CN114813998A - Polymer detection method - Google Patents
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- CN114813998A CN114813998A CN202210296693.8A CN202210296693A CN114813998A CN 114813998 A CN114813998 A CN 114813998A CN 202210296693 A CN202210296693 A CN 202210296693A CN 114813998 A CN114813998 A CN 114813998A
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Abstract
The invention discloses a method for detecting the content of polymer Azide- (Amide-linker) -PEG-b-PLA, which is a gel exclusion chromatography method and uses a high performance liquid chromatograph for detection.
Description
Technical Field
The invention relates to the technical field of chromatography, in particular to a method for detecting purity of a polymer.
Background
Polylactic acid is a biological material with no toxicity, no irritation, good biological safety and degradability, and is widely applied to the fields of biomedical materials such as bone fixation and repair materials, assembled engineering scaffold materials, drug controlled release carriers and the like. However, polylactic acid material is highly hydrophobic, which reduces its biocompatibility with other substances.
People develop an Azide-Amide-linker) -PEG-b-PLA polymer with a structural formula of
The biological composite material not only keeps the original excellent properties of easy degradation, no toxicity and no irritation of the polylactic acid, but also introduces PEG group into a linker, greatly improves the hydrophilicity of the polylactic acid polymer, and further widens the application fields of the polymer, such as surface modification, adhesion, drug delivery and the like, due to the introduction of azide group.
However, in the prior art, there is no suitable detection method for the above-mentioned polymers.
If a conventional chromatographic column is adopted for reverse phase liquid phase detection, the phenomenon of poor main peak type can occur, the aim of effectively separating the main peak type from other impurities can not be achieved, and therefore the purity of the polymer can not be effectively determined.
Exclusion chromatography is also known as size exclusion chromatography or gel permeation chromatography. Is a chromatographic technique that performs separation based on the size of the sample molecules. The separation mechanism of exclusion chromatography is steric exclusion, a phenomenon in which there is no interaction between the sample components and the stationary phase. The packing material for chromatographic columns is a gel, which is a surface inert material containing many pores or three-dimensional networks of different sizes. The pore size of the gel corresponds to the size of the sample to be separated. Only component molecules having a diameter smaller than the opening of the pores, which are so large for mobile phase molecules, are allowed to enter that the mobile phase molecules can freely diffuse out of the person. For component molecules with different sizes, the component molecules can respectively permeate into different depths in the gel pores, and large component molecules can permeate into the large pores of the gel but can not enter the small pores or even be completely rejected. Small component molecules and big holes and small holes can be infiltrated in the porous membrane, even enter deeply, and are not easy to elute out in a short time. Thus, large component molecules have a short residence time in the column, are washed out quickly, and have a small elution volume (i.e., retention time). The retention time of small component molecules in the chromatographic column is longer, and the retention time of the elution volume is longer), until the minimum molecules in all pores reach the outlet of the column, the elution process of separating according to the molecular size is completed.
However, no report related to the detection of the polymers Azide (Amide-linker) -PEG-b-PLA by using gel permeation chromatography has been found yet in the prior art.
Disclosure of Invention
In order to overcome the defects of the prior art, the invention aims to provide a method for detecting the content of the polymer Azide- (Amide-linker) -PEG-b-PLA, which can effectively reflect the purity of the polymer and is beneficial to controlling the purity of the polymer, and the determination method has good separation degree and repeatability and high column efficiency.
The invention provides a method for detecting the content of a polymer, wherein the polymer is Azide to (Amide-linker) -PEG-b-PLA, and the structural formula is
Wherein the molecular weight of the PEG part is 4500-5500 daltons, preferably the molecular weight of the PEG part is 5000 daltons; the molecular weight of the PLA part is 10000-20000 daltons, and preferably, the molecular weight of the PLA part is 16000 daltons; n is the degree of polymerization of the polyethylene glycol residue, and m is the degree of polymerization of the polylactic acid residue.
The method is gel exclusion chromatography, and high performance liquid chromatograph is used for detection, and the chromatographic conditions are as follows:
the high performance liquid chromatograph adopts porous gel as a filler, the gel is one of resin-based gel, sephadex, polyacrylamide gel, agarose gel, polystyrene gel, sephadex LH-20 and silica gel, and preferably, the gel is high-performance resin-based gel.
Further, the mobile phase in the gel exclusion chromatography is one of acetonitrile water solution, methanol water solution and tetrahydrofuran water solution, and preferably the acetonitrile water solution.
Further, the volume concentration of the mobile phase is 5-30% (v/v, such as 5%, 10%, 11%, 12%, 13%, 14%, 15%, 15.5%, 16%, 16.5%, 17%, 17.1%, 17.2%, 17.3%, 17.4%, 17.5%, 17.6%, 17.7%, 17.8%, 17.9%, 18%, 18.5%, 19%, 19.5%, 20%, 25%, 30%), preferably, the volume concentration of the mobile phase is 10-20%.
In one embodiment of the invention, the mobile phase has a volume concentration of 15%.
Further, the detection adopts an isocratic elution program.
Further, the elution time is 20-60 min (e.g., 20, 25, 30, 35, 40, 45, 50, 55, 60min), preferably 30-50 min.
In one embodiment of the present invention, the elution time is 40 min.
Furthermore, the exclusion molecular weight of a gel chromatographic column in the gel exclusion chromatography is 100-50000 daltons.
Further, the length of the gel chromatographic column is 30-350 mm (such as 33, 150 and 300mm), and preferably the length of the gel chromatographic column is 300 mm.
Further, the diameter of the gel chromatographic column is 4-8 mm (such as 4.6, 7.8mm), preferably, the diameter of the gel chromatographic column is 7.8 mm.
Further, the pore diameter of the filler is
(e.g. using
) Preferably, the pore size of the filler is
Further, the particle size of the filler is 3-7 μm (such as 3 μm, 4 μm, 5 μm, 6 μm, 7 μm), preferably, the particle size of the filler is 5 μm.
Further, the flow rate of the high performance liquid chromatograph is 0.1-1.0 mL/min (such as 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0mL/min), and preferably the flow rate of the high performance liquid chromatograph is 0.5-0.8 mL/min.
In one embodiment of the present invention, the flow rate of the high performance liquid chromatograph is 0.5 mL/min.
Further, the detector of the high performance liquid chromatograph may be an electrospray detector (CAD), and the parameters are:
acquisition frequency: 5 Hz;
filtration constant: 3.6 s;
temperature: 35 ℃ is carried out.
Further, the column temperature of the HPLC is 25 to 45 ℃ (such as 25, 28, 30, 32, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45 ℃), preferably 38 to 42 ℃.
In one embodiment of the invention, the column temperature of the high performance liquid chromatograph is 40 ℃;
the sample injection amount of the high performance liquid chromatograph is 10-30 μ L (such as 10, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30 μ L), preferably 15-25 μ L.
In one embodiment of the present invention, the sample volume of the high performance liquid chromatograph is 20 μ L.
The method for determining the contents of the polymers Azide to (Amide-linker) PEG-b to PLA is an external standard method, and specifically comprises the following steps:
(1) making a standard curve: taking polymers Azide-Amide-linker) -PEG-b-PLA with known contents as a reference, performing gradient dilution by using a solvent to obtain a linear reference solution, detecting by using gel exclusion chromatography, sucking the linear reference solution, injecting into a high performance liquid chromatograph, passing through a gel chromatographic column, detecting by using an electrospray detector (CAD), recording a chromatogram, and obtaining parameters such as retention time, peak height, peak area and the like to obtain a standard curve;
(2) the determination method comprises the following steps: putting a sample to be detected, namely, Azide-linker) -PEG-b-PLA, into a volumetric flask, adding a solvent to perform constant volume to obtain a sample solution, detecting by using gel exclusion chromatography, sucking the sample solution, injecting the sample solution into a high performance liquid chromatograph, passing through a gel column chromatographic column, detecting by using an electrospray detector (CAD), recording a chromatogram, obtaining parameters such as peak height, peak area and the like, substituting relevant parameters corresponding to the polymers, namely, Azide-linker) -PEG-b-PLA into a standard curve, and calculating to obtain the contents of the polymers, namely, Azide-linker) -PEG-b-PLA.
Further, the step of preparing a reference stock solution is also included before obtaining the linear reference solution in the step (1).
Further, the preparation of the reference stock solution comprises the steps of weighing polymers Azide to (Amide-linker) -PEG-b-PLA with known contents into a volumetric flask, adding a solvent into the volumetric flask for constant volume, and preparing the reference stock solution.
Further, the detection method of the polymer also comprises the steps of preparing a blank solution and preparing a quantitative limiting solution.
Further, the preparation of the blank solution comprises the step of selecting a solvent as the blank solution.
Further, the preparation of the quantitative limiting solution comprises the steps of weighing polymers Azide to (Amide to linker) PEG-b to PLA with known contents into a volumetric flask, adding a mobile phase into the volumetric flask for constant volume, and preparing the quantitative limiting solution.
Further, the solvent in steps (1) and (2) may be one of acetonitrile, methanol and tetrahydrofuran, preferably acetonitrile.
Further, the high performance liquid chromatograph may be a Thermo U3000 high performance liquid chromatograph.
Further, the chromatographic column of the high performance liquid chromatograph can be Acclaim SEC-300,7.8mm multiplied by 300 mm; 5 μm;
a size exclusion chromatography column.
On the other hand, the invention provides the application of the detection method in the quality evaluation of the polymers Azide to (Amide to linker) PEG5000 to b to PLA 16K.
In another aspect, the invention provides the use of the above detection method in the detection of high molecular polymers.
The detection method can be used for quickly and accurately separating products and impurities through the porous gel chromatographic column, meets the standards in the aspects of quantification, linear range and the like, can be used for accurately measuring the product purity in the linear range through an area normalization method, is convenient to operate, is good in separation, and has high durability.
Drawings
FIG. 1 shows nuclear magnetic spectra of polymers Azide to (Amide to linker) PEG5000 to b to PLA 16K.
FIG. 2 is a liquid chromatogram of detection using a chromatographic column of type SEC-1000.
FIG. 3 is a liquid chromatogram when the detection was performed at a flow rate of 0.4 mL/min.
FIG. 4 is a liquid chromatogram of a sample solution.
FIG. 5 is a liquid chromatogram of a blank solution.
FIG. 6 is a liquid chromatogram of a limiting quantitation solution.
FIG. 7 is a graph of the linear relationship of the linear control solutions.
FIG. 8 is a liquid chromatogram of the polymer Azide- (Amide-linker) -PEG5000-b-PLA16K when the reversed phase liquid phase detection is carried out by using octadecylsilane chemically bonded silica gel column.
Detailed Description
The technical solutions of the present invention will be described clearly and completely with reference to the following embodiments of the present invention, and it should be understood that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
The polymers Azide to (Amide to linker) -PEG5000 to b to PLA16K, the structure formula of which is Ke science and technology company of origin bond
Wherein n is the degree of polymerization of polyethylene glycol residues, m is the degree of polymerization of polylactic acid residues, and the molecular weight of the PEG part is 5000 daltons; the molecular weight of the PLA fraction is 16000 daltons.
The results of characterization of the polymers Azide to (Amide to linker) PEG5000 to b to PLA16K are as follows:
1. nuclear magnetic characterization:
the nuclear magnetic spectra of the polymers Azide to (Amide to linker) PEG5000 to b to PLA16K are shown in FIG. 1.
2. And (3) viscosity characterization:
the viscosity of the polymers Azide to (Amide to linker) PEG5000 to b to PLA16K is characterized by adopting a capillary method, and the viscosity is measured to be 0.32 dL/g.
Example 1
Screening of chromatographic column:
1. chromatographic conditions are as follows:
the instrument comprises the following steps: thermo U3000 high performance liquid chromatograph
A chromatographic column: size exclusion chromatography column with porous gel as stationary phase (Thermo, Acclaim SEC-1000,7.8mm × 300 mm; 5 μm; 1000A)
Mobile phase: the volume ratio of acetonitrile to water is 15: 85.
elution procedure: isocratic elution for 40 min.
Flow rate: 0.4 mL/min.
Column temperature: at 40 ℃.
Sample introduction amount: 20 μ L.
A workstation: chromeleon.
2. Preparing a solution:
sample solution: precisely weighing about 40mg of Azide- (Amide-linker) -PEG5000-b-PLA16K, placing in a 10mL volumetric flask, adding a proper amount of acetonitrile for dissolving, adding acetonitrile for constant volume to scale, and shaking up to obtain a sample solution of 4 mg/mL.
3. And (3) determination: precisely absorbing the prepared solution by adopting a high performance liquid chromatograph and using gel exclusion chromatography for detection, passing through a gel column chromatographic column, detecting by using an electrospray detector (CAD), recording a chromatogram, and obtaining parameters such as peak height, peak area and the like.
As shown in FIG. 2, the chromatographic column of this type has a wide peak shape.
Example 2
Screening of chromatographic condition flow rate:
1. chromatographic conditions are as follows:
the instrument comprises the following steps: thermo U3000 high performance liquid chromatograph
A chromatographic column: size exclusion chromatography column with porous gel as stationary phase (Thermo, Acclaim SEC-300,7.8 mm. times.300 mm; 5 μm; 300A)
Mobile phase: the volume ratio of acetonitrile to water is 15: 85.
elution procedure: isocratic elution for 40 min.
Flow rate: 0.4 mL/min.
Column temperature: at 40 ℃.
Sample introduction amount: 20 μ L.
A workstation: chromeleon.
2. Preparing a solution:
sample solution: precisely weighing about 40mg of Azide- (Amide-linker) -PEG5000-b-PLA16K, placing in a 10mL volumetric flask, adding a proper amount of acetonitrile for dissolving, adding acetonitrile for constant volume to scale, and shaking up to obtain a sample solution of 4 mg/mL.
3. Measurement of
Precisely absorbing the prepared solution by adopting a high performance liquid chromatograph and using gel exclusion chromatography for detection, passing through a gel column chromatographic column, detecting by using an electrospray detector (CAD), recording a chromatogram, and obtaining parameters such as peak height, peak area and the like. As shown in fig. 3, the flow rate separation is poor.
Example 3:
1. chromatographic conditions are as follows:
the instrument comprises the following steps: thermo U3000 high performance liquid chromatograph
A chromatographic column: size exclusion chromatography column with porous gel as stationary phase (Thermo, Acclaim SEC-300,7.8 mm. times.300 mm; 5 μm; 300A)
Mobile phase: the volume ratio of acetonitrile to water is 15: 85.
Elution procedure: isocratic elution for 40 min.
Flow rate: 0.5 mL/min.
Column temperature: at 40 ℃.
Sample injection amount: 20 μ L.
A workstation: chromeleon.
Blank solution: and (3) acetonitrile.
2. Preparation of the solution
Control stock solutions: precisely weighing about 200mg of Azide- (Amide-linker) -PEG5000-b-PLA16K, placing in a 10mL volumetric flask, adding a proper amount of acetonitrile for dissolving, adding acetonitrile for constant volume until the volume is scaled, and shaking up.
Sample solution: precisely weighing about 40mg of Azide- (Amide-linker) -PEG5000-b-PLA16K, placing in a 10mL volumetric flask, adding a proper amount of acetonitrile for dissolving, adding acetonitrile for constant volume to scale, and shaking up to obtain a sample solution of 4 mg/mL.
Limit of quantitation (LOQ) solution: precisely weighing about 10mg of Azide- (Amide-linker) -PEG5000-b-PLA16K, placing in a 10mL volumetric flask, adding a proper amount of acetonitrile to dissolve, then adding acetonitrile to a constant volume to scale, shaking up to obtain a 1mg/mL solution, accurately sucking 0.4mL of the solution into the 10mL volumetric flask, and diluting with acetonitrile to scale.
Linear control solution: control stock solutions (20mg/mL) were accurately pipetted into 0.02mL, 0.04mL, 0.05mL, 0.1mL, 0.25mL, 1.0mL, 1.5mL, and 2.0 mL-10 mL volumetric flasks, and acetonitrile was added to the flask to obtain solutions of 0.04mg/mL, 0.08mg/mL, 0.1mg/mL, 0.2mg/mL, 0.5mg/mL, 2mg/mL, 3mg/mL, and 4 mg/mL.
3. Measurement of
Precisely absorbing the prepared solutions by adopting a high performance liquid chromatograph and using gel exclusion chromatography for detection, passing through a gel column chromatographic column, detecting by using an electrospray detector (CAD), recording a chromatogram, and obtaining parameters such as peak height, peak area and the like.
FIGS. 4 to 5 are liquid chromatogram diagrams of the sample solution, the blank solution, and the quantitative limit solution, respectively.
Table 1 shows the linearity results of the polymer Azide- (Amide-linker) -PEG5000-b-PLA16K linearity control solution.
TABLE 1
Plotting the results of Table 1 yields a standard curve as shown in FIG. 7, which has regression coefficients R corresponding to the linear equations 2 Is 0.9970, which shows that the fitting degree of the linear equation is better.
Comparative example 1:
and (3) carrying out reverse phase liquid phase detection on the polymer Azide- (Amide-linker) -PEG5000-b-PLA16K by using an octadecylsilane chemically bonded silica gel column.
1. Chromatographic conditions are as follows:
the instrument comprises the following steps: high performance liquid chromatograph for SHIMADZU
A chromatographic column: chromatographic column using octadecylsilane bonded silica as stationary phase (Thermo, Hypersil GOLD C18,4.6 mm. times.250 mm; 5 μm)
Mobile phase: mobile phase a was water and mobile phase B was acetonitrile.
Elution procedure: gradient elution, 20 min. 0min, wherein the volume ratio of the mobile phase A to the mobile phase B is 90: 10; performing linear elution with the volume ratio of mobile phase A to mobile phase B gradually changed to 30:70 for 0-2 min; carrying out linear elution for 2-10min, wherein the volume ratio of the mobile phase A to the mobile phase B is linearly gradually changed to 5: 95; performing isocratic elution for 10-16min with the volume ratio of mobile phase A to mobile phase B being 5: 95; performing linear elution after the volume ratio of the mobile phase A to the mobile phase B is linearly gradually changed to 90:10 for 16-17 min; performing isocratic elution for 17-20min, wherein the volume ratio of the mobile phase A to the mobile phase B is 90: 10;
flow rate: 1 mL/min.
Column temperature: at 40 ℃.
Sample introduction amount: 10 μ L.
A workstation: and (4) Labsolution.
2. Preparing a solution:
sample solution: precisely weighing about 20mg of Azide- (Amide-linker) -PEG5000-b-PLA16K, placing in a 10mL volumetric flask, adding a proper amount of acetonitrile for dissolving, adding acetonitrile for constant volume to scale, and shaking up to obtain a sample solution of 2 mg/mL.
3. Measurement of
And (3) precisely absorbing the prepared solution for detection by adopting a high performance liquid chromatograph and using an octadecylsilane chemically bonded silica gel column, detecting by using an electrospray detector (CAD), recording a chromatogram, and acquiring parameters such as peak height, peak area and the like. As shown in FIG. 8, when the polymer Azide- (Amide-linker) -PEG5000-b-PLA16K was subjected to reversed-phase liquid phase detection by using an octadecylsilane chemically bonded silica gel column, the chromatogram obtained was poor in peak shape and poor in effect.
Claims (10)
1. The method for detecting the content of the polymer is characterized in that the polymer is Azide to (Amide-linker) -PEG-b-PLA, and the structural formula of the polymer is shown in the specification
Wherein n is the polymerization degree of polyethylene glycol residues, m is the polymerization degree of polylactic acid residues, and the molecular weight of the PEG part is 4500-5500 daltons; the molecular weight of the PLA part is 10000-20000 daltons, the method is gel exclusion chromatography, a high performance liquid chromatograph is used for detection, and the chromatographic conditions are as follows:
the high performance liquid chromatograph adopts porous gel as a filler, the gel is one of resin-based gel, sephadex, polyacrylamide gel, agarose gel, polystyrene gel, sephadex LH-20 and silica gel, and preferably the gel is resin-based gel.
2. The method for detecting the content of the polymer according to claim 1, wherein the mobile phase in the gel exclusion chromatography is one of acetonitrile, methanol and tetrahydrofuran, and preferably the mobile phase is acetonitrile in water.
3. The method for detecting the content of the polymer according to claim 2, wherein the volume concentration of the mobile phase is 5 to 30%, preferably 10 to 20%, and more preferably 15%.
4. The method for detecting the content of the polymer according to claim 1, wherein the detection is performed by an isocratic elution procedure; the elution time is 20-60 min, preferably 30-50 min, and more preferably 40 min.
5. The method for detecting the content of the polymer in claim 1, wherein the gel exclusion chromatography is characterized in that the exclusion molecular weight of a gel chromatographic column is 100-50000 daltons.
6. The method of claim 1, wherein the filler has a pore size of
Preferably, the pore size of the filler is
7. The method for detecting the content of the polymer according to claim 1, wherein the particle size of the filler is 3 to 7 μm, preferably 5 μm.
8. The method for detecting the content of the polymer according to claim 1, wherein the flow rate of the high performance liquid chromatograph is 0.1 to 1.0mL/min, preferably the flow rate of the high performance liquid chromatograph is 0.5 to 0.8mL/min, and more preferably the flow rate of the high performance liquid chromatograph is 0.5 mL/min.
9. The method for detecting the content of the polymer according to claim 1, wherein the column temperature of the high performance liquid chromatograph is 25 to 45 ℃, preferably 38 to 42 ℃, and more preferably 40 ℃.
10. The method for detecting the content of the polymer in claim 1, wherein the molecular weight of the PEG moiety is 5000 daltons, the molecular weight of the PLA moiety is 16000 daltons, and the method for determining the content of the polymers from Azide (Amide-linker) -PEG-b-PLA is an external standard method, which specifically comprises the following steps:
(1) making a standard curve: taking polymers Azide-Amide-linker) -PEG-b-PLA with known contents as a reference, performing gradient dilution by using a solvent to obtain a linear reference solution, detecting by using gel exclusion chromatography, sucking the linear reference solution, injecting into a high performance liquid chromatograph, passing through a gel chromatographic column, detecting under a detector, recording a chromatogram, and obtaining retention time, peak height and peak area to obtain a standard curve;
(2) the determination method comprises the following steps: putting a sample to be detected, namely Azide (Amide-linker) to PEG-b-PLA, into a volumetric flask, adding a solvent to perform constant volume to obtain a sample solution, detecting by using gel exclusion chromatography, sucking the sample solution, injecting the sample solution into a high performance liquid chromatograph, passing through a gel column chromatographic column, detecting under a detector, recording a chromatogram, obtaining the peak height and the peak area, substituting the relevant parameters corresponding to the polymers, namely Azide (Amide-linker) to PEG-b-PLA, into a standard curve, and calculating to obtain the contents of the polymers, namely Azide (Amide-linker) to PEG-b-PLA.
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