JP6541949B2 - Spectrofluorometer and method of acquiring three-dimensional fluorescence spectrum using the same - Google Patents

Description

  The present invention relates to a spectrofluorimeter equipped with a mechanism in which a cut filter is automatically inserted, and in order to suppress the generation of high-order light unnecessary for fluorescence measurement of a measurement sample, the cut filter is inserted according to measurement conditions Determining and executing the wavelength and measurement sequence contributes to shortening the measurement time of the three-dimensional fluorescence spectrum.

  The spectrofluorometer comprises a photometer unit, a data processing unit, and an interface unit. Continuous light from a light source is irradiated to a measurement sample as excitation light dispersed by an excitation side spectroscope. The fluorescence emitted from the sample is split into monochromatic light by the fluorescence spectrometer, detected by the detector, passed through the A / D converter, taken into the computer as signal intensity, and the measurement result is displayed on the monitor. Be done.

  In general, the excitation spectrum for measuring the fluorescence intensity when the excitation wavelength is changed with respect to the measurement sample, changes the excitation side spectroscope from the measurement start wavelength to the measurement end wavelength, and irradiates the measurement sample with excitation light of each wavelength. At that time, the change in the fluorescence intensity of the specific wavelength obtained through the fluorescence side spectroscope set to the fixed wavelength is detected by the detector, taken into the computer as the signal intensity through the A / D converter, and the measurement result in the monitor A two-dimensional spectrum of excitation wavelength and fluorescence intensity is displayed as

  In addition, the fluorescence spectrum for measuring the fluorescence intensity for each wavelength when irradiating the excitation light of the fixed wavelength to the measurement sample and changing the fluorescence wavelength is the excitation light from the excitation side spectroscope set to the fixed wavelength The measurement sample is irradiated, the fluorescence at that time is changed from the measurement start wavelength to the measurement end wavelength by the fluorescence side spectroscope, the change of the fluorescence for each wavelength is detected by the detector, and the computer passes through the A / D converter The measurement results are displayed as a two-dimensional spectrum of fluorescence wavelength and fluorescence intensity on a monitor.

  On the other hand, the three-dimensional fluorescence spectrum measures the fluorescence spectrum when the excitation wavelength is fixed, and when the fluorescence spectrum scanning is finished, the fluorescence wavelength is returned to the measurement start wavelength, the excitation wavelength is driven by a predetermined wavelength interval, and the next excitation Measure the fluorescence spectrum at the wavelength. The three-dimensional fluorescence spectrum is acquired by storing the obtained fluorescence spectrum in three dimensions of the excitation wavelength, the fluorescence wavelength, and the fluorescence intensity and repeating the excitation wavelength until reaching the final wavelength. The obtained three-dimensional fluorescence spectrum is drawn as a contour map or a bird's-eye view by connecting the same fluorescence intensities respectively. The excitation wavelength and the fluorescence wavelength that form a mountain in the contour line become the optimum excitation wavelength and the characteristic fluorescence wavelength of the measurement sample, and the fluorescence characteristics of the excitation wavelength and the fluorescence wavelength within the measurement range of the measurement sample can be seen at a glance. There is an advantage that much information such as the number of components of the fluorescent substance in the measurement sample and the identification of the components can be obtained from the three-dimensional fluorescence spectrum.

  When measuring an excitation spectrum, a fluorescence spectrum, and a three-dimensional fluorescence spectrum with a spectrofluorimeter, the generation of high-order light becomes a problem as light other than the target fluorescence. Light such as scattered light of excitation light, Raman scattered light, and fluorescence having a wavelength shorter than the target wavelength is incident on the fluorescence-side spectroscope to generate high-order light. In general, a cut filter such as a long pass filter or a band pass filter is used to suppress high-order light by guiding the target fluorescence to a spectroscope without transmitting light on the short wavelength side which is a factor of high-order light. A plurality of cut filters should be prepared for each transmission limit wavelength, and it is necessary to select a filter that blocks light as a generation factor and to insert it between the sample and the fluorescence side spectrometer. On the other hand, in the excitation-side spectroscope, high-order light generated on the shorter wavelength side than the set excitation wavelength becomes a problem. When measuring the fluorescence spectrum, if the measurement sample is irradiated with high-order light other than the excitation light, there is a problem that it is not possible to distinguish between the fluorescence emitted from the target excitation light and the fluorescence emitted from the high-order light. Do. Therefore, in order to prevent high-order light generated on the shorter wavelength side than the set excitation wavelength, a cut filter that transmits the target excitation light without transmitting high-order light is inserted between the excitation-side spectroscope and the fluorescence-side spectroscope. Conventionally, based on the knowledge of higher-order light, analysts have selected and inserted appropriate cut filters in order to obtain only the fluorescence of the sample. However, there are cases where the cut filters are mistaken, and measurement in a wide wavelength range At that time, it was also troublesome to replace the cut filter in the middle of the measurement.

  In order to prevent this problem, in the case of a spectrophotometer provided with an automatic insertion mechanism, a control method is employed in which a high-order light filter is switched in conjunction with wavelength scanning of a spectrometer. At that time, the wavelength scanning is temporarily stopped, the cut filter is switched, and the wavelength scanning is restarted. (Patent Document 1)

  In addition, in order to identify whether the peak in the obtained spectrum is a peak showing fluorescence or a peak showing Rayleigh scattered light, a method of discriminating using a spectrofluorimeter equipped with a cut filter is known. (Patent Document 2). Based on the excitation wavelength, an optional cut filter is placed in front of the fluorescence side spectroscope, and while rotating the diffraction grating, a light intensity signal (in the present application, fluorescence intensity and By acquiring the mark, control is performed to acquire a spectrum from which secondary light of Rayleigh scattering emitted from the sample is removed.

JP 61-105430 JP 2012-631438

  When measuring an excitation spectrum, a fluorescence spectrum, and a three-dimensional fluorescence spectrum with a spectrofluorimeter, the generation of high-order light becomes a problem as light other than the target fluorescence.

  In the case of the excitation spectrum, one cut filter corresponding to a fixed fluorescence wavelength is selected, and the cut filter is inserted between the measurement sample and the fluorescence-side spectroscope to obtain an excitation spectrum, whereby high-order light is suppressed. In the case of a fluorescence spectrum, one cut filter corresponding to a fixed excitation wavelength is selected, and the cut filter is inserted between the measurement sample and the fluorescence side spectroscope to obtain a fluorescence spectrum, whereby high-order light is suppressed. In order to suppress high-order light in the excitation spectrum and fluorescence spectrum, the measurement itself is easy because one cut filter is selected and inserted.

  On the other hand, for the three-dimensional fluorescence spectrum, it is necessary to acquire fluorescence spectra for excitation wavelength intervals of predetermined wavelength intervals, and it is necessary to select and insert cut filters one by one for each excitation wavelength set by the analyst. It is very complicated. In addition, since fluorescence spectra at a plurality of excitation wavelengths are acquired, analysis is more time-consuming than ordinary fluorescence spectrum measurement, and thus there is a disadvantage that the analyst is restricted for a long time.

For example, in the case of using the high-order light cut filter according to the method of Patent Document 1, it is necessary to temporarily stop the wavelength scanning and switch to the cut filter corresponding to the excitation wavelength during measurement of the fluorescence spectrum. At this time, filter switching corresponding to the number of times of measurement corresponding to a predetermined excitation wavelength interval occurs, which causes a problem of prolonging the measurement time. In addition, when high-speed wavelength scanning conditions are used to shorten the measurement time, there are problems such as load on the wavelength drive system and a step to the wavelength and fluorescence intensity value due to the temporary stop.

Patent Document 2 describes a configuration in which a single Rayleigh cut filter selected from among a plurality of filters is disposed in front of a fluorescence side spectrometer. The Rayleigh cut filter is disposed for the purpose of blocking light having a cutoff wavelength or less based on the set excitation wavelength. Then, in the determination of the fluorescence peak, an arbitrary Rayleigh cut filter is disposed in front of the fluorescence side spectroscope, and then the sample is irradiated while changing stepwise the light on the long wavelength side different from the set excitation wavelength, It is described that the fluorescence spectrum emitted from the sample is measured. In addition, it is possible to make a more accurate determination by selecting and using a Rayleigh cut filter having a wavelength difference longer than the set excitation wavelength and having the smallest number of wavelengths with the excitation wavelength from among a plurality of Rayleigh cut filters. It is stated that it can.

  However, when a cut filter is selected by this method, it is possible to determine a peak including high-order light derived from Rayleigh scattering and a peak including high-order light derived from Raman scattering, but the present invention has the following problems. The data which suppressed the influence of the high-order light which becomes unnecessary in the fluorescence measurement of the measurement sample which is the objective of is not obtained.

  As a first problem, secondary light of fluorescence is not considered. For example, when a sample having a fluorescence peak of 300 nm is measured at an excitation wavelength of 200 nm, the Rayleigh cut light with an excitation wavelength of 200 nm is not incident on a fluorescence spectrometer by a Rayleigh cut filter, so 2 of the Rayleigh scattered light appearing at 400 nm Higher-order light derived from Rayleigh scattered light such as secondary light is not observed. However, in the fluorescence spectrometer, secondary light of fluorescence is observed at 600 nm due to the fluorescence of 300 nm.

  The second problem is that the influence of high-order light from the excitation spectrometer is not taken into consideration. For example, when the excitation wavelength is 440 nm, the excitation light includes light of 220 nm as 1⁄2-order light. At this time, when the sample shown in the first example is measured, fluorescence excited by 1⁄2-order light at a fluorescence wavelength of 300 nm and secondary light of fluorescence at 600 nm are observed. None of the fluorescence is excited and appeared at 440 nm.

  The third problem is that the transmittance of the cut filter is not taken into consideration. In general, long pass filters and band pass filters used for Rayleigh cut filters do not have 100% transmittance, so light loss occurs. In the case of a long pass filter, the transmittance is about 80 to 90% in the transmission region. In addition, since there is an abrupt change in transmittance in the wavelength range which shifts from the cutoff wavelength range to the transmission wavelength range, application to measurement data is not considered, although it is inappropriate.

In the three-dimensional fluorescence spectrum displayed by three-dimensionally displaying the excitation wavelength, the fluorescence wavelength, and the fluorescence intensity as described in Patent Document 2, the objects of the present invention can be achieved for the first to third problems described above. It is not possible to obtain three-dimensional fluorescence spectrum data that suppresses the generation of high-order light that is unnecessary for the fluorescence measurement of the sample.
As described above, the related art can be summarized as the problems as follows.

  That is, at the time of acquiring the three-dimensional fluorescence spectrum by the above-mentioned conventional spectrofluorimeter, there is a possibility that the analyst makes an erroneous selection because the cut filter is selected based on experience and knowledge. In addition, since wavelength scanning is temporarily stopped during measurement to insert and replace filters, it is inevitable to extend the measurement time, and if high-speed wavelength scanning is performed to reduce this, the load on the wavelength drive system There is a problem such as an increase in size and an error or a step in the wavelength or the fluorescence intensity. Furthermore, using a filter that blocks light below the excitation wavelength based on the set excitation wavelength makes it impossible to obtain a three-dimensional fluorescence spectrum in which the effects of secondary light of fluorescence and high-order light of the excitation spectrometer are suppressed. In addition, there is a problem that the transmittance of the cut filter is not added even at the time of wavelength synthesis.

In order to achieve the above object, in the spectrofluorimetric analyzer of the present invention, a plurality of excitation sides for blocking high-order light based on excitation light between an excitation side spectroscope for separating light of a light source into excitation light and a sample Excitation-side filter moving mechanism for optically arranging the excitation-side filter selected by the excitation-side filter selection means that selects the filter according to the measurement conditions from the filter, the sample, and fluorescence emitted from the sample by the irradiation of the excitation light The fluorescence side filter selected by the fluorescence side filter selection means which selects a filter according to measurement conditions from a plurality of fluorescence side filters that block high-order light based on fluorescence, between the fluorescence side spectroscope that disperses It has the fluorescence side filter moving mechanism to arrange. As described above, in the detector for detecting the fluorescence after passing through the fluorescence filter and the fluorescence spectrometer, it is possible to obtain three-dimensional fluorescence spectrum data in which the influence of the excitation high-order light and the fluorescence high-order light is suppressed.

The above three-dimensional fluorescence spectrum data acquisition method has the following steps. A measurement condition setting step of specifying conditions relating to excitation light and fluorescence; a filter condition setting step of specifying conditions of a plurality of excitation side and fluorescence side filters provided in the apparatus; a wavelength range of excitation light and a wavelength range of fluorescence; Based on the calculation from the measurement condition setting step and the filter condition setting step, the wavelength range determined by the filter is a set of filters consisting of a combination of the excitation-side and fluorescence-side filters and the range that can be measured by the filters A step of comparing the wavelength range in which the excitation light wavelength and the fluorescence wavelength related to the boundary of each region are recognized as the excitation side filter switching wavelength and the fluorescence side filter switching wavelength respectively Whether the region is included in the range from the measurement start wavelength to the measurement end wavelength for each of the excitation side and the fluorescence side A determination step of determining, a measurement step of measuring a region according to the determination step using a filter group determined by the collation step, and a filter correction step of correcting the transmittance of the filter used for the data acquired by the measurement step A data synthesis step of combining data of the respective areas after the correction in the correction step to obtain data of the entire measurement range.

In the above three-dimensional fluorescence spectrum data acquisition method, the decrease in the fluorescence intensity caused by the deterioration of the measurement sample due to the excitation light generated in the process of the filter correction step and the data synthesis step is intensity corrected.

In the above-described filter condition setting step, the measurement start wavelength and the measurement end wavelength on the fluorescence side can be determined by comparing the magnitudes of the arbitrarily selected measurement start wavelength and measurement end wavelength with the fluorescence side filter switching wavelength.

According to the spectrofluorimeter of the present invention, it is possible to achieve high efficiency by providing a mechanism for automatically inserting and replacing the necessary cut filter by determining and executing the filter insertion wavelength and the measurement sequence according to the measurement conditions. Thus, it is possible to suppress the generation of high-order light unnecessary for the fluorescence measurement of the sample. Therefore, it is possible to obtain a three-dimensional fluorescence spectrum in which the generation of high-order light is suppressed by effectively shortening the measurement time.
In addition, since the necessary cut filters can be inserted and exchanged automatically, the restraint time of the analyst can be reduced and human error can be prevented.

Device configuration in the embodiment of the present invention Illustration of excitation spectrum Illustration of fluorescence spectrum Illustration of 3D fluorescence spectrum Conceptual diagram of excitation side cut filter use wavelength range Conceptual diagram of the wavelength range used for the fluorescence side cut filter Configuration example of cut filter Determination flow chart of 3D fluorescence measurement Determination flow chart of three-dimensional fluorescence measurement in the second embodiment Correction concept of fluorescence intensity (before correction) Correction concept of fluorescence intensity (after correction)

  Hereinafter, one embodiment of a spectrofluorimeter according to the present invention will be described with reference to FIGS. 1 to 8.

  As shown in FIG. 1, the spectrofluorometer comprises a photometer unit 100, a data processing unit 101, and an interface unit 102. The continuous light from the light source 1 is split as excitation light by the excitation side spectroscope 2, and it is irradiated to the measurement sample 6 installed in the sample installation unit 5 through the beam splitter 3. At this time, the monitor detector 4 measures the light quantity of the excitation light partially divided by the beam splitter 3 to correct the fluctuation of the light source. The fluorescence emitted from the sample is split into monochromatic light by the fluorescence side spectroscope 7, the light is detected by the detector 8, passes through the A / D converter 9, and is taken into the computer 10 as a signal intensity and is sent to the monitor 17. Measurement results are displayed.

  The wavelength drive system will be described. The excitation side pulse motor 12 is driven by the command of the computer 10 to set the excitation side spectroscope 2 at the target wavelength position. In addition, the fluorescence side spectroscope 7 is set at a target wavelength position by driving the fluorescence side pulse motor 11 according to a command of the computer 10. The excitation side spectroscope 2 and the fluorescence side spectroscope 7 use optical elements such as a diffraction grating and a prism, and are driven by the excitation side pulse motor 12 and the fluorescence side pulse motor 11 by gears and cams. The spectrum is scanned by rotating it. An excitation filter 15 is disposed between the excitation spectrometer 2 and the measurement sample 6. The excitation filter 15 is provided with a plurality of cut filters, and a single cut filter is inserted in the excitation filter pulse motor 13. A fluorescence filter 16 is disposed between the measurement sample 6 and the fluorescence spectrometer 7. The fluorescence side filter 16 is provided with a plurality of cut filters, and a single cut filter is inserted in the fluorescence side filter pulse motor 14.

  Generally, the excitation spectrum for measuring the fluorescence intensity when changing the excitation wavelength to the measurement sample 6 changes the excitation side spectroscope 2 from the measurement start wavelength to the measurement end wavelength, and uses the excitation light of each wavelength as the measurement sample The change in fluorescence of a specific wavelength is detected by the detector 8 through the fluorescence side spectroscope 7 set to the fixed wavelength at that time, and the signal intensity is taken into the computer 10 through the A / D converter 9 and monitored. As a measurement result at 17, a two-dimensional spectrum as shown in FIG. 2 of the excitation wavelength and the fluorescence intensity is displayed.

  Further, the fluorescence spectrum for measuring the fluorescence intensity for each wavelength when irradiating the excitation light of the fixed wavelength to the measurement sample and changing the fluorescence wavelength is the excitation from the excitation-side spectroscope 2 set to the fixed wavelength The light is irradiated to the measurement sample, the fluorescence at that time is changed from the measurement start wavelength to the measurement end wavelength by the fluorescence side spectroscope 7, the change of the fluorescence for each wavelength is detected by the detector 8, and the A / D converter 9 The signal intensity is taken into the computer 10 as a signal intensity, and the monitor 17 displays a two-dimensional spectrum as shown in FIG.

In the three-dimensional fluorescence spectrum, the excitation light from the excitation-side spectroscope 2 set to a fixed wavelength is irradiated to the measurement sample 6, and the fluorescence-side spectroscope 7 is subjected to spectrum scanning to measure the fluorescence spectrum. Then, the fluorescence wavelength is returned to the start wavelength, the excitation wavelength is driven by a predetermined wavelength interval, and the fluorescence spectrum at the next excitation wavelength is measured. As shown in FIG. 4, the obtained fluorescence spectrum is stored in three dimensions of excitation wavelength, fluorescence wavelength, and fluorescence intensity, and the above operation is repeated until the fluorescence spectrum for the final wavelength of the predetermined range is obtained. 3D fluorescence spectrum is displayed as a simple contour map.
Next, selection of the excitation filter 15 will be described with reference to the drawings.
First, a method of selecting an excitation-side filter in the present invention will be described.
FIG. 5 shows a conceptual diagram of the excitation-side filter used wavelength range in the present invention.

  The broken line Wrs extending obliquely upward to the right from the origin is the Rayleigh scattering wavelength, Wex1-3 on the excitation wavelength axis is the switching wavelength of the excitation filter 15, and Aex0-3 divided by the switching wavelength is the wavelength range of the excitation side filter 15 used. Show. Here, the excitation side filter 15 includes excitation side filter no. 0 (without excitation filter) or excitation filter No. The structure which is any one of 1-3 is shown. It is inserted by the excitation side filter pulse motor 13.

The excitation side filter is selected according to the used wavelength of excitation light according to the specification (type) of each filter. (1) Absorbing and shielding the wavelength region of half-order light of excitation light (absorption determination), and (2) being a wavelength region of high transmittance (high transmission determination) The condition is to simultaneously satisfy one judgment.
The above two determinations of the corresponding excitation filter 15 will be described below.

(1) Absorption judgment

よって、式１を満たすカットフィルタは、Ｗｅｘ（ｎｍ）以下の励起光波長であれば、不要な１／２次光に該当する信号が除去できることになる。Selection of the excitation-side filter 15 in the absorption determination is on the condition that the following determination formula is satisfied. Any filter may be used as long as the filter satisfies the determination formula.
[Equation 1]

Therefore, the cut filter satisfying Expression 1 can remove the signal corresponding to the unnecessary half-order light if the excitation light wavelength is less than Wex (nm).

係数α（例えば０〜２０ｎｍ）を用い、設定された励起波長Ｗｅｘ（ｎｍ）における励起側フィルタの完全吸収される波長域を導くことが必要となる。As the excitation-side filter 15, a sharp cut filter or equivalent product defined in JIS B7113 can be used. The absorption limit wavelength described in JIS B7113 is a wavelength having a transmittance of 5%, which can not be applied to the determination because half-order light of excitation light is transmitted. for that reason,

It is necessary to use a coefficient α (for example, 0 to 20 nm) to derive a wavelength range in which the excitation-side filter is completely absorbed at the set excitation wavelength Wex (nm).

(2) High Transmission Judgment Selection of the excitation-side filter 15 for transmission of excitation light in high transmission judgment is made on the condition that the following judgment formula is satisfied.
[Formula 2]

  Therefore, the cut filter satisfying Expression 2 has a high transmittance of about 90% or more with respect to the excitation wavelength Wex (nm), and it is possible to obtain a signal appearing in the corresponding region with low loss. This determination leads to the high transmission wavelength range of the excitation-side filter at the set excitation wavelength Wex (nm). Therefore, at the excitation wavelength Wex (nm), a filter satisfying the excitation side filter short wavelength determination condition and the high transmission determination condition is selected.

  Here, the half-order light of the excitation light does not appear due to the strong absorption of nitrogen in the air when it is shorter than 185 nm corresponding to the vacuum ultraviolet region. Therefore, when the 1⁄2-order light exists at 185 nm or less, the excitation-side filter 15 that absorbs and blocks the region becomes unnecessary.

  Based on such a concept, the cut filters used in the wavelength regions Aex0 to Aex3 of the excitation side filter of FIG. 5 are selected from the filters on the excitation side described in FIG. 7. These filters used what was marketed.

Ｇ２９５フィルタ）にて、調整係数をα７．５ｎｍとした場合、励起波長Ｗｅｘ（ｎｍ）に相当する励起側フィルタ１切替波長Ｗｅｘ１（ｎｍ）は、式２より３４０ｎｍに設定可能である。また、式１より５２０ｎｍ以下の励起光であれば１／２次光を透過しない。従って、ＷＧ２９５フィルタは、励起光波長が３４０〜５２０ｎｍの領域において、吸収判定の式１および高透過判定の式２を同時に満たすため励起側フィルタ１５として利用できる。More specifically, the maximum excitation slit width of 20 nm of a general spectrofluorimeter

In the G295 filter, when the adjustment coefficient is α7.5 nm, the excitation-side filter 1 switching wavelength Wex1 (nm) corresponding to the excitation wavelength Wex (nm) can be set to 340 nm from Expression 2. Further, according to Equation 1, if the excitation light is 520 nm or less, the half-order light is not transmitted. Therefore, the WG 295 filter can be used as the excitation-side filter 15 because it simultaneously satisfies Equation 1 for absorption determination and Equation 2 for high transmission determination in a region where the excitation light wavelength is 340 to 520 nm.

  In addition, if the excitation-side filter 1 switching wavelength Wex1 is 340 (nm), the half-order light with a smaller excitation light wavelength is smaller than approximately 200 nm, and is outside the usable range as general excitation light. When irradiating excitation light having a shorter wavelength than the excitation light, the excitation filter is not necessary.

（Ｙ４４フィルタ）にて調整係数αを７．５ｎｍとした場合、励起波長Ｗｅｘ（ｎｍ）に相当する波長は式２より４８５ｎｍ、また、１／２次光を遮蔽可能な波長は式１より８１０ｎｍとなる。従って、Ｙ４４フィルタは、励起光波長が４８５〜８１０ｎｍの励起光波長の範囲において、吸収判定の式１および高透過判定の式２を同時に満たすため励起側フィルタ１５として利用できる。ここで、ＷＧ２９５フィルタとの組み合わせにおいては、当該フィルタの式１に基づく５２０ｎｍの励起光波長をＹ４４フィルタへの切替波長Ｗｅｘ２に設定することができる。

Assuming that the adjustment coefficient α is 7.5 nm in (Y44 filter), the wavelength corresponding to the excitation wavelength Wex (nm) is 485 nm according to Equation 2, and the wavelength capable of shielding the 1⁄2-order light is 810 nm according to Equation 1. It becomes. Therefore, the Y44 filter can be used as the excitation-side filter 15 because it simultaneously satisfies Equation 1 for absorption determination and Equation 2 for high transmission determination in the excitation light wavelength range of 485 to 810 nm. Here, in the combination with the WG 295 filter, the excitation light wavelength of 520 nm based on Equation 1 of the filter can be set as the switching wavelength Wex2 for switching to the Y44 filter.

ルタ（Ｙ５０フィルタ）にて調整係数αを７．５ｎｍとした場合、式２より５４５ｎｍ及び式１より９３０ｎｍとなり、励起光波長が５４５〜９３０ｎｍの励起光波長の範囲において励起側フィルタ１５として利用できる。そして、Ｙ４４フィルタとの組み合わせにおいては、当該フィルタの式１に基づいて８００ｎｍの励起光波長をＹ５０フィルタへの切替波長Ｗｅｘ３に設定することができる。

When the adjustment coefficient α is 7.5 nm in Luta (Y50 filter), it becomes 545 nm from Equation 2 and 930 nm from Equation 1, and can be used as the excitation side filter 15 in the excitation light wavelength range of 545 to 930 nm. . Then, in combination with the Y44 filter, the excitation light wavelength of 800 nm can be set to the switching wavelength Wex3 for switching to the Y50 filter based on Expression 1 of the filter.

From the above, the excitation side filterless wavelength range Aex0 is set as the excitation wavelength range from the smallest excitation wavelength settable by the spectrofluorimeter to the excitation side filter 1 switching wavelength Wex1, and the excitation side filter 15 is used as the excitation side filter pulse motor 13 Excitation side filter No. 0 (without excitation filter) is inserted on the optical system. The excitation-side filter 1 wavelength region Aex1 is in the excitation wavelength range from the excitation-side filter 1 switching wavelength Wex1 to the excitation-side filter 2 switching wavelength Wex2. 1 (WG 295 filter) is inserted on the optical system. The excitation-side filter 2 wavelength region Aex2 is in the excitation wavelength range from the excitation-side filter 2 switching wavelength Wex2 to the excitation-side filter 3 switching wavelength Wex3. 2 (Y44 filter) is inserted on the optical system. The excitation side filter 3 wavelength range Aex3 is a wavelength range from the excitation side filter 3 switching wavelength Wex3 to the longest excitation wavelength settable by the spectrofluorometer, and the excitation side filter 15 is the excitation side filter at the excitation side filter pulse motor 13 No. 3 (Y50 filter) is inserted on the optical system.
Next, fluorescence measurement will be described.

FIG. 6 is a conceptual view of the wavelength range used by the fluorescence filter in the present invention.
The dashed-dotted line Wrs extending obliquely upward to the right from the origin is the wavelength of the Rayleigh scattered light, the broken line Wre extending obliquely from the center to the upper right of the fluorescence wavelength axis is the wavelength corresponding to the secondary light of the Rayleigh scattered light, Wex12 on the excitation wavelength axis The reference numeral 13 denotes an excitation wavelength related to the switching determination of the fluorescence side filter 16, and Aem0 to Aem3 divided by the switching wavelength indicate the wavelength range of the fluorescence side filter 16 to be used. Here, the fluorescence side filter 16 is the same as the fluorescence side filter no. 0 (no fluorescence side filter) or fluorescence side filter No. 1 to No. The structure which is any of 3 is shown. Of these filters on the fluorescence side, an appropriate one is selected by determination described later, and the filter on the fluorescence side is inserted into the optical system between the sample to be measured and the spectroscope on the fluorescence side by the fluorescence side filter pulse motor 14.

  The fluorescence side filter 16 is selected according to the use wavelength and fluorescence wavelength of excitation light by the specification (type) of each filter. (1) Absorbing and shielding the wavelength range of the scattered light of excitation light (fluorescence side absorption determination), (2) A wavelength range transmitting fluorescence at a high ratio (fluorescence side high transmission determination The condition is to simultaneously satisfy the two judgments of. Hereinafter, the above two determinations of the fluorescence side filter 16 will be described in detail.

(1) Fluorescence-side absorption determination The selection of the fluorescence-side filter 16 in absorption determination is based on the condition that the following determination formula is satisfied. The absorption determination formula is as follows.
[Equation 3]

された励起波長Ｗｅｘ（ｎｍ）における蛍光側フィルタ１６の完全吸収される波長域を導くことが必要となる。As with the excitation-side filter 15, a sharp cut filter defined in JIS B7113 or the like can be used for the fluorescence-side filter 16. As the absorption limit wavelength described in JIS B7113 is not 0% (5%), as described for the excitation filter, scattered light of excitation light is transmitted and can not be applied for determination. Therefore, for the determination of the fluorescence side filter 16,

It is necessary to derive a wavelength range in which the fluorescence-side filter 16 is completely absorbed at the specified excitation wavelength Wex (nm).

なお、“β”は、装置間の２次光の裾の出現状況の差異を調整する係数であり、蛍光調整係数（例えば、０〜２０ｎｍ）である。(2) Determination of high-transmission on the fluorescence side When selecting the fluorescence-side filter 16 for transmission of fluorescence in high-transmission determination, it is assumed that the following determination formula is satisfied.
[Equation 4]

Note that “β” is a coefficient for adjusting the difference in appearance of secondary light tails between devices, and is a fluorescence adjustment coefficient (for example, 0 to 20 nm).

  Therefore, the cut filter satisfying Equation 4 has high transmittance of about 90% or more for sample-based fluorescence at the excitation wavelength Wex (nm), and can obtain a signal appearing in the corresponding region with low loss. become. Therefore, this determination leads to the high transmission wavelength range of the fluorescence-side filter 16 for sample-based fluorescence at the set excitation wavelength Wex.

  Based on such a concept, the cut filters used in the wavelength regions Aem0 to Aem3 of the fluorescence side filter of FIG. 6 are selected from the fluorescence side filters described in FIG. These also used what was marketed similarly to the excitation side filter. The specific selection will be described using the above two judgment formulas (Equations 3 and 4).

ルタを用い、蛍光調整係数βを７．５ｎｍとした場合、蛍光側フィルタ２切替判定のための励起波長Ｗｅｘ１２は２５０ｎｍに設定可能である。蛍光側フィルタ

を用い、調整係数αを７．５ｎｍとした場合、蛍光側フィルタ３切替判定のための励起波長Ｗｅｘ１３は３５０ｎｍに設定可能である。Based on the maximum excitation slit width of 20 nm of a common spectrofluorometer,

When the fluorescence adjustment coefficient β is set to 7.5 nm using a filter, the excitation wavelength Wex12 can be set to 250 nm for the fluorescence side filter 2 switching determination. Fluorescent side filter

If the adjustment coefficient α is set to 7.5 nm using the above, the excitation wavelength Wex 13 for the fluorescence side filter 3 switching determination can be set to 350 nm.

できる。The fluorescence side filter 1 switching determination wavelength Wem1 is determined by (excitation wavelength Wex (nm)-excitation slit width Sex (nm)) x 2-fluorescence adjustment coefficient β (nm). The fluorescence side filter 2 switching judgment wavelength Wem2 is a fluorescence wavelength corresponding to the intersection of the excitation wavelength Wex12 for the fluorescence side filter 2 switching judgment and the fluorescence side filter 1 switching judgment wavelength Wem1. The fluorescence side filter 2 switching determination wavelength Wem2 uses a filter which does not transmit fluorescence half of the longest fluorescence wavelength which can be set by the spectrofluorimeter. For example, when the maximum fluorescence wavelength is 750 nm, the fluorescence side filter No. Transparent as 2

it can.

  In the fluorescence side filterless wavelength region Aem 0, the fluorescence wavelength corresponds to a shorter wavelength than the fluorescence side filter 1 switching determination wavelength Wem 1, and the fluorescence side filter 16 controls the fluorescence side filter No. 0 (no fluorescence side filter) is inserted on the optical system. The fluorescence side filter 1 wavelength region Aem1 has a fluorescence wavelength longer than that of the fluorescence side filter 1 switching judgment wavelength Wem1, is a shorter wavelength than the fluorescence side filter 2 switching judgment wavelength Wem2, and the excitation wavelength is a spectrofluorimeter The fluorescence side filter 16 corresponds to the wavelength range of the excitation wavelength Wex 12 for determining the switching of the fluorescence side filter 2 from the smallest possible excitation wavelength, and the fluorescence side filter 16 uses the fluorescence side filter No. 1 is inserted on the optical system. The fluorescence side filter 2 wavelength region Aem2 has a fluorescence wavelength longer than that of the fluorescence side filter 1 switching judgment wavelength Wem1, and a wavelength longer than that of the fluorescence side filter 2 switching judgment wavelength Wem2, and the excitation wavelength is a spectrofluorimeter The fluorescence side filter 16 corresponds to the wavelength range of the excitation wavelength Wex 13 for determining the switching of the fluorescence side filter 3 from the smallest possible excitation wavelength. 2 is inserted on the optical system. In the fluorescence side filter three wavelength region Aem3, the fluorescence wavelength is longer than the fluorescence side filter 1 switching determination wavelength Wem1, and the excitation wavelength is in the wavelength range longer than the excitation wavelength Wex13 for the fluorescence side filter 3 switching determination. The fluorescence side filter 16 corresponds to the fluorescence side filter No. 1 by the fluorescence side filter pulse motor 14. 3 is inserted on the optical system.

  FIG. 7 shows a configuration example of the cut filter in the present invention. The excitation-side filter absorption determination (formula 1) and the excitation-side filter high transmission determination (formula 2) of the excitation-side filter 15, the fluorescence-side filter absorption determination (formula 3) of the fluorescence-side filter 16 and the fluorescence side filter high transmission determination (formula It is configured to satisfy the judgment condition of 4). The excitation-side filter absorption determination of the excitation-side filter 15 (formula 1) and the excitation-side filter high transmission determination (formula 2), the fluorescence-side filter absorption determination of the fluorescence side filter 16 (formula 3), and the fluorescence side filter high transmission If the judgment condition of the judgment (equation 4) is satisfied, it is not this limitation.

ィルタを例示する。このフィルタの透過限界波長λＴは４４０ ｎｍ、波長傾斜

ｎｍである。透過スペクトルを測定し、目的の波長域を遮蔽していることを確認する。The excitation side filter shown in FIG. 7 will be described. Excitation side filter No. Example 1 WG 295 filter is illustrated. The transmission limit wavelength λT of this filter is 295 n

The filter is illustrated. Transmission limit wavelength λT of this filter is 440 nm, wavelength tilt

nm. Measure the transmission spectrum and confirm that the target wavelength range is blocked.

４２フィルタを例示する。このフィルタの透過限界波長λＴは４２０ ｎｍ、波

は２５ ｎｍである。励起側フィルタと同様に透過スペクトルを測定し、目的の波

と５％に該当する波長の波長間隔で得る。透過限界波長λＴは、波長傾斜幅の中点に該当する波長で得る。
図８に本発明における３次元蛍光スペクトル測定のフローチャートを記す。Similarly, the fluorescence side filter shown in FIG. 7 will be described. Fluorescence side filter No. Example 1 WG 295 filter is illustrated. The transmission limit wavelength λT of this filter is 29

42 filters are illustrated. Transmission limit wavelength λT of this filter is 420 nm, wave

Is 25 nm. Measure the transmission spectrum in the same way as the excitation filter, and

And at wavelength intervals of wavelengths corresponding to 5%. The transmission limit wavelength λT is obtained at a wavelength corresponding to the middle point of the wavelength tilt width.
FIG. 8 shows a flowchart of three-dimensional fluorescence spectrum measurement in the present invention.

As step 501, from the operation panel 18, as a condition of the three-dimensional fluorescence spectrum measurement of the photometer unit 100, the excitation side measurement start wavelength Wexs, the excitation side measurement end wavelength Wexe (nm), the excitation data interval Dex (nm), the fluorescence side The measurement start wavelength Wems (nm), the fluorescence measurement end wavelength Weme (nm), the fluorescence data interval Dem (nm), the excitation slit width Sex (nm), and the fluorescence slit width Sem (nm) are input.

ｍ）、調整係数αｅｘｘとする。予め搭載された蛍光側フィルタ１６におけるＹ番目

蛍光調整係数βｅｍｙとする。入力されたフィルタ条件より、励起側遮断／透過波長および蛍光側遮断／透過波長が導かれ、Ａｅｘ１励起側フィルタ１波長領域Ａｅｘ２ 励起側フィルタ２波長領域、Ａｅｘ３ 励起側フィルタ３波長領域、Ａｅｍ０ 蛍光側フィルタ無し波長領域、Ａｅｍ１ 蛍光側フィルタ１波長領域、Ａｅｍ２ 蛍光側フィルタ２波長領域、Ａｅｍ３ 蛍光側フィルタ３波長領域が決定される。ここで、ステップ５０１とステップ５０２の順番は問わない。In step 502, filter conditions corresponding to the mounted filter are input. At this time, the filter condition is X in the excitation-side filter 15 mounted.

m) Let an adjustment coefficient α exx. Y-th in the fluorescence side filter 16 mounted in advance

The fluorescence adjustment coefficient is βemy. The excitation side cutoff / transmission wavelength and the fluorescence side cutoff / transmission wavelength are derived from the input filter conditions, Aex1 excitation side filter 1 wavelength range Aex2 excitation side filter 2 wavelength range, Aex3 excitation side filter 3 wavelength range, Aem0 fluorescence side A non-filtered wavelength region, Aem1 fluorescence side filter 1 wavelength region, Aem2 fluorescence side filter 2 wavelength region, Aem3 fluorescence side filter 3 wavelength region are determined. Here, the order of step 501 and step 502 does not matter.

  Step 503 is the collation of the wavelength range. Aem0 fluorescence side unfiltered wavelength region, Aem1 fluorescence side filter 1 wavelength region, Aem2 fluorescence side filter 2 wavelength region, Aem3 fluorescence side filter 3 wavelength region determined by the measurement wavelength range input in 501 and the input value in 502 After collating and 504 measurement start is executed, it moves to the judgment step of 505-512.

  When the measurement start which is step 504 is executed from the operation panel 18, as the determination 1 (step 505), the excitation side measurement start wavelength Wexs, the excitation side measurement end wavelength Wexe (nm), the fluorescence side measurement start wavelength Wems (nm It is determined whether the fluorescence side unfiltered wavelength region Aem0 is included in the measurement wavelength range of the three-dimensional fluorescence spectrum consisting of the fluorescence side measurement end wavelength Weme (nm). Here, in the case where the fluorescence side filterless wavelength region Aem0 is included in the measurement wavelength range of the three-dimensional fluorescence spectrum, the fluorescence side filter 16 controls the fluorescence side filter No. 1 by the fluorescence side filter pulse motor 14. 0 (no fluorescence side filter) is inserted on the optical system. A three-dimensional fluorescence spectrum of the entire measurement wavelength range of the measurement conditions input without the fluorescence side filter 16 is acquired (step 506). At this time, the excitation-side filter 15 switches the excitation-side filter 15 in accordance with the excitation wavelength based on the excitation-side cut filter use wavelength range in FIG. 5 to acquire a three-dimensional fluorescence spectrum. If the fluorescence side unfiltered wavelength region Aem0 is not included in the measurement wavelength range of the three-dimensional fluorescence spectrum, the process proceeds to the next determination.

  Next, as determination 2 (step 507), it is determined whether the fluorescence-side filter one wavelength region Aem1 is included in the measurement wavelength range of the three-dimensional fluorescence spectrum. When the fluorescence side filter 1 wavelength region Aem1 is included in the measurement wavelength range of the three-dimensional fluorescence spectrum, the fluorescence side filter No. 1 is inserted in the fluorescence side filter 16 by the fluorescence side filter pulse motor 14, and the three-dimensional fluorescence spectrum The three-dimensional fluorescence spectrum of the wavelength range in which the fluorescence side filter one wavelength region Aem1 is included in the measurement wavelength range is acquired. At this time, the excitation-side filter 15 switches the excitation-side filter 15 in accordance with the excitation wavelength based on the excitation-side cut filter use wavelength region in FIG. 5 to acquire a three-dimensional fluorescence spectrum (step 508). When the fluorescence side filter 1 wavelength region Aem1 is not included in the measurement wavelength range of the three-dimensional fluorescence spectrum, the process proceeds to the next determination.

  Next, as determination 3 (step 509), it is determined whether the fluorescence side filter two wavelength region Aem2 is included in the measurement wavelength range of the three-dimensional fluorescence spectrum. When the fluorescence side filter 2 wavelength region Aem2 is included in the measurement wavelength range of the three dimensional fluorescence spectrum, the fluorescence side filter No. 2 is inserted by the fluorescence side filter pulse motor 14 of the fluorescence side filter 16 and the three dimensional fluorescence spectrum A three-dimensional fluorescence spectrum (step 510) of the wavelength range in which the fluorescence side filter two wavelength region Aem2 is included in the measurement wavelength range is acquired. When the fluorescence side filter two wavelength region Aem2 is not included in the measurement wavelength range of the three-dimensional fluorescence spectrum, the process proceeds to the next determination.

  Next, as determination 4 (step 511), it is determined whether the fluorescence side filter three wavelength region Aem3 is included in the measurement wavelength range of the three-dimensional fluorescence spectrum. When the fluorescence side filter three wavelength region Aem3 is included in the measurement wavelength range of the three dimensional fluorescence spectrum, the fluorescence side filter No. 3 is inserted in the fluorescence side filter 16 by the fluorescence side filter pulse motor 14, and the three dimensional fluorescence spectrum A three-dimensional fluorescence spectrum (step 512) of the wavelength range in which the fluorescence side filter three wavelength region Aem3 is included in the measurement wavelength range is acquired. When the fluorescence side filter three wavelength region Aem3 is not included in the measurement wavelength range of the three-dimensional fluorescence spectrum, the process proceeds to the next step.

  In the next step (step 513), correction of the filter transmittance is performed. The three-dimensional fluorescence spectrum measured in steps 2 to 4 (steps 507 to 512) is used as the fluorescence side filter No. In the state in which 1 to 3 cut filters are inserted, a loss of fluorescence intensity occurs due to the absorption of the cut filters. Therefore, the reciprocal of the filter transmittance of each wavelength is multiplied by the fluorescence spectrum to correct the loss of the fluorescence intensity. Similarly, in the excitation side filter 15, the excitation side filter No. In the wavelength range measured by inserting one to three cut filters, the reciprocal of the filter transmittance of each wavelength of the excitation-side filter 15 is multiplied by the fluorescence spectrum to correct the loss of the fluorescence intensity.

In the next step (step 515), the three-dimensional fluorescence spectrum of each wavelength range acquired in determination 1 to determination 4 (steps 505 to 512) is synthesized, and a single three-dimensional fluorescence spectrum in the entire range of measurement wavelengths is obtained. It is taken as measurement data.
The effects of the present invention will be described.

Excitation side measurement start wavelength 200 nm, excitation side measurement end wavelength 750 nm, excitation wavelength interval 5 nm, fluorescence side measurement start wavelength 200 nm, fluorescence side measurement end wavelength 750 nm, fluorescence wavelength interval 5 nm, fluorescence side measurement end wavelength 750 nm and high-order light cut filter Assuming that the cross point of the switching wavelength 200 is the excitation wavelength of 400 nm, when the three-dimensional fluorescence spectrum is measured by the method of Patent Document 1 which is the conventional filter control method, the number of filter switching is (400−200) / 5 + 1 = 41 ( Times). Assuming that the stop time associated with the filter switching time is 3 seconds, it takes 123 seconds to switch the filter. When the entire wavelength range is measured at a wavelength scanning speed of 60,000 nm / min, the actual measurement time without filter switching is 235 seconds, and adding this to the filter switching time results in 358 seconds.

  In the case of the present invention, in the case of measuring the entire wavelength range without the fluorescence side filter of the determination 1 (step 505), the number of times of switching of the excitation side filter 15 is two. The filter switching time of 6 seconds is added to the actual measurement time of 235 seconds, and the measurement time without the fluorescence-side filter of determination 1 is 241 seconds.

Determination 2 to determination 4 (steps 507 to 512) in the fluorescence filter has an excitation measurement start wavelength of 200 nm, an excitation measurement end wavelength of 400 nm, an excitation wavelength interval of 5 nm, a fluorescence measurement start wavelength of 200 nm, and a fluorescence measurement end A fluorescence wavelength range longer than the fluorescence side filter 1 switching judgment wavelength Wem 1 is measured at a wavelength of 750 nm and a fluorescence wavelength interval of 5 nm. The number of times the filter is switched is one excitation filter and three fluorescence filters. When the filter switching time of 12 seconds is added to 44 seconds, the measurement time of determination 2 to determination 4 (steps 507 to 512) is 56 seconds. It becomes 297 seconds if the measurement time of the determination 1 (steps 505 and 506) and the determination 2 to the determination 4 (steps 507 to 512) is added. The conventional filter control method requires 358 seconds, but according to the present invention, the measurement time is reduced by 61 seconds and can be measured in 297 seconds.
Next, a second embodiment will be described with reference to FIGS.

  The fluorescence side filter No. 1 in the judgment 1 (step 505). Measurement with no fluorescence side filter at 0 (Step 506) and judgments from 2 to 4 for the fluorescence side filter No. When performing the fluorescence side filter presence measurement (steps 507 to 511) in 1 to 3, the fluorescence intensity may decrease due to the deterioration of the measurement sample by the excitation light. If data synthesis is performed in this state, a step occurs in the fluorescence intensity based on the result of measurement without a fluorescence filter (step 506) in judgment 1 (step 505) and the result of measurement with a fluorescence filter in judgments 2 to 4 (steps 507 to 512) .

  FIG. 9 shows a determination flowchart of three-dimensional fluorescence measurement in the second embodiment. In such a case, the step of the fluorescence intensity due to the deterioration of the measurement sample is eliminated by performing the intensity correction in the process (step 514) between the filter correction and the data synthesis.

10 and 11 show conceptual diagrams of fluorescence intensity correction. The two-dimensional fluorescence spectrum at the excitation wavelength Wex (nm) cut out from the three-dimensional fluorescence spectrum is described for conceptual explanation. At this time, the synthetic wavelength Wem3 shown in FIG. 10 is the fluorescence side filter 1 switching judgment wavelength Wem1 or the fluorescence side filter 1 switching judgment wavelength Wem2. When the synthetic wavelength Wem3 is the fluorescence side filter 1 switching judgment wavelength Wem1, the fluorescence side filter absence measurement (step 506) in the judgment 1 (step 505) at the synthetic wavelength Wem3 is on the short wavelength side, the fluorescence side in the judgment 2 to 4 The measurement data with filter (steps 507 to 512) is displayed in spectrum on the long wavelength side. When the synthetic wavelength Wem3 is the fluorescence side filter 2 switching judgment wavelength Wem2, the fluorescence wavelength filter present measurement in the judgment 2 (step 507) at the synthetic wavelength Wem3 (step 508) is on the short wavelength side in the judgment 3 (step 509) The fluorescence side filter presence measurement (step 510) data is spectrally displayed on the long wavelength side.

At this time, fluorescence intensity data Fs on the short wavelength side and fluorescence intensity data Fl on the long wavelength side exist in the synthetic wavelength Wem3. As shown in FIG. 10, when a step of the fluorescence intensity occurs, Fs / Fl is acquired as the fluorescence intensity correction value fc. The fluorescence intensity is corrected by multiplying the fluorescence intensity correction value fc by the fluorescence intensity obtained on the long wavelength side (FIG. 11 ). The fluorescence intensity correction value fc is acquired at each excitation wavelength Wex (nm) of the three-dimensional fluorescence spectrum, and the fluorescence intensity is corrected. Since the influence may differ depending on the measurement conditions and the measurement sample, the presence or absence of the fluorescence intensity correction sequence may be selected from software.
A third embodiment will now be described.

In Example 1, as the determination 2 (step 507), when the fluorescence side filter 1 wavelength range Aem1 is included in the measurement wavelength range of the three-dimensional fluorescence spectrum, the fluorescence side filter No1 is inserted, and 3 of the wavelength range At this time, the fluorescence side measurement start wavelength Wems (nm) in the fluorescence side filter 1 wavelength region Aem1 is determined as the measurement start wavelength and measurement end wavelength that the analyst intends to set, and the fluorescence side filter switching The magnitudes of the wavelengths are compared to determine the actual fluorescence-side measurement start wavelength in steps 507, 509, and 511.
Wems W Wem1
Wems: Fluorescence start measurement wavelength nm
Wem1: Fluorescence side filter 1 switching judgment wavelength nm
In this case, the fluorescence side measurement start wavelength Wems (nm) set by the analyst is selected.
Wems <Wem1
In the case of the above, the determination wavelength Wem1 is set as the fluorescence side measurement start wavelength.
Similarly, the fluorescence measurement end wavelength Weme (nm) is
Weme W Wem 2
In the case of, it becomes Wem2 (nm). Weme <Wem2
In the case of, the fluorescence side measurement start wavelength Weme (nm) set by the analyzer is obtained.
By applying the determination wavelength Wem1 to the fluorescence side measurement start wavelength Wems (nm), the measurement time can be shortened.

  Here, the fluorescence side measurement start wavelength Wems (nm) may not be applied to the fluorescence side measurement start wavelength Wems (nm), and the fluorescence side measurement start wavelength Wems (nm) set by the analyst may be applied.

If the measurement wavelength range is different, mechanical wavelength error derived from the fluorescence side spectroscope 7 may occur. By applying the fluorescence side measurement start wavelength Wems (nm) set by the analyst without applying the fluorescence side filter 1 switching judgment wavelength Wem1 to the fluorescence side measurement start wavelength Wems (nm), the excitation wavelength Wex (nm) The mechanical wavelength accuracy can be kept constant because the fluorescence wavelength range is always constant. As a result, errors in data synthesis can be reduced. The same applies to determination 3 (step 509) and determination 4 (step 511). In the case of determination 3 (step 509), Wems (nm) set to the fluorescence-side measurement start wavelength is applied according to the following two equations.
Wems <Wem1
Wems <Wem2

  The setting of the application value of the fluorescence-side measurement start wavelength in the first embodiment and the third embodiment may be selected from software because the influence may differ depending on the measurement conditions and the measurement sample.

As described above, the technical scope of the present invention is not limited to the above embodiment, and various modifications can be made without departing from the scope of the present invention.
For example, in the embodiment of the present application, an example in which three types are provided for each of the excitation-side cut filter and the fluorescence-side cut filter has been shown, but more may be provided.

1 light source 2 excitation side spectroscope 3 beam splitter 4 monitor detector 5 sample setting part (cell)
6 measurement sample 7 fluorescence side spectroscope 8 detector 9 A / D converter 10 computer 11 fluorescence side pulse motor 12 excitation side pulse motor 13 excitation side filter pulse motor 14 fluorescence side filter pulse motor 15 excitation side filter 16 fluorescence side filter 17 Monitor 18 Operation panel 100 Photometer unit 101 Data processing unit 102 Interface Wrs Rayleigh scattering wavelength Wre Wavelength equivalent to secondary light of Rayleigh scattered light Wex1 Excitation filter 1 switching wavelength Wex2 Excitation filter 2 switching wavelength Wex3 Excitation filter 3 switching Wavelength Wem1 Fluorescence side filter 1 switching judgment wavelength Wem2 Fluorescence side filter 2 switching judgment wavelength Aex0 Excitation side filter without wavelength range Aex1 Excitation side filter 1 wavelength range Aex2 Excitation side filter 2 wavelength range Aex3 Excitation side filter 3 wavelength range Aem0 Fluorescence side filterless wavelength range Aem1 Fluorescence side filter 1 wavelength range Aem2 Fluorescence side filter 2 wavelength range Aem3 Fluorescence side filter 3 wavelength range Wex12 Excitation wavelength Wex13 for fluorescence side filter 2 switching judgment Excitation wavelength Wex13 for fluorescence side filter 3 switching judgment Excitation wavelength Wem3 synthetic wavelength

Claims (6)

A light source of excitation light to be irradiated to a sample to be measured, and a sample setting unit for setting the sample;
An excitation-side spectroscope that splits the light of the light source into the excitation light;
A plurality of excitation side filters for blocking high order light based on the excitation light;
(1) Absorb and block the wavelength region of half-order light of excitation light from the plurality of excitation side filters according to measurement conditions (absorption determination) (2) a wavelength region of high transmittance ((1) Excitation-side filter selection means for selecting the excitation-side filter simultaneously satisfying the two determinations (high transmission determination) and excitation-side filter movement for arranging the selected excitation side filter in the optical system between the sample and the light source Mechanism,
A fluorescence side spectroscope that disperses fluorescence emitted from the sample by the irradiation of the excitation light;
A plurality of fluorescent filters that block high-order light based on the fluorescence;
According to the measurement conditions, (1) absorbing and shielding the wavelength range of the scattered light of the excitation light from the plurality of fluorescence side filters (fluorescence side absorption determination), (2) a wavelength range transmitting the fluorescence at a high ratio it is placed in the optical system between the (fluorescent side high transmittance determination), the fluorescence-side filter and the selected selection means for selecting simultaneously satisfy the fluorescence side filter two determined and the sample of the fluorescence side spectroscope unit Fluorescence side filter moving mechanism,
A detector for detecting fluorescence after passing through the fluorescence side filter;
Equipped with
What is claimed is: 1. A spectrofluorimetric analyzer that suppresses the effects of high-order light based on the excitation light and high-order light based on the fluorescence. The range of the wavelength by the said excitation side spectroscope is 200-750 nm, The spectrofluorescent analyzer of Claim 1 provided with three types of said excitation side filters. The spectral fluorescence analyzer according to claim 1 or 2, wherein the wavelength range of the fluorescence side spectroscope is 200 to 750 nm, and three types of the fluorescence side filters are provided. In the method of acquiring a three-dimensional fluorescence spectrum using the spectrofluorimeter according to any one of claims 1 to 3.
A measurement condition setting step for specifying conditions related to excitation light and fluorescence;
A filter condition setting step of specifying the conditions of a plurality of filters on the excitation side and the fluorescence side provided in the apparatus;
A filter group comprising a combination of the excitation-side and the fluorescence-side filters based on calculations from the measurement condition setting step and the filter condition setting step, a region determined by the wavelength range of the excitation light and the wavelength range of the fluorescence And the range that can be measured by the filter are divided into a plurality of regions as one region, and the excitation light wavelength and the fluorescence wavelength related to the boundary of each region are respectively set as the excitation side filter switching wavelength and the fluorescence side filter switching wavelength Matching step of the wavelength range to be recognized;
A determination step of determining whether or not the area is included in the range from the measurement start wavelength to the measurement end wavelength on the excitation side and the fluorescence side for each of the plurality of divided areas;
Measuring the area according to the determining step using the filter group determined by the comparing step;
A filter correction step of correcting the transmittance based on the fluorescence intensity value of the filter used for the data acquired in the measurement step;
A data combining step of combining data of the respective areas after the correction in the correction step to obtain data of the entire measurement range;
A method of acquiring a three-dimensional fluorescence spectrum using a spectrofluorimeter comprising: The three-dimensional fluorescence spectrum acquisition method according to claim 4, characterized in that the reduction of the fluorescence intensity generated in the process of the filter correction step and the data synthesis step is intensity corrected.
How to obtain 3D fluorescence spectrum. The filter condition setting step according to claim 4 compares the measurement start wavelength and the measurement end wavelength arbitrarily selected with the fluorescence side filter switching wavelength, and measures the measurement start wavelength and the measurement end wavelength on the fluorescence side. decide,
How to obtain 3D fluorescence spectrum.

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