JP2009180706A - Spectrofluorometric detection apparatus for liquid chromatograph - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To easily find analytical conditions in a short time such as an excitation light wavelength and a fluorescence wavelength optimal for highly accurate determination of each sample component separated by a column. <P>SOLUTION: A target sample while being fixed to a prescribed excitation light wavelength and a prescribed fluorescence wavelength is analyzed by liquid chromatography (S1 and S2). When an intensity value takes a threshold value or greater, and its amount of change becomes small, a sample component is considered to have appeared (S3-S6) to obtain a retention time at the time and temporarily halt liquid feeding to the column (S7 and S8). With an eluate containing the sample component retained in a flow cell, an excitation light wavelength and a fluorescence wavelength are each scanned at high speed to obtain a schematic three-dimensional spectrum and store it with the retention time (S9-S10). At each time a sample component separated by the column has appeared, this is repeated to determine a three-dimensional spectrum corresponding to each sample component. A peak top having maximum intensity in the three-dimensional spectrum is searched for to find the excitation light wavelength and the fluorescence wavelength that provide the peak and display them with the retention time. <P>COPYRIGHT: (C)2009,JPO&INPIT

Description

  The present invention relates to a spectroscopic fluorescence detection apparatus for sequentially detecting each component in a sample separated in a time direction by a liquid chromatograph column.

  Although spectrofluorimetric analysis is limited in that it can detect only fluorescent or fluorescent substances, it can perform analysis with much higher sensitivity than absorption spectrophotometry using light absorption. It is widely used as a detector for liquid chromatographs. FIG. 8 is a schematic configuration diagram of a general spectral fluorescence detection apparatus conventionally known in, for example, Patent Document 1.

  In this spectral fluorescence detection apparatus 1, light emitted from a light source 10 such as a xenon lamp having a wide continuous spectrum from the ultraviolet region to the near infrared region is introduced into an excitation-side spectroscope 11 and a specific wavelength (excitation light wavelength EX ) Is extracted, and is irradiated as excitation light to the sample cell 12 in which the sample solution 13 is stored. The weak fluorescence emitted from the sample solution 13 by the irradiation of this excitation light is introduced into the fluorescence side spectroscope 14, and only the fluorescence having a specific wavelength (fluorescence wavelength EM) is taken out and incident on the photomultiplier tube 15. . The photomultiplier tube 15 outputs a current signal corresponding to the intensity of incident light, and the voltage signal converted by the current / voltage (I / V) converter 16 is converted into a digital value by the A / D converter 17. The data is input to the data processing unit 18. The data processing unit 18 analyzes the detection data to calculate a quantitative value of the specific component in the sample solution 13.

  In order to perform high-precision quantification in spectrofluorimetric analysis, it is necessary to determine appropriate excitation light wavelength EX and fluorescence wavelength EM as analysis conditions according to the type of sample. In general, determination of such analysis conditions requires sufficient experience and skill, and an inexperienced operator must find the optimum wavelength condition by performing preliminary measurements several times while changing the set wavelength condition. Therefore, analysis takes time and efficiency is poor. In addition, a large amount of sample is required for preliminary measurement.

  When a sample separated by a liquid chromatograph column is detected by a spectrofluorescence detection apparatus, determination of the analysis conditions as described above is a more complicated and time-consuming work. This is because the sample to be analyzed usually contains a large number of sample components, and the appropriate excitation light wavelength and fluorescence wavelength are different for each sample component. Further, in a liquid chromatograph, it takes several minutes at least for a single measurement, and it takes tens of minutes if it is long, and it is not practical to determine the optimal analysis conditions by repeating preliminary measurements many times. Therefore, in the end, it is the actual situation that the operator has to rely on the experience and intuition of the operator, and this also affects the accuracy of the analysis.

Japanese Unexamined Patent Publication No. 2006-300632 (paragraph 0002, FIG. 2)

  The present invention has been made to solve the above-mentioned problems, and the object of the present invention is to easily set an appropriate excitation light wavelength and fluorescence wavelength for each sample component even for those who have little experience. Another object of the present invention is to provide a spectrofluorometric detection apparatus for liquid chromatography that can be determined efficiently.

The present invention made in order to solve the above-mentioned problems is a spectral fluorescence detection device for detecting a sample component separated by a column of a liquid chromatograph, wherein a light source and light emitted from the light source are dispersed to obtain a predetermined wavelength. Excitation-side spectroscopic means for taking out the light from the sample; fluorescence-side spectroscopic means for separating the fluorescence obtained from the sample with respect to the excitation light extracted by the excitation-side spectroscopic means; A fluorescence detector for liquid chromatography, comprising: a photodetector for detecting fluorescence extracted by the light source; and a wavelength changing means for changing the wavelength of light extracted for each of the excitation-side spectroscopic means and the fluorescence-side spectroscopic means. In the device
a) component detection means for detecting the eluate from the column in a state where the excitation light wavelength and the fluorescence wavelength predetermined by the wavelength changing means are set, and detecting the appearance of a sample component based on the detection signal; ,
b) an instruction means for giving an instruction to temporarily stop the liquid feeding to the column when the appearance of the sample component is detected by the component detection means;
c) In a state where liquid feeding is stopped according to the instruction from the instruction means, the wavelength changing means scans the excitation light wavelength and the fluorescence wavelength, respectively, and uses the excitation light wavelength, the fluorescence wavelength and the signal intensity as dimensions. Spectrum acquisition means for acquiring a spectrum;
d) search means for searching for a combination of an excitation light wavelength and a fluorescence wavelength that gives a peak having a maximum signal intensity or a relatively large signal intensity with respect to the three-dimensional spectrum;
e) an output means for outputting the excitation light wavelength and the fluorescence wavelength obtained by the search means together with the time information at which the sample component from which the three-dimensional spectrum was acquired appears;
It is characterized by having.

  In order to temporarily stop the liquid feeding to the column in response to the instruction from the instruction means, for example, the operation of the liquid feeding pump for feeding the mobile phase to the column is stopped, or the flow to the column is switched by switching the flow path. A method such as diverting the inflow of the mobile phase to another flow path may be employed.

  When the liquid supply to the column is temporarily stopped as described above, the flow of the eluate from the column temporarily stays. Therefore, the spectrofluorescence detection apparatus according to the present invention detects the same sample component in the eluate. Will continue. The scanning of the excitation light wavelength and the fluorescence wavelength by the wavelength changing means takes a certain amount of time, but during that time, the same sample component remains at the detection position, so that a three-dimensional spectrum for the sample component can be acquired. When the liquid feeding is stopped, the separated components are diffused in the flow path, so that the separation ability is lowered and the peak is displaced. Therefore, although it is not preferable for measurement that requires accuracy, the purpose here is to find the excitation light wavelength and fluorescence wavelength that are optimal or close to each sample component. Misalignment is hardly a problem.

  In the spectral fluorescence detection apparatus according to the present invention, for example, the component detection means starts observing the temporal change amount of the signal when the level of the detection signal is equal to or higher than a predetermined threshold, and the change amount is large. Therefore, it can be determined that the sample component has appeared when it becomes smaller than a certain threshold value. Then, the three-dimensional spectrum is acquired in a state where the detected sample component is retained at the detection position by stopping the liquid supply. When the acquisition is completed, the liquid supply is resumed, and then the sample component eluted from the column. Wait for the appearance of. By repeating such processing, a three-dimensional spectrum is separately created for each of a plurality of sample components contained in the sample, and the excitation light wavelength that gives a peak having the maximum signal intensity or a relatively large signal intensity is obtained. Combinations with fluorescence wavelengths can be found. Of course, the holding time is not included in the time during which the liquid feeding is stopped.

  If only one liquid chromatographic measurement is performed on the target sample, the appropriate excitation light wavelength and fluorescence wavelength for all the sample components contained in the sample can be determined. Based on the information provided by the output means The second detailed liquid chromatographic measurement conditions can be set for the target sample. Of course, such condition setting may be performed automatically without bothering the user. However, if there are multiple peaks on the three-dimensional spectrum, the excitation light wavelength and fluorescence wavelength of the peak showing the maximum signal intensity may not be optimal as a condition. It is also possible to allow the user to make a selection.

  Further, as one aspect of the spectral fluorescence detection apparatus according to the present invention, a display means for rendering the three-dimensional spectrum acquired by the spectrum acquisition means and recognizing the peaks found by the search means, and the display On the three-dimensional spectrum displayed on the means, the operation means for the user to select a peak, and the excitation light wavelength and the fluorescence wavelength that give the peak specified according to the selection instruction by the operation means are set as the next analysis conditions It is good also as a structure further provided with the analysis condition setting means to do.

  According to this configuration, since the user can perform an operation while visually confirming the three-dimensional spectrum, an operation error is reduced. Further, for example, it is possible to easily recognize that some trouble has occurred in the measurement (for example, a large amount of unintended impurities are included), and it is possible to avoid performing unnecessary detailed measurement.

  The acquisition of the three-dimensional spectrum by the spectrum acquisition means is only for the purpose of determining analysis conditions, so it is desirable to reduce the time required for it as much as possible. Therefore, for example, the wavelength setting unit is further provided for the user to set the wavelength range of the excitation light wavelength and the fluorescence wavelength when the three-dimensional spectrum is acquired by the spectrum acquisition unit, and the user limits the wavelength range as necessary. By doing so, the measurement time should be shortened. It is also preferable that the user can set the wavelength step during scanning.

  According to the spectrofluorometric detection apparatus for a liquid chromatograph according to the present invention, various components contained in the target sample can be quantitatively analyzed with high accuracy by performing only one liquid chromatographic measurement on the target sample. It is possible to automatically collect information on the optimum excitation light wavelength and fluorescence wavelength. Thereby, even an inexperienced operator can perform analysis efficiently and with high accuracy. Further, since it is not necessary to perform many preliminary measurements, the amount of the target sample can be reduced, and wasteful consumption of the consumables of the apparatus (for example, a liquid chromatograph column) can be suppressed.

  Hereinafter, an embodiment of a liquid chromatograph using the spectral fluorescence detection apparatus according to the present invention will be described in detail with reference to the drawings. FIG. 1 is a configuration diagram of a main part of the liquid chromatograph of the present embodiment. In addition, the same code | symbol is attached | subjected to the same component as the structure demonstrated with FIG. 8, and description is abbreviate | omitted.

  In this liquid chromatograph, the mobile phase stored in the mobile phase container 2 is sucked by the liquid feed pump 3 and fed to the column 5 through the injector 4 at a substantially constant flow rate. When a sample is injected into the mobile phase by the injector 4, the sample is introduced into the column 5 along the flow of the mobile phase, and various components contained in the sample are separated while passing through the column 5. The difference eluates from the outlet end of the column 5 and is discharged after passing through the flow cell 6. The spectroscopic fluorescence detection apparatus 1 detects sample components in the eluate passing through the flow cell 6. The operation of the liquid feed pump 3 and the sample injection operation by the injector 4 are controlled by the analysis control unit 7.

  In the spectral fluorescence detection apparatus 1, a data processing unit 18 that receives detection data from the A / D conversion unit 17 includes a scanning timing determination processing unit 181, a data storage unit 182, a peak search unit 183, and the like. A control signal from the scanning timing determination processing unit 181 is sent from a scanning control unit 19 that controls a motor 112 that rotates the diffraction grating 111 in the excitation side spectroscope 11 and a motor 142 that rotates the diffraction grating 141 in the fluorescence side spectroscope 14. And input to the analysis control unit 7. Further, the data processing unit 18 is provided with an operation unit 20 and a display unit 21 operated by a user. Note that all or part of the functions of the data processing unit 18, the operation unit 20, the display unit 21, etc. may be embodied by a personal computer.

  Next, a characteristic analysis operation in the liquid chromatograph of the present embodiment will be described. In this liquid chromatograph, first, in order to determine the conditions for appropriate quantitative analysis of various components contained in the target sample, the first measurement is performed, and after the conditions are determined, detailed measurement is performed on the same target sample. Execute. FIG. 2 is a flowchart showing the procedure and processing in the first measurement.

  First, the operator inputs appropriate excitation light wavelength EX and fluorescence wavelength EM from the operation unit 20 as measurement conditions. Since this wavelength does not have to be so strict, it may be appropriately determined according to the type of the target sample. The scanning control unit 19 controls the motors 112 and 142 of the spectroscopes 11 and 14 so that the wavelengths corresponding to the instructions are selected (step S1). Next, the analysis control unit 7 operates the liquid feed pump 3 to feed the mobile phase to the column 5 at a constant flow rate, and the injector 4 injects the target sample into the mobile phase at a predetermined timing. Thereby, a liquid chromatograph (LC) analysis is started (step S2). With the start of the LC analysis, in the spectral fluorescence detection apparatus 1, the scanning timing determination processing unit 181 starts monitoring the intensity value of the detection data input (step S3).

  When the detection data for the eluate passing through the flow cell 6 is obtained, the scanning timing determination processing unit 181 determines whether the intensity value is equal to or greater than a preset threshold value TH1 (step S4). If the intensity value is less than the threshold value TH1, the process proceeds from step S4 to S13, and if the analysis is not completed, the process returns to step S3. For example, as shown in FIG. 5, when a sample component elutes from the outlet of the column 5 and is introduced into the flow cell 6, the intensity value starts to rise and exceeds the threshold value TH1 (at time t1 in FIG. 5). Then, the process proceeds from step S4 to S5, and this time, it starts to repeatedly calculate the temporal change amount of the intensity value. As shown in FIG. 5, the temporal change amount is the change amount ΔI of the intensity value during a certain minute time Δt.

  In general, when a peak due to a certain component appears in the chromatogram, the temporal change amount is large at the rising portion of the peak, and the temporal change amount becomes smaller as the peak top approaches. Therefore, it is determined whether or not the temporal change amount is equal to or less than a predetermined threshold value (step S6). If the temporal change amount is equal to or less than the threshold value (at time t2 in FIG. 5), the appearance of the sample component appears. Is detected, and the retention time (retention time) at that time is obtained (step S7). Further, a control signal is sent to the analysis control unit 7 to temporarily stop the operation of the liquid feeding pump 3 (step S8). Thereby, the supply of the mobile phase to the column 5 is stopped, and the eluate introduced immediately before in the flow cell 6 is held as it is.

  In place of the on / off control of the liquid feed pump 3, the flow of the mobile phase fed to the column 5 is changed to flow through another flow path by switching the flow path so that the sample in the column 5 is flown. The feeding may be temporarily stopped. In addition, during the period in which the liquid feeding is stopped in this way, the time measurement of the timer for measuring the holding time is also stopped to prevent the holding time from shifting.

  In the state where the eluate stays in the flow cell 6 as described above, the scanning control unit 19 performs wavelength scanning with the excitation side spectroscope 11 and the fluorescence side spectroscope 14 based on an instruction from the scanning timing determination processing unit 181. Perform (step S9). The data processing unit 18 acquires the three-dimensional spectrum by collecting the detection data obtained along with this wavelength scanning (step S10). This will be described in detail later. The obtained three-dimensional spectrum data and the holding time are stored in the data storage unit 182 in association with each other (step S11).

  After that, the analysis control unit 7 resumes the supply of the mobile phase by the liquid feed pump 3, and the LC analysis is performed again from the previous time (step S12). Then, for example, it is determined whether or not the analysis end condition is satisfied, for example, a predetermined end time has passed (step S13). If the analysis has not ended yet, the process returns to step S3. Therefore, until the end of the analysis, a three-dimensional spectrum is acquired every time a change occurs such that the intensity value of the detected data exceeds the threshold value TH1, and the three-dimensional spectrum is stored in the data storage unit 182 together with the holding time. In other words, as long as there is no temporal overlap of sample components, or there are no sample components that cannot be detected at the excitation light wavelength and fluorescence wavelength set at the beginning, three-dimensional spectra are created for the number of sample components. Become.

  Next, a procedure for acquiring a three-dimensional spectrum accompanying the wavelength scanning in steps S9 and S10 will be described with reference to FIG. In this example, it is assumed that wavelength scanning is performed at a wavelength step of 1 nm in a wavelength range of 200 to 750 nm (ultraviolet region to visible region).

  First, the motors 112 and 142 of the spectrometers 11 and 14 are controlled so that both the excitation light wavelength EX and the fluorescence wavelength EM are set to the start wavelength (200 nm) (step S21). Next, it is determined whether or not the excitation light wavelength EX at that time is equal to or less than the end wavelength (750 nm) (step S22). If it is equal to or less than the end wavelength, then the fluorescence wavelength EM is equal to or less than the end wavelength (750 nm). It is determined whether or not there is (step S23). If the fluorescence wavelength EM is less than or equal to the end wavelength, then the detection data obtained from the A / D converter 17 is read and stored (step S24), and the fluorescence wavelength EM is increased by 1 nm (step S25). Return to. Therefore, first, under the excitation condition where the excitation light wavelength EX is 200 nm, the detection data, that is, the fluorescence intensity value is changed while the wavelength range where the fluorescence wavelength EM is 200 to 750 nm is changed by 1 nm by repeating steps S23 to S25. collect.

  When the fluorescence wavelength EM exceeds 750 nm, the process proceeds from step S23 to S26, the fluorescence wavelength EM is returned to the start wavelength, this time the excitation light wavelength EX is increased by 1 nm (step S27), and the process returns to step S22. Accordingly, the fluorescence wavelength EM is scanned in a 1 nm step in the wavelength range of 200 to 750 nm under the excitation condition of the excitation light wavelength EX = 200 nm and then EX = 201 nm, and the detection data is collected. Thus, the fluorescence spectrum data in the wavelength range of 200 to 750 nm is collected while increasing the excitation light wavelength EX by 1 nm. When the excitation light wavelength EX exceeds 750 nm, the process proceeds from step S22 to S28, and the excitation light wavelength EX and the fluorescence wavelength are collected. The EM is returned to the original state and the process is terminated.

  By collecting data with wavelength scanning as described above, 3 having three dimensions of excitation light wavelength EX with a wavelength range of 200 to 750 nm, fluorescence wavelength EM with a wavelength range of 200 to 750 nm, and signal intensity 3 A dimensional spectrum is obtained and stored in the data storage unit 182. An example of the three-dimensional display of this three-dimensional spectrum is shown in FIG. As described above, the sample component is eluted from the column 5 and introduced into the flow cell 6 every time the sample is injected by the injector 4 of the liquid chromatograph, and the three-dimensional spectrum as shown in FIG. Acquisition is performed.

  The above three-dimensional spectrum is used exclusively for obtaining a rough waveform for determining the analysis conditions (excitation light wavelength and fluorescence wavelength). Therefore, the intensity value and the wavelength resolution are not important, and the scanning speed should be emphasized. Is preferred. Therefore, although the charge accumulation time at each wavelength may be short and the wavelength step may be large, the operation of the motors 112 and 142 that drive the diffraction gratings 111 and 141 and the timing of signal sampling in the A / D converter 17 Need to be synchronized.

As an example, the wavelength resolution of the motors (stepping motors) 112 and 142 for rotating the diffraction gratings 111 and 141 in the spectroscopes 11 and 14 is 1 step = 1 nm, and the motors 112 and 142 are operated at a constant speed of 2000 pps during scanning. Assume a case. In this case, if the wavelength step of the fluorescence wavelength EM is 1 nm and the wavelength step of the excitation light wavelength EX is 2 nm, the time required for scanning the fluorescence wavelength EX in the range of 200 to 750 nm is:
(1/2000 [pps]) × (750 [step] −200 [step] +1) = 275.5 [ms]
It is. Since it is necessary to return the fluorescence wavelength EM to 200 nm after one fluorescence wavelength scan and increase the excitation light wavelength EX by 2 nm, the time required for this is assumed to be 74.5 ms, and the excitation light wavelength EX is set to 200 to 750 nm. When scanning every 2 nm in the range,
(275.5 [ms] +74.5 [ms]) × {(750 [step] −200 [step]) / 2 + 1) = 96.5 [s]
It becomes. This is the time required to acquire one three-dimensional spectrum. However, since it is sufficient to perform the actual fluorescence analysis only in the range of the fluorescence wavelength EM> the excitation light wavelength EX, the scanning range of the fluorescence wavelength EM can be limited according to the excitation light wavelength EX, and the time actually taken Is shorter than the above estimated value.

  Now, after the three-dimensional spectrum as described above is obtained, a peak top determination process is executed by the peak search unit 183 for the three-dimensional spectrum, and appropriate excitation light wavelength and fluorescence wavelength are searched. This peak top determination process may be executed sequentially for all three-dimensional spectra after one LC analysis as shown in FIG. 2 is completed (so-called batch process), or LC You may be made to perform immediately (what is called sequential processing) whenever a new three-dimensional spectrum is acquired during analysis execution. Unlike wavelength scanning, the peak top determination process does not involve mechanical driving, so it can be performed in a short time, and there is no time problem even with sequential processing.

FIG. 4 is a flowchart showing a procedure of peak top determination processing for one three-dimensional spectrum.
First, both the excitation light wavelength EX and the fluorescence wavelength EM are set to the start wavelength (200 nm), and the maximum intensity value MAX, the excitation light wavelength EXmax giving the maximum intensity value, and the fluorescence wavelength EMmax giving the maximum intensity value are all zero. Initialization is performed (step S31). Next, it is determined whether or not the excitation light wavelength EX at that time is equal to or less than the end wavelength (750 nm) (step S32). If it is equal to or less than the end wavelength, then the fluorescence wavelength EM is equal to or less than the end wavelength (750 nm). Is determined (step S33). If the fluorescence wavelength EM is equal to or less than the end wavelength, the intensity value D for the excitation light wavelength and the fluorescence wavelength at that time is read from the data storage unit 182 in the three-dimensional spectrum (step S34), and the intensity value D is the maximum intensity value. It is determined whether it is larger than MAX (step S35).

  If the intensity value D is larger than the maximum intensity value MAX, the maximum intensity value MAX is updated to the intensity value D, and the excitation light wavelength EX and the fluorescence wavelength EM at that time are stored in EXmax and EMmax, respectively (step S36). On the other hand, if the intensity value D is less than or equal to the maximum intensity value MAX in step S35, it is not necessary to update MAX, EXmax, and EMmax, so step S36 is passed, and all increase the fluorescence wavelength EM by 1 nm (step S37). ) Return to Step S33. Therefore, first, under the condition of the excitation light wavelength EX = 200 nm, the maximum intensity value is searched by repeating Steps S33 to S37, and the fluorescence wavelength EM giving the maximum intensity value is obtained.

  When the fluorescence wavelength EM exceeds 750 nm, the process proceeds from step S33 to S38, the fluorescence wavelength EM is returned to the start wavelength, this time the excitation light wavelength EX is increased by 1 nm (step S39), and the process returns to step S32. Therefore, next to the excitation light wavelength EX = 200 nm, with respect to EX = 201 nm, it is searched whether there is any fluorescent spectrum in the wavelength range of 200 to 750 nm that exceeds the previously determined maximum intensity value. . If the excitation light wavelength EX exceeds 750 nm, NO is determined in step S32 and the process ends. Accordingly, the maximum intensity value in the three-dimensional spectrum is stored in MAX at the end of the process, and the excitation light wavelength EX and the fluorescence wavelength EM at that time are stored in EXmax and EMmax, respectively. That is, since this is a condition of the excitation light wavelength and the fluorescence wavelength that gives a peak having the maximum signal intensity, the combination of these wavelengths and the retention time when the three-dimensional spectrum is acquired are displayed on the display unit 21. Tell the user.

  Since the peak top determination process as described above is performed for each three-dimensional spectrum, for example, as shown in FIG. 7, information including a holding time t, an excitation light wavelength EX, and a fluorescence wavelength EM is created, This is output from the display unit 21. Based on this, a program for setting the excitation light wavelength and the fluorescence wavelength was assembled, and the second detailed analysis was performed on the target sample, thereby accurately reflecting the contents of various components contained in the target sample. An intensity value can be obtained, and high-precision quantitative analysis can be performed from this intensity value. Further, the excitation light wavelength and the fluorescence wavelength may be automatically changed for each holding time based on the analysis condition information as shown in FIG.

  In the above peak top determination process, only one peak having the maximum intensity value is searched for one three-dimensional spectrum. It is also possible to search for a plurality of peaks such as a large one and inform the user of the excitation light wavelength and the fluorescence wavelength for obtaining the peak. In this case, since a plurality of pairs of excitation light wavelengths and fluorescence wavelengths are presented for the same holding time, it is necessary for the user to select which combination is to be used when performing the detailed analysis. Therefore, it is advisable to perform a display that prompts such a selection or display in an easy-to-select manner.

  For example, a three-dimensional spectrum as shown in FIG. 6 is displayed on the screen of the display unit 21 and a peak having a large intensity value can be identified (for example, a display color is changed). By selecting and instructing with a pointing device such as a mouse, the excitation light wavelength and the fluorescence wavelength corresponding to the instructed peak may be automatically set as analysis conditions. By using such a graphical user interface, operational errors can be reduced.

  Each of the above embodiments is merely an example of the present invention, and it will be understood that the present invention is encompassed by the scope of the claims of the present application, even if appropriate changes, modifications, additions, etc. are made within the scope of the present invention.

The block diagram of the principal part of the liquid chromatograph by one Example of this invention. The flowchart which shows the procedure and process in the case of the measurement in order to determine the analysis conditions in the liquid chromatograph of a present Example. The flowchart which shows the procedure of acquisition of the three-dimensional spectrum accompanying wavelength scanning. The flowchart which shows the procedure of the peak top determination process with respect to one three-dimensional spectrum. Schematic for demonstrating the detection operation of appearance of a sample component. The figure which shows an example of a three-dimensional spectrum. The figure which shows the example of an output of the result of a peak top determination process. 1 is a schematic configuration diagram of a general spectral fluorescence detection apparatus.

Explanation of symbols

DESCRIPTION OF SYMBOLS 1 ... Spectral fluorescence detection apparatus 10 ... Light source 11 ... Excitation side spectroscope 12 ... Sample cell 14 ... Fluorescence side spectroscope 111, 141 ... Diffraction gratings 112, 142 ... Motor 15 ... Photomultiplier 16 ... I / V conversion part 17 ... A / D conversion unit 18 ... data processing unit 181 ... scanning timing determination processing unit 182 ... data storage unit 183 ... peak search unit 19 ... scanning control unit 20 ... operation unit 21 ... display unit 2 ... mobile phase container 3 ... liquid feeding Pump 4 ... Injector 5 ... Column 6 ... Flow cell 7 ... Analysis control unit

Claims (3)

A spectrofluorescence detection device for detecting a sample component separated by a column of a liquid chromatograph, comprising: a light source; an excitation-side spectroscopic means for separating light emitted from the light source and extracting light of a predetermined wavelength; and the excitation side A fluorescence-side spectroscopic means for separating the fluorescence obtained from the sample with respect to the excitation light extracted by the spectroscopic means and extracting light of a predetermined wavelength; a photodetector for detecting the fluorescence extracted by the fluorescence-side spectroscopic means; In the spectral fluorescence detection apparatus for a liquid chromatograph, comprising: a wavelength changing unit that changes a wavelength of light extracted for each of the excitation side spectroscopic unit and the fluorescence side spectroscopic unit,
a) component detection means for detecting the eluate from the column in a state where the excitation light wavelength and the fluorescence wavelength predetermined by the wavelength changing means are set, and detecting the appearance of a sample component based on the detection signal; ,
b) an instruction means for giving an instruction to temporarily stop the liquid feeding to the column when the appearance of the sample component is detected by the component detection means;
c) In a state where liquid feeding is stopped according to the instruction from the instruction means, the wavelength changing means scans the excitation light wavelength and the fluorescence wavelength, respectively, and uses the excitation light wavelength, the fluorescence wavelength and the signal intensity as dimensions. Spectrum acquisition means for acquiring a spectrum;
d) search means for searching for a combination of an excitation light wavelength and a fluorescence wavelength that gives a peak having a maximum signal intensity or a relatively large signal intensity with respect to the three-dimensional spectrum;
e) an output means for outputting the excitation light wavelength and the fluorescence wavelength obtained by the search means together with the time information at which the sample component from which the three-dimensional spectrum was acquired appears;
A spectral fluorescence detection apparatus for liquid chromatography, comprising:   2. The spectral fluorescence detection apparatus for liquid chromatograph according to claim 1, wherein a user sets a wavelength range of an excitation light wavelength and a fluorescence wavelength when acquiring a three-dimensional spectrum by the spectrum acquisition unit. A spectral fluorescence detection apparatus for liquid chromatography, further comprising:   3. The spectrofluorescence detection apparatus for liquid chromatograph according to claim 1 or 2, wherein the three-dimensional spectrum acquired by the spectrum acquisition means is rendered and the peak found by the search means is displayed in a recognizable manner. Means, an operation means for a user to select a peak on the three-dimensional spectrum displayed on the display means, and an excitation light wavelength and a fluorescence wavelength that give a peak specified according to the selection instruction by the operation means And an analysis condition setting means for setting as an analysis condition for the liquid chromatograph.

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