CN219657486U - Multi-angle light scattering detector based on static light scattering technology - Google Patents
Multi-angle light scattering detector based on static light scattering technology Download PDFInfo
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- CN219657486U CN219657486U CN202320204700.7U CN202320204700U CN219657486U CN 219657486 U CN219657486 U CN 219657486U CN 202320204700 U CN202320204700 U CN 202320204700U CN 219657486 U CN219657486 U CN 219657486U
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- 238000001370 static light scattering Methods 0.000 title claims abstract description 33
- 238000000569 multi-angle light scattering Methods 0.000 title claims abstract description 32
- 238000001514 detection method Methods 0.000 claims abstract description 20
- 238000005227 gel permeation chromatography Methods 0.000 claims description 13
- 238000000926 separation method Methods 0.000 claims description 12
- 238000001641 gel filtration chromatography Methods 0.000 claims description 8
- 238000001542 size-exclusion chromatography Methods 0.000 claims description 4
- 238000009792 diffusion process Methods 0.000 claims description 3
- 230000000694 effects Effects 0.000 claims description 3
- 238000013213 extrapolation Methods 0.000 claims description 3
- 102000004169 proteins and genes Human genes 0.000 abstract description 2
- 108090000623 proteins and genes Proteins 0.000 abstract description 2
- 230000008054 signal transmission Effects 0.000 description 6
- 238000000034 method Methods 0.000 description 4
- 230000008901 benefit Effects 0.000 description 3
- 238000004364 calculation method Methods 0.000 description 3
- 238000010586 diagram Methods 0.000 description 3
- 229920000642 polymer Polymers 0.000 description 3
- 230000032683 aging Effects 0.000 description 2
- 238000011088 calibration curve Methods 0.000 description 2
- 238000002347 injection Methods 0.000 description 2
- 239000007924 injection Substances 0.000 description 2
- 230000003287 optical effect Effects 0.000 description 2
- 239000002245 particle Substances 0.000 description 2
- 238000005070 sampling Methods 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- 238000010521 absorption reaction Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 229960000074 biopharmaceutical Drugs 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 102000034238 globular proteins Human genes 0.000 description 1
- 108091005896 globular proteins Proteins 0.000 description 1
- 150000004676 glycans Chemical class 0.000 description 1
- 229920000587 hyperbranched polymer Polymers 0.000 description 1
- 230000010354 integration Effects 0.000 description 1
- 230000001678 irradiating effect Effects 0.000 description 1
- 229920002521 macromolecule Polymers 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 239000002861 polymer material Substances 0.000 description 1
- 229920001282 polysaccharide Polymers 0.000 description 1
- 239000005017 polysaccharide Substances 0.000 description 1
- 239000010453 quartz Substances 0.000 description 1
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N silicon dioxide Inorganic materials O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 1
- 238000004448 titration Methods 0.000 description 1
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Abstract
The utility model discloses a multi-angle light scattering detector based on a static light scattering technology. The multi-angle light scattering detector comprises a circulating sample cell, PD1-PD5 detectors, a laser, a plano-convex lens a, a plano-convex lens b, a data acquisition card and a control unit, wherein the PD1-PD5 detectors are arranged at different angles and used for static light scattering acquisition, laser emitted by the laser irradiates on a sample in the circulating sample cell through a lens group, and meanwhile, the PD1-PD5 detectors receive scattered light of the sample and transmit signals to the control unit through the data acquisition card; the utility model realizes the accurate and high-resolution detection of the molecular weight and the distribution of the high-molecular and protein samples.
Description
Technical Field
The utility model relates to a multi-angle light scattering detector, in particular to a multi-angle light scattering detector based on a static light scattering technology.
Background
Macromolecules are a class of substances with relatively high molecular weights, generally referred to as compounds with molecular weights up to several thousands to millions. The molecular weight and molecular weight distribution of the polymer have a close correlation with their properties. There are currently various test techniques for detecting molecular weight of high molecular substances, such as end group titration (detection of number average molecular weight Mn), viscosity (detection of viscosity average molecular weight Mv), single tube static light scattering (detection of weight average molecular weight Mw), which are all capable of detecting only average molecular weight.
Gel permeation chromatography GPC can yield molecular weight distribution. Conventional gel permeation chromatography GPC uses a differential refractive index detector or an ultraviolet detector to detect the flow-out time and concentration information of a sample, by plotting a calibration curve using a series of narrow distribution standard samples of different molecular weights, separating the sample by a chromatographic column during the test, obtaining the molecular weight of each component sample from the calibration curve, and further obtaining the molecular weight distribution and average molecular weight (Mw, mn, mz) relative to the standard samples. Conventional gel permeation chromatography detection yields relative molecular weight information, not absolute molecular weight information of the sample, which, although widely used, has significant limitations. Firstly, the correlation between the relative molecular weight and various properties of the sample (such as strength, toughness, viscosity, etc.) is poor, and secondly, when the sample has additional structural information (such as branched, hyperbranched polymer, or protein sample), there may be several times of difference between the relative molecular weight and absolute molecular weight, and the test result of the relative molecular weight is easily affected by factors such as chromatographic pump precision, chromatographic column brands and combinations, chromatographic column aging, column temperature fluctuation, etc.
Disclosure of Invention
Aiming at the problem that the existing molecular weight detection technology can only obtain average molecular weight or relative molecular weight distribution, the utility model provides a multi-angle light scattering detector based on a static light scattering technology and a detection method thereof, which can obtain the absolute molecular weight and molecular weight distribution of a sample.
In order to solve the technical problems, the utility model adopts the following technical scheme:
the utility model provides a multi-angle light scattering detector based on a static light scattering technology, which is used for detecting the molecular weight and the distribution of a sample and is suitable for being connected with a complete front-end separation device gel permeation chromatography GPC, size exclusion chromatography SEC and gel filtration chromatography GFC, wherein the front-end separation device at least comprises a differential refraction detector or an ultraviolet detector, and each component can be separated by utilizing a chromatographic column according to the size of the sample component and sequentially flows out. The multi-angle light scattering detector comprises a circulating sample cell, PD1-PD5 photodiode detectors, a laser, a plano-convex lens a, a plano-convex lens b, a data acquisition card and a control unit, wherein the PD1-PD5 photodiode detectors are arranged at different angles and used for static light scattering acquisition, laser emitted by the laser irradiates on a sample in the circulating sample cell through a lens group, and the PD1-PD5 detectors simultaneously receive scattered light of the sample and transmit signals to the control unit through the data acquisition card.
According to the multi-angle light scattering detector based on the static light scattering technology, the optimal scheme is that 1-5 PD detectors can be configured on different angles to meet detection requirements of different molecular weight ranges, wherein PD1 is arranged in the direction which is orthogonal to laser light and is 90 degrees, PD2 is arranged in the direction which is less than or equal to 9 degrees, PD3 is arranged in the direction which is more than or equal to 170 degrees, PD4 is arranged in the direction which is 35-55 degrees, and PD5 is arranged in the direction which is 125-145 degrees.
A detection method of a multi-angle light scattering detector based on a static light scattering technology comprises the steps that PD1-PD5 detectors collect static light scattering signals of different angles, absolute molecular weight information of each outflow component is obtained through calculation through a Rayleigh scattering equation by combining signals of a differential refraction detector or an ultraviolet detector, then weight average molecular weight Mw, number average molecular weight Mn, Z-average molecular weight Mz and molecular weight distribution coefficient PD=Mw/Mn are obtained, and meanwhile actual molecular weight distribution curve information can be obtained.
In the detection method of the multi-angle light scattering detector based on the static light scattering technology, when only one detector PD1 which is arranged at the angle of 90 degrees and orthogonal to laser light is configured, only a uniformly scattered sample with scattering independent of angles can be accurately detected. According to the Rayleigh scattering equation, the molecular size (mean square rotation radius Rg) of the sample is not more than 1/20 of the laser wavelength, namely the sample can be regarded as uniform scattering; taking 635nm incident light as an example, the upper limit of the size of a sample corresponding to uniform scattering is that Rg is not more than 31.8nm, the theoretical calculated value of the molecular weight of the corresponding globular protein is about 2000 kiloDa, the theoretical calculated value of the linear polysaccharide molecule is about 50 kiloDa, and the theoretical calculated value of the hyperbranched high molecular polymer depends on the structure of the hyperbranched high molecular polymer to be 1500-25000 kiloDa.
According to the detection method of the multi-angle light scattering detector based on the static light scattering technology, the optimal scheme is that when one detector PD5 and any one detector PD1-PD4 which are arranged in the direction less than or equal to 9 degrees is configured, scattering information of an angle of 0 degrees can be obtained through extrapolation, and the upper limit of the molecular weight of a sample which can be detected according to a Rayleigh scattering equation is not limited.
According to the detection method of the multi-angle light scattering detector based on the static light scattering technology, when two or more PD detectors are configured, the information of the mean square rotation radius Rg of a sample can be obtained through drawing a Zimm curve according to the slope of the Zimm curve.
According to the detection method of the multi-angle light scattering detector based on the static light scattering technology, the preferable scheme is that under the premise that the refractive index increment dn/dc and the ultraviolet absorption increment dA/dc of a sample are known, the absolute concentration of the sample in a corresponding range can be calculated by setting a base line and an integral range for signals of a differential refraction detector or an ultraviolet detector, and the concentration unit is mg/mL.
According to the detection method of the multi-angle light scattering detector based on the static light scattering technology, the optimal scheme is that the flow sample cell has extremely low volume (less than 30 mu L) and can prevent diffusion effect of samples in the flow cell to the greatest extent.
The detection method of the multi-angle light scattering detector based on the static light scattering technology has the preferable scheme that the detection method has extremely high sampling rate when tested by adopting the static light scattering technology, and the sampling time of the fastest data point is 0.2 seconds, namely the molecular weight result of an outflow component can be obtained through the test of 0.2 seconds.
According to the detection method of the multi-angle light scattering detector based on the static light scattering technology, the data acquisition card can receive the trigger signal of the analog or digital signal sent by the front-end separation equipment, and the test can be automatically started by the trigger signal command. The time when the software receives the trigger signal is the test starting time, and the total test duration is determined by an operator according to the actual sample outflow time.
The utility model has the following beneficial effects and advantages:
1. the apparatus and method of the present utility model are suitable for use in connection with a complete front-end separation apparatus, gel permeation chromatography GPC or size exclusion chromatography SEC or gel filtration chromatography GFC, wherein the front-end separation apparatus comprises at least one differential refractive detector or one ultraviolet detector, and wherein each component can be separated by a chromatographic column depending on the size of the sample component and flowed out sequentially. The multi-angle light scattering detector collects static light scattering signals of different angles, combines signals of a differential refraction detector or an ultraviolet detector, calculates absolute molecular weight information of each outflow component through a Rayleigh scattering equation, and then obtains weight average molecular weight Mw, number average molecular weight Mn, Z-average molecular weight Mz and molecular weight distribution coefficient PD=Mw/Mn, and meanwhile can obtain actual molecular weight distribution curve information;
2. the multi-angle light scattering detector is connected with the front-end chromatographic device, a series of narrow-distribution standard samples with different molecular weights are not required to be used for drawing a correction curve in the test process, the test result does not depend on the outflow time of a sample signal, and the absolute molecular weight result obtained by the test is slightly influenced by factors such as chromatographic pump precision, chromatographic column marks and combinations, chromatographic column aging, column temperature fluctuation and the like. The result has high accuracy, good repeatability and simpler operation;
3. the utility model is widely applied to the research and application fields of biopharmaceuticals, high polymer materials, foods, agricultural scientific research, electronics, environment and the like. The equipment has good popularization and practical value, and can generate good economic benefit and social benefit after wide popularization and application.
Drawings
FIG. 1 is an electrical schematic diagram of a multi-angle light scattering detector based on static light scattering technology;
FIG. 2 is a schematic diagram of the utility model in combination with a front end separation device;
FIG. 3 is a schematic top view of the multi-angle detector core optical path;
FIG. 4 is a schematic side view of a flow-through sample cell;
FIG. 5 is a schematic diagram of a diaphragm structure;
FIG. 6 is a Zimm plot of scattered light signals from a multi-angle light scattering detector by a PD detector disposed at multiple angles;
FIG. 7 is a graph showing the static light scattering results and the calculated molecular weight results.
In the figure: 1-laser, 2-flow cell, 3-flow cell inlet, 4-flow cell outlet, 5-diaphragm a, 6-diaphragm b, 7-mirror a, 8-mirror b, 9-plano-convex lens a, 10-plano-convex lens b,11-PD1 direction scattered light, 12-PD1 detector, 13-PD2 direction scattered light, 14-PD2 detector, 15-PD3 direction scattered light, 16-PD3 detector, 17-PD4 direction scattered light, 18-PD4 detector, 19-PD5 direction scattered light, 20-PD5 detector, 21-PD signal transmission line, 22-sample, 30-flow path, 51-incident light through aperture, 52-scattered light through aperture.
Detailed Description
The utility model is further elucidated below in connection with the drawings of the specification.
As shown in fig. 1, the utility model provides a multi-angle light scattering detector based on a static light scattering technology, which comprises a flow sample cell, a PD1-PD5 photodiode detector, a laser, a plano-convex lens a, a plano-convex lens b and a data acquisition card, wherein laser emitted by the laser irradiates on a sample in the flow sample cell through the plano-convex lens a, the PD1-PD5 detector simultaneously receives scattered light signals of the sample, and analog signals of a differential refraction detector or an ultraviolet detector all transmit the signals to a computer through the data acquisition card.
As shown in fig. 2, the multi-angle light scattering detector according to the present utility model is suitable for use in connection with a complete front-end separation device (gel permeation chromatography GPC or size exclusion chromatography SEC or gel filtration chromatography GFC) comprising at least one differential refractive detector or one ultraviolet detector. The front end separation device can carry out sample injection, samples are separated by the size of the sample components through a chromatographic column in the flowing process and flow out sequentially, and the separated sample components sequentially flow through a differential refraction detector or an ultraviolet detector and a multi-angle light scattering detector. The trigger signal of the front-end separation device and the analog signal output by the differential refraction detector or one ultraviolet detector can be input into the multi-angle light scattering detector, collected by the data acquisition card and transmitted to the software system for calculation. The chromatographic pump and the automatic sampler at the front end are directly communicated with PC end software and are used for controlling the flow speed and sample injection of the chromatographic pump.
As shown in fig. 3, a top view of a core optical path of a multi-angle detector according to the present utility model is shown, which comprises a laser 1 for irradiating a sample, a flow cell 2, a sample entering the flow cell 2 through an inlet end 3 of the flow cell, and a flow cell 2 through an outlet end 4 of the flow cell, a diaphragm a5 disposed at an incident end of the laser and a diaphragm b6 disposed at an exit end of the flow cell are used for passing the laser and restricting scattered light from a specific angle, a through-hole-equipped mirror a7 disposed at the incident end of the laser and a through-hole-equipped mirror b8 disposed at the exit end of the laser are used for passing the laser and reflecting the scattered light in directions of PD2 and PD3 into the PD2 and PD3 detectors, a plano-convex lens a9 and a plano-convex lens b10 disposed at both ends of the flow cell are used for converging the laser and converting the scattered light emitted from the detection point into parallel light, the PD1 detector 12 is arranged in a direction perpendicular to the laser beam by 90 DEG, receives 90 DEG angle PD1 direction scattered light 11, and transmits the 90 DEG angle PD1 direction scattered light to the signal acquisition card through a PD signal transmission line 21, the PD2 detector 14 is arranged in a direction less than or equal to 9 DEG, receives forward angle PD2 direction scattered light 13, and transmits the angle PD2 direction scattered light to the signal acquisition card through the PD signal transmission line 21, the PD3 detector 16 is arranged in a direction more than or equal to 170 DEG, receives backward angle PD3 direction scattered light 15, and transmits the angle PD3 direction scattered light to the signal acquisition card through the PD signal transmission line 21, the PD4 detector 18 is arranged in a direction of 35 DEG to 55 DEG, receives side angle PD4 direction scattered light 17, and transmits the angle PD5 direction scattered light 19 through the PD signal transmission line 21, and transmits the angle PD5 direction scattered light 19 to the signal acquisition card through the PD signal transmission line 21.
As shown in fig. 4, in the side view of the flow cell used in the present utility model, the sample 22 flows in through the inlet end 3 of the flow cell and flows out through the outlet end 4 of the flow cell, the flow path 30 of the sample 22 is a through hole, both right and left ends of the through hole are in plane contact with the plano-convex lens a9 and the plano-convex lens b10, and the plane seal is used, and laser light is incident from left to right. The side wall of the flow sample cell 2 is made of transparent quartz material, and scattered light can be emitted through the side wall and the front and back laser light passing directions. The flow cell has a positive low capacity of not more than 30 mu L, and can prevent the influence on the detection resolution caused by the diffusion effect to the greatest extent.
As shown in fig. 5, a diaphragm used in the present utility model is schematically structured, in which an incident light passing hole 51 is formed in the middle of the diaphragm for passing laser light, and a scattered light passing hole 52 is formed in the outer side of the diaphragm for passing scattered light of a sample.
FIG. 6 is a schematic view of Zimm curve used in the present utility model to calculate the molecular weight and mean square radius of rotation Rg. Scattered light signals and differential refraction detector (or ultraviolet detector) signals obtained by each PD detector, corresponding Kc/R values are calculated and are measured at an angle Sin 2 Extrapolation is performed with an intercept corresponding to 0 DEG of 1/Mw, the slope of which is proportional to the mean square radius of rotation Rg 2 。
As shown in fig. 7, during the test, the PD detector is used to collect the sample scattered light intensity signal and at the same time the differential refraction detector or uv detector signal is collected. The collection frequency of the static light scattering scattered light is 1-5Hz (0.2 second point at maximum), and the collection frequency of the differential refraction detector or the ultraviolet detector signal is the same as that of the static light scattering signal. After the test is finished, baseline setting and integral setting are carried out on the static light scattering signal and the differential refraction detector or the ultraviolet detector signal, and an area needing to be calculated is defined. By subtracting the baseline value of the corresponding outflow volume from the actual detected response signal value to obtain the net response value of the signal, and combining the dn/dc (differential refraction detector) or dA/dc (ultraviolet detector) of the sample, the concentration information C of the sample under the corresponding particle size can be calculated i C is carried out by i Brought into Rayleigh scattering equation, combined with the scattered light intensity LS at the corresponding outflow volume i Obtaining the molecular weight M of the effluent component corresponding to the effluent volume i . A set of component weights and corresponding concentration data C i ,M i ]Plotting the molecular weight on the abscissa and the concentration (or relative concentration) on the ordinate to obtain the molecular weight distribution curve of the sample, which can be obtained by the following formula
Calculating the number average molecular weight M n Weight average molecular weight M w Z-average molecular weight M z Molecular weight distribution pd=m w /M n . The integral range of the outflow volume can also be set by the particle size analyzer software, the molecular weight distribution in the integral range can be calculated, and the concentration C can be used for the calculation of the molecular weight distribution i The sum gives the total concentration of the sample over the integration range.
Claims (5)
1. A multi-angle light scattering detector based on static light scattering technology, characterized in that: the detector is used for detecting the molecular weight and the distribution of the sample and is suitable for being connected with a complete front-end separation device gel permeation chromatography GPC or size exclusion chromatography SEC or gel filtration chromatography GFC, wherein the front-end separation device at least comprises one differential refraction detector or one ultraviolet detector, and each component can be separated by utilizing a chromatographic column according to the size of the component of the sample and flows out in sequence; the multi-angle light scattering detector comprises a flow sample cell, PD1-PD5 photodiode detectors, a laser, a plano-convex lens a, a plano-convex lens b, a data acquisition card and a control unit, wherein the PD1-PD5 photodiode detectors are arranged at different angles and used for static light scattering acquisition, laser emitted by the laser irradiates on a sample in the flow sample cell through a lens group, and the PD1-PD5 detectors simultaneously receive scattered light of the sample and transmit signals to the control unit through the data acquisition card;
1-5 PD detectors are arranged at different angles for meeting detection requirements of different molecular weight ranges, wherein PD1 is arranged at 90 DEG orthogonal to the laser, PD2 is arranged at a direction less than or equal to 9 DEG, PD3 is arranged at a direction less than or equal to 170 DEG, PD4 is arranged at a direction of 35 DEG-55 DEG, and PD5 is arranged at a direction of 125 DEG-145 deg.
2. A multi-angle light scattering detector based on static light scattering technology as claimed in claim 1, wherein: when only one detector PD1 disposed at 90 ° orthogonal to the laser light is provided, only a uniformly scattered sample whose scattering is independent of angle can be accurately detected; according to the Rayleigh scattering equation, the molecular size of the sample, namely the mean square rotation radius Rg, is not more than 1/20 of the laser wavelength, namely uniform scattering.
3. A multi-angle light scattering detector based on static light scattering technology as claimed in claim 1, wherein: when one detector PD5 and any one of PD1 to PD4 arranged in the direction of 9 ° or less is configured, scattering information of 0 ° can be obtained by extrapolation, and the upper limit of the molecular weight of the sample detected according to the rayleigh scattering equation is not limited.
4. A multi-angle light scattering detector based on static light scattering technology as claimed in claim 1, wherein: the volume of the flow-through sample cell is less than 30 mu L, so that the diffusion effect of the sample in the flow-through cell can be prevented to the greatest extent.
5. A multi-angle light scattering detector based on static light scattering technology as claimed in claim 1, wherein: the data acquisition card receives a trigger signal of an analog or digital signal sent by the front-end separation equipment, and the test is automatically started by a command of the trigger signal.
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