JP2012058047A - Fluorospectro-photometer, measuring method for fluorospectro-photometer, and sample cell switch device - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a fluorospectro-photometer for reducing influences by self-absorption without diluting a sample in fluorometry in a transmission direction of a high concentration sample where the influences by the self-absorption are problematic.SOLUTION: The fluorescence spectrum in a reflection direction is measured in processing 1 and peak wavelength is detected in decision 1. When there is peak wavelength, on the ground that the sample has fluoresncence, identification wavelength is calculated from the fluorescence spectrum in the reflection direction obtained in the processing 1 in processing 2. Next, in processing 3, a transmission spectrum when an optical path length is changed is measured, and a cell having optical path length appropriate for measuring the fluorescence spectrum in the transmission direction is decided in decision 2. Finally, in processing 4, the fluorescence spectrum in the transmission direction in the cell having the optical path length accepted in the decision 2 is measured.

Description

  The present invention relates to a spectrofluorometer, and more particularly to measurement of a fluorescence spectrum in the transmission direction of a solution sample, and further to a sample cell switching device.

  The spectrofluorometer is composed of a photometer unit, a data processing unit, and an operation / processing unit. A measurement sample is irradiated with continuous light from a light source as excitation light separated by an excitation-side spectroscope. The fluorescence emitted from the sample is split into monochromatic light by the fluorescence side spectrograph, and the electrical signal detected by the detector is captured as signal intensity by the computer via the analog-digital converter, and the measurement result is displayed on the display. Is displayed.

  Measure the excitation light from the excitation-side spectrometer set to the fixed wavelength for the fluorescence spectrum that measures the fluorescence intensity for each wavelength when the measurement sample is irradiated with the excitation light of the fixed wavelength and the fluorescence wavelength is changed. The sample is irradiated, and the fluorescence at that time is changed from the measurement start wavelength to the measurement end wavelength by the fluorescence side spectrograph, and the electrical signal detected by the detector at each wavelength is signaled to the computer via the analog-digital converter. The intensity is taken in, and the measurement result is displayed on the display as a two-dimensional spectrum of fluorescence wavelength and fluorescence intensity.

  Here, since fluorescence appears on a longer wavelength side than the excitation wavelength as a wavelength different from that of the excitation light, transmitted light, reflected light, and scattered light resulting from the excitation light become unnecessary light. Therefore, for example, as shown in Non-Patent Document 1, in a solution sample measurement of a general fluorometer, a sample is put in a cell having four surfaces that are transparent and in a direction of 90 degrees with respect to excitation light. A detection optical system is installed to detect lateral fluorescence at a position that minimizes transmitted light, reflected light, and scattered light of excitation light. This sample placement method can obtain the relationship between the sample concentration and the fluorescence intensity as long as the sample is in a low concentration region having an absorbance of 0.5 or less at the excitation wavelength.

  However, since a general fluorometer detects fluorescence emitted from the central portion of the sample cell, in a high concentration region where the absorbance is 0.5 or more, the light emitting portion is irradiated with excitation light more than the central portion of the sample cell. Since an internal shielding effect is generated near the surface, an error occurs in the relationship between the sample concentration and the fluorescence intensity.

  Therefore, the fluorescence in the reflection direction has been detected by installing the sample cell tilted with respect to the excitation light. This fluorescence detection in the reflection direction is a useful method for measuring a high concentration sample that does not transmit excitation light. However, when measuring a low-concentration sample that transmits excitation light, the influence of reflected light and scattered light due to the excitation light is greater than the detection position of the side fluorescence, and thus it is not often used.

  On the other hand, the fluorescence in the transmission direction is also measured by folding the excitation light in a 90-degree direction with a reflecting plate such as a mirror and installing a sample cell between the reflecting plate and the detection optical system. In the fluorescence detection in this transmission direction, the influence of transmitted light and scattered light caused by excitation light is larger than the detection position of side fluorescence, and when measuring a high concentration sample that does not transmit excitation light, Since the fluorescence in the transmission direction is absorbed by the sample itself during the transmission process, the fluorescence spectrum shape changes depending on the sample concentration and the absorption spectrum, so that it is rarely used.

  However, the fluorescence in the transmission direction is important measurement data for grasping the total amount of fluorescence emitted by the sample. The fluorescence of the solution sample appears radially in all directions, but in a general fluorescence measurement, the relative fluorescence intensity in only a normal direction in the side, reflection, and transmission directions in a cell with four transparent surfaces This is because the spectral distribution is measured.

  Within the same measurement system, it is possible to compare samples by measuring only in a certain normal direction for the purpose of grasping relative fluorescence intensity and fluorescence spectrum shape. However, it is impossible with this measurement system to measure the total amount of fluorescence that appears radially in all directions.

  In this case, an integrating sphere is used for setting the sample. The integrating sphere is coated with a white material having a high reflectivity on the inner surface, and the light from the sample is diffusely reflected and has the effect of making the light generated in all directions uniform and leading it to the detection optical system. For example, as described in Patent Document 1, when measuring the fluorescence of a solid sample such as a powder or a substrate, the sample is placed at the position of the exit port of the integrating sphere, and the influence of the unevenness of the sample surface unique to the solid sample is affected. The direction dependency of fluorescence generated by the above can be made uniform and guided to the detection optical system.

  However, in the case of a solution sample, since excitation light is transmitted, fluorescence in the transmission direction is generated, and thus there is a problem that it is difficult to accurately measure transmitted fluorescence.

JP 2008-70172 A

Kinoshita and Mihashi, “The Spectroscopical Society of Japan Series 3 Fluorescence Measurement-Application to Biological Sciences” (.45-63), Academic Publishing Center (published first edition on January 20, 1983)

  As described above, in the measurement of the fluorescence spectrum in the transmission direction, when measuring a high-concentration sample that does not transmit excitation light, the fluorescence in the transmission direction is self-absorbed by the sample itself during the transmission process. Varies with sample concentration and absorption spectrum. In order to solve this, there is a method to dilute the sample and prepare a concentration with less influence of self-absorption. However, it takes time and effort to dilute, and by diluting, the fluorescence intensity decreases and the measurement accuracy decreases. There is a problem that the fluorescence spectrum at the target concentration cannot be evaluated.

  In the fluorescence measurement in the transmission direction of a high-concentration sample in which the influence of self-absorption is a problem, the present invention aims at the transmission direction in which the influence of self-absorption can be reduced without diluting the sample. It is an object of the present invention to provide a spectrofluorometer that obtains a fluorescence spectrum and a measuring method thereof.

  Another object of the present invention is to provide a measurement method for comprehensively grasping a three-dimensional fluorescence spectrum in the transmission direction, and a sample cell switching device and the like that can switch a sample cell with a simple mechanism.

  A feature of the present invention is that, in a spectrofluorometer equipped with a cell switching device in which a plurality of cells having different optical path lengths from an integrating sphere are installed in a sample installation portion, a first process for measuring a fluorescence spectrum in the reflection direction; First determination to detect the peak wavelength based on the fluorescence spectrum data in the reflection direction obtained in the first process, and the peak wavelength from the fluorescence spectrum data in the reflection direction obtained in the first process A second process for calculating an identification wavelength shorter than a fluorescence peak having a fluorescence intensity multiplied by an arbitrary coefficient, and a third process for measuring a transmission spectrum in a plurality of optical path lengths by a cell switching device. A second determination is made as to whether or not the transmittance at the processing identification wavelength obtained in the second process is equal to or greater than an arbitrary value in the cell having the corresponding optical path length from the transmission spectrum obtained in the process and the third process. Judgment and judgment In the fourth processing mechanism for measuring the fluorescence spectrum of the transmission direction in a cell having an optical path length that pass, further, the procedure of the operation.

  Another feature of the present invention is that three-dimensional fluorescence is obtained by measuring fluorescence spectra in the transmission direction in a plurality of cells having different optical path lengths and displaying the fluorescence wavelength on the X axis, the optical path length on the Y axis, and the fluorescence intensity on the Z axis. The spectrum measurement and the mechanism for switching the sample cell, etc., are described in detail in the embodiments of the invention described below.

  According to the present invention, in the fluorescence measurement in the transmission direction of a high-concentration sample in which the influence of self-absorption occurs, it is possible to measure the fluorescence spectrum with reduced influence of self-absorption without diluting the sample. Therefore, a suitable sample cell switching device or the like can be realized with a simple mechanism.

The block diagram of the spectrofluorometer which concerns on 1st Example of this invention The block diagram which shows the detail of the cell switching apparatus of FIG. The block diagram which shows the other example of the cell switching apparatus of FIG. Flow chart of fluorescence spectrum measurement in the transmission direction according to this example The block diagram which shows the structure of the fluorescence spectrum to the reflection direction which concerns on a present Example The graph which shows an example of the fluorescence spectrum to the reflection direction of a present Example The graph which shows an example of the transmission spectrum of a present Example The graph which shows an example of the three-dimensional transmission spectrum of a present Example The graph which shows an example which measured the fluorescence spectrum to the permeation | transmission direction of a present Example The graph which shows an example which measured the fluorescence spectrum to the permeation | transmission direction of a present Example The graph which shows an example of the three-dimensional fluorescence spectrum which concerns on 2nd Example of this invention Flow chart for carrying out the three-dimensional fluorescence spectrum of this example Configuration diagram of a penta-arranged sample cell switching device according to a third embodiment of the present invention Configuration diagram of belt-type sample cell switching device of this embodiment

  Hereinafter, embodiments of the present invention will be described in detail with reference to the drawings.

FIG. 1 shows a configuration diagram of a spectrofluorometer according to a first embodiment of the present invention.
First, basic components of the spectrofluorometer will be described. As shown in FIG. 1, the spectrofluorometer 100 includes a photometer unit 110, a data processing unit 120, and an operation / display unit 130. Further, the photometer unit 110 is roughly divided into an excitation side optical system 111, a sample chamber 112, a fluorescence side optical system 113, and a drive unit 114. Here, the arrows indicate the flow of light delivery, and the solid line indicates the connection of electrical signals. The continuous light generated from the light source 1 is split as excitation light by the excitation-side spectroscope 2 and irradiated through the beam splitter 3 to the measurement sample 6 placed in the transmission fluorescence sample cell 16.

  At this time, a part of the excitation light split by the beam splitter 3 is measured by the monitor detector 4 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, is taken in as a signal intensity by the computer 10 through the analog-digital converter 9, and is displayed on the display 13. The measurement result is displayed at.

  Next, the wavelength driving system will be described. The excitation-side spectroscope 2 is set at a target wavelength position by driving the excitation-side pulse motor 12 in accordance with a command from the computer 10. Further, the fluorescence side spectroscope 7 is set at a target wavelength position by driving the fluorescence side pulse motor 11 in accordance with a command from the computer 10. Optical elements such as a diffraction grating and a prism are used for the excitation side spectroscope 2 and the fluorescence side spectroscope 7, and the excitation side pulse motor 12 and the fluorescence side pulse motor 11 are used as power, and they are driven by a gear and a cam. The spectrum is scanned by rotating it.

  In such a basic configuration, a feature of the present embodiment is a configuration for detecting transmitted fluorescence. Regarding the transmitted fluorescence for transmitted fluorescence, since the fluorescence generated on the back surface is generated in all directions with respect to the back surface direction, the integrating sphere 15 is used in the sample chamber 112. The sample 6 is placed in a transmission fluorescence sample cell 16 having an area larger than the opening of the integrating sphere 15 in order to efficiently cause the fluorescence transmitted through the back surface to enter the integrating sphere. The transmission fluorescence sample cell 16 is installed in a sample cell switching device 18 in which a plurality of sample cells having different optical path lengths are installed on a disk.

  FIG. 2 is a configuration diagram showing the configuration of the cell switching device 18, in which FIG. 2 (A) shows a front view and FIG. 2 (B) shows a side view. The sample cell switching device 18 in the present embodiment has a function of selecting an arbitrary cell by rotating the cell switching device by the sample cell switching pulse motor 19 through the computer 10.

  The sample cell switching device of FIG. 2 has six cell installation bases from cell bases 51 to 56. The number of cell stands is determined by the size of the sample chamber 112. In this example, six cell mounts are installed. Here, the optical path length of the transmission fluorescence sample cell 16 is registered in the computer 10 in advance.

  The transmitted fluorescence sample cell 16 needs to be in close contact with the opening of the integrating sphere 15 in order to efficiently enter the fluorescence into the integrating sphere 15. Therefore, the sample cell switching device 18 has a function of moving the cell switching device 18 through the computer 10 by the position adjusting pulse motor 20 and the position adjusting turret rotating shaft 21 so that the transmitted fluorescence sample cell 16 is closely attached to the opening of the integrating sphere. Have A distance L (mm) is adjusted to adjust the transmission fluorescence sample cell 16 in close contact.

  Further, the distance O (mm) is registered in advance in the computer as the origin position of the cell switching device 18 shown in FIG. At this time, when the optical path length of the transmission fluorescence sample cell 16 is D (mm), the distance L (mm) is a value obtained by subtracting the optical path length D (mm) from the distance O (mm) at the origin position.

  For example, when the distance O between the opening of the integrating sphere 15 and the origin position is 20 mm and the optical path length D of the transmission fluorescence sample cell 16 is 5 mm, the distance L to be adjusted is 15 mm. By obtaining this distance L, it is possible to avoid breakage of the transmission fluorescence sample cell 16 from an excessive load.

  Further, when the distance between the transmission fluorescence sample cell 16 and the cell switching device 18 is not sufficient, the integrating sphere 15 and the transmission fluorescence sample cell 16 come into physical contact with each other by the rotation of the cell switching device 18, and the transmission fluorescence sample cell 16. Therefore, when the cell switching device 18 is rotated, the cell switching device 18 is once returned to the original position and then rotated.

  FIG. 3 is a configuration diagram when the cell switching device 18 is installed in an opening that is a pair of positions with respect to the opening into which excitation light is incident. FIG. 3 (A) is a front view, and FIG. 3 (B). Shows a side view. When measuring the fluorescence spectrum in the reflection direction, the cell switching device 18 is provided with a reflection fluorescence sample cell 17 containing the sample 6 and a reflection fluorescence sample cell 17 containing a blank solvent, which are switched arbitrarily. Used to measure.

  FIG. 4 is a flowchart in the fluorescence spectrum measurement in the transmission direction according to the present embodiment. Fluorescence always appears longer than the excitation wavelength due to Stokes' law. This indicates that the absorption wavelength of the sample is present at a wavelength shorter than the fluorescence wavelength, and the transmission spectrum on the long wavelength side is close to 100% transmittance as long as there is no influence of impurities. That is, if the wavelength at which absorption is started is found by changing the optical path length, there will be no influence of absorption on the longer wavelength side, leading to accurate measurement of the fluorescence spectrum in the transmission direction.

  FIG. 4 is a flowchart for realizing this, and each process and determination contents are as follows. Process 1 is measurement of the fluorescence spectrum in the reflection direction. Judgment 1 is detection of the peak wavelength from the fluorescence spectrum in the reflection direction obtained in Process 1. If there is a peak wavelength, the sample is assumed to have fluorescence, and the process proceeds to process 2. If there is no peak wavelength, the sequence ends, assuming that the sample has no fluorescence. Process 2 is a process for calculating the identification wavelength λrf from the fluorescence spectrum in the reflection direction obtained in Process 1.

  Process 3 is measurement of the transmission spectrum when the optical path length is changed. Determination 2 is determination of a cell having an optical path length suitable for measuring a fluorescence spectrum in the transmission direction. Process 4 is the measurement of the fluorescence spectrum in the transmission direction in the cell having the optical path length passed in the determination 2.

Hereinafter, details of each process and determination contents will be described with reference to the drawings.
First, processing 1 will be described in detail. Process 1 is measurement of the fluorescence spectrum in the reflection direction. In the process 1, the measurement of the sample 6 and the measurement of the blank solvent of the sample 6 are performed.
FIG. 5 is a configuration diagram showing the configuration of the fluorescence spectrum in the reflection direction according to the present embodiment. When measuring the fluorescence spectrum in the reflection direction of the process 1, the cell switching device 18 is provided in the integrating sphere 15 installed in the sample chamber 112 at the opening that is paired with the opening through which the excitation light is incident. The sample cell 17 for reflection fluorescence which put the sample 6 or the blank solvent in is installed.

  FIG. 6 is a graph showing an example of the fluorescence spectrum in the reflection direction. The vertical axis represents relative fluorescence intensity, and the horizontal axis represents wavelength (nm).

  The determination 1 will be described in detail. Judgment 1 is peak detection from the fluorescence spectrum in the reflection direction obtained in the process 1. Here, when the fluorescence intensity Frf at the peak top of the fluorescence spectrum in the reflection direction of the sample 6 is set to be the fluorescence intensity Fbkg of the same wavelength of the blank solvent, the fluorescence intensity Frf is sufficiently larger than the fluorescence intensity Fbkg. There are requirements. In general, it is considered that a peak is obtained when the fluorescence intensity of the sample 6 is three times or more than the standard deviation σbkg of the multiple measurements of the fluorescence intensity Fbkg. Ideally, it is desirable that the fluorescence intensity is 10 times or more.

  Here, assuming that the coefficient for peak determination is k, the wavelength having the fluorescence intensity Frf larger than the fluorescence intensity Fbkg × k is regarded as the peak wavelength, and when there is a plurality, the wavelength with the highest fluorescence intensity is the fluorescence in the reflection direction. The peak of the spectrum is the fluorescence wavelength wavelength λft. If this fluorescence wavelength λft exists, the determination is passed and the process proceeds to the next process 2. On the other hand, if this fluorescence wavelength λft does not exist, the fluorescence from the sample is not confirmed, so the determination is rejected and the sequence ends.

  Processing 2 will be described. The fluorescence wavelength having the fluorescence intensity of Frf × α obtained by multiplying the fluorescence intensity Frf at the peak fluorescence wavelength λft of the fluorescence spectrum in the reflection direction confirmed in the determination 1 by the sensitivity coefficient α is identified as the identification wavelength λrf. And Here, the sensitivity coefficient α is a positive number smaller than 1. The identification wavelength λrf is searched from the peak top fluorescence wavelength λft to the short wavelength side, and is the wavelength at which the value of Frf × α initially matches the fluorescence spectrum data. This is because, when searching from the short wavelength side where the measurement is started, in the case of a spectrum with weak fluorescence intensity and large noise, a region where no fluorescence is generated is set as the identification wavelength λrf to prevent an erroneous result.

  By searching from the peak top fluorescence wavelength λft to the short wavelength side, it is possible to recognize the rising wavelength of the fluorescence spectrum in the reflection direction with high accuracy and less influence of the above-mentioned misidentification. The sensitivity coefficient α is preferably set in the range of about 0.1 to 0.01 for the purpose of recognizing the rising wavelength of the fluorescence spectrum in the reflection direction. For example, if the sensitivity coefficient α = 0.1, the wavelength of the fluorescence intensity that is 0.1 times the peak-top fluorescence intensity Frf is obtained as the identification wavelength λrf.

  Processing 3 will be described. Process 3 is a process of measuring transmission spectra in a plurality of cells having different optical path lengths. The transmission spectrum is obtained from a synchronous spectrum that scans the excitation side spectroscope 2 and the fluorescence side spectroscope 7 at the same wavelength. First, cells having respective optical path lengths in which the blank solvent of the sample 6 is put in the cell switching device 18 are installed, and a base line with the light amount for each wavelength as 100% transmittance is set to the apparatus for each cell having the respective optical path lengths. Remember. For example, a long optical path cell having an optical path length of about 50 mm can be applied to the cell. However, when using a general optical path length of 10 mm or less and using the sample cell switching device 18, six cells can be installed. It is desirable to select the optical path length of the cell at intervals of about 0.5 mm, 1 mm, 3 mm, 5 mm, 7 mm, and 10 mm. Next, a cell of each optical path length in which the sample 6 is put in the cell switching device is installed, and the transmission spectrum is obtained by dividing the light amount for each wavelength by the light amount of the baseline.

  FIG. 7 is a graph showing an example of a transmission spectrum. The vertical axis represents transmittance (%), and the horizontal axis represents wavelength (nm). The spectra obtained at the respective optical path lengths are overlaid and displayed as (1) to (6).

  The determination 2 will be described. Determination 2 is determination of the optical path length γ suitable for measuring the fluorescence spectrum in the transmission direction. The optical path length γ is determined by the transmittance at the identification wavelength λrf obtained in the process 2. This transmittance is the identification transmittance β. If the transmission spectrum obtained with each optical path length is a determination target and the identification transmittance at the identification wavelength λrf is equal to or greater than β%, the determination is acceptable and the process proceeds to the next process 4. On the other hand, if the identification transmittance is β% or less, it means that light does not transmit in the region where fluorescence occurs, and the fluorescence in the transmission direction may be absorbed by the sample itself, so the determination is rejected. The sequence in the cell having the corresponding optical path length ends. The optical path length γ at this time is an optical path length suitable for measuring the fluorescence spectrum in the transmission direction. For example, in FIG. 7, the optical path lengths (1) and (2) correspond. The identification transmittance β is expressed as a percentage (%) and is a numerical value of 0 to 100. It is desirable that β is a numerical value of 80 to 100 in order to grasp the rate of absorption of the sample.

  FIG. 8 is a graph showing an example of a three-dimensional transmission spectrum. The three-dimensional transmission spectrum is drawn with a contour map or a bird's eye view. Here, the obtained transmission spectrum is obtained by three events of transmittance (%), wavelength (nm), and optical path length (mm), and can be displayed three-dimensionally. The X axis indicates the wavelength (nm), the Y axis indicates the optical path length (mm), and the Z axis indicates the transmittance (%). Here, in FIG. 6, the measurement value having the same transmittance is drawn with contour lines on the X axis and the Y axis as the Z axis. By displaying in three dimensions, the optimum optical path length γ can be obtained from the intersection of the identification wavelength λrf and the identification wavelength β%, and the information can be captured visually. At this time, when the optical path length of the cell is changed at intervals of about 0.5 mm, 1 mm, 3 mm, 5 mm, 7 mm, and 10 mm, for example, by interpolating the data between the optical path lengths, The transmission spectrum of the optical path length not actually acquired can be grasped by calculation. From these points, the three-dimensional transmission spectrum when the optical path length is changed is useful for obtaining the optimum optical path length γ in detail.

  Processing 4 will be described. Process 4 is the measurement of the fluorescence spectrum in the transmission direction in the cell having the optical path length γ that passed in the determination 2.

  FIG. 9 is a graph showing an example of measuring the fluorescence spectrum in the transmission direction. As shown in FIG. 7, by using a cell having an optical path length γ, it is possible to obtain a fluorescence spectrum in the transmission direction without being affected by self-absorption. If the optical path length γ is extremely small, there is a concern that the amount of fluorescence from the sample will be insufficient and the spectrum will have a large noise. Therefore, it is not necessary to use a cell with a short optical path length.

  FIG. 10 is a graph showing an example of measuring the fluorescence spectrum in the transmission direction. It is an example at the time of using the cell of the optical path length which became the pass and failure in the determination 2. As shown in FIG. 10, when a cell having an optical path length longer than γ is used, the short wavelength side of the fluorescence peak is self-absorbed, so that the spectrum shape is shifted to the long wavelength side.

  As described above, according to the present embodiment, by performing these processes and determinations, it is possible to measure without diluting the sample when measuring the fluorescence spectrum in the transmission direction.

As a second embodiment of the present invention, an example in which a process 5 is added between the process 3 and the determination 2 will be described.
Processing 5 will be described. Process 5 measures the fluorescence spectrum in the transmission direction by changing the optical path length in the sample cell switching device 18, the fluorescence wavelength (nm) on the X axis, the optical path length (mm) on the Y axis, and the fluorescence on the Z axis. It is a three-dimensional fluorescence spectrum measurement in the transmission direction showing intensity (arbitrary unit).

  FIG. 11 is a graph showing an example of a three-dimensional fluorescence spectrum when the optical path length is changed. The three-dimensional fluorescence spectrum is drawn with a contour map or a bird's eye view. When the fluorescence intensity differs for each optical path length, standardization of the peak top facilitates comparison even at different intensities.

  FIG. 12 is a flowchart for implementing a three-dimensional fluorescence spectrum when the optical path length is changed. A process 5 is added between the process 3 and the determination 2. The determination 2 is executed after the processing 5. At this time, attention is paid to the optical path length γmm derived in the determination 2 on the three-dimensional fluorescence spectrum. For example, if the optical path length is γmm or less in a three-dimensional fluorescence spectrum, contour lines at regular intervals where the spectrum rises from the identification wavelength λrfnm can be obtained. On the other hand, when the optical path length is longer than the optical path length γmm, the spectrum absorbs fluorescence having a short wavelength, so that the rise of the spectrum shifts to a longer wavelength than the identification wavelength λrf nm.

  Note that there may be a plurality of cells having the corresponding optical path length if the optical path length is γ or less. However, there is no measurement problem because the spectrum shape does not change in the fluorescence spectrum measurement in the transmission direction. . In addition, if the optical path length γmm is extremely small, the amount of fluorescence from the sample may be insufficient, resulting in a spectrum with a large noise. Therefore, evaluate the noise from the three-dimensional fluorescence spectrum and find the optimum optical path length. You can also.

  This three-dimensional fluorescence spectrum in the transmission direction is also useful for confirming the validity of the identification wavelength λrf nm and the optical path length γmm, and it is possible to comprehensively grasp changes in the fluorescence spectrum for each optical path length. .

Another example of the cell switching device 18 is shown as a third embodiment of the present invention.
FIG. 13 is a configuration diagram showing the configuration of the penta-arranged sample cell switching device 22, and FIG. 13 (A) shows a front view and FIG. 13 (B) shows a side view. There are a total of four openings of the integrating sphere 15 as the excitation light entrance and its counter electrode, and the fluorescence exit and its counter electrode. Among these, the excitation light entrance is used for sample setting for transmission spectrum measurement and fluorescence spectrum measurement in the transmission direction. Moreover, the opening part of the counter electrode of excitation light incident light is used for the sample installation for the fluorescence spectrum measurement to a reflection direction. The fluorescence outlet is always left open to guide the fluorescence to the fluorescence side spectroscope and detector. Further, the opening of the counter electrode of the fluorescent side exit is not used in this measurement, and is covered with an aluminum oxide white plate or the like.

Here, the configuration of the penta-arranged sample cell switching device 22 will be described. The penta-arranged sample cell switching device 22 has a rotation axis on the bottom surface of the integrating sphere 15. In addition, five sample mounting bases from the cell base 51 to the cell base 55 are mounted, and are located at the center of each side of a regular pentagon having the integrating sphere 15 as an inscribed circle. Each cell stage is installed at a position where the transmitted fluorescence sample cell 16 or the reflected fluorescence sample cell 17 is in close contact with the integrating sphere 15. The opening of the integrating sphere 15 is located at the center of each side of a square with the integrating sphere 15 as an inscribed circle.

  When the sample is placed at the excitation light entrance, the other four cell bases are positioned so as not to overlap with the other three openings of the integrating sphere 15 and do not interfere with the measurement. Similarly, when the cell stage moves to the opening of the counter electrode at the excitation light entrance, the other four cell stages are positioned so as not to overlap with the other three openings of the integrating sphere 15, thereby disturbing the measurement. Not. In the penta-arranged sample cell switching device 22, the number of cells that can be installed is limited to five, but the bottom surface of the integrating sphere 15 is the axis of rotation, and the sample cell rotates at a predetermined radius. Even if it uses, there exists an advantage installed in the state which contacted the opening part of the integrating sphere, and the sample cell. Further, there is an advantage that the position adjusting pulse motor 20 and the position adjusting turret rotating shaft 21 used in the sample cell switching device 18 are not necessary.

  FIG. 14 is a configuration diagram showing the configuration of the belt type sample cell switching device 23. In the belt-type sample cell switching device 23, for example, a belt on which seven sample tables 51 to 57 are installed moves using the sample cell switching pulse motor 19 as power. When the sample is placed at the excitation light entrance, the belt-type sample cell switching device 23 carries the cell stage 52 to the position of the origin 1 in the integrating sphere opening of the excitation light incidence shown in FIG. The belt moves using the pulse motor 24 for sample cell loading as power, and the cell stage 52 is carried to the integrating sphere opening where the excitation light is incident.

  Similarly, when carrying the cell base to the opening of the counter electrode of the excitation light entrance, the belt moves using the reflected fluorescence sample cell loading pulse motor 25 as power, and the cell base 52 is integrated with the counter electrode of the excitation light entrance. Carry to sphere opening. This belt-type sample cell switching device 23 has a number of parts, but has a feature that the number of cell bases can be increased or decreased by adjusting the length of the belt.

DESCRIPTION OF SYMBOLS 1 ... Light source 2 ... Excitation side spectroscope 3 ... Beam splitter 4 ... Monitor detector 5 ... Sample 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 ... Display display 14 ... Operation panel 15 ... Integrating sphere 16 ... Sample cell for transmitted fluorescence 17 ... Sample cell for reflected fluorescence 18 ... Turret type sample cell switching device 19 ... Sample cell switching pulse motor 20 ... Position adjustment pulse motor 21 ... Position Adjusting turret rotating shaft 22 ... Pentar arrangement sample cell switching device 23 ... Belt-type sample cell switching device 24 ... Transmission fluorescence sample cell loading pulse motor 25 ... Reflection fluorescence sample cell loading buffer Motors 51 to 57... Cell stand 100... Spectrofluorometer 110... Photometer unit 111... Excitation side optical system 112. Drive unit 120 ... data processing unit 130 ... operation / display unit

Claims (6)

  In a spectrofluorometer equipped with a cell switching device in which a plurality of cells having different optical path lengths and integrating spheres are installed in the sample installation part, obtained by the first process and the first process for measuring the fluorescence spectrum in the reflection direction The first determination to detect the peak wavelength based on the fluorescence spectrum data in the reflection direction and the peak wavelength from the fluorescence spectrum data in the reflection direction obtained in the first process is multiplied by an arbitrary coefficient. A second process for calculating an identification wavelength shorter than the fluorescence peak having the combined fluorescence intensity, a third process for measuring transmission spectra in a plurality of optical path lengths in the cell switching device, and a third process A second determination for determining whether or not the transmittance at the processing identification wavelength obtained in the second processing is an arbitrary value or more from the transmission spectrum obtained by the processing in the cell of the corresponding optical path length; Light path passed in Spectrofluorometer, wherein the first performing fourth processing of measuring the fluorescence spectrum of the transmission direction in a cell having a.   The first process of measuring the fluorescence spectrum in the reflection direction is performed, the first determination for detecting the peak wavelength is performed based on the fluorescence spectrum data in the reflection direction obtained in the first process, and the first A second process of calculating an identification wavelength shorter than the fluorescence peak having a fluorescence intensity obtained by multiplying the peak wavelength by an arbitrary coefficient from the fluorescence spectrum data in the reflection direction obtained by the process. The cell switching device performs the third process of measuring the transmission spectrum in a plurality of optical path lengths, and the transmittance at the processing identification wavelength obtained in the second process is obtained from the transmission spectrum obtained in the third process. 4th process which performs the 2nd determination which determines whether it becomes more than arbitrary values in the cell of the applicable optical path length, and measures the fluorescence spectrum to a permeation | transmission direction in the cell which has the optical path length which passed by the determination 2. It is characterized by performing Method of measuring the fluorescence spectrophotometer.   In a spectrofluorometer equipped with a cell switching device in which a plurality of cells with different optical path lengths and integrating spheres are installed in the sample setting section, the fluorescence spectrum in the transmission direction of the plurality of cells with different optical path lengths is measured by the sample cell switching device. A measuring method of a spectrofluorometer, characterized by measuring and measuring a three-dimensional fluorescence spectrum in a transmission direction in which a fluorescence wavelength is displayed on the X axis, an optical path length on the Y axis, and a fluorescence intensity on the Z axis.   A cell base on which a plurality of transmission fluorescence sample cells and reflection fluorescence sample cells having different optical path lengths arranged on the circumference are installed, and a sample cell switching that serves as a power for moving the cell stage by rotating and switching the cells. Controls the pulse motor, the position adjustment pulse motor that is the power to bring the integrating sphere opening, the transmission fluorescence sample cell and the reflection fluorescence sample cell into close contact, and the position adjustment turret rotation shaft for position adjustment. The distance to be in close contact with the integrating sphere opening is calculated by subtracting the origin position information input to the computer and the optical path length of the sample cell, and integration is performed by the position adjustment pulse motor and the position adjustment turret rotation axis. A sample cell switching device characterized in that a sample cell is brought into close contact with a spherical opening. Five cell bases in which a transmission fluorescence sample cell or a reflection fluorescence sample cell located in the center of each side of a regular pentagon having an integrating sphere as an inscribed circle is placed in close contact with the integration sphere, and the cell base is rotated. A sample cell switching device comprising a sample cell switching pulse motor which is a motive power for power supply and a computer for controlling them.   Multiple cell stands installed on the belt, sample cell switching pulse motor to be the power to move the belt, and sample cell loading for transmitted fluorescence to be the power to move the cell stage to the sample position to measure the transmitted fluorescence of the integrating sphere A sample cell equipped with a pulse motor for loading, a pulse motor for loading a sample cell for reflected fluorescence, which is a power for moving the cell base to the sample position where the reflected fluorescence of the integrating sphere is measured, and a computer for controlling these Switching device.

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