CN219694830U - Flow mode nanometer particle analyzer based on dynamic and static light scattering technology - Google Patents
Flow mode nanometer particle analyzer based on dynamic and static light scattering technology Download PDFInfo
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- CN219694830U CN219694830U CN202320204536.XU CN202320204536U CN219694830U CN 219694830 U CN219694830 U CN 219694830U CN 202320204536 U CN202320204536 U CN 202320204536U CN 219694830 U CN219694830 U CN 219694830U
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- 238000001370 static light scattering Methods 0.000 title claims abstract description 42
- 238000002296 dynamic light scattering Methods 0.000 title claims abstract description 39
- 238000000034 method Methods 0.000 claims abstract description 12
- 238000005227 gel permeation chromatography Methods 0.000 claims abstract description 11
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- 102000004169 proteins and genes Human genes 0.000 claims description 5
- 108090000623 proteins and genes Proteins 0.000 claims description 5
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- 238000001514 detection method Methods 0.000 abstract description 10
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- RRZXIRBKKLTSOM-XPNPUAGNSA-N avermectin B1a Chemical compound C1=C[C@H](C)[C@@H]([C@@H](C)CC)O[C@]11O[C@H](C\C=C(C)\[C@@H](O[C@@H]2O[C@@H](C)[C@H](O[C@@H]3O[C@@H](C)[C@H](O)[C@@H](OC)C3)[C@@H](OC)C2)[C@@H](C)\C=C\C=C/2[C@]3([C@H](C(=O)O4)C=C(C)[C@@H](O)[C@H]3OC\2)O)C[C@H]4C1 RRZXIRBKKLTSOM-XPNPUAGNSA-N 0.000 description 1
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Abstract
The utility model discloses a flow mode nanometer particle analyzer based on dynamic and static light scattering technology and a detection method thereof. The technique is suitable for use in connection with a complete front-end separation device-field flow separation system or gel permeation chromatography, where the front-end separation device can separate each component depending on the size of the sample component and flow out sequentially. The nanometer particle analyzer comprises a flow sample cell, an APD detector for dynamic light scattering technology, a PD detector for static light scattering technology, a laser, a lens group, a first data acquisition card, a second data acquisition card and a control unit, wherein laser emitted by the laser irradiates on a sample in the flow sample cell through the lens group, and the two detectors simultaneously receive scattered light of the sample and transmit signals to the control unit through the first data acquisition card and the second data acquisition card respectively; the utility model realizes accurate and high-resolution detection of the size and the molecular weight of the sample, and can obtain the intrinsic viscosity information at the same time.
Description
Technical Field
The utility model relates to a nanometer particle analyzer, in particular to a flow mode nanometer particle analyzer based on dynamic and static light scattering technology.
Background
Conventional nanoparticle sizers are based on dynamic light scattering techniques, using a beam of laser light to illuminate a sample, and detecting fluctuations in scattered light caused by brownian motion of particles suspended in a liquid by a photodetector. The original fluctuation signal of the scattered light intensity along with time is calculated to obtain a correlation curve of the system through correlation, and then the particle size and the particle size distribution of the particles are obtained through different mathematical models, such as a cumulative method or a multi-exponential method.
Generally, a nanoparticle analyzer has dynamic light scattering and static light scattering testing capabilities. Particle systems with the particle size range of about 1 nanometer to 1000 nanometers can be effectively detected by a dynamic light scattering technology, and the method has the characteristics of high testing speed, wide range, good repeatability, good accuracy and the like, and is widely applied, but a traditional quartz or plastic cuvette testing mode (commonly called batch mode in literature) is widely adopted by a nanometer particle analyzer, the particle size distribution testing resolution of a wide distribution sample is lower, and the limit resolution can only distinguish narrow distribution single components with the particle size difference of 2.5-3 times, so that the quantification of the particle size distribution result is greatly limited; the nanometer particle analyzer can also utilize static light scattering (Rayleigh scattering equation) to detect the net scattered light intensity by configuring a series of protein or macromolecule solution, then drawing a Debye curve and performing linear extrapolation to obtain the average molecular weight Mw of the sample, and because the mode has complicated steps, has extremely high requirements on the concentration accuracy of the sample and the cleanliness of the sample, has higher requirements on operators and operating environments, leads to larger error (10% or higher) of detection results, has lower repeatability (10% or higher) and can not give out molecular weight distribution results, the practical application and literature report are less.
Disclosure of Invention
Aiming at the problems that the particle size detection resolution of a wide distribution sample in the existing nano-particle size analyzer technology is low, the molecular weight detection is high, the result error is high, the repeatability is low, and the molecular weight distribution result cannot be given, the utility model aims to provide a flow mode nano-particle size analyzer based on dynamic and static light scattering technology and a detection method thereof.
In order to solve the technical problems, the utility model adopts the following technical scheme:
the utility model provides a flow mode nanometer particle analyzer based on dynamic and static light scattering technology, which is suitable for being connected with a complete front-end particle separating device-field flow separating system FFF or a macromolecule and protein separating device-gel permeation chromatography GPC/SEC, wherein the front-end separating device at least comprises a differential refraction detector or an ultraviolet detector, and each component can be separated according to the size of a sample component and sequentially flows out. The nanometer particle analyzer comprises a flow sample cell, an APD detector for dynamic light scattering technology, a PD detector for static light scattering technology, a laser, a focusing lens, a first data acquisition card, a control unit, a second data acquisition card and a control unit, wherein laser emitted by the laser irradiates on a sample in the flow sample cell through the focusing lens, and the two detectors simultaneously receive scattered light of the sample and transmit signals to the control unit through the first data acquisition card and the second data acquisition card respectively.
The flow mode nanometer particle analyzer based on dynamic and static light scattering technology has the preferable scheme that an APD detector is connected with 90 degrees or an optical fiber receiving component arranged back to collect dynamic light scattering light fluctuation signals, a correlation curve is calculated, a diffusion coefficient of sample particles is calculated, the particle size of particles of each outflow component is obtained through a Stokes Einstein equation, and a concentration signal obtained by a differential refraction detector or an ultraviolet detector is combined to obtain high-resolution particle size distribution independent of a calculation model. The 90-degree optical fiber receiving assembly is suitable for detecting samples with moderate concentration range, strong scattered light and no multiple light scattering, and the optical fiber receiving assembly arranged in a back direction can detect samples with weaker scattered light or higher concentration and multiple light scattering on the basis of the detection capability of the 90-degree optical fiber receiving assembly.
The flow mode nanometer particle analyzer based on dynamic and static light scattering technology has the preferable scheme that a PD detector is arranged at an angle of 90 degrees of an incident laser beam, static light scattering signals are collected, signals of a differential refraction detector or an ultraviolet detector are combined, and absolute molecular weight and molecular weight distribution information of each outflow component are calculated through a Rayleigh scattering equation. The utility model realizes more accurate and high-resolution detection of the size distribution of the particles, and can obtain the molecular weight and the molecular weight distribution information of the sample.
According to the flow mode nanometer particle analyzer based on the dynamic and static light scattering technology, the first data acquisition card can collect analog signals output by the differential refraction detector or the ultraviolet detector and trigger signals of front-end separation equipment, scattered light fluctuation of each outflow component is detected through dynamic light scattering, particle size information is obtained through calculation, concentration corresponding to each outflow component is calculated through signals of the differential refraction detector or the ultraviolet detector, and high-resolution particle size distribution information is obtained through calculation by combining the particle size information and concentration information of the outflow components.
According to the flow mode nanometer particle analyzer based on the dynamic and static light scattering technology, the second data acquisition card can collect analog signals output by the differential refraction detector or the ultraviolet detector and trigger signals of front-end separation equipment, scattered light intensity information of each outflow component is detected through static light scattering, concentration corresponding to each outflow component is calculated through the differential refraction detector or the ultraviolet detector signals, absolute molecular weight of each outflow component is calculated through combining the scattered light intensity information and the concentration information of the outflow component, and then weight average molecular weight Mw, number average molecular weight Mn, Z-average molecular weight Mz and molecular weight distribution information are obtained.
The flow mode nanometer particle analyzer based on dynamic and static light scattering technology adopts the preferable scheme that the static light scattering principle is adopted to uniformly scatter based on sample scattering independent of angles. According to the Rayleigh scattering equation, the molecular size of the sample, the mean square rotation radius Rg, is not more than 1/20 of the laser wavelength, and can be regarded as uniform scattering. Taking 671nm incident light as an example, the upper limit of the size of a sample corresponding to uniform scattering is not more than 33.5nm, the theoretical calculated value of the molecular weight of the corresponding globular protein is about 2220 Da, the theoretical calculated value of the linear polysaccharide molecule is about 56 Da, and the theoretical calculated value of the hyperbranched high molecular polymer depends on the structure of the hyperbranched high molecular polymer and is 1553 Da to 27400 Da.
In the flow mode nanometer particle analyzer based on dynamic and static light scattering technology, in a testing process, for a suitable sample, such as a high molecular solution or a protein solution, dynamic light scattering and static light scattering can be simultaneously carried out to obtain high-resolution particle size distribution and molecular weight of the sample, wherein the molecular weight comprises Mw, mn and Mz, and a molecular weight distribution coefficient PD=Mw/Mn and molecular weight actual distribution curve information.
According to the flow mode nanometer particle analyzer based on the dynamic and static light scattering technology, the preferable scheme is that for a proper sample, such as a high polymer solution, the intrinsic viscosity IV of the corresponding component can be calculated by combining the particle size and molecular weight information of each component, and the Mark-Houwink curve, the Mark-Houwink alpha value and the K value are further obtained by plotting the intrinsic viscosity IV on the molecular weight, so that the information of the molecular structure of the high polymer is obtained.
The flow mode nanometer particle analyzer based on dynamic and static light scattering technology has the preferable scheme that under the premise of knowing the refractive index increment dn/dc and the ultraviolet absorption increment dA/dc of a sample, 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.
The flow mode nanometer particle analyzer based on dynamic and static light scattering technology has the preferable scheme that the flow sample cell has extremely low volume (less than 30 mu L) and can prevent the diffusion effect of the sample in the flow sample cell to the maximum extent.
The flow mode nanometer particle analyzer based on dynamic and static light scattering technology has the preferable scheme that a flow sample cell is provided with a standard chromatographic pipeline interface, and is suitable for various pipeline connection requirements of 1/16 inch outer diameter and 0.01 inch-0.04 inch inner diameter.
The flow mode nanometer particle analyzer based on dynamic and static light scattering technology has the preferable scheme that the flow mode nanometer particle analyzer has extremely high sampling rate when tested by adopting the dynamic light scattering technology, and the sampling time of the fastest data point is 0.4 seconds, namely, the particle diameter result of an outflow component can be obtained through the test of 0.4 seconds.
The flow mode nanometer particle analyzer based on dynamic and static light scattering technology has the preferable scheme that the flow mode nanometer particle analyzer 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.
The flow mode nanometer particle analyzer based on dynamic and static light scattering technology has the preferable scheme that the data acquisition card 1 and the data acquisition card 2 can both receive trigger signals of analog or digital signals sent by front-end separation equipment, and the test is automatically started by the trigger signals. 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.
Advantageous effects
1. The device and the method are suitable for being connected with a complete front-end particle separating device-field flow separating system FFF or a high molecular and protein separating device-gel permeation chromatography GPC/SEC, wherein the separating device can separate each component according to the size of a sample component and sequentially flow out, the theoretical resolution of the FFF and the GPC/SEC separating device is better than 1.1 times of particle or molecular size difference, and the high-resolution particle size distribution test result and the molecular weight distribution test result can be obtained through the detection of each flowing-out component. The equipment has good popularization and practical value, and good economic benefit and social benefit can be generated after wide popularization and application;
2. according to the static light scattering test, namely the molecular weight test, a series of samples with the concentration are not required to be configured, the net scattered light intensity of each concentration sample is detected, so that the molecular weight information is extrapolated, only one sample with the concentration is required to be configured, the sample is separated and flows out through GPC/SEC after being sampled, the concentration information of each flowing-out component is calculated through a differential refraction detector or an ultraviolet detector signal in a flow path and is used for calculating the molecular weight, so that not only the weight average molecular weight Mw, but also the number average molecular weight Mn and the Z-average molecular weight Mz can be obtained, the molecular weight distribution coefficient PD=Mw/Mn, and meanwhile, the actual molecular weight distribution curve information can be obtained;
3. according to the technology, for a suitable sample such as a high polymer solution, the intrinsic viscosity IV of a corresponding outflow component is calculated, and a Mark-Houwink curve, a Mark-Houwink alpha value and a K value are further obtained by plotting the intrinsic viscosity IV on the molecular weight, so that information on the molecular structure of the high polymer such as the molecular density, the branching degree and the like is obtained;
4. the utility model is widely applied to the research and application fields of biopharmaceuticals, high polymer materials, foods, agricultural scientific research, electronics, environment, instruments and meters and the like.
Drawings
FIG. 1 is an electrical schematic diagram of a flow mode nano-particle analyzer based on dynamic and static light scattering techniques;
FIG. 2 is a schematic diagram of the utility model in combination with a front end separation device;
FIG. 3 is a schematic diagram of a dynamic light scattering test using a 90℃optical path and a static light scattering test using a 90℃optical path;
FIG. 4 is a schematic diagram of a dynamic light scattering test using a back-to-back light path and a static light scattering test using a 90℃light path;
FIG. 5 is a graph showing the original dynamic light scattering results and the calculated particle size distribution results
FIG. 6 is a graph showing static light scattering results and calculated molecular weight results;
FIG. 7 is a graph showing the correlation curves of dynamic light scattering;
FIG. 8 is a schematic diagram of a flow-through sample cell;
FIG. 9 is a Mark-Houwink graph.
In the figure: 1-laser, 2-optical fiber, 3-flow sample cell, 4-flow sample cell inlet, 5-optical trap, 6-sample, 7-90 DEG angle direction scattered light, 8-PD detector, 9-PD signal transmission line, 10-flow sample cell outlet, 11-back scattered light, 12-focusing lens, 13-optical fiber bracket, 14-motor bearing.
Detailed Description
The utility model is further elucidated below in connection with the drawings of the specification.
As shown in fig. 1, the present utility model provides a flow mode nano-particle analyzer based on dynamic and static light scattering technology, comprising a flow sample cell 3, an APD detector connected with an optical fiber 2, a PD detector 8, a laser 1, a focusing lens 12, a first data acquisition card, and a data acquisition card, wherein laser 1 emitted from the laser irradiates onto a sample 6 in the flow sample cell 3 through the focusing lens, and the APD detector and the PD detector 8 connected with the optical fiber 2 receive scattered light of the sample at the same time and transmit signals to a computer through the first data acquisition card and the second data acquisition card, respectively.
As shown in fig. 3 and 4, the APD detector is used for dynamic light scattering test, the APD detector receives scattered light of a sample through an optical fiber 2 connected with the APD detector, the optical fiber 2 is arranged on one side of the optical fiber bracket 13 (fig. 3) which is arranged at an angle of 90 degrees with the scattered light 7, or is opposite to the APD detector (fig. 4), and APD signals are transmitted to a computer through a first data acquisition card; the PD detector 8 is used for static light scattering test, is arranged on one side of the 90-degree angle of the laser beam, and PD signals are transmitted to a computer through the second data acquisition card by the PD signal transmission line 9.
As shown in FIG. 2, the nano-particle analyzer of the present utility model is suitable for use in connection with a complete front-end particle separation device-field flow separation system FFF or a polymer and protein separation device-gel permeation chromatography GPC/SEC, wherein the front-end separation device comprises at least one differential refractive index detector or one ultraviolet detector. The front-end separation device can carry out sample injection, a sample can separate each component according to the size of the sample component through an FFF field flow separation channel or a gel permeation chromatographic column in the flowing process and sequentially flows out, and the separated sample component sequentially flows through a differential refraction detector or an ultraviolet detector and the nano-particle analyzer. 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 nano-particle analyzer, collected by the first data acquisition card or the second data acquisition card, and transmitted to the software of the nano-particle analyzer 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. 5, in a dynamic light scattering test in flow mode, the APD detector is used to collect sample scattered light fluctuation signals and at the same time to collect differential refraction detector or ultraviolet detector signals. With a certain period of time (fastest0.4 second for a period, and the period is not more than 10 seconds), and obtaining a correlation curve of a sample outflow component flowing through the nanometer particle analyzer in the period through correlation calculation of a time fluctuation signal of scattered light in the period, and obtaining a corresponding particle size d through calculation by an accumulation method i Information. Baseline setting is carried out on signals of the differential refraction detector or the ultraviolet detector, the baseline value corresponding to the outflow volume is subtracted from the actually detected response signal value to obtain the net response value of the signal, and the concentration information C of the sample under the corresponding particle size can be calculated by combining dn/dc (differential refraction detector) or dA/dc (ultraviolet detector) of the sample i . A group of particle sizes and corresponding concentration data [ C ] i ,d i ]The particle size distribution curve of the sample is obtained by plotting the particle size on the abscissa and the concentration (or relative concentration) on the ordinate. The integral range of the outflow volume can also be set by the particle size analyzer software, the particle size distribution in the integral range can be calculated, and the concentration C can be used for the particle size distribution i The sum gives the total concentration of the sample over the integration range.
As shown in fig. 7, a normalized correlation curve in the dynamic light scattering test by the method of the present utility model is schematically shown.
As shown in fig. 6, in the static light scattering test in the flow mode, the PD detector 8 is used to collect a sample scattered light intensity signal and at the same time to collect a differential refraction detector or an ultraviolet detector signal. 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 an effluent component corresponding to the effluent volumeMolecular weight M of (2) i . A set of molecular 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.
As shown in fig. 8, the flow sample cell 3 has a four-sided light-passing structure, and the scattered light can be emitted from both sides by the scattered light 7 in the 90 ° angular direction, and the back-emitted back-scattered light 11, so that the sample flows in through the flow sample cell inlet 4 and flows out through the flow sample cell outlet 10, the volume of the flow sample cell 3 is low, and the influence on the detection resolution caused by the diffusion effect can be prevented to the maximum extent. The flow-through sample cell is compatible with all pipelines with the outer diameter of 1/16 inch and the inner diameter of 0.01-0.04 inch.
As shown in fig. 9, the equation iv= (5/12) · (N A ·π·D i 3 /M i ) Combining particle sizes D corresponding to each component i And molecular weight M i The information can calculate the intrinsic viscosity IV of the corresponding component, where N A Is the avermectin constant. Further bisecting at intrinsic viscosity IVAnd (5) carrying out molecular weight mapping to obtain a Mark-Houwink curve. Using formula iv=km α Fitting the curve to obtain Mark-Houwink alpha value and K value, thereby obtaining the information of the molecular structure of the polymer.
Claims (1)
1. A flow mode nano-particle sizer based on dynamic and static light scattering techniques, characterized by: the nanometer particle analyzer is suitable for being connected with a complete front-end particle separating device-field flow separating system FFF or a macromolecule and protein separating device-gel permeation chromatography GPC/SEC, wherein the front-end separating device at least comprises a differential refraction detector or an ultraviolet detector, and each component is separated according to the size of the sample component and flows out in sequence; the nanometer particle analyzer comprises a flow sample cell, an APD detector for dynamic light scattering technology, a PD detector for static light scattering technology, a laser, a focusing lens, a first data acquisition card, a control unit, a second data acquisition card and a control unit, wherein laser emitted by the laser irradiates on a sample in the flow sample cell through the focusing lens, and the two detectors simultaneously receive scattered light of the sample and transmit signals to the control unit through the first data acquisition card and the second data acquisition card respectively;
the PD detector is disposed at the 90 ° laser beam and collects a static light scattering signal.
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