Raman analysis plate, Raman analysis device, analysis system, and Raman analysis method

Abstract

To provide a Raman analysis plate that is capable of stably holding a liquid sample separated through chromatography, in this invention, a Raman analysis plate 21 used for Raman spectroscopy of a liquid sample S separated through chromatography is made to comprise metal exposure areas 211 that are provided in a plurality of locations on the surface of the plate and hold the sample S and a hydrophobic area 212 that is provided on the surface of the plate, surrounds the metal exposure areas 211, and is more hydrophobic than the metal exposure areas 211.

Description

[Technical field]

[0001] The present invention relates to a plate for Raman analysis and a Raman analysis device using the same. [Background technology]

[0002] As disclosed in Patent Document 1, there is a Raman analysis apparatus configured to analyze a liquid sample separated by chromatography using Raman spectroscopy.

[0003] Specifically, this device dispenses the separated liquid sample into multiple locations on a glass plate or a water-repellent plate, dries it up, and obtains the Raman spectrum of the dried sample.

[0004] However, as mentioned above, when using a glass plate or a water-repellent plate, the separated liquid sample moves around on the plate (for example, if the sample is an organic solvent, it becomes wet and the droplets spread), making it difficult to hold it stably. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] International Publication No. 2014 / 027652 Summary of the Invention [Problem to be solved by the invention]

[0006] Therefore, a main object of the present invention is to provide a plate for Raman analysis that can stably hold a liquid sample separated by chromatography. [Means for solving the problem]

[0007] In other words, the Raman analysis plate of the present invention is a Raman analysis plate used for analyzing a liquid sample separated by chromatography by Raman spectroscopy, and is characterized in that it comprises metal exposed regions provided at multiple locations on the plate surface for holding the sample, and hydrophobic regions provided on the plate surface, surrounding the metal exposed regions, and having higher hydrophobicity than the metal exposed regions.

[0008] In the Raman analysis plate configured in this way, the metal exposed regions provided at multiple locations on the plate surface are surrounded by the more hydrophobic surface region, so that by separating a liquid sample into the metal exposed regions, the separated sample can be retained in the metal exposed regions, thereby making it possible to stably hold the liquid sample separated by chromatography, thereby improving the analytical accuracy. Furthermore, since the sample is held in the exposed metal area and the Raman light generated from the sample is reflected by the exposed metal area, the Raman light can be directed to the photodetector more efficiently than when, for example, a glass plate is used as the Raman analysis plate. Moreover, since the sample is held in the exposed metal area, the background from the glass that occurs when a glass plate is used can be suppressed, and the analytical accuracy can be further improved.

[0009] In order to impart high reflectance to the exposed metal area, the exposed metal area is preferably made of gold, silver, or aluminum.

[0010] Furthermore, the Raman analysis apparatus according to the present invention is characterized by comprising the above-mentioned Raman analysis plate, a light irradiator that irradiates light onto the sample on the Raman analysis plate, a photodetector that detects Raman light generated from the sample when irradiated with light, and an analysis unit that analyzes the sample based on the detected Raman spectrum. Such a Raman analysis device is equipped with the above-mentioned Raman analysis plate, and is therefore able to stably hold a liquid sample separated by chromatography, thereby improving the analytical accuracy.

[0011] When using the above-mentioned Raman analysis device, the liquid sample dispensed onto the Raman analysis plate is waited for to dry up, and then the dried up sample is irradiated with light for analysis. Generally, however, the measurement range in Raman analysis is determined visually, and it is difficult to find a dried up sample by visual inspection, so it takes time to determine the measurement range.

[0012] Therefore, it is preferable that the apparatus further includes an imaging device for imaging the surface of the Raman analysis plate, and the analysis unit determines a measurement range including the sample dried on the Raman analysis plate based on the imaging data obtained by the imaging device, and analyzes the measurement range. With this configuration, the measurement range can be determined in a short time without relying on visual inspection.

[0013] The present invention also provides an analytical system comprising the above-mentioned Raman analysis device and a separation mechanism that includes a separation column through which the liquid sample is delivered by a mobile phase and separates the sample by chromatography. According to such an analysis system, since it is equipped with the above-mentioned plate for Raman analysis, it is possible to stably hold a liquid sample separated by chromatography. Furthermore, by drying the separated liquid sample on a plate for Raman analysis, the sample is concentrated, making it possible to improve the detection sensitivity of low molecular weight compounds contained in the sample, for example, to 1 μM or less.

[0014] However, when the signal derived from the mobile phase and the signal derived from the sample overlap, if the signal derived from the sample is weak, it will be buried and accurate analysis will not be possible. Therefore, it is preferable that the analysis section analyzes the sample based on a plurality of Raman spectra obtained by changing the mobile phase. With this configuration, for example, by performing measurements multiple times with different mobile phase concentrations and comparing the Raman spectra obtained from the multiple measurements, it is possible to determine that peaks with no or small fluctuations are due to the sample, thereby isolating the spectrum due to the sample from the obtained Raman spectrum and improving the analytical accuracy.

[0015] It is preferable that the separation mechanism separates from the sample a fluorescent substance that emits light in a wavelength band that overlaps at least a portion of the wavelength band of the Raman light generated from the sample. With this configuration, light with the excitation wavelength of the fluorescent substance can be used as the light to irradiate the sample, which is useful for various analyses according to various purposes.

[0016] Furthermore, the Raman analysis method according to the present invention is a Raman analysis method in which a liquid sample separated by chromatography is held on a Raman analysis plate and analyzed by Raman spectroscopy, characterized in that the Raman analysis plate comprises metal exposed regions provided at multiple locations on the plate surface for holding the sample, and a hydrophobic surface region provided on the plate surface, surrounding the metal exposed regions, and having a higher hydrophobicity than the metal exposed regions. According to this Raman analysis method, similar to the effect achieved by the above-mentioned plate for Raman analysis, it is possible to stably hold a liquid sample separated by chromatography. Effect of the Invention

[0017] According to the present invention configured in this manner, a liquid sample separated by chromatography can be stably held on a plate for Raman analysis, thereby improving the analytical accuracy of Raman analysis. [Brief description of the drawings]

[0018] [Figure 1] 1 is a schematic diagram showing the overall configuration of an analysis system according to an embodiment of the present invention; [Diagram 2]FIG. 2 is a schematic diagram showing the configuration of a plate for Raman analysis according to the embodiment. [Diagram 3] FIG. 2 is a schematic diagram for explaining an imaging device constituting the Raman spectrometer of the embodiment. [Figure 4] FIG. 2 is a functional block diagram showing functions of an analysis unit in the embodiment. [Diagram 5] FIG. 11 is a diagram for explaining an aspect of an analysis unit according to another embodiment. [Figure 6] FIG. 13 is a schematic diagram showing the configuration of a plate for Raman analysis according to another embodiment. [Explanation of symbols]

[0019] 100...Analysis System S: Sample Z...mobile phase 10...Separation mechanism 20. Raman analyzer 21 Raman analysis plate 211...metal exposed area 212...Hydrophobic region 213 Plate body 214...Liquid repellent film 215 Metal Spot 216 Metal Film DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0020] An embodiment of an analysis system according to the present invention will be described below with reference to the drawings.

[0021] The analysis system 100 of this embodiment is a system that utilizes both chromatography and Raman spectroscopy, and specifically includes a separation mechanism 10 that separates a liquid sample S by liquid chromatography, and a Raman analyzer 20 that analyzes the separated sample S by Raman spectroscopy, as shown in Fig. 1. The separation mechanism 10 may be one that separates the liquid sample S by supercritical fluid chromatography.

[0022] As shown in FIG. 1, the separation mechanism 10 is configured to pump the mobile phase Z stored in a storage section 11 into a flow path 13 using a pump 12, inject a liquid sample S into the flow path 13, and deliver the sample S together with the mobile phase Z to a separation column 14, thereby separating the sample S into its components. The mobile phase Z is, for example, a mixture of multiple types of liquids, and is a mixture of water and an organic solvent such as ethanol in this example. However, the mobile phase Z may be a single liquid or a gradient solvent having a concentration gradient.

[0023] 1, the Raman analysis device 20 includes a Raman analysis plate 21 that holds a sample S, a light irradiator 22 that irradiates excitation light such as laser light onto the sample S on the Raman analysis plate 21, a spectrometer 23 that disperses Raman scattered light generated from the sample S by being irradiated with the excitation light, a photodetector 24 that detects the dispersed Raman scattered light, and an analysis unit 25 that analyzes the sample S based on the detected Raman spectrum. A specific embodiment of the analysis unit 25 will be described later.

[0024] As shown in FIG. 1, the analysis system 100 of this embodiment is configured to dispense the liquid sample S separated by the separation mechanism 10 onto a plate 21 for Raman analysis, dry the sample S, and analyze the dried sample S by a Raman analysis device 20.

[0025] 2, the Raman analysis plate 21 is used for analyzing a liquid sample S separated by chromatography by Raman spectroscopy, and the separated liquid sample S is dropped one after another. Note that the Raman analysis plate 21 here is, for example, a rectangular flat plate, but the shape is not limited to this, and may be appropriately changed to, for example, a disk or block shape, as long as it has a surface onto which the liquid sample S can be dropped.

[0026] 2, the plate 21 for Raman analysis includes exposed metal regions 211 provided at multiple locations on the plate surface for holding the sample S, and hydrophobic regions 212 provided on the plate surface, surrounding the exposed metal regions 211, and having higher hydrophobicity than the exposed metal regions 211. The plate surface referred to here is shown in the upper part of FIG. 2 and is a surface visible from the outside of the plate 21 for Raman analysis, in other words, an exposed surface of the plate 21 for Raman analysis.

[0027] In this embodiment, a plurality of metal exposed regions 211 and hydrophobic regions 212 surrounding each of the metal exposed regions 211 are provided on one surface of a flat plate body 213 made of glass or the like.

[0028] More specifically, in this embodiment, first, liquid-repellent film 214 is formed on one side of plate body 213, and then, for example, metal is spot-welded to the surface of liquid-repellent film 214. As a result, the surface of metal spot 215 is formed as exposed metal region 211, and the exposed portion of liquid-repellent film 214 around metal spot 215 is formed as hydrophobic region 212.

[0029] The exposed metal regions 211 are made of a metal with high reflectivity, such as gold, silver, or aluminum, and have a circular shape with a diameter of, for example, about 100 μm to 300 μm. Here, the multiple exposed metal regions 211 are disposed on one surface of the plate body 213 at equal intervals along the vertical direction and at equal intervals along the horizontal direction.

[0030] The hydrophobic region 212 is made of a liquid-repellent film 214 formed by, for example, chemical modification or a periodic microstructure, and here, is provided on the entire region except for the metal exposed region 211 on one side of the plate body 213. However, it is sufficient that the hydrophobic region 212 is provided so as to surround the periphery of each of the metal exposed regions 211, and it is not necessarily required that the hydrophobic region 212 is provided on the entire region except for the metal exposed region 211.

[0031] Incidentally, the liquid sample S dispensed onto the Raman analysis plate 21 is dried before analysis, as described above. This allows the sample S to be concentrated, and as a result, the Raman analysis device 20 in the analysis system 100 of this embodiment can improve the detection sensitivity of low molecular weight compounds contained in the sample S to, for example, 1 μM or less.

[0032] On the other hand, it is difficult to visually confirm the dried sample S, and if an attempt is made to visually determine the measurement area for the Raman analyzer 20, it takes a long time to do so.

[0033] Therefore, the Raman analysis apparatus 20 of this embodiment is further provided with an imaging device that images the surface of the Raman analysis plate 21, as shown in Figure 3, and is configured to output the imaging data obtained by this imaging device to the analysis unit 25, and the analysis unit 25 determines the measurement area based on the imaging data.

[0034] More specifically, the imaging device is configured to be movable in the vertical and horizontal directions along the plate surface relative to the Raman analysis plate 21, or the Raman analysis plate 21 is configured to be movable relative to the imaging device. With this movement, the imaging device sequentially captures images or videos of each of the above-mentioned exposed metal regions 211, and sequentially outputs the captured image data to the analysis unit 25.

[0035] The analysis unit 25 is equipped with a CPU, memory, etc., and performs the functions of at least a measurement area determination unit 251 and an analysis execution unit 252, as shown in Figure 4, by each device working together in accordance with a program stored in the memory.

[0036] The measurement area determination unit 251 acquires imaging data obtained by an imaging device, and identifies the sample S that has been dried on the Raman analysis plate 21 by, for example, performing image analysis on the imaging data, and determines the area including the sample S as the measurement area. As a specific method for identifying the sample S, there may be mentioned a method in which the liquid sample S is held in the metal exposed region 211 of the Raman analysis plate 21 and dried, and the position of the dried sample S is identified, for example, by utilizing the coffee ring effect. More specifically, there may be mentioned a method in which the brightness or the like in the bright field image is compared with a threshold value to distinguish between the sample S and other locations, or an image shown by the imaging data is compared with a reference image prepared in advance to distinguish between the sample S and other locations.

[0037] The analysis execution section 252 sequentially performs Raman analysis on each of the measurement regions determined by the measurement region determination section 251 . More specifically, when the measurement area determination unit 251 determines the measurement areas, light from the light irradiator 22 is sequentially irradiated onto those measurement areas, and the Raman scattered light generated from the sample S in each measurement area is dispersed by the spectrometer 23 and detected by the photodetector 24. Then, based on the Raman spectrum detected from one measurement area, the analysis execution unit 252 analyzes, for example, the molecular structure or physical properties of the sample S in that measurement area, performs this Raman analysis sequentially on the sample S in each measurement area, and outputs the analysis results to a display or the like.

[0038] According to the analysis system 100 configured in this manner, the metal exposed regions 211 provided at multiple locations on the plate surface of the Raman analysis plate 21 are surrounded by hydrophobic surface regions having higher hydrophobicity, so that by separating the liquid sample S into the metal exposed regions 211, the separated sample S can be retained in the metal exposed regions 211. This allows the liquid sample S separated by chromatography to be stably held, thereby improving the analysis accuracy.

[0039] Furthermore, since the sample S is held in the exposed metal region 211 and the Raman light generated from the sample S is reflected by the exposed metal region 211, the Raman light can be directed toward the photodetector 24 more efficiently than when, for example, a glass plate is used as the Raman analysis plate 21.

[0040] Furthermore, since the measurement range in each metal exposed region 211 is determined based on the imaging data obtained by the imaging device, the measurement range can be determined in a short time without relying on visual inspection.

[0041] The present invention is not limited to the above-described embodiment.

[0042] For example, when a signal derived from mobile phase Z and a signal derived from sample S overlap, if the signal derived from sample S is weak, it may be buried and may not be possible to perform an accurate analysis. Therefore, the analysis section 25 may be configured to analyze the sample S based on a plurality of Raman spectra obtained by changing the mobile phase Z, as shown in FIG.

[0043] One example of such an analysis section 25 is a mode in which the ratio of multiple types of liquid (water and organic solvent in the above embodiment) that make up the mobile phase Z is changed, and the sample S is analyzed based on multiple Raman spectra obtained when each mobile phase Z is used. Specifically, among the multiple Raman spectra obtained, for example, peaks with large fluctuations can be considered to be derived from mobile phase Z, and peaks with no or small fluctuations can be considered to be derived from sample S. This makes it possible to isolate the spectrum of sample S from the obtained Raman spectra, improving the analytical accuracy.

[0044] To give a more specific example, if the solvent is composed of liquid A and liquid B, the composition ratio of liquid A to liquid B is, for example, 100:0, 80:20, 40:60, 20:80, and 0:100, and the Raman spectrum of sample S is obtained using each solvent. Then, the Raman spectra are measured by focusing on peaks that appear at the same time in each of the obtained Raman spectra. In this case, it can be determined that a peak whose height varies depending on the composition ratio of the solvent is derived from the solvent, and conversely, a peak whose height does not vary is derived from the sample.

[0045] Another example of an analytical mode using multiple Raman spectra is a mode in which, when mobile phase Z is a mixture of multiple types of liquid, each liquid is used alone as mobile phase Z, and sample S is analyzed based on the multiple Raman spectra obtained when each liquid is used. Specifically, the peaks originating from sample S can be analyzed by subtracting the Raman spectrum obtained when a mobile phase Z made up of multiple types of liquids is used alone from the Raman spectrum obtained when a mobile phase Z made up of multiple types of liquids is used. In addition, the peaks originating from the sample S can also be analyzed by subjecting these multiple spectra to, for example, singular value decomposition or multivariate analysis.

[0046] Furthermore, the analysis section 25 may use a plurality of types of gradient solvents having different concentration gradients, and analyze the sample S based on the Raman spectrum obtained when each gradient solvent is used.

[0047] In the case where the sample S does not emit autofluorescence, the separation mechanism 10 may be one that separates from the sample S a fluorescent substance that emits light in a wavelength band that overlaps at least a part of the wavelength band of the Raman light generated from the sample S. The wavelength of the emitted fluorescence of the fluorescent substance to be separated (for example, an aromatic compound or a compound with a ring structure) is known in advance. With this configuration, light with an excitation wavelength of the fluorescent substance (e.g., 480 nm to 550 nm) can be used as the light to be irradiated onto the sample S, which contributes to various analyses according to various purposes, etc. Before measuring the sample S, the fraction after separation of the fluorescent substance may be subjected to detection of the fluorescent substance using a detector such as a UV / UV-VIS detector, a photodiode array detector (PDA), or a diode array (DAD) to confirm that the fluorescent substance has been separated.

[0048] In the embodiment of the Raman analysis plate 21, a liquid-repellent film 214 is formed on one side of the plate body 213, and metal spots 215 are formed on the surface of the liquid-repellent film 214 to form the metal exposed region 211 and the hydrophobic region 212. However, as shown in Figure 6, a metal film 216 may be formed on one side of the plate body 213, for example by vapor deposition, and a liquid-repellent film 214 may be formed on the surface of the metal film 216 to form the metal exposed region 211 and the hydrophobic region 212.

[0049] Furthermore, the Raman analysis plate 21 in the above embodiment is provided with the metal exposed region 211 and the hydrophobic region 212 on the plate body 213 made of glass or the like, but if the plate body 213 is made of metal, for example, by forming a liquid-repellent film 214 on a part of the surface, the region of the plate body 213 on which the liquid-repellent film 214 is not formed becomes the metal exposed region 211, and the surface of the liquid-repellent film 214 becomes the hydrophobic region 212. With such a configuration, it is possible to eliminate the need for the glass plate that is the plate body 213 in the above embodiment.

[0050] In the above embodiment, a liquid sample separated by chromatography is held on the Raman analysis plate 21 and analyzed. However, a liquid sample before separation by chromatography may be held on the Raman analysis plate 21 and analyzed.

[0051] Furthermore, the present invention is not limited to the above-described embodiment, and it goes without saying that various modifications are possible without departing from the spirit and scope of the present invention. [Industrial Applicability]

[0052] According to the present invention, a liquid sample separated by chromatography can be stably held.

Claims

1. A Raman analyzer for analyzing a liquid sample separated by chromatography using Raman spectroscopy, comprising: A plate for Raman analysis; an imaging device for imaging the surface of the Raman analysis plate; a light irradiator that irradiates the sample on the Raman analysis plate with light; a photodetector for detecting Raman light generated from the sample when the sample is irradiated with light; and an analysis unit for analyzing the sample based on the detected Raman spectrum, The Raman analysis plate is A plurality of exposed metal areas are provided on the surface of the plate to hold the samples; a hydrophobic region provided on the plate surface, surrounding the metal exposed region, and having a higher hydrophobicity than the metal exposed region; A Raman analysis apparatus, wherein the analysis unit determines a measurement range including the sample dried on the Raman analysis plate based on imaging data obtained by the imaging device, and analyzes the measurement range.

2. 2. The Raman analysis apparatus according to claim 1, further comprising the plate for Raman analysis, wherein the exposed metal region is made of gold, silver, or aluminum.

3. The Raman analyzer according to claim 1 or 2, An analytical system comprising: a separation column through which the liquid sample is delivered by a mobile phase; and a separation mechanism for separating the sample by chromatography.

4. The analytical system according to claim 3 , wherein the analytical section analyzes the sample based on a plurality of Raman spectra obtained by changing the mobile phase.

5. 5. The analytical system according to claim 3, wherein the separation mechanism separates from the sample a fluorescent substance that emits light in a wavelength band that overlaps at least a portion of a wavelength band of Raman light generated from the sample.

6. A Raman analysis method in which a liquid sample separated by chromatography is held on a Raman analysis plate and analyzed by Raman spectroscopy, comprising the steps of: The Raman analysis plate is A plurality of exposed metal areas are provided on the surface of the plate to hold the samples; a hydrophobic region provided on the plate surface, surrounding the metal exposed region, and having a higher hydrophobicity than the metal exposed region; imaging the surface of the plate for Raman analysis with an imaging device; determining a measurement range including the sample dried on the Raman analysis plate by an analysis unit based on the imaging data obtained by the imaging device; Irradiating the measurement area on the Raman analysis plate with light using a light irradiator; Detecting Raman light generated from the sample by irradiating the sample with light using a photodetector; A Raman analysis method, comprising: analyzing the measurement range by the analysis unit based on the detected Raman spectrum.

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