CN113281337B - A kind of extraction method of complex compound Raman spectrum - Google Patents

A kind of extraction method of complex compound Raman spectrum Download PDF

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CN113281337B
CN113281337B CN202110663665.0A CN202110663665A CN113281337B CN 113281337 B CN113281337 B CN 113281337B CN 202110663665 A CN202110663665 A CN 202110663665A CN 113281337 B CN113281337 B CN 113281337B
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raman spectrum
raman
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CN113281337A (en
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冯继宏
朱书缘
鲁贞贞
赵蒙蒙
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Beijing University of Technology
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    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/84Systems specially adapted for particular applications
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/65Raman scattering

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Abstract

An extraction method of a complex compound Raman spectrum relates to the fields of analytical chemistry and pharmaceutical ingredients. The method is an extraction method of the complex compound high-quality Raman spectrum based on the combination of the optical microscopic imaging technology with high spatial resolution and the Raman spectrum technology. The method is simple to operate, is non-contact and harmless, can be used for products, only needs trace substances to be carried out under the irradiation of light waves, can be applied to analysis of complex compounds, and does not change the physical and chemical properties of the complex compounds to be detected.

Description

一种复杂化合物拉曼光谱的提取方法A kind of extraction method of complex compound Raman spectrum

技术领域technical field

本发明涉及分析化学领域和药物成分领域,具体而言,涉及一种基于光学显微成像结合拉曼光谱的复杂化合物高质量拉曼光谱的提取方法。The invention relates to the field of analytical chemistry and the field of pharmaceutical ingredients, in particular to a method for extracting high-quality Raman spectra of complex compounds based on optical microscopic imaging combined with Raman spectra.

背景技术Background technique

交联、聚合反应生成的高分子量复杂化合物,广泛地应用于饮料、医药行业中。但是这种化合物成分复杂,包含简单的初始物质、中间产物、最终产物,而交联、聚合反应生成的中产物又是分子量处于一定区间的交联体,因此对于这种物质的单晶分析困难,尤其是在不改变其物理和化学性质的情况下,如不溶于水、不加热制冷、不采用变色反应,那么光学检测是一种技术手段。High-molecular-weight complex compounds produced by cross-linking and polymerization reactions are widely used in the beverage and pharmaceutical industries. However, the composition of this compound is complex, including simple initial substances, intermediate products, and final products, and the intermediate products generated by crosslinking and polymerization reactions are crosslinked products with a molecular weight in a certain range, so it is difficult to analyze single crystals of this substance , especially without changing its physical and chemical properties, such as being insoluble in water, not heating and cooling, and not using color-changing reactions, then optical detection is a technical means.

鉴于交联化合物在饮料、医药行业中的重要作用,亟需建立一种快速、无接触、保留复杂化合物化学和物理性质的检测方法,目前已有的检测方法采用热熔点法、高分辨质谱法破坏物理性质;采用核磁共振技术无法分辨。近年来,高空间分辨率的光学显微成像技术发展迅速,为空间分辨复杂化合物中的单晶提供可行性;同时拉曼光谱具有分子标签的作用,使鉴定复杂化合物的成分成为可能。而通过光谱方法鉴定复杂化合物成分拉曼光谱需要得到高质量拉曼光谱。鉴于此,本发明基于高空间分辨率的光学显微成像技术与拉曼光谱技术结合建立了一种非接触、无损伤的复杂化合物高质量拉曼光谱的提取方法。In view of the important role of cross-linked compounds in the beverage and pharmaceutical industries, it is urgent to establish a rapid, non-contact detection method that retains the chemical and physical properties of complex compounds. The existing detection methods use thermal melting point method and high-resolution mass spectrometry Destruction of physical properties; indistinguishable by nuclear magnetic resonance techniques. In recent years, high-spatial-resolution optical microscopy imaging technology has developed rapidly, providing feasibility for spatially resolving single crystals in complex compounds; at the same time, Raman spectroscopy has the role of molecular labels, making it possible to identify the components of complex compounds. However, the identification of complex compound components by Raman spectroscopy requires high-quality Raman spectroscopy. In view of this, the present invention establishes a non-contact, non-damaging high-quality Raman spectrum extraction method for complex compounds based on the combination of optical microscopic imaging technology with high spatial resolution and Raman spectroscopy technology.

发明内容Contents of the invention

本发明的目的是提供一种基于高空间分辨率的光学显微成像技术与拉曼光谱技术结合建立了一种的复杂化合物高质量拉曼光谱的提取方法。该方法操作简单、非接触、无损伤,可以对产品进行,只需要微量的物质在光波的照射下进行,可以应用于复杂化合物分析,而不改变待测复杂化合物的物理和化学性质。The purpose of the present invention is to provide a method for extracting high-quality Raman spectra of complex compounds based on the combination of optical microscopic imaging technology with high spatial resolution and Raman spectroscopy technology. The method is simple, non-contact, non-destructive, and can be carried out on products. It only needs a small amount of substances to be carried out under the irradiation of light waves, and can be applied to the analysis of complex compounds without changing the physical and chemical properties of the complex compounds to be tested.

为实现上述目的,本发明提供了一种基于高空间分辨率的光学显微成像技术与拉曼光谱技术结合的一种的化合物高质量拉曼光谱的提取方法,包括以下步骤:In order to achieve the above object, the present invention provides a method for extracting a high-quality Raman spectrum of a compound based on the combination of high spatial resolution optical microscopic imaging technology and Raman spectrum technology, comprising the following steps:

(1)取1-5mg待检测的粉末样品,放置在载玻片上,裸露直径3~5mm,或者3-5mm矩形,或者3-5毫米长方形的表面压实成为一个薄层,制成待测样品;(1) Take 1-5mg of the powder sample to be tested, place it on a glass slide, expose a diameter of 3-5mm, or a 3-5mm rectangle, or compact a 3-5mm rectangle surface into a thin layer, and make it to be tested sample;

(2)在光学显微镜下对待测样品进行观察,寻找成像清晰并且具有广泛形貌特征的一个分散颗粒,对分散颗粒进行明场成像,选择成像图像中颗粒上表示光亮度的灰度级最高的一点进行作为感兴趣点;(2) Observe the sample to be tested under an optical microscope, look for a dispersed particle with a clear image and a wide range of morphological characteristics, perform bright-field imaging on the dispersed particle, and select the highest gray level on the particle in the imaging image that represents the brightness One point proceeds as a point of interest;

(3)对步骤(2)感兴趣点在颗粒上的对应位置点进行特定波长的光照射,测量得到在该颗粒上对应位置点的该激发波长的拉曼光谱,拉曼光谱包含两个参数k和I,k为拉曼光谱中的拉曼位移波数,I为拉曼光谱中波数k对应的光谱强度;(3) Perform light irradiation of a specific wavelength on the corresponding position of the point of interest in step (2) on the particle, and measure the Raman spectrum of the excitation wavelength at the corresponding position on the particle. The Raman spectrum contains two parameters k and I, k is the Raman shift wavenumber in the Raman spectrum, and I is the spectral intensity corresponding to the wavenumber k in the Raman spectrum;

(4)多次重复步骤(3),得到多个对应位置点上该激发波长的拉曼光谱,再对多个拉曼光谱中相同波数k对应的光谱强度I进行平均计算得到平均拉曼光谱;(4) Repeat step (3) multiple times to obtain the Raman spectrum of the excitation wavelength on multiple corresponding positions, and then average the spectral intensity I corresponding to the same wave number k in multiple Raman spectra to obtain the average Raman spectrum ;

(5)在步骤(4)计算得到平均拉曼光谱中找到峰值高且峰形尖锐的拉曼光谱特征峰作为该待测物的拉曼光谱特征峰,记录该拉曼光谱特征峰对应的波数k0(5) In the average Raman spectrum calculated in step (4), find the Raman spectrum characteristic peak with high peak and sharp peak shape as the Raman spectrum characteristic peak of the analyte, and record the wave number corresponding to the Raman spectrum characteristic peak k 0 ;

(6)再次在光学显微镜下对待测样品进行观察寻找成像清晰并且具有广泛形貌特征的一个分散颗粒,或直接使用步骤(2)中寻找到的颗粒,对该颗粒进行明场成像,将包含该颗粒整体或局部的一个矩形区域作为感兴趣区域;(6) Observe the sample to be tested again under the optical microscope to find a dispersed particle with clear imaging and extensive morphology features, or directly use the particle found in step (2) to perform bright field imaging on the particle, which will contain A rectangular area of the whole or part of the particle is used as the area of interest;

(7)对步骤(6)感兴趣区域对应样品上区域进行共焦扫描得到一个拉曼光谱数据集,拉曼光谱数据集包含四个参数,分别为x、y、k和I,其中x和y对应感兴趣区域内一个点的平面坐标,k为在一个点测量的拉曼光谱的拉曼位移波数,I为一个点的拉曼光谱中一个波数k对应的光谱强度;(7) Carry out confocal scanning to step (6) region of interest corresponding to the area on the sample to obtain a Raman spectrum data set, the Raman spectrum data set contains four parameters, are respectively x, y, k and I, wherein x and y corresponds to the plane coordinates of a point in the region of interest, k is the Raman shift wavenumber of the Raman spectrum measured at a point, and I is the spectral intensity corresponding to a wavenumber k in the Raman spectrum of a point;

(8)对步骤(7)得到的拉曼光谱数据集每点拉曼光谱进行去除宇宙噪声和消除背景噪声处理,得到处理后的拉曼光谱数据集;(8) each point Raman spectrum of the Raman spectrum data set obtained in step (7) is processed to remove cosmic noise and eliminate background noise, and obtain the processed Raman spectrum data set;

(9)在步骤(8)处理后的拉曼光谱数据集中,将每点拉曼光谱在波数k0对应光谱强度I作为平面图像中该点的灰度值生成拉曼特征峰的拉曼图像;(9) In the Raman spectrum data set after step (8) processing, the Raman image of the Raman characteristic peak is generated as the gray value of the point in the plane image at the wavenumber k of each point of the Raman spectrum corresponding to the spectral intensity I ;

(10)在步骤(9)得到的拉曼图像中选择灰度值最高点作为感兴趣点;(10) in the Raman image that step (9) obtains, select the highest point of the gray value as the point of interest;

(11)根据步骤(10)得到的感兴趣点在样品中所在位置,在步骤(8)处理后的拉曼光谱数据集中进行区域定域提取对应位置的高质量的拉曼光谱。(11) According to the position of the point of interest obtained in step (10) in the sample, perform region-specific extraction of a high-quality Raman spectrum at the corresponding position in the Raman spectrum data set processed in step (8).

本发明待检测的粉末样品为单一化合物或可能存在多种成分的聚合物。The powder sample to be tested in the present invention is a single compound or a polymer with multiple components.

在本发明的第一个具体实施中,待检测的粉末样品使用从德国公司巴斯夫购买的聚乙烯醇吡咯烷酮(PVP)K25粉末样品,批号61861347G0。In the first implementation of the present invention, the powder sample to be tested is a polyvinylpyrrolidone (PVP) K25 powder sample purchased from the German company BASF, batch number 61861347G0.

在本发明的第一个具体实施中,采用共焦拉曼光谱显微镜WITec Alpha300-RAMAN进行样品颗粒光学显微镜观察、明场图像采集、拉曼光谱测量以及共焦扫描。使用WITecProject FIVE软件对明场图像、拉曼光谱、拉曼图像和感兴趣区域以及感兴趣点进行显示,并且对拉曼光谱和拉曼光谱数据集进行数据处理,以及在拉曼光谱数据集中提取高质量拉曼光谱。In the first implementation of the present invention, a confocal Raman spectroscopy microscope WITec Alpha300-RAMAN is used for optical microscope observation of sample particles, bright field image acquisition, Raman spectroscopy measurement and confocal scanning. Use WITecProject FIVE software to display bright field images, Raman spectra, Raman images, regions of interest, and points of interest, and perform data processing on Raman spectra and Raman spectral datasets, and extract data from Raman spectral datasets High quality Raman spectroscopy.

在本发明的实施例一中,在步骤(1)中,取PVP粉末样品2-5mg置于载玻片上,轻微摇晃使样品粉末颗粒尽量分散,盖上盖玻片后轻微碾压盖玻片,使样品粉末颗粒进一步分散。In Embodiment 1 of the present invention, in step (1), take 2-5 mg of PVP powder sample and place it on a glass slide, shake it slightly to disperse the sample powder particles as much as possible, cover the cover glass and then slightly roll the cover glass , to further disperse the sample powder particles.

在本发明的实施例一中,步骤(2)中,使用50倍物镜明场显微镜观察样品选择进行拉曼扫描的样品颗粒。In the first embodiment of the present invention, in step (2), a 50 times objective lens is used to observe the sample with a bright-field microscope to select sample particles for Raman scanning.

在本发明的实施例一中,步骤(2)中,观察发现PVP样品中颗粒形状均不规则,根据观察结果,在PVP样品中选择一个不规则形状颗粒进行成像,并通过视觉观察选择成像图像中颗粒上灰度级最高的一点进行作为感兴趣点。In the first embodiment of the present invention, in step (2), it was observed that the shape of the particles in the PVP sample was irregular. According to the observation results, an irregularly shaped particle was selected in the PVP sample for imaging, and the imaging image was selected by visual observation. The point with the highest gray level on the middle particle is taken as the point of interest.

在本发明的实施例一中,步骤(3)中,使用共焦拉曼光谱显微镜WITec Alpha300-RAMAN进行拉曼光谱测量,测量采用激发波长为532nm、激光功率为10mw、光栅为1200g/mm、物镜50倍、数值孔径为0.75以及波数范围为0~1900cm-1In Embodiment 1 of the present invention, in step (3), a confocal Raman spectrum microscope WITec Alpha300-RAMAN is used for Raman spectrum measurement, and the measurement uses an excitation wavelength of 532nm, a laser power of 10mw, and a grating of 1200g/mm. The objective lens is 50 times, the numerical aperture is 0.75, and the wavenumber range is 0-1900cm -1 .

在本发明的实施例一中,步骤(4)中,使用共焦拉曼光谱显微镜对所述PVP样品颗粒上位置进行10次拉曼光谱测量并进行平均计算得到平均拉曼光谱。In Embodiment 1 of the present invention, in step (4), the Raman spectrum is measured 10 times on the position on the PVP sample particle using a confocal Raman spectrum microscope, and the average Raman spectrum is calculated to obtain the average Raman spectrum.

在本发明的实施例一中,步骤(5)中,记录的拉曼光谱特征峰对应波数k为934.2cm-1In the first embodiment of the present invention, in step (5), the recorded characteristic peak of the Raman spectrum corresponds to a wave number k of 934.2 cm −1 .

在本发明的实施例一中,步骤(6)中,得到的感兴趣区域为包含步骤(2)中寻找到的PVP颗粒局部的20μm*20μm的正方形区域。In the first embodiment of the present invention, in step (6), the region of interest obtained is a square region of 20 μm*20 μm including the local PVP particles found in step (2).

在本发明的实施例一中,步骤(7)中,使用共焦拉曼光谱显微镜WITec Alpha300-RAMAN进行共焦扫描,扫描采用激发波长为532nm,激光功率为10mw,光栅为1200g/mm,物镜倍数为50,数值孔径为0.75以及波数为0~1900cm-1In the first embodiment of the present invention, in step (7), a confocal Raman spectrum microscope WITec Alpha300-RAMAN is used for confocal scanning, the scanning adopts an excitation wavelength of 532nm, a laser power of 10mw, a grating of 1200g/mm, and an objective lens The multiple is 50, the numerical aperture is 0.75 and the wave number is 0-1900 cm -1 .

在本发明的第二实施例中,待检测的粉末样品使用从德国公司巴斯夫购买的交联聚乙烯醇吡咯烷酮(PVPP)粉末作为样品,批号92441577L0。In the second embodiment of the present invention, the powder sample to be tested is cross-linked polyvinylpyrrolidone (PVPP) powder purchased from the German company BASF as the sample, batch number 92441577L0.

在本发明的第二实施例中,采用共焦拉曼光谱显微镜WITec Alpha300-RAMAN进行样品颗粒光学显微镜观察、明场图像采集、拉曼光谱测量以及共焦扫描。使用WITecProject FIVE软件对明场图像、拉曼光谱、拉曼图像和感兴趣区域以及感兴趣点进行显示,并且对拉曼光谱和拉曼光谱数据集进行数据处理,以及在拉曼光谱数据集中提取高质量拉曼光谱。In the second embodiment of the present invention, a confocal Raman spectroscopy microscope WITec Alpha300-RAMAN is used for optical microscope observation of sample particles, bright field image acquisition, Raman spectroscopy measurement and confocal scanning. Use WITecProject FIVE software to display bright field images, Raman spectra, Raman images, regions of interest, and points of interest, and perform data processing on Raman spectra and Raman spectral datasets, and extract data from Raman spectral datasets High quality Raman spectroscopy.

在本发明的实施例二中,在步骤(1)中,取PVPP粉末样品2-5mg置于载玻片上,轻微摇晃使样品粉末颗粒尽量分散,盖上盖玻片后轻微碾压盖玻片,使样品粉末颗粒进一步分散。In the second embodiment of the present invention, in step (1), take 2-5 mg of the PVPP powder sample and place it on a glass slide, shake it slightly to disperse the sample powder particles as much as possible, cover the cover glass and then slightly roll the cover glass , to further disperse the sample powder particles.

在本发明的实施例二中,步骤(2)中,使用50倍物镜明场显微镜观察样品选择进行拉曼扫描的样品颗粒。In the second embodiment of the present invention, in step (2), a bright field microscope with a 50 times objective lens is used to observe the sample and select sample particles for Raman scanning.

在本发明的实施例二中,步骤(2)中,观察发现PVPP样品中颗粒绝大部分形状为球形颗粒,极小部分为很小的不规则颗粒,根据观察结果,在PVPP样品中选择一个球形颗粒进行成像,并通过视觉观察选择成像图像中颗粒上灰度级最高的一点进行作为感兴趣点。In the second embodiment of the present invention, in step (2), it is observed that most of the particles in the PVPP sample are spherical particles, and a very small part is a very small irregular particle. According to the observation results, one of the PVPP samples is selected. Spherical particles are imaged, and the point with the highest gray level on the particle in the imaging image is selected as the point of interest by visual observation.

在本发明的实施例二中,步骤(3)中,使用共焦拉曼光谱显微镜WITec Alpha300-RAMAN进行拉曼光谱测量,测量采用激发波长为532nm、激光功率为10mw、光栅为1200g/mm、物镜50倍、数值孔径为0.75以及波数范围为0~1900cm-1In the second embodiment of the present invention, in step (3), a confocal Raman spectrum microscope WITec Alpha300-RAMAN is used for Raman spectrum measurement, and the measurement adopts an excitation wavelength of 532nm, a laser power of 10mw, and a grating of 1200g/mm. The objective lens is 50 times, the numerical aperture is 0.75, and the wavenumber range is 0-1900cm -1 .

在本发明的实施例二中,步骤(4)中,使用共焦拉曼光谱显微镜对所述PVPP样品颗粒上位置进行10次拉曼光谱测量并进行平均计算得到平均拉曼光谱。In the second embodiment of the present invention, in step (4), a confocal Raman spectrum microscope is used to perform 10 Raman spectrum measurements on the position on the PVPP sample particle and perform average calculation to obtain an average Raman spectrum.

在本发明的实施例二中,步骤(5)中,记录的拉曼光谱特征峰对应波数k为934.2cm-1In the second embodiment of the present invention, in step (5), the recorded characteristic peak of the Raman spectrum corresponds to a wave number k of 934.2 cm −1 .

在本发明的实施例二中,步骤(6)中,得到的感兴趣区域为包含寻找到的另一PVPP样品颗粒局部的10μm*10μm的正方形区域。In the second embodiment of the present invention, in step (6), the region of interest obtained is a square region of 10 μm*10 μm including the part of another PVPP sample particle found.

在本发明的实施例二中,步骤(7)中,使用共焦拉曼光谱显微镜WITec Alpha300-RAMAN进行共焦扫描,扫描采用激发波长为532nm,激光功率为10mw,光栅为1200g/mm,物镜倍数为50,数值孔径为0.75,以及波数为0~1900cm-1In the second embodiment of the present invention, in step (7), a confocal Raman spectrum microscope WITec Alpha300-RAMAN is used for confocal scanning, the scanning adopts an excitation wavelength of 532nm, a laser power of 10mw, a grating of 1200g/mm, and an objective lens The magnification is 50, the numerical aperture is 0.75, and the wave number is 0 to 1900 cm −1 .

与现有技术相比,本发明的有益效果为:Compared with prior art, the beneficial effect of the present invention is:

本发明提供的方法,以一种基于光学显微成像结合拉曼光谱的复杂化合物的分析方法,建立化学和药物成分高质量拉曼光谱提取方法。对化合物颗粒光学显微成像,根据获得的图像识别图像中的颗粒;将图像颗粒区域进行特定波长的光照射,得到拉曼光谱;分析拉曼特征峰并作出拉曼特征峰的拉曼图像;根据拉曼图像进行区域定域提取高质量拉曼光谱。该方法精准、非接触、非损伤、快速、不改变待测物质的物理性质与化学性质,可以用于不同化学物质的成分拉曼光谱分析。The method provided by the present invention establishes a high-quality Raman spectrum extraction method for chemical and pharmaceutical components with an analysis method based on optical microscopic imaging combined with Raman spectrum for complex compounds. Optical microscopic imaging of compound particles, identifying the particles in the image according to the obtained image; irradiating the image particle area with light of a specific wavelength to obtain a Raman spectrum; analyzing the Raman characteristic peaks and making a Raman image of the Raman characteristic peaks; Based on the Raman image, perform region-specific extraction of high-quality Raman spectra. The method is accurate, non-contact, non-damaging, fast, and does not change the physical and chemical properties of the substance to be measured, and can be used for Raman spectroscopic analysis of the components of different chemical substances.

本发明的方法直接对化合物高分辨率成像与拉曼光谱结合,非接触、不改变化合物的物理和化学性质。The method of the invention directly combines the high-resolution imaging of the compound with Raman spectroscopy, without contact and without changing the physical and chemical properties of the compound.

附图说明Description of drawings

图1PVP K25粉末样品临时装片;Figure 1 PVP K25 powder sample temporary loading;

图2中(a)为PVP样品颗粒明场图像,十字线标记位置为感兴趣点;(b)为平均计算后的PVP样品的拉曼光谱;Among Fig. 2 (a) is the bright-field image of the PVP sample particle, and the crosshair mark position is the point of interest; (b) is the Raman spectrum of the PVP sample after the average calculation;

图3中(a)为PVP样品中不规则颗粒明场图像,(b)为PVP样品中不规则颗粒的拉曼特征峰的拉曼图像;Among Fig. 3 (a) is the bright-field image of the irregular particle in the PVP sample, (b) is the Raman image of the Raman characteristic peak of the irregular particle in the PVP sample;

图4中(a)PVP样品中不规则颗粒的拉曼特征峰的拉曼图像,其中感兴趣点使用十字线标记;(b)PVP样品颗粒拉曼光谱数据集中提取的高质量拉曼光谱。In Fig. 4 (a) Raman image of Raman characteristic peaks of irregular particles in PVP samples, where points of interest are marked with crosshairs; (b) high-quality Raman spectra extracted from the Raman spectrum dataset of PVP sample particles.

图5粉末样品临时装片;Figure 5 Temporary loading of powder samples;

图6中(a)为PVPP颗粒明场图像,十字线标记位置为感兴趣点;(b)为平均计算后的PVPP样品的拉曼光谱;Among Fig. 6 (a) is the bright-field image of PVPP particles, and the position marked by the crosshair is the point of interest; (b) is the Raman spectrum of the PVPP sample after average calculation;

图7中(a)为PVPP样品中球形颗粒明场图像,其中实线框内区域为包含颗粒局部的感兴趣区域;(b)为PVPP样品中球形颗粒的拉曼特征峰的拉曼图像;Among Fig. 7 (a) is the bright-field image of the spherical particle in the PVPP sample, wherein the region in the solid line frame is the region of interest that contains the local particle; (b) is the Raman image of the Raman characteristic peak of the spherical particle in the PVPP sample;

图8中(a)为PVPP样品中颗粒的拉曼特征峰拉曼图像,其中感兴趣点分别使用十字线标记;(b)为PVPP样品颗粒拉曼光谱数据集中提取的高质量拉曼光谱。Figure 8 (a) is the Raman image of the Raman characteristic peaks of the particles in the PVPP sample, where the points of interest are marked with cross lines; (b) is the high-quality Raman spectrum extracted from the Raman spectrum data set of the PVPP sample particles.

具体实施方式Detailed ways

下面结合实施例对本发明作进一步说明,但本发明并不限于以下实施例。The present invention will be further described below in conjunction with the examples, but the present invention is not limited to the following examples.

实施例1:Example 1:

使用从德国公司巴斯夫购买的PVP K25粉末样品,批号61861347G0。采用共焦拉曼光谱显微镜WITec Alpha300-RAMAN进行样品颗粒光学显微镜观察、明场图像采集、拉曼光谱测量以及共焦扫描。使用WITec Project FIVE软件对明场图像、拉曼光谱、拉曼图像和感兴趣区域以及感兴趣点进行显示,并且对拉曼光谱和拉曼光谱数据集进行数据处理,以及在拉曼光谱数据集中提取高质量拉曼光谱。A PVP K25 powder sample purchased from the German company BASF, lot number 61861347G0 was used. Confocal Raman spectroscopy microscope WITec Alpha300-RAMAN was used for optical microscope observation of sample particles, bright field image acquisition, Raman spectroscopy measurement and confocal scanning. Use WITec Project FIVE software to display bright field images, Raman spectra, Raman images, regions of interest, and points of interest, and perform data processing on Raman spectra and Raman spectral datasets, and in Raman spectral datasets Extract high-quality Raman spectra.

将PVP粉末样品制作成临时装片,临时装片如图1所示。The PVP powder sample was made into a temporary loading sheet, as shown in Figure 1.

将制备好的临时装片置于共焦拉曼显微镜样品台上进行成像,通过50倍物镜分别观察PVP样品中的微粒。观察发现PVP样品中颗粒形状均为不规则颗粒。根据观察结果,在PVP样品中选择一个不规则形状颗粒进行明场成像,并通过视觉观察分别选择明场成像图像中颗粒上灰度级最高的一点进行作为感兴趣点。PVP样品颗粒明场成像图像分别如图2中(a)所示,图像中感兴趣点为十字线标注位置。对得到的感兴趣点对应颗粒上所在位置进行特定激发波长的光照射,测量得到该单晶在该点的该激发波长的拉曼光谱,测量采用激发波长为532nm、激光功率为10mw、光栅为1200g/mm、物镜50倍、数值孔径为0.75以及波数范围为0~1900cm-1。对PVP样品颗粒明场图像中感兴趣点对应颗粒上所在位置分别进行10次拉曼光谱测量后,对得到的10个拉曼光谱进行光谱平均计算得到平均拉曼光谱作为PVP样品的拉曼光谱,PVP样品拉曼光谱如图2中(b)所示。观察图2中(b)中PVP样品的拉曼光谱信号在k=934.2cm-1(此记为k0)位置存在锋利的拉曼特征峰。The prepared temporary mount was placed on the sample stage of a confocal Raman microscope for imaging, and the particles in the PVP sample were observed through a 50x objective lens. It was observed that the particle shapes in the PVP samples were all irregular particles. According to the observation results, an irregular-shaped particle was selected in the PVP sample for bright-field imaging, and the point with the highest gray level on the particle in the bright-field imaging image was selected as the point of interest through visual observation. The bright-field imaging images of PVP sample particles are shown in (a) in Figure 2, and the points of interest in the images are marked by cross lines. Light irradiation of a specific excitation wavelength is carried out on the position corresponding to the obtained point of interest on the particle, and the Raman spectrum of the single crystal at this excitation wavelength is measured. The measurement adopts an excitation wavelength of 532nm, a laser power of 10mw, and a grating of 1200g/mm, objective lens 50 times, numerical aperture 0.75 and wave number range 0-1900cm -1 . After performing 10 Raman spectrum measurements on the positions of the particles corresponding to the points of interest in the bright field image of the PVP sample particles, the average Raman spectrum of the obtained 10 Raman spectra was calculated to obtain the average Raman spectrum as the Raman spectrum of the PVP sample. , the Raman spectrum of the PVP sample is shown in Figure 2 (b). Observe that the Raman spectrum signal of the PVP sample in (b) in Figure 2 has a sharp Raman characteristic peak at the position k=934.2cm -1 (this is denoted as k 0 ).

随后,再次对PVP样品在步骤(2)中寻找到不规则颗粒进行明场成像,明场图像如图3中(a)所示,图3的(a)中实线框内区域为包含颗粒局部的感兴趣区域,感兴趣区域大小为20μm*20μm。在感兴趣区域对应样品上区域进行共焦扫描,分别得到PVP样品颗粒的拉曼光谱数据集。扫描采用激发波长为532nm,激光功率为10mw,光栅为1200g/mm,物镜倍数为50,数值孔径为0.75,以及波数为0~1900cm-1。对得到的拉曼光谱数据集每点拉曼光谱进行去除宇宙噪声和消除背景噪声处理,得到处理后的PVP样品颗粒拉曼光谱数据集。随后,在处理后的拉曼光谱数据集中,将每点拉曼光谱在波数k0=934.2cm-1的光谱强度I作为平面图像该点的灰度值生成拉曼特征峰的拉曼图像,PVP样品颗粒的拉曼特征峰的拉曼图像如图3中(b)所示。Subsequently, the irregular particles found in step (2) of the PVP sample were again subjected to bright-field imaging. The bright-field image is shown in Figure 3 (a), and the area in the solid line frame in Figure 3 (a) contains particles Local ROI, the ROI size is 20μm*20μm. Confocal scanning was performed on the area corresponding to the sample in the region of interest, and the Raman spectrum data sets of the PVP sample particles were obtained respectively. For scanning, the excitation wavelength is 532nm, the laser power is 10mw, the grating is 1200g/mm, the objective lens multiple is 50, the numerical aperture is 0.75, and the wave number is 0-1900cm -1 . The Raman spectrum of each point of the obtained Raman spectrum data set is processed to remove cosmic noise and background noise, and the processed Raman spectrum data set of PVP sample particles is obtained. Subsequently, in the processed Raman spectrum data set, the spectral intensity I of each point of the Raman spectrum at the wave number k 0 =934.2 cm -1 is used as the gray value of the point in the plane image to generate a Raman image of the Raman characteristic peak, The Raman image of the Raman characteristic peaks of the PVP sample particles is shown in Figure 3 (b).

在得到的PVP样品颗粒的拉曼特征峰的拉曼图像中选择灰度值最高点作为感兴趣点,如图4中(a)所示,感兴趣点使用十字线标记。根据感兴趣点对应样品中所在位置,在处理后的PVP样品颗粒拉曼光谱数据集中进行区域定域提取高质量的拉曼光谱,提取的高质量拉曼光谱如图4中(b)所示。In the obtained Raman image of the Raman characteristic peak of the PVP sample particles, select the point with the highest gray value as the point of interest, as shown in (a) in Figure 4, and the point of interest is marked with a cross. According to the position in the sample corresponding to the point of interest, a high-quality Raman spectrum is extracted in the processed PVP sample particle Raman spectrum data set, and the extracted high-quality Raman spectrum is shown in Figure 4 (b) .

实施例2:Example 2:

使用从德国公司巴斯夫购买的PVPP粉末作为样品,批号92441577L0。方案采用共焦拉曼光谱显微镜WITec Alpha300-RAMAN进行样品颗粒光学显微镜观察、明场图像采集、拉曼光谱测量以及共焦扫描。使用WITec Project FIVE软件对明场图像、拉曼光谱、拉曼图像和感兴趣区域以及感兴趣点进行显示,并且对拉曼光谱和拉曼光谱数据集进行数据处理,以及在拉曼光谱数据集中提取高质量拉曼光谱。As a sample, PVPP powder purchased from the German company BASF, lot number 92441577L0 was used. The scheme uses confocal Raman spectroscopy microscope WITec Alpha300-RAMAN for optical microscope observation of sample particles, bright field image acquisition, Raman spectroscopy measurement and confocal scanning. Use WITec Project FIVE software to display bright field images, Raman spectra, Raman images, regions of interest, and points of interest, and perform data processing on Raman spectra and Raman spectral datasets, and in Raman spectral datasets Extract high-quality Raman spectra.

将PVPP粉末样品分别制作成临时装片,临时装片如图5所示。The PVPP powder samples were made into temporary mounts, as shown in Figure 5.

将制备好的临时装片置于共焦拉曼显微镜样品台上进行成像,通过50倍物镜观察PVPP样品中的微粒。观察发现PVPP样品中颗粒绝大部分形状为球形颗粒,极小部分为很小的不规则颗粒。根据观察结果,在PVP样品中选择一个不规则形状颗粒进行明场成像,在PVPP样品中选择一个球形颗粒进行明场成像,并通过视觉观察分别选择明场成像成像图像中颗粒上灰度级最高的一点进行作为感兴趣点。PVPP样品颗粒明场成像图像如图6中(a)所示,图像中感兴趣点为十字线标记位置。对得到的感兴趣点对应颗粒上所在位置进行特定激发波长的光照射,测量得到该单晶在该点的该激发波长的拉曼光谱,测量采用激发波长为532nm、激光功率为10mw、光栅为1200g/mm、物镜50倍、数值孔径为0.75以及波数范围为0~1900cm-1。对PVPP样品颗粒明场图像中感兴趣点对应颗粒上所在位置分别进行10次拉曼光谱测量后,对得到的10个拉曼光谱进行光谱平均计算得到平均拉曼光谱作为PVPP样品的拉曼光谱,PVPP样品拉曼光谱如图6中(b)。观察(b)中PVPP样品的拉曼光谱信号在k=934.2cm-1(记为k0)位置附近存在锋利的拉曼特征峰。The prepared temporary mount was placed on the sample stage of a confocal Raman microscope for imaging, and the particles in the PVPP sample were observed through a 50x objective lens. It was observed that most of the particles in the PVPP sample were spherical particles, and a very small part was small irregular particles. According to the observation results, select an irregular particle in the PVP sample for bright field imaging, select a spherical particle in the PVPP sample for bright field imaging, and select the particle with the highest gray level in the bright field imaging image by visual observation A point of is performed as a point of interest. The bright-field imaging image of PVPP sample particles is shown in Fig. 6(a), and the point of interest in the image is the position marked by the crosshair. Light irradiation of a specific excitation wavelength is carried out on the position corresponding to the obtained point of interest on the particle, and the Raman spectrum of the single crystal at this excitation wavelength is measured. The measurement adopts an excitation wavelength of 532nm, a laser power of 10mw, and a grating of 1200g/mm, objective lens 50 times, numerical aperture 0.75 and wave number range 0-1900cm -1 . After performing 10 Raman spectrum measurements on the positions of the particles corresponding to the points of interest in the bright field image of the PVPP sample particles, the average Raman spectrum of the obtained 10 Raman spectra was calculated to obtain the average Raman spectrum as the Raman spectrum of the PVPP sample. , Raman spectrum of the PVPP sample is shown in Figure 6(b). Observe that there is a sharp Raman characteristic peak near the position of k=934.2cm -1 (denoted as k 0 ) in the Raman spectrum signal of the PVPP sample in (b).

随后,使用明场显微镜观察PVPP样品中的分散颗粒在PVPP样品中找到一个球形颗粒,并进行明场成像,明场图像如图7中(a)所示,图7的(a)中实线框内区域为包含颗粒局部的感兴趣区域,明场图像中感兴趣区域大小为10μm*10μm。在感兴趣区域对应样品上区域进行共焦扫描,得到PVPP样品颗粒的拉曼光谱数据集。其中,扫描采用激发波长为532nm,激光功率为10mw,光栅为1200g/mm,物镜倍数为50,数值孔径为0.75,以及波数为0~1900cm-1。对得到的拉曼光谱数据集每点拉曼光谱进行去除宇宙噪声和消除背景噪声处理,得到处理后的PVPP样品颗粒拉曼光谱数据集。随后,在处理后的拉曼光谱数据集中,将每点拉曼光谱在波数k0=934.2cm-1的光谱强度I作为平面图像该点的灰度值生成拉曼特征峰的拉曼图像,PVPP样品颗粒的拉曼特征峰的拉曼图像分别如图7中(b)所示。Subsequently, use a bright-field microscope to observe the dispersed particles in the PVPP sample. Find a spherical particle in the PVPP sample and perform bright-field imaging. The bright-field image is shown in Figure 7 (a), and the solid line in Figure 7 (a) The area in the frame is the region of interest including the local particles, and the size of the region of interest in the bright field image is 10 μm*10 μm. Confocal scanning is performed on the area corresponding to the sample area of interest to obtain the Raman spectrum data set of PVPP sample particles. Among them, the scanning adopts an excitation wavelength of 532nm, a laser power of 10mw, a grating of 1200g/mm, an objective lens multiple of 50, a numerical aperture of 0.75, and a wavenumber of 0-1900cm -1 . The Raman spectrum of each point of the obtained Raman spectrum data set is processed to remove cosmic noise and background noise, and the processed Raman spectrum data set of PVPP sample particles is obtained. Subsequently, in the processed Raman spectrum data set, the spectral intensity I of each point of the Raman spectrum at the wave number k 0 =934.2 cm -1 is used as the gray value of the point in the plane image to generate a Raman image of the Raman characteristic peak, The Raman images of the Raman characteristic peaks of the PVPP sample particles are shown in (b) in Figure 7, respectively.

在得到的PVPP样品颗粒的拉曼特征峰的拉曼图像中选择灰度值最高点作为感兴趣点,如图8中(a)所示,其中感兴趣点使用十字线标记。根据感兴趣点对应样品中所在位置,在处理后的PVPP样品颗粒拉曼光谱数据集中进行区域定域提取高质量的拉曼光谱,提取的高质量拉曼光谱分别如图8中(b)所示。In the obtained Raman image of the Raman characteristic peak of the PVPP sample particle, select the point with the highest gray value as the point of interest, as shown in (a) in Figure 8, where the point of interest is marked with a cross. According to the position in the sample corresponding to the point of interest, high-quality Raman spectra are extracted in the processed PVPP sample particle Raman spectrum data set, and the extracted high-quality Raman spectra are shown in (b) in Figure 8. Show.

Claims (3)

1.一种复杂化合物拉曼光谱的提取方法,其特征在于,是一种基于高空间分辨率的光学显微成像技术与拉曼光谱技术结合的一种的化合物高质量拉曼光谱的提取方法,包括以下步骤:1. A method for extracting Raman spectra of complex compounds, characterized in that it is a method for extracting high-quality Raman spectra of compounds based on high spatial resolution optical microscopic imaging technology and Raman spectroscopy technology , including the following steps: (1)取1-5mg待检测的粉末样品,放置在载玻片上,裸露直径3~5mm,或者3-5mm矩形,或者3-5毫米长方形的表面压实成为一个薄层,制成待测样品;(1) Take 1-5mg of the powder sample to be tested, place it on a glass slide, expose a diameter of 3-5mm, or a 3-5mm rectangle, or compact a 3-5mm rectangle surface into a thin layer, and make it to be tested sample; (2)在光学显微镜下对待测样品进行观察,寻找成像清晰并且具有广泛形貌特征的一个分散颗粒,对分散颗粒进行明场成像,选择成像图像中颗粒上灰度级最高的一点进行作为感兴趣点;(2) Observe the sample to be tested under an optical microscope, look for a dispersed particle with a clear image and a wide range of morphological characteristics, perform bright field imaging on the dispersed particle, and select the point with the highest gray level on the particle in the imaging image as the sensor Points of Interest; (3)对步骤(2)感兴趣点在颗粒上的对应位置点进行特定激发波长的光照射,测量得到在该颗粒上对应位置点的该激发波长的拉曼光谱,拉曼光谱包含两个参数k和I,k为拉曼光谱中的拉曼位移波数,I为拉曼光谱中波数k对应的光谱强度;(3) Light irradiation with a specific excitation wavelength is performed on the corresponding position of the point of interest in step (2) on the particle, and the Raman spectrum of the excitation wavelength at the corresponding position on the particle is measured. The Raman spectrum includes two Parameters k and I, k is the Raman shift wavenumber in the Raman spectrum, and I is the spectral intensity corresponding to the wavenumber k in the Raman spectrum; (4)多次重复步骤(3),得到多个对应位置点上该激发波长的拉曼光谱,再对多个拉曼光谱中相同波数k对应的光谱强度I进行平均计算得到平均拉曼光谱;(4) Repeat step (3) multiple times to obtain the Raman spectrum of the excitation wavelength on multiple corresponding positions, and then average the spectral intensity I corresponding to the same wave number k in multiple Raman spectra to obtain the average Raman spectrum ; (5)在步骤(4)计算得到平均拉曼光谱中找到峰值高且峰形尖锐的拉曼光谱特征峰作为该待测物的拉曼光谱特征峰,记录该拉曼光谱特征峰对应的波数k0(5) In the average Raman spectrum calculated in step (4), find the Raman spectrum characteristic peak with high peak and sharp peak shape as the Raman spectrum characteristic peak of the analyte, and record the wave number corresponding to the Raman spectrum characteristic peak k 0 ; (6)再次在光学显微镜下对待测样品进行观察寻找成像清晰并且具有广泛形貌特征的一个分散颗粒,或直接使用步骤(2)中寻找到的颗粒,对该颗粒进行明场成像,将包含该颗粒整体或局部的一个矩形区域作为感兴趣区域;(6) Observe the sample to be tested again under the optical microscope to find a dispersed particle with clear imaging and extensive morphology features, or directly use the particle found in step (2) to perform bright field imaging on the particle, which will contain A rectangular area of the whole or part of the particle is used as the area of interest; (7)对步骤(6)感兴趣区域对应样品上区域进行共焦扫描得到一个拉曼光谱数据集,拉曼光谱数据集包含四个参数,分别为x、y、k和I,其中x和y对应感兴趣区域内一个点的平面坐标,k为在一个点测量的拉曼光谱的拉曼位移波数,I为一个点的拉曼光谱中一个波数k对应的光谱强度;(7) Carry out confocal scanning to step (6) region of interest corresponding to the area on the sample to obtain a Raman spectrum data set, the Raman spectrum data set contains four parameters, are respectively x, y, k and I, wherein x and y corresponds to the plane coordinates of a point in the region of interest, k is the Raman shift wavenumber of the Raman spectrum measured at a point, and I is the spectral intensity corresponding to a wavenumber k in the Raman spectrum of a point; (8)对步骤(7)得到的拉曼光谱数据集每点拉曼光谱进行去除宇宙噪声和消除背景噪声处理,得到处理后的拉曼光谱数据集;(8) each point Raman spectrum of the Raman spectrum data set obtained in step (7) is processed to remove cosmic noise and eliminate background noise, and obtain the processed Raman spectrum data set; (9)在步骤(8)处理后的拉曼光谱数据集中,将每点拉曼光谱在波数k0对应光谱强度I作为平面图像中该点的灰度值生成拉曼特征峰的拉曼图像;(9) In the Raman spectrum data set after step (8) processing, the Raman image of the Raman characteristic peak is generated as the gray value of the point in the plane image at the wavenumber k of each point of the Raman spectrum corresponding to the spectral intensity I ; (10)在步骤(9)得到的拉曼图像中选择灰度值最高点作为感兴趣点;(10) in the Raman image that step (9) obtains, select the highest point of the gray value as the point of interest; (11)根据步骤(10)得到的感兴趣点在样品中所在位置,在步骤(8)处理后的拉曼光谱数据集中进行区域定域提取对应位置的高质量的拉曼光谱;(11) According to the position of the point of interest obtained in step (10) in the sample, perform regional localization in the Raman spectrum data set processed in step (8) to extract the high-quality Raman spectrum of the corresponding position; 待检测的粉末样品为交联、聚合反应生成的高分子量复杂化合物,包含简单的初始物质、中间产物、最终产物,而交联、聚合反应生成的产物又是分子量处于一定区间的交联体。The powder sample to be tested is a high-molecular-weight complex compound generated by cross-linking and polymerization reactions, including simple initial materials, intermediate products, and final products, and the products generated by cross-linking and polymerization reactions are cross-linked bodies with molecular weights in a certain range. 2.按照权利要求1所述的一种复杂化合物拉曼光谱的提取方法,其特征在于,待检测的粉末样品为聚乙烯醇吡咯烷酮(PVP);2. according to the extraction method of a kind of complex compound Raman spectrum according to claim 1, it is characterized in that, the powder sample to be detected is polyvinylpyrrolidone (PVP); 采用共焦拉曼光谱显微镜WITec Alpha300-RAMAN进行样品颗粒光学显微镜观察、明场图像采集、拉曼光谱测量以及共焦扫描;使用WITec Project FIVE软件对明场图像、拉曼光谱、拉曼图像和感兴趣区域以及感兴趣点进行显示,并且对拉曼光谱和拉曼光谱数据集进行数据处理,以及在拉曼光谱数据集中提取高质量拉曼光谱;The confocal Raman spectrum microscope WITec Alpha300-RAMAN was used for the optical microscope observation of sample particles, bright field image acquisition, Raman spectrum measurement and confocal scanning; the bright field image, Raman spectrum, Raman image and Display the region of interest and the point of interest, and perform data processing on the Raman spectrum and the Raman spectrum dataset, and extract high-quality Raman spectra from the Raman spectrum dataset; 在步骤(1)中,取PVP粉末样品2-5mg置于载玻片上,轻微摇晃使样品粉末颗粒尽量分散,盖上盖玻片后轻微碾压盖玻片,使样品粉末颗粒进一步分散;In step (1), take 2-5 mg of the PVP powder sample and place it on a glass slide, shake it slightly to disperse the sample powder particles as much as possible, cover the cover glass and then slightly roll the cover glass to further disperse the sample powder particles; 步骤(2)中,使用50倍物镜明场显微镜观察样品选择进行拉曼扫描的样品颗粒;观察发现PVP样品中颗粒形状均不规则,根据观察结果,在PVP样品中选择一个不规则形状颗粒进行成像,并通过视觉观察选择成像图像中颗粒上灰度级最高的一点进行作为感兴趣点;In step (2), use a 50 times objective lens bright field microscope to observe the sample particles selected for Raman scanning; observations find that the particle shapes in the PVP sample are irregular, and according to the observation results, select an irregular shape particle in the PVP sample for Imaging, and select the point with the highest gray level on the particle in the imaging image as the point of interest through visual observation; 步骤(3)中,使用共焦拉曼光谱显微镜WITec Alpha300-RAMAN进行拉曼光谱测量,测量采用激发波长为532nm、激光功率为10mw、光栅为1200g/mm、物镜50倍、数值孔径为0.75、以及波数范围为0~1900cm-1In step (3), use the confocal Raman spectrum microscope WITec Alpha300-RAMAN to perform Raman spectrum measurement. The measurement uses an excitation wavelength of 532nm, a laser power of 10mw, a grating of 1200g/mm, an objective lens of 50 times, and a numerical aperture of 0.75, And the range of wave number is 0~1900cm -1 ; 步骤(4)中,使用共焦拉曼光谱显微镜对所述PVP样品颗粒上位置进行10次拉曼光谱测量并进行平均计算得到平均拉曼光谱;In step (4), use a confocal Raman spectrum microscope to perform 10 Raman spectrum measurements on the position on the PVP sample particle and perform an average calculation to obtain an average Raman spectrum; 步骤(5)中,记录的拉曼光谱特征峰对应波数k0为934.2cm-1In step (5), the corresponding wavenumber k 0 of the recorded Raman spectrum characteristic peak is 934.2cm −1 ; 步骤(6)中,得到的感兴趣区域为包含步骤(2)中寻找到的PVP颗粒局部的20μm*20μm的正方形区域;In step (6), the obtained region of interest is a square area of 20 μm*20 μm including the local PVP particles found in step (2); 步骤(7)中,使用共焦拉曼光谱显微镜WITec Alpha300-RAMAN进行共焦扫描,扫描采用激发波长为532nm,激光功率为10mw,光栅为1200g/mm,物镜倍数为50,数值孔径为0.75,以及波数为0~1900cm-1In step (7), use the confocal Raman spectroscopy microscope WITec Alpha300-RAMAN to perform confocal scanning. The scanning adopts an excitation wavelength of 532nm, a laser power of 10mw, a grating of 1200g/mm, an objective lens multiple of 50, and a numerical aperture of 0.75. And the wave number is 0 to 1900 cm -1 . 3.按照权利要求1所述的一种复杂化合物拉曼光谱的提取方法,其特征在于,待检测的粉末样品为交联聚乙烯醇吡咯烷酮(PVPP)粉末;3. according to the extraction method of a kind of complex compound Raman spectrum according to claim 1, it is characterized in that, the powder sample to be detected is cross-linked polyvinylpyrrolidone (PVPP) powder; 采用共焦拉曼光谱显微镜WITec Alpha300-RAMAN进行样品颗粒光学显微镜观察、明场图像采集、拉曼光谱测量以及共焦扫描;使用WITec Project FIVE软件对明场图像、拉曼光谱、拉曼图像和感兴趣区域以及感兴趣点进行显示,并且对拉曼光谱和拉曼光谱数据集进行数据处理,以及在拉曼光谱数据集中提取高质量拉曼光谱;The confocal Raman spectrum microscope WITec Alpha300-RAMAN was used for the optical microscope observation of sample particles, bright field image acquisition, Raman spectrum measurement and confocal scanning; the bright field image, Raman spectrum, Raman image and Display the region of interest and the point of interest, and perform data processing on the Raman spectrum and the Raman spectrum dataset, and extract high-quality Raman spectra from the Raman spectrum dataset; 在步骤(1)中,取PVPP粉末样品2-5mg置于载玻片上,轻微摇晃使样品粉末颗粒尽量分散,盖上盖玻片后轻微碾压盖玻片,使样品粉末颗粒进一步分散;In step (1), take 2-5 mg of the PVPP powder sample and place it on a glass slide, shake slightly to disperse the sample powder particles as much as possible, cover the cover glass and then slightly roll the cover glass to further disperse the sample powder particles; 步骤(2)中,使用50倍物镜明场显微镜观察样品选择进行拉曼扫描的样品颗粒;观察发现PVPP样品中颗粒绝大部分形状为球形颗粒,极小部分为很小的不规则颗粒,根据观察结果,在PVPP样品中选择一个球形颗粒进行成像,并通过视觉观察选择成像图像中颗粒上灰度级最高的一点进行作为感兴趣点;In step (2), use a 50 times objective lens bright-field microscope to observe the sample particles selected for Raman scanning; observe that most of the particles in the PVPP sample are spherical particles, and a very small part is very small irregular particles, according to Observe the results, select a spherical particle in the PVPP sample for imaging, and select the point with the highest gray level on the particle in the imaging image as the point of interest through visual observation; 步骤(3)中,使用共焦拉曼光谱显微镜WITec Alpha300-RAMAN进行拉曼光谱测量,测量采用激发波长为532nm、激光功率为10mw、光栅为1200g/mm、物镜50倍、数值孔径为0.75以及波数范围为0~1900cm-1In step (3), use the confocal Raman spectrum microscope WITec Alpha300-RAMAN to perform Raman spectrum measurement. The measurement uses an excitation wavelength of 532nm, a laser power of 10mw, a grating of 1200g/mm, an objective lens of 50 times, and a numerical aperture of 0.75 and The range of wave number is 0~1900cm -1 ; 步骤(4)中,使用共焦拉曼光谱显微镜对所述PVPP样品颗粒上位置进行10次拉曼光谱测量并进行平均计算得到平均拉曼光谱;In step (4), use a confocal Raman spectrum microscope to perform 10 Raman spectrum measurements on the PVPP sample particles and perform an average calculation to obtain an average Raman spectrum; 步骤(5)中,记录的拉曼光谱特征峰对应波数k0为934.2cm-1In step (5), the corresponding wavenumber k 0 of the recorded Raman spectrum characteristic peak is 934.2cm −1 ; 步骤(6)中,得到的感兴趣区域为包含寻找到的另一PVPP样品颗粒局部的10μm*10μm的正方形区域;In step (6), the obtained region of interest is a square region of 10 μm*10 μm that contains another PVPP sample particle that is found; 步骤(7)中,使用共焦拉曼光谱显微镜WITec Alpha300-RAMAN进行共焦扫描,扫描采用激发波长为532nm,激光功率为10mw,光栅为1200g/mm,物镜倍数为50,数值孔径为0.75,以及波数为0~1900cm-1In step (7), use the confocal Raman spectroscopy microscope WITec Alpha300-RAMAN to perform confocal scanning. The scanning adopts an excitation wavelength of 532nm, a laser power of 10mw, a grating of 1200g/mm, an objective lens multiple of 50, and a numerical aperture of 0.75. And the wave number is 0 to 1900 cm -1 .
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