CN113281337B - Extraction method of complex compound Raman spectrum - Google Patents
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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
Technical Field
The invention relates to the fields of analytical chemistry and pharmaceutical compositions, in particular to an extraction method of a complex compound high-quality Raman spectrum based on combination of optical microscopic imaging and Raman spectrum.
Background
The high molecular weight complex compound produced by crosslinking and polymerization reaction is widely applied to beverage and medicine industries. However, the compound has complex components, including simple initial substances, intermediate products and final products, and the intermediate products generated by crosslinking and polymerization are crosslinked bodies with molecular weights in a certain range, so that single crystal analysis of the substances is difficult, and especially optical detection is a technical means under the condition of not changing physical and chemical properties of the substances, such as insolubility in water, no heating and refrigeration and no color change reaction.
In view of the important roles of crosslinking compounds in the beverage and pharmaceutical industries, there is a need to establish a rapid, non-contact detection method which retains the chemical and physical properties of complex compounds, and the existing detection methods currently use a thermal melting point method and a high resolution mass spectrometry method to destroy the physical properties; indistinguishable using nuclear magnetic resonance techniques. In recent years, the development of high-spatial resolution optical microscopic imaging technology is rapid, and feasibility is provided for single crystals in spatially resolved complex compounds; at the same time, the Raman spectrum has the function of molecular labels, so that the identification of the components of complex compounds is possible. And the identification of the raman spectrum of the complex compound component by a spectroscopic method requires the acquisition of high quality raman spectrum. In view of the above, the invention establishes a non-contact and nondestructive extraction method of the high-quality Raman spectrum of the complex compound based on the combination of the high-spatial-resolution optical microscopic imaging technology and the Raman spectrum technology.
Disclosure of Invention
The invention aims to provide an extraction method of a complex compound high-quality Raman spectrum based on the combination of a high-spatial-resolution optical microscopic imaging technology and a 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.
In order to achieve the above object, the present invention provides a method for extracting a compound high quality raman spectrum based on a combination of a high spatial resolution optical microscopy imaging technique and a raman spectrum technique, comprising the steps of:
(1) Taking 1-5mg of powder sample to be detected, placing the powder sample on a glass slide, compacting the surface of a rectangle with the exposed diameter of 3-5mm or 3-5mm into a thin layer, and preparing the sample to be detected;
(2) Observing a sample to be detected under an optical microscope, searching for a dispersed particle which is clear in imaging and has wide morphological characteristics, performing bright field imaging on the dispersed particle, and selecting a point with highest gray level, which represents brightness, on the particle in an imaging image as an interesting point;
(3) Carrying out light irradiation of specific wavelength on the corresponding position point of the interest point on the particle in the step (2), and measuring to obtain a Raman spectrum of the excitation wavelength of the corresponding position point on the particle, wherein the Raman spectrum comprises two parameters k and I, k is a Raman shift wave number in the Raman spectrum, and I is spectrum intensity corresponding to the wave number k in the Raman spectrum;
(4) Repeating the step (3) for a plurality of times to obtain Raman spectra of the excitation wavelength at a plurality of corresponding position points, and carrying out average calculation on the spectrum intensity I corresponding to the same wave number k in the plurality of Raman spectra to obtain an average Raman spectrum;
(5) In the step (4), a Raman spectrum characteristic peak with a high peak value and a sharp peak shape is found in the average Raman spectrum obtained by calculation and is used as the Raman spectrum characteristic peak of the object to be detected, and the wave number k corresponding to the Raman spectrum characteristic peak is recorded 0 ；
(6) Observing a sample to be detected under an optical microscope to find a dispersed particle with clear imaging and wide morphology characteristics, or directly using the particle found in the step (2), carrying out bright field imaging on the particle, and taking a rectangular region containing the whole or part of the particle as a region of interest;
(7) Carrying out confocal scanning on the region on the sample corresponding to the region of interest in the step (6) to obtain a Raman spectrum data set, wherein the Raman spectrum data set comprises four parameters, namely x, y, k and I, respectively, wherein x and y correspond to the plane coordinates of a point in the region of interest, k is the Raman shift wave number of the Raman spectrum measured at the point, and I is the spectrum intensity corresponding to one wave number k in the Raman spectrum of the point;
(8) Performing the processing of removing universe noise and eliminating background noise on each point Raman spectrum of the Raman spectrum data set obtained in the step (7) to obtain a processed Raman spectrum data set;
(9) In the Raman spectrum data set processed in the step (8), each point of Raman spectrum is recorded in the wave number k 0 Generating a Raman image of a Raman characteristic peak by taking the corresponding spectral intensity I as a gray value of the point in the plane image;
(10) Selecting a point with the highest gray value from the Raman image obtained in the step (9) as an interesting point;
(11) And (3) carrying out regional localization extraction on the Raman spectrum data set processed in the step (8) according to the position of the interest point in the sample obtained in the step (10) to obtain high-quality Raman spectrum of the corresponding position.
The powder sample to be tested according to the invention is a single compound or a polymer with possibly multiple components.
In a first embodiment of the invention, the powder sample to be tested is a polyvinylpyrrolidone (PVP) K25 powder sample purchased from Basoff, germany, lot 61861347G0.
In a first embodiment of the invention, confocal RAMAN spectroscopy microscopy, WITec Alpha300-RAMAN, is used for sample particle optical microscopy, bright field image acquisition, RAMAN spectroscopy, and confocal scanning. The bright field image, raman spectrum, raman image and region of interest and points of interest are displayed using WITec Project FIVE software, and the raman spectrum and raman spectrum dataset are data processed and high quality raman spectrum is extracted in the raman spectrum dataset.
In the first embodiment of the present invention, in the step (1), 2-5mg of PVP powder sample is placed on a glass slide, and the sample powder particles are dispersed as much as possible by slightly shaking, and the glass slide is lightly crushed after the glass slide is covered, so that the sample powder particles are further dispersed.
In a first embodiment of the present invention, in step (2), the sample particles selected for raman scanning are observed using a 50-fold objective bright field microscope.
In the first embodiment of the present invention, in the step (2), the particles in the PVP sample are observed to be irregular in shape, and according to the observation result, an irregularly shaped particle is selected in the PVP sample for imaging, and a point with the highest gray level on the particle in the imaged image is selected as the point of interest by visual observation.
In a first embodiment of the present invention, in step (3), raman spectroscopy is performed using a confocal Raman spectroscopy microscope WITec Alpha300-RAMAN, using an excitation wavelength of 532nm, a laser power of 10mw, a grating of 1200g/mm, an objective lens of 50 times, a numerical aperture of 0.75, and a wavenumber range of 0 to 1900cm -1 。
In the first embodiment of the present invention, in the step (4), a confocal raman spectrum microscope is used to perform 10 raman spectrum measurements on the positions on the PVP sample particles and perform an average calculation to obtain an average raman spectrum.
In the first embodiment of the present invention, in the step (5), the recorded characteristic peak of the raman spectrum corresponds to a wavenumber k of 934.2cm -1 。
In a first embodiment of the present invention, in step (6), the region of interest obtained is a square region of 20 μm x 20 μm containing the local PVP particles found in step (2).
In the first embodiment of the present invention, in the step (7), confocal scanning is performed by using a confocal RAMAN spectrum microscope WITec Alpha300-RAMAN, wherein the scanning adopts an excitation wavelength of 532nm, a laser power of 10mw, a grating of 1200g/mm, and an objective lens multiple50, a numerical aperture of 0.75 and a wave number of 0 to 1900cm -1 。
In a second example of the invention, the powder sample to be tested used as sample was crosslinked polyvinylpyrrolidone (PVPP) powder purchased from Basoff, germany, lot 92441577L0.
In a second embodiment of the invention, confocal RAMAN spectroscopy microscopy, WITec Alpha300-RAMAN, is used for sample particle optical microscopy, bright field image acquisition, RAMAN spectroscopy, and confocal scanning. The bright field image, raman spectrum, raman image and region of interest and points of interest are displayed using WITec Project FIVE software, and the raman spectrum and raman spectrum dataset are data processed and high quality raman spectrum is extracted in the raman spectrum dataset.
In the second embodiment of the present invention, in the step (1), 2-5mg of PVPP powder sample is placed on a glass slide, and the sample powder particles are dispersed as much as possible by slightly shaking, and the glass slide is covered and then slightly crushed, so that the sample powder particles are further dispersed.
In the second embodiment of the present invention, in the step (2), the sample particles selected for raman scanning are observed using a 50-fold objective bright field microscope.
In the second embodiment of the present invention, in the step (2), most of the particles in the PVPP sample are observed to be spherical particles, the very small part is very small irregular particles, one spherical particle is selected in the PVPP sample to image according to the observation result, and the point with the highest gray level on the particle in the imaged image is selected as the point of interest through visual observation.
In the second embodiment of the present invention, in the step (3), raman spectrum measurement is performed using a confocal Raman spectrum microscope WITec Alpha300-RAMAN, wherein the measurement employs an excitation wavelength of 532nm, a laser power of 10mw, a grating of 1200g/mm, an objective lens of 50 times, a numerical aperture of 0.75, and a wave number range of 0 to 1900cm -1 。
In the second embodiment of the present invention, in the step (4), a confocal raman spectrum microscope is used to perform 10 raman spectrum measurements on the positions on the PVPP sample particles, and an average raman spectrum is obtained by performing an average calculation.
In the second embodiment of the present invention, in the step (5), the recorded characteristic peak of the raman spectrum corresponds to a wavenumber k of 934.2cm -1 。
In a second embodiment of the present invention, in the step (6), the obtained region of interest is a square region of 10 μm by 10 μm containing the found part of the other PVPP sample particle.
In the second embodiment of the present invention, in the step (7), confocal scanning is performed using a confocal Raman spectrum microscope WITec Alpha300-RAMAN, the scanning is performed with an excitation wavelength of 532nm, a laser power of 10mw, a grating of 1200g/mm, an objective lens magnification of 50, a numerical aperture of 0.75, and a wave number of 0 to 1900cm -1 。
Compared with the prior art, the invention has the beneficial effects that:
the method provided by the invention establishes a high-quality Raman spectrum extraction method of chemical and pharmaceutical components by an analysis method of complex compounds based on optical microscopic imaging combined with Raman spectrum. Optical microscopic imaging of the compound particles, identifying particles in the image from the obtained image; carrying out light irradiation of specific wavelength on the image particle region to obtain a Raman spectrum; analyzing the Raman characteristic peak and making a Raman image of the Raman characteristic peak; and carrying out regional localization extraction on the high-quality Raman spectrum according to the Raman image. The method is accurate, non-contact, non-damaging, rapid and free from changing the physical property and chemical property of the substance to be detected, and can be used for the component Raman spectrum analysis of different chemical substances.
The method of the invention directly combines high-resolution imaging and Raman spectrum of the compound, and does not contact or change the physical and chemical properties of the compound.
Drawings
FIG. 1PVP K25 powder sample temporary mounting;
in fig. 2, (a) is a bright field image of PVP sample particles, with reticle mark locations being points of interest; (b) is the raman spectrum of the PVP sample averaged;
fig. 3 (a) shows a bright field image of irregular particles in a PVP sample, and (b) shows a raman image of raman characteristic peaks of irregular particles in a PVP sample;
fig. 4 (a) raman image of raman characteristic peaks of irregular particles in a PVP sample, wherein the point of interest is marked with a cross-hair; (b) High quality raman spectra extracted in the PVP sample particle raman spectrum dataset.
FIG. 5 powder sample temporary mounting;
in fig. 6, (a) is a PVPP particle bright field image, and the reticle mark position is the point of interest; (b) is the raman spectrum of the average calculated PVPP sample;
fig. 7 (a) is a bright field image of spherical particles in a PVPP sample, wherein the solid line in-frame region is the region of interest comprising the particle fraction; (b) Raman images of raman characteristic peaks of spherical particles in the PVPP sample;
fig. 8 (a) is a raman image of raman characteristic peaks of particles in a PVPP sample, wherein the points of interest are marked with cross hairs, respectively; (b) High quality raman spectra were extracted for the PVPP sample particle raman spectrum dataset.
Detailed Description
The present invention will be further illustrated with reference to the following examples, but the present invention is not limited to the following examples.
Example 1:
PVP K25 powder samples purchased from Basoff, germany, lot number 61861347G0 were used. And (3) carrying out optical microscopic observation of the sample particles by adopting a confocal Raman spectrum microscope WITec Alpha300-RAMAN, acquiring bright field images, measuring Raman spectrum and carrying out confocal scanning. The bright field image, raman spectrum, raman image and region of interest and points of interest are displayed using WITec Project FIVE software, and the raman spectrum and raman spectrum dataset are data processed and high quality raman spectrum is extracted in the raman spectrum dataset.
The PVP powder samples were prepared into temporary patches, as shown in fig. 1.
And (3) placing the prepared temporary device sheet on a confocal Raman microscope sample stage for imaging, and respectively observing particles in the PVP sample through a 50-time objective lens. The particle shape of PVP sample is observed to be irregularAnd (5) granulating. According to the observation result, selecting an irregularly-shaped particle in the PVP sample for bright-field imaging, and respectively selecting a point with highest gray level on the particle in the bright-field imaging image as a point of interest through visual observation. The bright field imaging images of PVP sample particles are respectively shown in (a) of fig. 2, and the interesting points in the images are cross line labeling positions. The obtained position of the interested point corresponding to the particle is irradiated with light of specific excitation wavelength, the Raman spectrum of the single crystal at the excitation wavelength of the point is measured, the excitation wavelength is 532nm, the laser power is 10mw, the grating is 1200g/mm, the objective lens is 50 times, the numerical aperture is 0.75, and the wave number range is 0-1900 cm -1 . And (2) respectively carrying out 10 times of Raman spectrum measurement on the positions of the particles corresponding to the interest points in the bright field image of the PVP sample particles, and carrying out spectrum average calculation on the obtained 10 Raman spectra to obtain an average Raman spectrum as the Raman spectrum of the PVP sample, wherein the Raman spectrum of the PVP sample is shown in (b) of fig. 2. Observe the raman spectrum signal at k= 934.2cm for the PVP sample in fig. 2 (b) -1 (this is denoted as k 0 ) Sharp raman characteristic peaks exist at the locations.
Then, the PVP sample is subjected to bright field imaging again after finding irregular particles in step (2), the bright field image is shown in fig. 3 (a), the solid line in-frame region in fig. 3 (a) is the region of interest containing the particles locally, and the size of the region of interest is 20 μm×20 μm. Confocal scanning is carried out on the region of interest corresponding to the sample upper region, and Raman spectrum data sets of PVP sample particles are respectively obtained. Scanning with excitation wavelength 532nm, laser power 10mw, grating 1200g/mm, objective lens multiple 50, numerical aperture 0.75, and wave number 0-1900 cm -1 . And carrying out the treatment of removing cosmic noise and eliminating background noise on the Raman spectrum of each point of the obtained Raman spectrum data set to obtain the processed PVP sample particle Raman spectrum data set. Then, in the processed Raman spectrum data set, each point of Raman spectrum is recorded in wave number k 0 ＝934.2cm -1 As the gray value of the point of the planar image, a raman image of the raman characteristic peak of the PVP sample particle is generated, as shown in fig. 3 (b).
The highest gray value point is selected as the point of interest in the raman image of the raman characteristic peak of the obtained PVP sample particle, and as shown in fig. 4 (a), the point of interest is marked with a reticle. And (3) carrying out regional localization on the processed PVP sample particle Raman spectrum data set according to the position of the interest point in the corresponding sample, and extracting high-quality Raman spectrum, wherein the extracted high-quality Raman spectrum is shown in (b) of fig. 4.
Example 2:
PVPP powder purchased from Basoff, germany was used as sample, lot 92441577L0. The scheme adopts a confocal Raman spectrum microscope WITec Alpha300-RAMAN to carry out sample particle optical microscope observation, bright field image acquisition, raman spectrum measurement and confocal scanning. The bright field image, raman spectrum, raman image and region of interest and points of interest are displayed using WITec Project FIVE software, and the raman spectrum and raman spectrum dataset are data processed and high quality raman spectrum is extracted in the raman spectrum dataset.
The PVPP powder samples were individually prepared into temporary patches, which are shown in FIG. 5.
The prepared clinical device sheet is placed on a confocal Raman microscope sample stage for imaging, and particles in PVPP samples are observed through a 50-time objective lens. The particles in the PVPP sample were observed to be mostly spherical in shape and very small in size as irregular particles. According to the observation result, selecting an irregularly-shaped particle in the PVP sample for bright field imaging, selecting a spherical particle in the PVPP sample for bright field imaging, and respectively selecting a point with highest gray level on the particle in the bright field imaging image as a point of interest through visual observation. The PVPP sample particle bright field imaging image is shown in (a) of FIG. 6, and the interesting point in the image is a cross line mark position. The obtained position of the interested point corresponding to the particle is irradiated with light of specific excitation wavelength, the Raman spectrum of the single crystal at the excitation wavelength of the point is measured, the excitation wavelength is 532nm, the laser power is 10mw, the grating is 1200g/mm, the objective lens is 50 times, the numerical aperture is 0.75, and the wave number range is 0-1900 cm -1 . For PVPP samplesAfter carrying out 10 raman spectrum measurements on the positions of the particles corresponding to the points of interest in the particle bright field image, carrying out spectrum average calculation on the obtained 10 raman spectra to obtain an average raman spectrum as a raman spectrum of a PVPP sample, wherein the Raman spectrum of the PVPP sample is shown in (b) of fig. 6. Observing the raman spectrum signal of the PVPP sample in (b) at k= 934.2cm -1 (denoted as k 0 ) Sharp raman characteristic peaks exist near the location.
Then, the dispersed particles in the PVPP sample were observed with a bright field microscope to find a spherical particle in the PVPP sample, and bright field imaging was performed, as shown in fig. 7 (a), in which the solid line in-frame region in fig. 7 (a) is a region of interest including a part of the particle, and the size of the region of interest in the bright field image was 10 μm×10 μm. And carrying out confocal scanning on the region of interest corresponding to the sample to obtain a Raman spectrum data set of PVPP sample particles. Wherein the scanning adopts excitation wavelength of 532nm, laser power of 10mw, grating of 1200g/mm, objective lens multiple of 50, numerical aperture of 0.75, and wave number of 0-1900 cm -1 . And carrying out the processing of removing cosmic noise and eliminating background noise on the Raman spectrum of each point of the obtained Raman spectrum data set to obtain the processed PVPP sample particle Raman spectrum data set. Then, in the processed Raman spectrum data set, each point of Raman spectrum is recorded in wave number k 0 ＝934.2cm -1 The raman image of the raman characteristic peak of the PVPP sample particle is generated as the gray value of the point of the planar image, and the raman image of the raman characteristic peak of the PVPP sample particle is shown in fig. 7 (b), respectively.
The highest gray value point is selected as the point of interest in the raman image of the raman characteristic peak of the obtained PVPP sample particle, as shown in fig. 8 (a), wherein the point of interest is marked with a cross-hair. And (3) carrying out regional localization on the processed PVPP sample particle Raman spectrum data set according to the position of the interest point in the corresponding sample, and extracting high-quality Raman spectra, wherein the extracted high-quality Raman spectra are respectively shown in (b) of FIG. 8.
Claims (3)
1. The extraction method of the compound Raman spectrum is characterized by being a compound high-quality Raman spectrum extraction method based on the combination of a high-spatial-resolution optical microscopic imaging technology and a Raman spectrum technology, and comprises the following steps:
(1) Taking 1-5mg of powder sample to be detected, placing the powder sample on a glass slide, compacting the surface of a rectangle with the exposed diameter of 3-5mm or 3-5mm into a thin layer, and preparing the sample to be detected;
(2) Observing a sample to be detected under an optical microscope, searching for a dispersed particle which is clear in imaging and has wide morphological characteristics, performing bright field imaging on the dispersed particle, and selecting a point with highest gray level on the particle in an imaging image as an interesting point;
(3) Carrying out light irradiation of specific excitation wavelength on the corresponding position point of the interest point on the particle in the step (2), and measuring to obtain a Raman spectrum of the excitation wavelength of the corresponding position point on the particle, wherein the Raman spectrum comprises two parameters k and I, k is a Raman shift wave number in the Raman spectrum, and I is spectrum intensity corresponding to the wave number k in the Raman spectrum;
(4) Repeating the step (3) for a plurality of times to obtain Raman spectra of the excitation wavelength at a plurality of corresponding position points, and carrying out average calculation on the spectrum intensity I corresponding to the same wave number k in the plurality of Raman spectra to obtain an average Raman spectrum;
(5) In the step (4), a Raman spectrum characteristic peak with a high peak value and a sharp peak shape is found in the average Raman spectrum obtained by calculation and is used as the Raman spectrum characteristic peak of the object to be detected, and the wave number k corresponding to the Raman spectrum characteristic peak is recorded 0 ；
(6) Observing a sample to be detected under an optical microscope to find a dispersed particle with clear imaging and wide morphology characteristics, or directly using the particle found in the step (2), carrying out bright field imaging on the particle, and taking a rectangular region containing the whole or part of the particle as a region of interest;
(7) Carrying out confocal scanning on the region on the sample corresponding to the region of interest in the step (6) to obtain a Raman spectrum data set, wherein the Raman spectrum data set comprises four parameters, namely x, y, k and I, respectively, wherein x and y correspond to the plane coordinates of a point in the region of interest, k is the Raman shift wave number of the Raman spectrum measured at the point, and I is the spectrum intensity corresponding to one wave number k in the Raman spectrum of the point;
(8) Performing the processing of removing universe noise and eliminating background noise on each point Raman spectrum of the Raman spectrum data set obtained in the step (7) to obtain a processed Raman spectrum data set;
(9) In the Raman spectrum data set processed in the step (8), each point of Raman spectrum is recorded in the wave number k 0 Generating a Raman image of a Raman characteristic peak by taking the corresponding spectral intensity I as a gray value of the point in the plane image;
(10) Selecting a point with the highest gray value from the Raman image obtained in the step (9) as an interesting point;
(11) Carrying out regional localization extraction on the Raman spectrum data set processed in the step (8) according to the position of the interest point in the sample, which is obtained in the step (10), to obtain high-quality Raman spectrum of the corresponding position;
the powder sample to be detected is a high molecular weight complex compound generated by crosslinking and polymerization reaction, which comprises simple initial substances, intermediate products and final products, and the product generated by crosslinking and polymerization reaction is a crosslinked body with a certain molecular weight.
2. The method for extracting a complex compound raman spectrum according to claim 1, wherein the powder sample to be detected is polyvinyl pyrrolidone (PVP);
performing sample particle optical microscope observation, bright field image acquisition, raman spectrum measurement and confocal scanning by adopting a confocal Raman spectrum microscope WITec Alpha300-RAMAN, displaying bright field images, raman spectra, raman images, regions of interest and points of interest by using WITec Project FIVE software, performing data processing on the Raman spectra and a Raman spectrum data set, and extracting high-quality Raman spectra in the Raman spectrum data set;
in the step (1), 2-5mg of PVP powder sample is taken and placed on a glass slide, the sample powder particles are dispersed as much as possible by slight shaking, and the glass slide is slightly rolled after the glass slide is covered, so that the sample powder particles are further dispersed;
in the step (2), observing sample particles selected for Raman scanning by using a 50-time objective bright field microscope; observing to find that the particle shapes in the PVP sample are irregular, selecting one irregularly-shaped particle in the PVP sample for imaging according to the observation result, and selecting a point with the highest gray level on the particle in the imaging image to serve as an interesting point through visual observation;
in the step (3), raman spectrum measurement is carried out by using a confocal Raman spectrum microscope WITec Alpha300-RAMAN, wherein the excitation wavelength is 532nm, the laser power is 10mw, the grating is 1200g/mm, the objective lens is 50 times, the numerical aperture is 0.75, and the wave number range is 0-1900 cm -1 ；
In the step (4), carrying out 10 times of Raman spectrum measurement on the positions on the PVP sample particles by using a confocal Raman spectrum microscope and carrying out average calculation to obtain an average Raman spectrum;
in the step (5), the recorded characteristic peak of the Raman spectrum corresponds to the wave number k 0 Is 934.2cm -1 ；
In the step (6), the obtained region of interest is a square region of 20 μm by 20 μm containing the local PVP particles found in the step (2);
in the step (7), confocal Raman spectrum microscope WITec Alpha300-RAMAN is used for confocal scanning, the scanning adopts excitation wavelength of 532nm, laser power of 10mw, grating of 1200g/mm, objective lens multiple of 50, numerical aperture of 0.75 and wave number of 0-1900 cm -1 。
3. The method for extracting a complex compound raman spectrum according to claim 1, wherein the powder sample to be detected is a crosslinked polyvinylpyrrolidone (PVPP) powder;
performing sample particle optical microscope observation, bright field image acquisition, raman spectrum measurement and confocal scanning by adopting a confocal Raman spectrum microscope WITec Alpha300-RAMAN, displaying bright field images, raman spectra, raman images, regions of interest and points of interest by using WITec Project FIVE software, performing data processing on the Raman spectra and a Raman spectrum data set, and extracting high-quality Raman spectra in the Raman spectrum data set;
in the step (1), 2-5mg of PVPP powder sample is taken and placed on a glass slide, the sample powder particles are dispersed as much as possible by slight shaking, and the glass slide is slightly rolled after the glass slide is covered, so that the sample powder particles are further dispersed;
in the step (2), observing sample particles selected for Raman scanning by using a 50-time objective bright field microscope; observing to find that most of particles in the PVPP sample are spherical particles and the smallest part of the particles are very small irregular particles, selecting one spherical particle in the PVPP sample for imaging according to an observation result, and selecting a point with the highest gray level on the particle in an imaging image as a point of interest through visual observation;
in the step (3), a confocal Raman spectrum microscope WITec Alpha300-RAMAN is used for Raman spectrum measurement, wherein the excitation wavelength is 532nm, the laser power is 10mw, the grating is 1200g/mm, the objective lens is 50 times, the numerical aperture is 0.75, and the wave number range is 0-1900 cm -1 ；
In the step (4), carrying out 10 times of Raman spectrum measurement on the positions on the PVPP sample particles by using a confocal Raman spectrum microscope and carrying out average calculation to obtain an average Raman spectrum;
in the step (5), the recorded characteristic peak of the Raman spectrum corresponds to the wave number k 0 Is 934.2cm -1 ；
In the step (6), the obtained region of interest is a square region of 10 μm by 10 μm containing the local part of the found other PVPP sample particle;
in the step (7), confocal Raman spectrum microscope WITec Alpha300-RAMAN is used for confocal scanning, the scanning adopts excitation wavelength of 532nm, laser power of 10mw, grating of 1200g/mm, objective lens multiple of 50, numerical aperture of 0.75 and wave number of 0-1900 cm -1 。
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| CN101432606A (en) * | 2004-06-30 | 2009-05-13 | 化学影像公司 | Spectroscopic methods for component particle analysis |
| CA2837463A1 (en) * | 2011-05-29 | 2012-12-06 | Korea Research Institute Of Chemical Technology | High-speed screening apparatus for a raman analysis-based high-speed multiple drug |
| CN112888934A (en) * | 2018-08-17 | 2021-06-01 | 方宁 | Integrated system of Raman spectroscopy and advanced optical microscopy and application thereof |
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| CN101432606A (en) * | 2004-06-30 | 2009-05-13 | 化学影像公司 | Spectroscopic methods for component particle analysis |
| CA2837463A1 (en) * | 2011-05-29 | 2012-12-06 | Korea Research Institute Of Chemical Technology | High-speed screening apparatus for a raman analysis-based high-speed multiple drug |
| CN112888934A (en) * | 2018-08-17 | 2021-06-01 | 方宁 | Integrated system of Raman spectroscopy and advanced optical microscopy and application thereof |
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| 岩心显微拉曼光谱成像方法的研究;黄乔松等;《光谱学与光谱分析》;20081215(第12期);第2880-2884页 * |
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