a plurality of excitation wavelengths by a user;

b) A measurement wavelength range determination unit that determines a transmission light measurement wavelength range and a light emission measurement wavelength range corresponding to each excitation wavelength in accordance with a predetermined rule in accordance with the plurality of excitation wavelengths;

c) A blank measurement execution unit that receives a blank measurement execution instruction from a user, and controls the light source unit and the detection unit to execute the following blank measurement: causing the light source unit to emit light of the plurality of excitation wavelengths in order, and measuring the intensity of light in the wavelength range of the transmitted light passing through the sample arrangement unit for each light of the excitation wavelength; and

d) An actual measurement execution unit that receives an instruction to execute an actual measurement by a user, and controls the light source unit and the detection unit to execute the actual measurement as follows: the light source unit is caused to emit light of the plurality of excitation wavelengths in order, and the intensity of light in the transmitted light measurement wavelength range that has passed through the sample arrangement unit and the intensity of light in the emission measurement wavelength range emitted from the sample arrangement unit are measured for each excitation wavelength of light.

ADVANTAGEOUS EFFECTS OF INVENTION

by using the spectrofluorometer, the spectroscopic measurement method, or the spectrofluorometer control software according to the present invention, blank measurement and actual measurement can be easily performed for excitation lights of a plurality of wavelengths.

Drawings

Fig. 1 is a schematic configuration diagram of an embodiment of a fluorescence spectrophotometer according to the present invention.

Fig. 2 is a diagram illustrating the arrangement of the measurement system in the fluorescence spectrophotometer according to the present embodiment.

fig. 3 is a flowchart of an embodiment of the spectrometry method according to the present invention.

Fig. 4 shows an example of a spectrum obtained by the fluorescence spectrophotometer and the spectroscopic measurement method of the present embodiment.

Fig. 5 is a list of expressions of the number of photons in the blank measurement and the actual measurement at a plurality of excitation wavelengths.

Fig. 6 is a list of calculation formulas for obtaining the external quantum efficiency and the internal quantum efficiency for a plurality of excitation wavelengths.

FIG. 7 shows an example of spectrum development in the fluorescence spectrophotometer and the spectrometry method of the present embodiment.

FIG. 8 is a display example of the measurement results in the fluorescence spectrophotometer and the spectroscopic measurement method of the present embodiment.

FIG. 9 is a display example of the measurement results of a plurality of samples in the fluorescence spectrophotometer and the spectroscopic measurement method according to the present embodiment.

Detailed Description

Next, an embodiment of a spectrofluorometer, a spectroscopic measurement method, and spectrofluorometer control software according to the present invention will be described with reference to the drawings.

Fig. 1 shows a main part structure of a spectrofluorometer 1 of the present embodiment. The fluorescence spectrophotometer roughly includes a measurement unit 10 and a control unit 20. The measurement unit 10 includes a light source unit 11, a sample arrangement unit 12, and a detection unit 13. The light source unit 11 includes a light source 111 and a beam splitter 112, and the light source 111 emits continuous light including light of a plurality of excitation wavelengths, which will be described later. The samples (blank sample and actual sample) are placed on the sample arrangement portion 12. The detector 13 includes a beam splitter 131 and a detector 132. The beam splitters 112 and 131 of this embodiment are diffraction gratings, and the detector 132 is a photodiode array detector. The continuous light emitted from the light source 111 is monochromatized by the beam splitter 112 and then irradiated to the sample 121. Of the light transmitted through the sample 121 and the light emitted from the sample 121, the light in the wavelength range selected by the spectroscope 131 is incident on the detector 132, and the intensity thereof is measured. The output signals from the detector 132 are sequentially transmitted and stored in the storage unit 21.

As shown in fig. 2, sample arrangement portion 12 is provided at the center in integrating sphere 100. In the integrating sphere 100, a light entrance window 101 and a first light exit window 102 are formed at positions facing each other with the sample arrangement portion 12 (the center of the integrating sphere 100) therebetween in the X-Y plane in the drawing, the light entrance window 101 being for entering light from the light source unit 11, and the first light exit window 102 being for emitting light that has passed through the sample 121 placed on the sample arrangement portion 12. In addition, a second exit window 103 is formed at the pole of the integrating sphere (directly above the sample arrangement portion; one of the intersection points of the integrating sphere 100 and the Z axis). The fluorescence or phosphorescence emitted from the sample 121 is repeatedly reflected inside the integrating sphere and emitted from the second exit window 103. The transmitted light emitted from the first light exit window 102 and the fluorescence or phosphorescence emitted from the second light exit window 103 are guided to the detection section 13 by an optical system not shown.

The control unit 20 includes, in addition to the storage unit 21, a wavelength range determination unit 221, a blank measurement execution unit 222, an actual measurement execution unit 223, a spectrum creation unit 224, a quantum efficiency calculation unit 225, and an analysis result display unit 226, which are functional blocks embodied by executing the control software 22 for a spectrophotometer. The control unit 20 is a personal computer, and is connected to an input unit 30 including a keyboard and a mouse, and a display unit 40 such as a liquid crystal display. The storage unit 21 stores the sample name and information on the measurement condition (a plurality of excitation wavelengths λ) for each of the plurality of actual samples_A、λ_BFirst wavelength λ₁A second wavelength lambda₂And a third wavelength lambda₃) Corresponding compound database 211. Further, the storage unit 21 stores photon count calculation information (a numerical expression and/or a correspondence table) for determining the number of photons from the detection intensity of the light by the detector 132.

First wavelength λ stored in compound database 211₁Is a value for determining a measurement wavelength range (transmission light measurement wavelength range) when measuring light (transmission light) that has passed through the blank sample and the actual sample, thereby determining the transmission light measurement wavelength range as the excitation wavelength ± λ₁. In addition, the second wavelength λ₂And a third wavelength lambda₃Is a value for determining a measurement wavelength range (luminescence measurement wavelength range) for measuring fluorescence or phosphorescence emitted from an actual sample, and thereby determines the lower limit wavelength of the luminescence measurement wavelength range as the excitation wavelength + λ₂and the upper limit wavelength is determined as lambda₃。

Next, the spectroscopic measurement by the spectrofluorometer 1 of the present example will be described. Fig. 3 is a flowchart illustrating a procedure of the spectrometry method according to the present embodiment. Here, the following case will be explained as an example: six samples of similar compounds were dissolved in the same solvent to prepare sample solutions, and three excitation wavelengths λ were obtained for each sample_A～λ_Cexternal quantum efficiency and internal quantum efficiency.

when the user instructs to start the spectrophotometric measurement by activating a predetermined operation such as the control software 22 for a spectrophotometer, a screen for inputting information (sample name, type, measurement type, and the like) and the number of measurement samples (actual samples) is displayed on the display unit 40, and the user is prompted to input the information.

When the user inputs the information and the number of actual samples (step S1), the wavelength range determination unit 221 searches whether or not information corresponding to the information of the actual samples input by the user is present in the compound database 211 stored in the storage unit 21. When the information corresponding to the information on the actual sample input by the user is not present in the compound database, the display unit 40 displays the information for inputting the excitation wavelengths λ_A～λ_CA first wavelength lambda₁A second wavelength lambda₂and a third wavelength lambda₃To allow the user to input (step S2). When the user finishes inputting, aiming at a plurality of excitation wavelengths lambda_A～λ_CSetting the transmission light measurement wavelength range lambda for each excitation wavelength in (1)_A-λ₁～λ_A+λ₁Etc. and luminescence measurement wavelength range lambda_A+λ₂～λ₃And so on (step S3). On the other hand, when information corresponding to the information on the actual sample input by the user is present in the compound database 211, a plurality of excitation wavelengths λ corresponding to the information on the actual sample are read_A～λ_CA first wavelength lambda₁A second wavelength lambda₂and a third wavelength lambda₃The transmitted light measurement wavelength range lambda is obtained for each of a plurality of excitation wavelengths_A-λ₁～λ_A+λ₁Etc. and luminescence measurement wavelength range lambda_A+λ₂～λ₃And displayed on the display unit 40. The user checks each displayed wavelength and measurement wavelength range, and appropriately changes these pieces of information as necessary.

Next, the user seals a blank sample (solvent only) in the cuvette and places the cuvette in the sample arrangement portion 12 (step S4), and instructs execution of blank measurement. Blank measurement is receivedupon receiving the instruction, the line unit 222 causes the light source 111 of the light source unit 11 to generate continuous light, and the first excitation wavelength λ is extracted by the beam splitter 112_AAnd irradiated to the blank sample. The spectroscope 131 of the detection unit 13 is rotated to measure the wavelength range λ with transmitted light_A-λ₁～λ_A+λ₁the transmitted light from the blank sample was separated in wavelength, and the intensity of light at each wavelength was measured. The spectroscope 131 is further rotated to measure the wavelength range lambda by luminescence_A+λ₂～λ₃The fluorescence emitted from the blank sample is separated in wavelength, and the intensity of light of each wavelength is measured (step S5). In the present embodiment, the spectroscope 131 is sequentially rotated to measure the light in the transmitted light measurement wavelength range and the light in the emission light measurement wavelength range, but when the light obtained by wavelength separation in these wavelength ranges can be simultaneously incident on the detector 132, these lights may be measured at once.

When aiming at the initial excitation wavelength lambda_AWhen the blank measurement is completed with the light of (2), the blank measurement execution unit 222 checks whether or not the blank measurement is performed for all the excitation wavelengths λ_A～λ_CThe blank measurement is completed (step S6). When the excitation wavelength remains unmeasured (step S6: NO), the spectroscope 112 of the light source unit 11 is operated to extract the next excitation wavelength λ_BAnd irradiated to the blank sample. The spectroscope 131 of the detection unit 13 is rotated to measure the wavelength range λ with transmitted light_B-λ₁～λ_B+λ₁Wavelength separation is performed and the intensity of transmitted light from the blank sample is measured to measure the wavelength range lambda by luminescence_B+λ₂～λ₃The wavelength separation is performed and the intensity of fluorescence from the actual sample is measured (step S5). In this manner, the blank measurement execution unit 222 controls the light source unit 11 and the detection unit 13 to perform a plurality of excitation wavelengths λ_A～λ_CThe light of (2) is sequentially irradiated to the blank sample, the light having passed through the blank sample is wavelength-separated for each excitation wavelength, and the intensity of the light of each wavelength is measured. When blank measurement is completed for all excitation wavelengths (step S6: "YES:)") is displayed on the display unit 40 to indicate that the blank measurement has been completed, and the user is prompted to place the actual sample in the sample arrangement unit 12.

Next, the user washes the cuvette in which the blank sample has been enclosed, and encloses the first actual sample (sample solution) in the cuvette and places it on the sample arrangement part 12 (step S7). Then, the actual measurement is instructed to be performed. Upon receiving the instruction, the actual measurement execution unit 223 causes the light source 111 of the light source unit 11 to generate continuous light, and the spectrometer 112 extracts the first excitation wavelength λ_Aand irradiated to the actual sample. The spectroscope 131 of the detection unit 13 is rotated to measure the wavelength range λ of the transmitted light from the actual sample_A-λ₁～λ_A+λ₁The light of (2) is incident on the detector 132, and the intensity of the light of each wavelength is measured. The spectroscope 131 is further rotated to measure the wavelength range lambda by luminescence_A+λ₂～λ₃the fluorescence emitted from the actual sample is separated in wavelength, and the intensity of light of each wavelength is measured (step S8).

when aiming at the initial excitation wavelength lambda₁When the actual measurement is completed, the actual measurement execution unit 223 checks whether or not the actual measurement is completed for all the excitation wavelengths (step S9). When the excitation wavelength remains unmeasured (step S9: NO), the spectroscope 112 of the light source unit 11 is operated to extract the next excitation wavelength λ_BAnd irradiated to the actual sample. The spectroscope 131 of the detection unit 13 is rotated to measure the wavelength range λ with transmitted light_A-λ₁～λ_A+λ₁transmitted light from an actual sample is separated in wavelength, and the intensity of light of each wavelength is measured. In addition, the wavelength range λ is measured by luminescence_B+λ₂～λ₃The fluorescence from the actual sample is separated into wavelengths, and the intensity of light of each wavelength is measured (step S8). In this manner, the actual measurement execution unit 223 controls the light source unit 11 and the detection unit 13 to set the plurality of excitation wavelengths λ_A～λ_CThe light of (2) is sequentially irradiated to the actual sample, and the transmitted light passing through the actual sample and the fluorescence emitted from the actual sample are analyzed for each excitation wavelengthWavelength separation was performed and measurement was performed.

When the actual measurement is completed for all the excitation wavelengths (step S9: YES), the actual measurement execution unit 223 confirms whether the actual measurement of all the actual samples is completed (step S10). If there is any actual sample that has not been measured, a message urging the user to set the next actual sample is displayed on the display unit 40. When the next actual sample has been placed, the actual measurement execution section 223 sequentially irradiates light of all excitation wavelengths through the same procedure as described above, and measures the intensities of the transmitted light and the fluorescence from the actual sample. When the actual measurement is completed for all samples (step S10: YES), the actual measurement execution unit 223 displays a message on the display unit 40 that all measurements have been completed.

When all the measurements are completed, the spectrum creation unit 224 reads the measurement data stored in the storage unit 21, creates spectrum data for each actual sample, and displays the spectrum on the display unit 40 (step S11). Fig. 4 shows an example of a spectrum obtained by performing blank measurement and actual measurement using three excitation wavelengths for one sample. FIG. 4 (a) is about the excitation wavelength λ_ASpecifically, the spectrum of the transmitted light intensity (intensity of the excitation light irradiated to the actual sample) in the blank measurement and the spectrum of the intensity obtained by subtracting the fluorescence intensity (intensity of the fluorescence from the solvent) in the blank measurement from the fluorescence intensity (intensity of the fluorescence from the sample and the solvent) in the actual measurement are described. FIG. 4 (b) is about the excitation wavelength λ_BThe same spectrum, FIG. 4 (c) is for the excitation wavelength λ_CThe same spectrum. In addition, FIG. 5 is a graph that will be described with respect to three excitation wavelengths λ_A、λ_B、λ_CThe spectra of (a) are superimposed and displayed. The display of the spectra in fig. 4 (a) to (c) and fig. 5 can be switched as appropriate in accordance with an instruction from the user. Although fig. 4 and 5 are spectra corresponding to the external quantum efficiency, instead of the excitation light irradiated to the sample, a spectrum corresponding to the internal quantum efficiency, which indicates the intensity of the excitation light absorbed by the sample (the intensity obtained by subtracting the intensity of the transmitted light at the time of actual measurement from the intensity of the transmitted light at the time of blank measurement), may be displayedSpectrum of intensity of light. These spectral data are prepared for all samples and stored in the storage unit 21.

When the spectral data of all the samples are prepared, the quantum efficiency calculation unit 225 obtains the external quantum efficiency and the internal quantum efficiency at each excitation wavelength for each sample (step S12). Specifically, the excitation wavelengths λ used are obtained based on the photon count calculation information stored in the storage unit 21 from the transmission light intensity and fluorescence intensity in the blank measurement stored in the storage unit 21 and the transmission light intensity and fluorescence intensity in the actual measurement_A、λ_B、λ_CNumber of photons F of transmitted light in blank measurement_EXA、F_EXB、F_EXCAnd the number of fluorescence photons F_EMA、F_EMB、F_EMCThe number of photons F of transmitted light in actual measurement_EXA’、F_EXB’、F_EXC' and number of fluorescence photons F_EMA’、F_EMB’、F_EMC' (refer to FIG. 6). Then, for example, for the excitation wavelength λ_AAccording to (F)_EMA’-F_EMA)/F_EXAObtaining external quantum efficiency according to (F)_EMA’-F_EMA)/(F_EXA-F_EXA') the internal quantum efficiency was determined (see FIG. 7). The external quantum efficiency and the internal quantum efficiency can be obtained for all samples and all excitation wavelengths.

When the processing by the quantum efficiency calculation section 225 is completed, the analysis result display section 226 uses a plurality of excitation wavelengths λ_A、λ_B、λ_CThe following values of the external quantum efficiency and the internal quantum efficiency are obtained by approximating the excitation wavelength dependence of the external quantum efficiency and the internal quantum efficiency by a method such as a curve, and are displayed on the display unit 40 (step S13). Fig. 8 is a display example of the excitation wavelength dependence. The analysis result display unit 226 displays a list of the quantum efficiencies of the respective samples (step S13). The list display may be: for example, as shown in fig. 9, a two-dimensional region in which samples are arranged in the vertical axis direction with the horizontal axis as the wavelength is divided into a plurality of sections, and the quantum efficiency in the section is expressed by the shade of color (shown by hatching in fig. 9). Thereby the device is provided withthe user can easily confirm the quantum efficiency of a plurality of samples. Of course, the display form can be changed as appropriate, and besides the shade of the color, the display can be made visually recognizable by coloring, hatching, or the like.

as described above, in the spectrofluorometer and the spectroscopic measurement method of the present embodiment, since blank measurement is sequentially performed for a plurality of excitation wavelengths and then actual measurement is sequentially performed for a plurality of excitation wavelengths, it is only necessary to replace the blank sample with the actual sample after the measurement of the blank samples concerning all the excitation wavelengths is completed, and the measurement can be performed easily. In addition, the analyzer inputs a plurality of excitation wavelengths λ just before the start of measurement_A～λ_CAnd a first wavelength lambda₁third wavelength λ₃(or information on the sample) can be input, and therefore, the labor and time of the analyst can be reduced as compared with the measurement using the conventional apparatus.

The above embodiment is an example, and can be modified as appropriate in accordance with the spirit of the present invention.

In the above embodiments, the first wavelength λ is adjusted₁And a second wavelength lambda₂as a relative wavelength with respect to the excitation wavelength and a third wavelength lambda₃Although the case where the input is made as the absolute wavelength has been described, the second wavelength λ may be set to be the absolute wavelength₂Input is made as absolute wavelength. Wherein in this case the second wavelength λ needs to be avoided₂Wavelength range lambda of transmitted light_A-λ₁～λ_A+λ₁And the like. Therefore, it is preferable that the wavelength is set to the second wavelength λ input by the user₂Present in the measurement wavelength range lambda_A-λ₁～λ_A+λ₁Or the like, to prompt the user to re-input. In addition, when the measurement is repeated only for the same type of sample, the configuration may be such that the first wavelength λ is set in advance in the fluorescence spectrophotometer₁Third wavelength λ₃As an initial value, the value is changed by the user only when necessary.

In the above-described embodiment, the sample solution (blank sample and actual sample) was sealed in the cuvette and measured, but the measurement may be performed while the cuvette is stored in another container. Further, the gas sample or the solid sample may be stored in a sample container and measured in the same manner as described above. In the case of a solid sample, measurement can also be performed by directly placing a blank sample (e.g., a substrate) and an actual sample (e.g., a functional substance coated on the substrate) in the sample arrangement part.

Further, the measurement can be performed by enclosing a gas sample in a sample container, not only in a liquid sample. Further, the solid sample can be directly measured (or stored in a sample container).

in the above examples, the intensity of fluorescence or phosphorescence from a blank sample (solvent) was measured in the blank measurement, but in the known blank sample, the intensity of fluorescence or phosphorescence at a plurality of excitation wavelengths λ was measured_A～λ_Cin the case where neither fluorescence nor phosphorescence is emitted, only the intensity of transmitted light from the blank sample may be measured at the time of blank measurement, and the intensity value and the number of photons of fluorescence or phosphorescence from the blank sample may be set to 0.

Description of the reference numerals

1: a fluorescence spectrophotometer; 10: a measurement section; 11: a light source unit; 111: a light source; 112: a light splitter; 12: a sample arrangement part; 121: a sample; 13: a detection unit; 131: a light splitter; 132: a detector; 20: a control unit; 21: a storage unit; 211: a compound database; 22: control software for a spectrophotometer; 221: a wavelength range determination unit; 222: a blank measurement execution unit; 223: an actual measurement execution unit; 224: a spectrum creation unit; 225: a quantum efficiency calculation unit; 226: an analysis result display unit; 30: an input section; 40: a display unit.