JP5152083B2 - Spectrofluorometer - Google Patents

Description

  The present invention relates to a spectrofluorometer that irradiates a sample with excitation light and detects fluorescence emitted from the sample with high sensitivity.

  A general spectrofluorometer irradiates a sample cell containing a sample solution to be measured with excitation light of a specific wavelength extracted by the excitation side spectrometer, and the fluorescence emitted from the sample solution passes through the fluorescence side spectrometer. It has a configuration in which it is wavelength-dispersed and introduced into a detector for detection. As the sample cell, a square cell made of quartz glass or the like is usually used, but when used as a detector of a liquid chromatograph, a flow cell is used.

  In order to improve measurement sensitivity in such a spectrofluorometer, it is necessary to efficiently make as much excitation light as possible incident on the sample solution in the sample cell, and to collect the generated fluorescence as efficiently as possible. Must be introduced into the detector. For this purpose, a high-sensitivity cell holder having reflection mirrors on the excitation light irradiation axis and the fluorescence detection axis has been conventionally known (see Patent Document 1).

  Fluorescence is emitted in all directions around 360 ° around the focal position of the excitation light in the square cell. If a high-sensitivity cell holder is not used, the fluorescence incident on the entrance slit of the fluorescence spectrometer is used. Only the component is reflected in the detected value. On the other hand, when a high sensitivity cell holder is used, the excitation light once irradiated on the sample is reflected by the reflection mirror to excite the sample again. Further, the fluorescence emitted from the sample in the direction opposite to the fluorescence spectrometer is folded by the reflection mirror and is incident on the entrance slit of the fluorescence spectrometer. As a result, it is possible to detect fluorescence that is two to three times stronger than a normal cell holder.

  Although the intensity of the fluorescence detected as described above is increased by using the high sensitivity cell holder, the reflection wavelength characteristic is included in the fluorescence spectrum because the reflection mirror is used. For this reason, the obtained fluorescence spectrum is not necessarily a true fluorescence spectrum. A reflection mirror that is generally used exhibits a relatively flat reflection wavelength characteristic in an ultraviolet / visible wavelength region. For this reason, the reflection wavelength characteristics of the reflection mirror are often not a problem when measuring in the ultraviolet / visible wavelength region.

  On the other hand, for example, as disclosed in Patent Document 2, when measuring photoluminescence with a spectrofluorometer for evaluating dispersion of carbon nanotubes, the wavelength region of the photoluminescence to be measured is 800 to 1600 [ nm] in the infrared to near-infrared wavelength region. In such a wavelength region, the reflection wavelength characteristic of the reflection mirror tends to vary greatly, so that the fluorescence spectrum obtained for the sample can deviate greatly from the true fluorescence spectrum. In addition, when using a mirror that is coated with an anti-oxidation film to increase durability as a reflection mirror used in high-sensitivity cell holders, the reflection wavelength characteristics fluctuate even in the ultraviolet wavelength region due to the effect of such coating. May increase and the deviation from the true fluorescence spectrum may increase.

Japanese Utility Model Publication No. 7-23242 JP 2009-31114 A

  The present invention has been made to solve the above-mentioned problems, and an object of the present invention is to provide a spectrofluorometer capable of obtaining a fluorescence spectrum with high sensitivity and close to the true wavelength characteristic of a sample. It is in.

In order to solve the above problems, the present invention includes an excitation light irradiating means for irradiating a sample with excitation light, a detection means for detecting and analyzing fluorescence emitted from the sample upon receiving the excitation light, and the excitation light. A first reflecting mirror that irradiates the sample by the irradiation means and passes through the sample and returns the excitation light to the sample side; a second reflecting mirror that returns the fluorescence emitted from the sample in the direction opposite to the direction of taking out the fluorescence to the sample side; In a spectrofluorometer comprising:
a) a light shielding means for preventing return of excitation light and fluorescence reflected by the first and second reflecting mirrors and returned to the sample;
b) Based on the detection signal obtained by the detection means, the same spectrum, a fluorescence spectrum in a predetermined wavelength range in a state where light is shielded by the light shielding means, and a predetermined wavelength range in a state where light is not shielded by the light shielding means Measurement execution means for acquiring each of the fluorescence spectra of
c) correction information acquisition means for calculating and storing correction information reflecting the influence of the first and second reflecting mirrors from two fluorescence spectra corresponding to the presence or absence of light shielding obtained by the measurement execution means;
d) correction processing means for correcting the spectrum using the correction information when obtaining a fluorescence spectrum in a predetermined wavelength range in a state where the target sample is not shielded by the light shielding means;
It is characterized by having.

  The light shielding means can be, for example, a shutter (light shielding plate) that is removably inserted into each optical path between the sample and the first and second reflecting mirrors. Also, by moving the positions of the first and second reflecting mirrors or changing their postures, the excitation light and fluorescence coming from the sample are prevented from hitting the reflecting mirrors, or It may be a drive mechanism that prevents the excitation light or fluorescence that hits from returning to the sample.

  The fluorescence spectrum in the predetermined wavelength range in a state where light is shielded by the light shielding means does not include the influence of the first and second reflecting mirrors, that is, the wavelength characteristics of the reflecting mirrors themselves. On the other hand, the fluorescence spectrum in a predetermined wavelength range in a state where light is not shielded by the light shielding means includes the wavelength characteristics of the first and second reflecting mirrors themselves. Therefore, the correction information acquisition means calculates correction information reflecting the wavelength characteristics of the first and second reflecting mirrors from the two fluorescence spectra corresponding to the presence or absence of light shielding, and stores this in a nonvolatile memory or the like.

  When the reflecting mirror itself is exchanged, it is necessary to update the correction information (that is, measure a predetermined sample by the measurement execution unit and calculate new correction information based on the result). In addition to this, it is also corrected if the reflection wavelength characteristics of the reflector may change, such as when the reflector becomes dirty, when the reflector deteriorates, or when the reflector is washed. It is preferable to update the information.

  As described above, the correction information reflects the wavelength characteristics of the first and second reflecting mirrors. Therefore, when a fluorescence spectrum in a predetermined wavelength range is obtained in a state where the target sample is not shielded by the light shielding means. By correcting the spectrum using the correction information by the correction processing means, it is possible to obtain a spectrum from which the influence of the wavelength characteristics of the first and second reflecting mirrors has been removed. Naturally, the fluorescence intensity detected by the detection means differs depending on whether or not the light is shielded by the light shielding means. However, by making information reflecting only the difference in wavelength characteristics as correction information, the sensitivity when using a reflecting mirror is high. The influence of the wavelength characteristic of the reflecting mirror can be removed while taking advantage of the above.

  According to the spectrofluorometer according to the present invention, it is possible to remove the influence of the reflection wavelength characteristic of the reflector itself arranged to increase the detection sensitivity, and to obtain a true or near fluorescence spectrum of the sample. . Thereby, it is possible to obtain an accurate fluorescence spectrum with high sensitivity.

1 is a schematic configuration diagram of a spectrofluorometer according to an embodiment of the present invention. Schematic which shows the measurement state in the spectrofluorometer by a present Example. The flowchart which shows the measurement procedure in the spectrofluorometer by a present Example. An actual measurement example of a fluorescence spectrum obtained with the spectrofluorometer according to this example. The example of the correction function obtained with the spectrofluorometer by a present Example.

  Hereinafter, a spectrofluorometer which is an embodiment of the present invention will be described with reference to the accompanying drawings. FIG. 1 is a schematic configuration diagram of a spectrofluorometer according to this embodiment, and FIG. 2 is a schematic diagram showing a measurement state in the spectrofluorometer according to this embodiment.

  In FIG. 1, light emitted from the light source unit 1 is introduced into the excitation-side spectroscope 2, monochromatic light having a specific wavelength set by the analysis control unit 10 is extracted, and a sample solution S is stored as excitation light. The cell 3 is irradiated. The fluorescence excited and emitted by the excitation light is introduced into the fluorescence spectrometer 6, and the fluorescence having a specific wavelength set by the analysis controller 10 is taken out and introduced into the detector 7. The detector 7 outputs a detection signal corresponding to the intensity of the incident fluorescence, and this detection signal is converted into a digital value by the A / D converter 8 and sent to the data processing unit 9 for predetermined data processing. Thus, a fluorescence spectrum is created.

  The data processing unit 9 includes a correction function calculation unit 91 and a correction function storage unit 92 as functional blocks characteristic of the present invention. These specific functions will be described later. As described above, the analysis control unit 10 controls the operation of each unit to perform measurement, such as setting the wavelength of excitation light and the wavelength of fluorescence to be detected. An operation unit 12 and a display unit 13 are connected to the central control unit 11, and reception of measurement condition input settings by the operation unit 12 and display control of measurement results on the display unit 13 are executed. All or part of the functions of the central control unit 11, the analysis control unit 10, and the data processing unit 9 should be achieved by executing dedicated control / processing software installed in the personal computer. Can do.

  The sample cell 3 is a square cell of 10 mm square, for example, and a concave mirror (corresponding to the first reflecting mirror of the present invention) 4 is arranged on the side opposite to the excitation light irradiation port, and emission of fluorescence to the fluorescence side spectroscope 6 is performed. A plane mirror (corresponding to the second reflecting mirror of the present invention) 5 is disposed on the side opposite to the mouth. As shown in FIG. 2A, in a normal measurement state, the excitation light irradiated on the sample cell 3 and transmitted through the sample cell 3 strikes the concave mirror 4 and is folded (and collected), and then enters the sample cell 3 again. To do. Therefore, as compared with the case where the concave mirror 4 is not provided, more excitation light hits the sample solution S, and the amount of fluorescence generated accordingly increases. On the other hand, of the fluorescence emitted from the sample solution S, the fluorescence emitted in the direction opposite to the fluorescence emission port is reflected by the plane mirror 5 and emitted together with the fluorescence originally emitted toward the fluorescence emission port. Therefore, more fluorescence is introduced into the fluorescence side spectroscope 6 than when the plane mirror 5 is not provided.

  Shutters 21 and 22 can be inserted on the optical path between the sample cell 3 and the concave mirror 4 and on the optical path between the sample cell 3 and the plane mirror 5, respectively. When the shutters 21 and 22 are inserted (see FIG. 2B), the shutters 21 and 22 completely block excitation light and fluorescence, respectively. There is no reflection by the shutters 21 and 22 themselves, or they are suppressed to a level that can be ignored. Each of the shutters 21 and 22 may be installed and removed by the measurer, or may be automatically opened and closed according to the operation of the measurer by the shutter opening / closing mechanism or in accordance with the measurement execution described later.

  A characteristic measurement operation of the spectrofluorometer of the present embodiment will be described with reference to the flowchart shown in FIG.

  The measurer prepares a standard sample having fluorescence in the same wavelength region as the sample (target sample) to be measured, and sets the sample cell 3 containing the standard sample at a predetermined position (step S1). After that, when the measurer gives an instruction to start the correction function acquisition process from the operation unit 12 (step S2), the analysis control unit 10 that has received this instruction via the central control unit 11 first closes both shutters 21 and 22. That is, each part is controlled to execute measurement in a state where light is shielded (step S3). In the case where the operator manually opens and closes the shutters 21 and 22 as described above, a display that prompts the user to perform an operation of closing the shutter is performed on the display unit 13 and, for example, the measurement is performed. When it is confirmed by the key operation by the person, the measurement may be actually started.

In the state where the light is blocked by the shutters 21 and 22, the excitation side spectroscope 2 sequentially sets the wavelength of the excitation light in a predetermined wavelength range λEX1 to λEX2, and the fluorescence side spectroscope 6 sets one excitation light wavelength λEXa, λEXb. ,... Are scanned in the predetermined wavelength range λEM1 to λEM2, and data based on the detection signal obtained by the detector 7 is input to the data processing unit 9. Therefore, in the data processing unit 9, the fluorescence spectrum over a predetermined wavelength range is obtained for each different excitation light wavelength λEXa, λEXb,. However, when a pair of excitation light wavelength and fluorescence wavelength used for measurement is determined in advance, it is sufficient to obtain the fluorescence intensity for only that wavelength pair. In this way, necessary fluorescence spectrum data Fclose (λEX, λEM) is obtained in a state where the light is blocked by the shutters 21 and 22 (step S4).

Next, the analysis control unit 10 controls each unit to perform measurement in a state where both the shutters 21 and 22 are opened, that is, not shielded from light (step S5). In a configuration in which the measurer manually opens and closes the shutters 21 and 22, a display for prompting the user to open the shutter is displayed on the display unit 13, and it is confirmed, for example, by a key operation by the measurer. And actually start the measurement. Necessary fluorescence spectrum data Fopen (λEX, λEM) is acquired in a state where light is not blocked by the shutters 21 and 22 (step S6).

  When the fluorescence spectrum data Fclose (λEX, λEM) is acquired, the concave mirror 4 and the plane mirror 5 are not present on the optical paths of the excitation light and the fluorescence, and thus the reflection wavelength characteristics of the mirrors 4 and 5 are not included. On the other hand, when the fluorescence spectrum data Fopen (λEX, λEM) is acquired, the concave mirror 4 and the plane mirror 5 exist on the optical paths of the excitation light and the fluorescence, and therefore the reflection wavelength characteristics of the mirrors 4 and 5 are included. The correction function calculator 91 calculates a correction function reflecting the reflection wavelength characteristics of the concave mirror 4 and the plane mirror 5 from the fluorescence spectrum data Fclose (λEX, λEM) and the fluorescence spectrum data Fopen (λEX, λEM) (step S7). Specifically, Fopen (λEX, λEM) / Fclose (λEX, λEM) is calculated and normalized so that the maximum value in the function becomes 1, and the correction function Q (λEX, λEM) is obtained. Note that Fopen (λEX, λEM) / Fclose (λEX, λEM) is an operation for obtaining a ratio at the same excitation light wavelength and fluorescence wavelength. By obtaining the normalized correction function as described above, the influence of the intensity difference depending on the sensitivity difference is eliminated, and the correction function Q (λEX, λEM) basically reflects only the reflection wavelength characteristics of the concave mirror 4 and the plane mirror 5. Will be. The correction function Q (λEX, λEM) thus determined is stored in the correction function storage unit 92.

  When measuring the fluorescence spectrum of the target sample under the condition where the correction function Q (λEX, λEM) is stored in the correction function storage unit 92 as described above, the process is performed from step S8. That is, the sample cell 3 containing the target sample is set at a predetermined position, and measurement is performed in a state where the shutters 21 and 22 are opened, that is, a state where the shutters 21 and 22 are not shielded (step S8). By this measurement, fluorescence spectrum data Funk (λEX, λEM) is acquired (step S9). Thereafter, the data processing unit 9 uses the correction function read from the correction function storage unit 92 to calculate Funk (λEX, λEM) / Q (λEX, λEM), and thereby the concave mirror 4 and the plane mirror 5 are operated. A true fluorescence spectrum excluding the influence of the reflection wavelength characteristic is obtained (step S10).

  For example, if the reflectivity for each wavelength of the concave mirror 4 and the plane mirror 5, that is, the theoretical (or design) reflection wavelength characteristic, is known, the theoretical correction function can be obtained therefrom. However, in actual devices, the mirror surface shape of the mirror to be used is deviated from an ideal state (distortion, etc.), and there is a positional deviation when the mirror is installed. It does not follow the desired condition. As a result, even if correction processing of the measured fluorescence spectrum is executed using such a correction function, a true fluorescence spectrum cannot be obtained. On the other hand, in the fluorescence spectrophotometer according to the present embodiment, a correction function for removing the influence of the reflection wavelength characteristics of the concave mirror 4 and the plane mirror 5 is calculated based on the result measured by an actual apparatus. For this reason, it is possible to obtain a true fluorescence spectrum or a fluorescence spectrum very close to it by executing correction processing of the measured fluorescence spectrum using this correction function.

  4A and 4B are actual measurement examples of the fluorescence spectrum obtained by the spectrofluorophotometer according to the present embodiment. FIG. 4A is a fluorescence spectrum when the shutters 21 and 22 are opened, and FIG. 4B is a state where the shutters 21 and 22 are closed. Is a fluorescence spectrum. This sample uses an aqueous solution in which single-walled carbon nanotubes (hereinafter referred to as SWNTs) are isolated and dispersed using a surfactant as a sample, and the excitation light is visible light (wavelength: 644 nm), and the infrared to near-infrared wavelength region. It is the result of having measured the fluorescence spectrum. In the figure, each peak top on the fluorescence spectrum indicates the SWNT diameter distribution (chirality).

  The dispersion ratio of SWNTs of different diameters dispersed in the solution can be evaluated by comparing the peak top height. Comparing FIGS. 4A and 4B, it can be seen that the peak top heights of SWNT chiralities (7, 5) and (7, 6) are reversed. This is because in FIG. 4A, the reflection wavelength characteristics of the mirrors 4 and 5 are included in the measured values, and in this state, the SWNT diameter distribution cannot be considered quantitatively and accurately. On the other hand, by correcting the fluorescence spectrum by applying the correction function obtained based on the actual measurement result as in this embodiment, the shape as shown in FIG. 4B and the shape shown in FIG. A highly sensitive true fluorescence spectrum having the intensity shown can be obtained.

  FIG. 5A shows the reflectance (nominal value) of the mirror used and a visible light cut filter that does not allow the excitation wavelength inserted in the fluorescence emission side optical path to pass (the spectrum of the secondary wavelength of the excitation wavelength is in the near infrared wavelength region). FIG. 5B is a diagram showing a theoretical correction function that combines the transmittance of a filter for preventing the light from overlapping with FIG. 5B, and FIG. 5B shows the fluorescence shown in FIGS. 4A and 4B, which are actual measurement examples. It is a figure which shows the correction function which combined the correction function calculated using the spectrum, and the transmittance | permeability of the visible light cut filter used for the measurement. It can be seen that there is a considerable difference between the two shapes. That is, in the spectrofluorometer of the present embodiment, the fluorescence spectrum is truly corrected by using a correction function that is based on the actual value as shown in FIG. Can do.

  In the above embodiment, the shutters 21 and 22 are inserted on the optical path between the sample cell 3 and the concave mirror 4 and the plane mirror 5 so as to shield the excitation light and fluorescence that are turned back. If the configuration does not return the excitation light transmitted through the sample cell 3 side and the fluorescence emitted from the sample solution S in the direction of the plane mirror 5 does not return to the sample cell 3 side, various configurations can be used instead of the shutters 21 and 22. Can be adopted. For example, the concave mirror 4 and the plane mirror 5 are configured to be able to stand up and fall, and when the concave mirror 4 and the plane mirror 5 are in the fallen state, the excitation light and the fluorescent light are not placed on the mirrors 4 and 5 and are installed, for example, behind them. What is necessary is just to make it strike and absorb a light absorber.

  Moreover, the said Example is an example of this invention, Even if it changes suitably, amends, and is added in the range of the meaning of this invention, it is naturally included in the claim of this application.

DESCRIPTION OF SYMBOLS 1 ... Light source part 2 ... Excitation side spectroscope 3 ... Sample cell 4 ... Concave mirror 5 ... Plane mirror 6 ... Fluorescence side spectroscope 7 ... Detector 8 ... A / D converter 9 ... Data processing part 91 ... Correction function calculating part 92 ... Correction function storage unit 10 ... analysis control unit S ... sample solution

Claims (1)

Excitation light irradiation means for irradiating the sample with excitation light, detection means for spectroscopic detection of fluorescence emitted from the sample upon receiving the excitation light, and excitation light that is irradiated to the sample by the excitation light irradiation means and transmitted through the sample A spectrofluorometer comprising: a first reflecting mirror that folds back to the sample side; and a second reflecting mirror that folds the fluorescence emitted from the sample in the direction opposite to the direction of taking out the fluorescence to the sample side.
a) a light shielding means for preventing return of excitation light and fluorescence reflected by the first and second reflecting mirrors and returned to the sample;
b) Based on the detection signal obtained by the detection means, the same spectrum, a fluorescence spectrum in a predetermined wavelength range in a state where light is shielded by the light shielding means, and a predetermined wavelength range in a state where light is not shielded by the light shielding means Measurement execution means for acquiring each of the fluorescence spectra of
c) correction information acquisition means for calculating and storing correction information reflecting the influence of the first and second reflecting mirrors from two fluorescence spectra corresponding to the presence or absence of light shielding obtained by the measurement execution means;
d) correction processing means for correcting the spectrum using the correction information when obtaining a fluorescence spectrum in a predetermined wavelength range in a state where the target sample is not shielded by the light shielding means;
A spectrofluorometer characterized by comprising:

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