HPLC Raman spectroscopy analyzer, HPLC Raman spectroscopy method

Abstract

By integrating an HPLC analyzer and a Raman spectrometer, and efficiently focusing Raman scattered light for analysis, the time required for sample analysis is reduced, and sample degradation during analysis is prevented. [Solution] The system comprises at least: a separation column that separates the components of the sample contained in a mobile phase containing the sample; a flow cell through which the eluate flowing out of the separation column flows; a laser light source that emits laser light, which is an irradiation light, toward the flow cell; a Raman probe disposed between the laser light source and the flow cell, which selectively transmits the Raman scattered light generated in the flow cell; a focusing means that focuses the Raman scattered light generated in the flow cell and reflects it toward the Raman probe; and a spectral measurement unit that performs analysis of the sample based on the spectrum of the Raman scattered light output from the Raman probe.

Description

Technical Field

[0001] The present invention relates to an HPLC Raman spectroscopic analyzer and an HPLC Raman spectroscopic analysis method.

Background Art

[0002] An HPLC (High Performance Liquid Chromatography) analyzer is column chromatography that separates and analyzes a sample containing a plurality of components for each constituent component, and is widely used for analyzing compounds in which a plurality of components are mixed. On the other hand, a Raman spectroscopic apparatus irradiates illumination light toward a sample, and is used for analyzing the structure of substances contained in the sample by using the spectrum of Raman scattered light having a wavelength different from that of the illumination light generated from the sample.

[0003] In studying carotenoid pigments, the inventors first isomerized an all-trans isomer solution and separated and fractionated a solution in which a plurality of isomers were mixed for each component using an HPLC analyzer in the isolation, purification, and fractionation of geometric isomers of carotenoids. However, since the amount of each isomer component obtained by one HPLC analysis is small, the amount of each isomer component required for Raman spectroscopic analysis was ensured by repeating the isolation to fractionation operations a plurality of times. However, some isomers are very unstable and some deteriorate during multiple fractionations, and there is also a problem that highly accurate isolation and purification is difficult.

[0004] Conventionally, in order to separate a sample for each component using an HPLC analyzer and perform Raman scattering measurement, since the Raman scattered light obtained by the Raman spectroscopic apparatus has a very weak light intensity, after isolating and fractionating the sample for each component using the HPLC analyzer, it was necessary to perform analysis for each component. In this case, the amount of the sample of each component required for analysis by the Raman spectroscopic apparatus is not sufficient with the amount obtained by one operation using the HPLC analyzer, and there is a problem that it is necessary to perform multiple operations.

[0005] Furthermore, there were challenges such as the difficulty of separating each component using an HPLC analyzer and the degradation of the sample over time due to the need for multiple separations. In addition, since the Raman scattered light obtained by a Raman spectrometer is very weak, there was a concern that increasing the light intensity to obtain appropriate analytical results would degrade or decompose the sample due to irradiation with high-intensity light.

[0006] For this reason, for example, Patent Document 1 discloses an analytical method for acquiring spectral data of an analyte using a spectral measuring device that includes a flow cell through which a sample solution flows and a photodetector for detecting the amount of light in each wavelength band from the flow cell, and performs spectral measurement of the sample solution flowing through the flow cell. Patent Document 1 states that spectral data of an analyte in a solution flowing through a flow cell can be acquired with high signal intensity.

[0007] Furthermore, Non-Patent Document 1 describes how to perform highly accurate Raman spectroscopy analysis of a sample by using a high-power semiconductor laser with a wavelength of 785 nm and an irradiation intensity of 300 mW to perform Raman scattering measurements under non-resonant conditions. [Prior art documents] [Patent Documents]

[0008] [Patent Document 1] Japanese Patent Publication No. 2023-131244 [Non-patent literature]

[0009] [Non-Patent Document 1] Online Liquid Chromatography - Raman Spectroscopy Using the Vertical Flow Method, Analytical Chemistry 92 (2020) 14601-14607 [Overview of the Initiative] [Problems that the invention aims to solve]

[0010] However, in the invention disclosed in Patent Document 1, the intensity of the Raman scattered light obtained is weak, so it is necessary to irradiate with a high-intensity laser light. However, there was a concern that the target component would be photodegraded if a high-intensity laser light was irradiated.

[0011] Furthermore, Non-Patent Document 1 describes performing highly accurate Raman spectroscopy analysis of a sample by using a high-power semiconductor laser to perform Raman scattering measurements under non-resonant conditions. However, there was a concern that the analyte would be photodegraded if high-intensity laser light from such a high-power semiconductor laser was irradiated onto the analyte.

[0012] The present invention has been proposed in view of the above problems, and aims to provide an HPLC-Raman spectrometer and an HPLC-Raman spectrometer that integrate an HPLC analyzer and a Raman spectrometer, and further efficiently collects Raman scattered light for analysis, thereby shortening the time required for sample analysis and preventing sample degradation during analysis. [Means for solving the problem]

[0013] The inventors have discovered a new apparatus and method that integrates an HPLC analyzer and a Raman spectrometer to reduce time constraints and human errors associated with preparative analysis of sample solutions. This simplifies the conventional analysis process of isolation → preparative → measurement, and adopts a consistent process of isolation → measurement, enabling real-time analysis while minimizing sample degradation.

[0014] Furthermore, by utilizing the resonance Raman effect, which is excited at a wavelength that resonates with the absorption of the sample, the problem of the low intensity of Raman scattered light is solved, and it was found that the resonance Raman effect makes it possible to measure Raman scattering even with trace amounts of components. In addition, because the intensity of scattered light is increased by the resonance Raman effect, the intensity of the irradiated light can be kept low. As a result, a configuration was found that minimizes the risk of sample degradation due to measurement.

[0015] To solve the above problems, the HPLC Raman spectroscopy apparatus and HPLC Raman spectroscopy method of one embodiment of the present invention propose the following means. (1) An HPLC Raman spectrometer according to embodiment 1 of the present invention comprises at least: a separation column for separating the components of a sample contained in a mobile phase containing the sample; a flow cell for carrying the eluate flowing out of the separation column; a laser light source for emitting laser light, which is an irradiation light, toward the flow cell; a Raman probe disposed between the laser light source and the flow cell for selectively transmitting Raman scattered light generated in the flow cell; a focusing means for focusing the Raman scattered light generated in the flow cell and reflecting it toward the Raman probe; and a spectrum measurement unit for performing analysis of the sample based on the spectrum of Raman scattered light output from the Raman probe.

[0016] (2) A second aspect of the present invention is the HPLC Raman spectrometer of the first aspect, wherein the Raman probe further comprises a notch filter that blocks light in the wavelength range of the laser light.

[0017] (3) Embodiment 3 of the present invention is an HPLC Raman spectrometer according to Embodiment 1 or 2, wherein the focusing means is composed of a concave mirror.

[0018] (4) Embodiment 4 of the present invention is an HPLC Raman spectrometer according to any one of Embodiments 1 to 3, wherein the sample comprises an olefin compound and an aromatic compound.

[0019] (5) The HPLC Raman spectroscopic analysis method of Embodiment 5 of the present invention is an HPLC Raman spectroscopic analysis method using any one of the HPLC Raman spectroscopic analyzers of Embodiments 1 to 4, and includes a separation step of separating the sample contained in the mobile phase containing the sample into components for each component, an irradiation step of irradiating the eluate flowing out from the separation column with the laser light to generate the Raman scattered light corresponding to the components of the sample, and a spectral measurement step of condensing the Raman scattered light and then analyzing the sample based on the spectrum of the Raman scattered light.

[0020] (6) Embodiment 6 of the present invention is the HPLC Raman spectroscopic analysis method of Embodiment 5, and in the spectral measurement step, resonance Raman scattering generated by making the wavelength of the laser light approach or coincide with the electronic absorption band of the sample is used.

[0021] (7) Embodiment 7 of the present invention is the HPLC Raman spectroscopic analysis method of Embodiment 5 or 6, and in the HPLC Raman spectroscopic analysis method, quantification is performed for each isomer of olefin compounds and aromatic compounds.

Advantages of the Invention

[0022] According to the present invention, by integrating an HPLC analyzer and a Raman spectroscopic measurement device, and further efficiently condensing and analyzing Raman scattered light, it is possible to provide an HPLC Raman spectroscopic analyzer capable of shortening the time related to the analysis of a sample and preventing deterioration of the sample during analysis, and an HPLC Raman spectroscopic analysis method using the same.

Brief Description of the Drawings

[0023] [Figure 1] It is a schematic configuration diagram showing an HPLC Raman spectroscopic analyzer of an embodiment of the present invention. [Figure 2] It is a flowchart showing step by step a Raman spectroscopic analysis method of an embodiment of the present invention. [Figure 3] It is a graph showing the results of an example. [Figure 4]This is a comparison between the results of quantum chemical calculations and the results obtained through actual testing. [Modes for carrying out the invention]

[0024] The following describes an HPLC Raman spectrometer and an HPLC Raman spectrometer according to one embodiment of the present invention, with reference to the drawings. The embodiments described below are provided specifically to better illustrate the spirit of the invention and do not limit the present invention unless otherwise specified. In addition, the drawings used in the following description may be enlarged for convenience to make the features of the present invention easier to understand, and the dimensional ratios of each component may not be the same as in reality.

[0025] (HPLC Raman spectroscopy analyzer) Figure 1 is a schematic diagram showing an HPLC Raman spectroscopy analyzer according to one embodiment of the present invention. The HPLC Raman spectroscopy analyzer 10 of this embodiment includes a solvent container 11, a sample injector 25, a liquid delivery pump 12, a separation column 13, a flow cell 14, a Raman probe 15, a notch filter 16 placed on the Raman probe 15, a concave mirror 17 which is a focusing means, a laser light source 18, a Raman light focusing optical system 19, and a spectrum measurement unit 21. Of these, the laser light source 18 and the Raman probe 15, and the Raman probe 15 and the Raman light focusing optical system 19 are connected by optical fibers Gf.

[0026] The spectrum measurement unit 21 may include a Raman light detection unit 22 and a calculation unit 23. Furthermore, a laser light focusing optical system 24 may be formed between the laser light source 18 and the Raman probe 15.

[0027] The solvent container 11 contains a solvent (developing solvent) for dissolving the component to be analyzed. Any liquid capable of dissolving the component to be analyzed can be used as the solvent; for example, any solvent such as water, alcohol, acetic acid, ethyl acetate, benzene, acetone, n-hexane, or cyclohexane can be used. Typically, the solvent is a mixture of two or more liquids.

[0028] The liquid transfer pump 12 is a pump that pushes the solvent (developing solvent) from the solvent container 11 and sends it towards the sample injector 25 at a predetermined pressure and flow rate. It can be any pump capable of delivering a small amount of liquid at a precise flow rate, such as a diaphragm pump, turbine pump, axial flow pump, or tube pump.

[0029] The sample injector 25 injects the component to be analyzed, and the component to be analyzed dissolves in the solvent (developing solvent) injected by the liquid delivery pump 12 to form a liquid sample. Examples of liquid samples include solutions containing multiple components. One example of such a liquid sample is a solution containing an unknown isomer of carotenoid (15-cis type, dic type, etc.) and other carotenoids. The HPLC Raman spectrometer 10 of this embodiment can also be suitably applied to the detection and analysis of such carotenoid isomers.

[0030] Carotenoids, an example of olefin compounds, are generally composed of eight 2-methyl-1,3-butadiene (isoprene) units linked together. 40 H 56 These are pigments that have a basic structural framework, and specific examples include beta-carotene, lycopene, lutein, astaxanthin, and zeaxanthin.

[0031] The separation column 13 consists of, for example, a cylindrical tube and a stationary phase packed inside this cylindrical tube. The stationary phase may, for example, contain spherical particles with chemically modified functional groups.

[0032] When a liquid sample is introduced into the separation column 13 from one end by the liquid delivery pump 12, the rate at which the sample flows out from the other end of the separation column 13 changes depending on the strength of the interaction between the analyte contained in the liquid sample and the mobile phase, which is the solvent in which the component is dissolved. In other words, if the interaction between the component and the mobile phase is strong, the sample flows out of the separation column 13 quickly, and if the interaction between the component and the stationary phase is strong, the sample flows out of the separation column 13 slowly.

[0033] In this way, the separation column 13 separates the components of the liquid sample by causing differences in the migration speed of the components through the interaction (adsorption, partitioning) between each component in the liquid sample and the stationary phase packed in the separation column 13. This causes the arrival time of each component from one end of the separation column 13 to the other to differ, thereby separating the components in the liquid sample from each other.

[0034] The flow cell 14 is made of a transparent tube, such as a quartz glass tube, through which the laser light emitted from the laser light source 18 and the Raman scattered light generated therein can pass. This flow cell 14 is connected to the separation column 13, and each separated component flowing out of the separation column 13 is sequentially introduced into it. Then, laser light emitted from the laser light source 18 is shone into it, generating Raman scattered light of different wavelengths for each component separated in the separation column 13.

[0035] The Raman probe 15 irradiates the flow cell 14 with laser light (excitation light) emitted from the laser light source 18, and simultaneously receives Raman scattered light of different wavelengths for each component passing through the flow cell 14, outputting it towards the Raman light focusing optical system 19.

[0036] The notch filter 16 placed on the Raman probe 15 is an optical filter that blocks the laser light (excitation light) and transmits only Raman scattered light, such as Stokes (long wavelength) scattered light and anti-Stokes (short wavelength) scattered light, toward the Raman light focusing optical system 19.

[0037] The laser light source 18 irradiates the flow cell 14 with laser light (excitation light) to generate a unique Raman scattering light for each component passing through it. The laser light (excitation light) to be irradiated can be selected according to the component to be analyzed and is not limited to any particular type, but examples include green laser light with a wavelength of 532 nm, blue laser light with a wavelength of 488 nm, and near-infrared laser light with a wavelength of 785 nm.

[0038] It is also preferable to set the wavelength of the laser light emitted from the laser light source 18 to approach or match the electron absorption band of a component expected to be contained in the liquid sample being analyzed. This makes it possible to generate resonant Raman scattered light, in which the intensity of the Raman scattered light is amplified by resonance.

[0039] The laser light focusing optical system 24 is an optical system that focuses the laser light (excitation light) emitted from the laser light source 18. By injecting the focused laser light (excitation light) into the flow cell 14, locally stronger Raman scattered light can be generated from the detection component.

[0040] The light-gathering means, a concave mirror (light-gathering means) 17, is an optical mirror that reflects and focuses the Raman scattered light emitted in the direction opposite to the Raman probe 15 from the Raman scattered light generated by the laser light (excitation light) irradiated onto the flow cell 14 via the Raman probe 15, and directs it into the Raman probe 15.

[0041] By using such a concave mirror (light-gathering means) 17, Raman scattered light other than the Raman scattered light directly emitted from the flow cell 14 toward the Raman probe 15 can also be used for measurement and analysis, and weak Raman scattered light can be efficiently utilized.

[0042] The Raman light focusing optical system 19 focuses the Raman scattered light emitted from the Raman probe 15, which differs in its component, and decomposes it into a spectrum using a spectrometer to generate a Raman spectrum.

[0043] The spectral measurement unit 21 detects the Raman spectrum with the Raman light detection unit 22, and the calculation unit 23 identifies each component based on the characteristics of the Raman spectrum for each component.

[0044] The Raman light detection unit 22 is preferably a photosensor with high quantum efficiency for the wavelength of the component to be detected. Furthermore, since it is preferable to simultaneously detect spectrally separated light of multiple wavelengths in Raman spectroscopy, a two-dimensional detector with an array of elements is preferably used.

[0045] Cooled CCDs (charge-coupled CDs) are commonly used as two-dimensional detectors in such Raman light detection units 22. Furthermore, when performing Raman spectroscopy in the near-infrared wavelength range to avoid fluorescence, InGaAs detectors or EMCCDs (electron-multiplier CCDs), which significantly increase detection sensitivity by multiplying electrons photoelectrically converted by the applied voltage, can also be used.

[0046] The calculation unit 23, for example, consists of a personal computer and an interface, and compares the Raman spectral data for each component with known Raman spectral data for each substance stored in the calculation unit 23's memory or on a network to identify the composition and types of isomers of each component contained in the liquid sample. These analysis results can be displayed on a display connected to the calculation unit 23.

[0047] (HPLC Raman spectroscopy method) Next, an embodiment of the operation of the HPLC Raman spectroscopy analyzer and the Raman spectroscopy method described above will be explained. Figure 2 is a flowchart illustrating the steps involved in a Raman spectroscopy analysis method according to one embodiment of the present invention.

[0048] When analyzing a liquid sample containing carotenoids (components) with multiple isomers using the HPLC Raman spectrometer 10 configured as described above, first, the liquid delivery pump 12 is operated to push the solvent out of the solvent container 11 and inject it into the sample injector 25 into which the component to be analyzed has been injected, dissolving the component to be analyzed in the solvent to form a liquid sample. Then, the formed liquid sample is injected into the separation column 13 at a predetermined pressure and flow rate (sample injection step S1).

[0049] The liquid sample injected into the separation column 13 can be any solution containing multiple components to be analyzed. For example, a solution (liquid sample) in which an unknown isomer of carotenoid (such as 15-cis or 15-discis form) and other carotenoids are dissolved in any solvent may be used.

[0050] Next, the separation column 13 separates each component contained in the injected liquid sample (separation step S2). In this separation step S2, the interaction (adsorption, partitioning) between the stationary phase packed in the separation column 13 and the multiple types of components contained in the liquid sample causes differences in the migration speed of each component within the separation column 13. That is, if the interaction between the component and the mobile phase is strong, it flows out of the separation column 13 faster, and if the interaction between the component and the stationary phase is strong, it flows out of the separation column 13 more slowly. By doing so, the time it takes for each component to reach the other end of the separation column 13 is made different for each component, thereby separating the various components contained in the liquid sample.

[0051] Next, laser light is irradiated onto the eluate, which has been separated by component and is flowing out of the separation column 13 and introduced into the flow cell 14, to generate Raman scattered light with different characteristics for each component of the liquid sample (irradiation step S3).

[0052] In this irradiation step S3, laser light (excitation light) is irradiated from the laser light source 18 towards the flow cell 14 via the Raman probe 15. At this time, the irradiated laser light is focused by the laser light focusing optical system 24 before being incident on the flow cell 14, thereby generating locally stronger Raman scattered light from the detection component.

[0053] Furthermore, by adjusting the wavelength of the laser light emitted from the laser light source 18 to, for example, approach or match the electron absorption band of a component expected to be contained in the liquid sample being analyzed, resonant Raman scattered light can be generated, in which the intensity of the Raman scattered light is amplified by resonance.

[0054] When laser light (excitation light) is incident on each component flowing through the flow cell 14, each component emits resonant Raman scattered light in a wavelength range different from that of the laser light. This resonant Raman scattered light is received by the Raman probe 15, but since resonant Raman scattered light is not emitted in only one direction, it is also emitted in directions other than the Raman probe 15.

[0055] Resonant Raman scattered light emitted in directions other than the Raman probe 15, for example, in the direction opposite to the Raman probe 15, is reflected and focused by the concave mirror (focusing means) 17 and incident on the Raman probe 15 (reflection and focusing step S4). This allows resonant Raman scattered light other than the resonant Raman scattered light emitted directly from the flow cell 14 toward the Raman probe 15 to be used for measurement and analysis, and enables efficient use of Raman scattered light with a low light intensity.

[0056] Of the light incident on the Raman probe 15, the laser light (excitation light) is blocked by the notch filter 16, so that only resonant Raman scattered light, such as Stokes (long wavelength) scattered light and anti-Stokes (short wavelength) scattered light corresponding to each component, is incident on the Raman light focusing optical system 19.

[0057] Then, the resonant Raman scattered light, which has different characteristics for each component, is incident on the Raman light focusing optical system 19 and is decomposed into spectra by a spectrometer, generating Raman spectra for each component (spectrum generation step S5).

[0058] Then, the Raman spectra, each with different characteristics for each component, that are incident from the Raman light focusing optical system 19 to the spectral measurement unit 21 are detected by a Raman light detection unit 22, which is, for example, a CCD (spectral measurement step S6).

[0059] Then, in the calculation unit 23 that constitutes the spectral measurement unit 21, the Raman spectral data detected for each component is compared with the known Raman spectral data for each substance. This identifies the composition and isomer types of each component contained in the liquid sample. These results are displayed, for example, on a display connected to the calculation unit 23.

[0060] As described above, the HPLC-Raman spectroscopy method using the HPLC-Raman spectrometer 10 of this embodiment utilizes the resonance Raman effect to select the excitation light, making it possible to perform measurements even with excitation light of much weaker intensity than that of conventionally known Raman spectrometers. This prevents degradation and decomposition of the sample (components) due to irradiation with high-intensity excitation light.

[0061] In the HPLC Raman spectrometer 10 of this embodiment described above, the wavelength of the excitation light can be selected, so any sample (component) that absorbs the excitation light and has low fluorescence can be measured and analyzed.

[0062] These properties can be applied to the measurement and analysis of olefin compounds such as carotenoids, as well as aromatic compounds such as anthocyanins and aromatic amines (and their derivatives), as mentioned as an example. Furthermore, since Raman spectra can be accurately reproduced by quantum chemical calculations, combining these calculations with other methods can increase the basis for structural analysis of components, and for example, it becomes possible to quantify cis-trans isomers of carotenoids. By using Raman bands with very little difference in Raman scattering intensity depending on the type of isomer as an internal standard, it also becomes possible to quantify the component proportion of each isomer in an isomer mixture.

[0063] Although embodiments of the present invention have been described above, these embodiments are presented as examples and are not intended to limit the scope of the invention. These embodiments can be carried out in various other forms, and various omissions, substitutions, and modifications can be made without departing from the spirit of the invention. These embodiments and their variations are included in the scope and spirit of the invention, as well as in the claims and their equivalents. [Examples]

[0064] An HPLC-Raman spectrometer with the configuration shown in Figure 1 was assembled. As the analyte, 10 mg of astaxanthin was dissolved in 10 mL of dichloromethane and sealed in a pressure-resistant container. This container was placed in an oil bath and heated at 80°C for 6 hours. Afterward, the container was immersed in ice water and allowed to cool to room temperature (25°C) and atmospheric pressure. The astaxanthin isomer mixture solution was removed from the pressure-resistant container, and the solvent was removed using an evaporator. This was dissolved in 2 mL of developing solvent (acetone:ethyl acetate:n-hexane = 1:1:8), and 0.02 mL of this solution was injected into a sample injector. The sample solution dissolved in the developing solvent was then injected into separation column 13. A CAPCELL PAK SILICA SG120 (manufactured by Osaka Soda Co., Ltd.: φ4.6 mm, length 250 mm) was used as the separation column, and measurements were taken at a flow rate of 0.8 mL for 40 minutes. Figure 3 shows the Raman spectra obtained using the mixture of nine different astaxanthin isomers. Figure 4 shows a comparison with the results obtained by quantum chemical calculations.

[0065] As shown in Figures 3 and 4, it was confirmed that by using the HPLC Raman spectroscopy analyzer of this embodiment, it is possible to measure and analyze samples (components) containing these multiple isomers with high accuracy. [Industrial applicability]

[0066] The HPLC-Raman spectroscopy apparatus and method of the present invention enable high-precision analysis using weak excitation light, even for samples that are easily altered by light. Therefore, it has industrial applicability. [Explanation of Symbols]

[0067] 10…HPLC Raman Spectrometer 11… Solvent container 12…Liquid transfer pump 13…Separation column 14…Flow Cell 15…Raman probe 16... Notch filter 17…Concave mirror (light-gathering means) 18…Laser light source 19…Raman light focusing optical system 21…Spectrum measurement section 22...Raman light detection unit 23...Arithmetic section 24…Laser light focusing optical system 25... Sample Injector

Claims

1. A separation column that separates the components of the sample contained in the mobile phase, A flow cell through which the eluate flowing out of the separation column flows, A laser light source that emits laser light, which is the irradiation light, toward the flow cell, A Raman probe is positioned between the laser light source and the flow cell, and selectively transmits the Raman scattered light generated in the flow cell. A focusing means for collecting the Raman scattered light generated in the flow cell and reflecting it toward a Raman probe, A spectral measurement unit that analyzes the sample based on the spectrum of Raman scattered light output from the Raman probe, An HPLC Raman spectrometer having at least the following.

2. The HPLC Raman spectrometer according to claim 1, further comprising a notch filter for blocking light in the wavelength range of the laser light in the Raman probe.

3. The HPLC Raman spectrometer according to claim 1 or 2, wherein the light-gathering means is composed of a concave mirror.

4. The HPLC Raman spectrometer according to claim 1 or 2, wherein the sample comprises an olefin compound and an aromatic compound.

5. An HPLC-Raman spectroscopy method using the HPLC-Raman spectroscopy apparatus described in claim 1 or 2, A separation step in which the sample contained in the mobile phase containing the sample is separated from each other according to its constituent components, An irradiation step in which the laser light is irradiated toward the eluate flowing out of the separation column to generate Raman scattered light corresponding to the constituent components of the sample, An HPLC-Raman spectroscopy method comprising: a spectral measurement step of focusing the Raman scattered light and then analyzing the sample based on the spectrum of the Raman scattered light.

6. The HPLC-Raman spectroscopy method according to claim 5, wherein the spectral measurement step uses resonant Raman scattering generated by bringing the wavelength of the laser light close to or matching the electron absorption band of the sample.

7. The HPLC Raman spectroscopy method according to claim 5, wherein the HPLC Raman spectroscopy method performs quantitative analysis of each isomer of the aromatic compound.

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