CN109459504B - Method for measuring weight average molecular weight and content of polyguluronic acid sulfate - Google Patents
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Abstract
The invention belongs to the field of natural medicinal chemistry and quality control, and relates to a method for determining the weight average molecular weight and the content of polyguluronic acid sulfate. The method of the invention can be used for rapidly and accurately measuring the weight average molecular weight and the content of polyguluronic acid sulfate.
Description
Technical Field
The invention belongs to the field of natural medicinal chemistry and quality control thereof, and relates to a method for measuring the weight average molecular weight and content of polyguluronic acid sulfate.
Background
Sodium alginate is classified by acidolysis to obtain polyguluronic acid, and the polyguluronic acid is sulfated to obtain polyguluronic acid sulfate which is a linear compound formed by connecting 2-OH/3-OH sulfated L-Guluronic acid (L-Guluronic acid, G) through alpha-1, 4-glycosidic bonds. At present, the polyguluronic acid sulfate is reported in the literature at home and abroad to be used for preventing and treating the urinary calculus and also has the biological activity of resisting tumors.
Polyguluronic acid sulfate salt structure
The weight average molecular weight and the content of the polyguluronic acid sulfate are important measurement parameters for developing polyguluronic acid sulfate drugs. At present, in chinese pharmacopoeia, high performance liquid Gel Permeation Chromatography (GPC) is a common method for determining molecular weight, which is based on the principle of exclusion Chromatography, polysaccharides with different molecular weights flow out of a chromatographic column in sequence according to the size sequence, and a calibration curve is measured by the correlation between standard polysaccharides with different molecular weights and retention time, and the relative molecular weight of the corresponding polysaccharide to be determined is calculated. However, the conventional method has certain limitations because of the necessity of a corresponding polysaccharide standard.
In addition, a common method for measuring the content of polysaccharide and oligosaccharide is a carbazole sulfate method, and the method firstly uses the concentration and the color development of a monosaccharide standard substance as a standard curve, and then reads the content of a sample to be measured on the standard curve. However, monosaccharide of the acidic sugar is not sold as a standard substance, and if glucuronic acid is used as the standard substance, the structure and color development efficiency of the monosaccharide are different from those of the acidic sugar, so that the monosaccharide cannot be accurately quantified.
The recent advent of Size Exclusion Chromatography coupled with Multi-Angle Laser Light Scattering (SEC-MALS) measurements of the absolute weight average molecular weight, molecular weight distribution and content of samples has attracted extensive attention in the industry. The working principle of the 18-angle laser light scattering instrument (MALS) is as follows: the laser beam generates light scattering through the solution, the scattered light intensity is in direct proportion to the molecular weight of the sample and the concentration of the solution, and the light intensity and the molecular size of different scattering angles are related, so that a plurality of detection probes (the measurement accuracy of the molecular weight is in direct proportion to the square root of the number of angles) are arranged at different positions around the sample cell and simultaneously receive scattered light intensity signals of different angles. According to the light scattering equation K c/R (theta) 1/MWP(θ)+2A2c, drawing Zimm graph to obtain the weight average absolute molecular weight Mw, mean square rotation radius rg and second dimensional coefficient A of macromolecule2. The combination of MALS and Size Exclusion Chromatography (SEC) allows for the direct determination of molecular weights and molecular weight distributions without relying on pump flow rates, calibration curves, and other false assumptions. MALS utilizes the light scattering quantity of samples separated by a chromatographic column at different angles, the mass is calculated by the eluent concentration and dn/dc value obtained by a differential Refraction (RI) detector, and the molecular weight of each section can be calculated by combining the two. The method does not need standard calibration, and overcomes the phase caused by different chemical compositions, molecular structures and sizes of the sample and the standardError in molecular weight determination. The method has been adopted by researchers to measure chitosan, average molecular weight of Hyaluronic acid (simple Fe, Neil Errington, Kornelia J, et al. correlation of SEC/MALLS with ultra front μ ge and virometry data for chips. Eur Biophys J (2003)32: 457-464; Sanya Hokputsa, Kornelia J, Catherine Alexander, et al. correlation of molecular mass determination of Hyaluronic acid viscosity SEC/MAS and viscosity determination of polysaccharide. Eur Biophys J (2003)32: 450-456), and a conventional technique (Kit-left chemistry, diameter Wu, J. Zo. correlation of polysaccharide viscosity determination) for quantifying natural neutral polysaccharide and predicting that this method will become polysaccharide quantification of polysaccharide (cement-viscosity analysis, viscosity Wo. J. roughness of viscosity analysis) 106: 1400. about. weight conversion of polysaccharide). This method has been adopted in the current European pharmacopoeia as a method for measuring the weight average molecular weight and molecular weight distribution of hydroxyethyl starch (European pharmacopoeia 8.0, p3307-3308, staghes hydroxyethenyl).
In conclusion, SEC-MALS can successfully determine the weight average molecular weight and the content of neutral polysaccharide.
Disclosure of Invention
The invention aims to provide a method for accurately, efficiently, simply and stably detecting the weight average molecular weight and the content of polyguluronic acid sulfate.
One aspect of the present invention provides a method for determining the weight average molecular weight and content of polyguluronic acid sulfate, wherein the method comprises the steps of:
(1) determining the weight percentage content A of metal ions in the polyguluronic acid sulfate sample;
(2) determination of the weight-average molecular weight Mw1 and of the content X using SEC-MALS;
(3) the weight average molecular weight and the content of polyguluronic acid sulfate were calculated using the following formula:
in the above equation (1): mw1 is the weight average molecular weight (Da) of polyguluronic acid sulfate salt directly obtained by SEC-MALS method; a is the content of metal ions in polyguluronic acid sulfate;
in the above equation (2): x is the mass calculated by the map processing software; y is the quality of sample introduction; a is the content of metal ions in polyguluronic acid sulfate.
The method provided by the invention can be used for accurately measuring the weight average molecular weight and the content of the polyguluronic acid sulfate, and successfully solves the problems of large deviation and inaccurate content quantification of the weight average molecular weight of the polyguluronic acid sulfate.
Drawings
FIG. 1 shows the SEC-ICP-MS spectra of the solvent water (A) and polyguluronic acid sulfate sodium salt (B), and the SEC-MALS difference spectrum of polyguluronic acid sulfate sodium salt (C)
Detailed Description
Definition of
All percentages, ratios or parts are by weight unless otherwise specified.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In addition, any methods and materials similar or equivalent to those described herein can be used in the methods of the present invention. The preferred embodiments and materials described herein are intended to be exemplary only.
As used herein, the term "dn/dc" refers to the refractive index increase, which can be determined using methods commonly used in the art (liuli et al. methods for determining the absolute molecular weight of medical sodium hyaluronate gels and their distribution [ J ] journal of drug analysis 2013, 33(8), 1435-; and can also be obtained by inquiring from related technical documents disclosed in the field.
As used herein, the term "SEC-MALS" refers to Size Exclusion Chromatography coupled with Multi-Angle Laser Scattering (Size-Exclusion Chromatography/Multi-Angle Laser Light Scattering), which has been widely used in recent years for determining the absolute weight average molecular weight, molecular weight distribution and content of a sample, and can be determined by methods known in the art (simple Fee, New Errington, Kornelia Jumel, et al. correlation of SEC/MALLS with atomic concentration and viscosity data for chips. Eur Biophys J (2003)32: 457-464; Sanya Hokputsa, Kornelia Jemel, Catheralex, et al. correlation of molecular concentration and viscosity, and molecular concentration of molecular concentration and viscosity of chemical absorption/molecular dispersion, and molecular concentration of molecular concentration and viscosity of molecular dispersion, respectively, and molecular concentration of molecular concentration and viscosity of molecular dispersion, respectively, molecular dispersion and viscosity of molecular dispersion, such as mentioned above, the term "SEC-MALL. refers to Size Exclusion Chromatography coupled with Multi-Angle Laser Scattering (particle Scattering and viscosity) 32. A. simulation, and viscosity of molecular dispersion, such as described in the description of molecular dispersion, molecular dispersion of molecular dispersion, such as described in the detection of molecular dispersion, molecular dispersion of molecular dispersion, molecular dispersion of molecular dispersion, molecular dispersion of molecular dispersion, molecular dispersion of molecular dispersion, molecular dispersion of molecular dispersion, molecular dispersion of molecular dispersion, molecular dispersion of A reliable index detector, journal of Chromatography A (2015)1400: 98-106).
As used herein, the term "ICP-MS" refers to Inductively coupled plasma mass spectrometry (ICP-MS), which is a major development in the present-generation elemental composition measurement technology, and has many advantages such as high sensitivity, less interference, ultra trace detection limit, and multi-element simultaneous analysis, and the specific measurement method can adopt methods known in the art, such as those described in Von advanced, Qutaigen, the latest application progress of ICP-MS, and Chinese inorganic chemical analysis (2011)1: 46-52.
As used herein, the term "ICP-OES" refers to Inductively Coupled Plasma Optical Emission Spectrometry (Inductively Coupled Plasma Optical Emission Spectrometry) which is a common method used in the art for measuring the content of metal ions, and a specific method may be a method well known in the art.
Discovery of technical problems
The inventor of the application finds that when the existing SEC-MALS is used for measuring the polyguluronic acid sulfate sample, an unknown peak exists after the sample peak, the content of polyguluronic acid sulfate calculated is unreasonable, and the weight average molecular weight obtained by MALS on the basis of the content also has deviation. In view of the above phenomenon, it is generally considered that the adsorption of the sample on the column causes the content to decrease, but the inventors found that this case is not solved even after the separation is performed a plurality of times using the column. In order to explore this problem, the inventors analyzed the unknown peak in SEC-MALS measurement of polyguluronic acid sulfate, and found that the unknown peak is important for obtaining the correct weight average molecular weight and content of polyguluronic acid sulfate.
The inventor of the present invention found that the peak is not known to be attributed to the metal ion in the polyguluronic acid sulfate structure, and therefore, the weight average molecular weight and content of polyguluronic acid sulfate should be corrected by considering the influence of the metal ion content, i.e., the weight average molecular weight and content measured by SEC-MALS are divided by the mass fraction (1-metal ion content) of the sugar chain portion in polyguluronic acid sulfate to obtain the weight average molecular weight and content of polyguluronic acid sulfate, i.e., the following calculation formula 1 and formula 2 are used for correction:
in the above equation (1): mw1 is the weight average molecular weight (Da) directly obtained by SEC-MALS method; a is the content of metal ions in polyguluronic acid sulfate;
in the above equation (2): x is the mass (mug) calculated by the map processing software; y is the mass (mug) of the sample; a is the content of metal ions in polyguluronic acid sulfate.
The method provided by the invention can be used for accurately measuring the weight average molecular weight and the content of the polyguluronic acid sulfate, and successfully solves the problems of large deviation and inaccurate content quantification of the weight average molecular weight of the polyguluronic acid sulfate.
In some embodiments of the invention, the polyguluronic acid sulfate salt is an alkali metal salt. In other embodiments of the present invention, the polyguluronic acid sulfate salt is a sodium, potassium, calcium or magnesium salt. In other embodiments of the present invention, the polyguluronic acid sulfate salt is the sodium salt.
Determination of the Metal ion content
In an embodiment of the invention, the polyguluronic acid sulfate has a content of metal ions determined by a method selected from the group consisting of: ICP-MS, ICP-OES, atomic absorption or ion chromatography. ICP-MS and ion chromatography are preferable, and ion chromatography is more preferable. These assays are described above. They are commonly used with SEC.
Inductively coupled plasma mass spectrometry (ICP-MS) is a great development in the present element composition determination technology, and has the advantages of high sensitivity, less interference, ultra trace detection limit, multi-element simultaneous analysis and the like (Von is advanced, Tatarian, Inductively coupled plasma mass spectrometry (ICP-MS) is the latest application progress, and Chinese inorganic chemical analysis (2011)1: 46-52). The inventor successfully confirms that an unknown peak after the peak of the polyguluronic acid sulfate sample is measured by SEC-MALS is a metal ion by combining SEC and ICP-MS for the first time, and can obtain related data of the type and the content of the metal ion.
On the basis, the weight average molecular weight and the content determined by the SEC-MALS method are corrected by determining the species and the content of ions: the weight average molecular weight and the content of the polyguluronic acid sulfate are obtained by dividing the measured weight average molecular weight and content by the mass fraction of the sugar chain moiety in the polyguluronic acid sulfate.
SEC-MALS method for determining molecular weight and content
In the present invention, the SEC-MALS method is used to determine the molecular weight and content of acidic sugars. The inventor finds that the polyguluronic acid sulfate sample is dissociated when entering the SEC column for analysis, and therefore, the SEC-MALS method obtains the weight average molecular weight and content of the acidic sugar which does not contain metal ions after dissociation, so the weight average molecular weight and content of the polyguluronic acid sulfate should also take the influence of the content of the metal ions into consideration, and the current SEC-MALS method has not been reported in the aspect.
In the SEC-MALS method employed in the present invention, a solution of alcohol + salt may be used as the mobile phase; the flow rate may be 0.05-0.2 mL/min; wherein the content of alcohol in the mobile phase is 0-20%. The alcohol may be methanol, ethanol, propanol, cyclohexanol or mixture thereof, and the salt is selected from NH4Ac. NaCl or Na2SO4The concentration of the salt solution is 0.05-0.15 mol/L.
In another preferred embodiment of the invention, the SEC-MALS assay uses 20% methanol + 80% 80mmol/L NH4Ac as a mobile phase; the flow rate was 0.1 mL/min.
In another preferred embodiment of the invention, the SEC-MALS assay employs a chromatographic column capable of good separation of 2k-30kDa polysaccharide; preferred include, but are not limited to, the following chromatography columns: waters ACQUITY
SEC 1.7um 4.6X 150mm column, TSK G4000PWXL 7.8X 300mm (TOSOH) Shodex Ohpak SB-803HQ 7.8X 300mm (Shodex) Advance Bio SEC
2.7 μm 4.6X 150mm column (Agilent).
In some embodiments of the invention, the method of determining further comprises the step of determining the increase in refractive index (dn/dc) of the polyguluronic acid sulfate.
In some embodiments of the invention, the measurement of the increase in refractive index (dn/dc) of the polyguluronic acid sulfate comprises the following steps:
(1) dissolving polyguluronic acid sulfate sample with mobile phase determined by SEC-MALS, and diluting the sample to obtain 5-6 concentration points of serial solutions within the concentration range of 0.1-2 mg/ml;
(2) sequentially injecting the mobile phase and the sample solution prepared in the step 2 by using a differential refraction detector (Wyatt, Optilab T-rEX (WTREX-08), and collecting signals;
(3) dn/dc was calculated by Astra software.
In one embodiment of the present invention, the weight average molecular weight and content of polyguluronic acid sulfate are determined as follows:
step 1: the weight percentage content A of metal ions in the polyguluronic acid sulfate was determined by ICP-MS, ICP-OES, atomic absorption or ion chromatography.
Step 2: determination of dn/dc of Polyguluronic acid sulfate
(1) The sample is weighed to constant weight and dissolved by adding SEC mobile phase (such as the mobile phase used in step 3 below) to prepare a series of solutions of 0.1-5mg/ml, preferably 0.2-2mg/ml, which may be at concentrations of, for example, 0.2mg/ml, 0.4mg/ml, 0.8mg/ml, 1.2mg/ml, 1.8mg/ml, 2 mg/ml.
(2) The mobile phase and the series of solutions prepared in the previous step are each sequentially injected with a differential refractive detector (e.g., Wyatt, Optilab T-rEX (WTREX-08)), and signals are collected,
(3) the dn/dc is calculated by Astra software to obtain the dn/dc.
And step 3: SEC-MALS determination of Polyguluronic acid sulfate molecular weight and content
(1) Selecting a proper chromatographic column according to the estimated molecular weight range of the polyguluronic acid sulfate to be detected;
(2) preparing a gradient solution of polyguluronic acid sulfate, wherein the range of the gradient solution is 2-500mg/ml, and the gradient solution is divided into 3-10 gradients; for example, about 2, 10, 50, 100, 300mg/mL (concentration-weight/volume) of the solution.
(3) Using a solution of alcohol + salt as a mobile phase; the flow rate may be 0.05-0.2 mL/min; wherein the content of alcohol in the mobile phase is 0-20%. The alcohol may be methanol, ethanol, propanol, cyclohexanol or mixture thereof, and the salt is selected from NH4Ac. NaCl or Na2SO4Concentration of salt solutionThe degree is 0.05-0.15mol/L, and the detector uses an eighteen-angle laser detector and a refractive index detector;
(4) the weight average molecular weight Mw1 of the polyguluronic acid sulfate directly obtained by SEC-MALS method;
(5) calculating the mass X of the polyguluronic acid sulfate in the obtained sample by using map processing software;
(6) the weight average molecular weight and the content of polyguluronic acid sulfate were calculated using the following formula:
in the above equation (1): mw1 is the weight average molecular weight (Da) of the mannooligosaccharide diacid soluble salt directly obtained by the SEC-MALS method; a is the content of metal ions in polyguluronic acid sulfate;
in the above equation (2): x is the mass calculated by the map processing software; y is the quality of sample introduction; a is the content of metal ions in polyguluronic acid sulfate.
The specific operating conditions for determining the weight average molecular weight and the content by the SEC-MALS method can be carried out according to conventional experimental protocols.
Compared with the prior art, the invention has the following advantages:
(1) the unknown peaks in the analysis of the polyguluronic acid sulfate sample SEC-MALS were explained.
(2) Can accurately determine the weight average molecular weight and the content of polyguluronic acid sulfate, and has good repeatability.
(3) The weight average molecular weight and the content can be obtained in one-time analysis, and the operation is simple.
(4) The accuracy of measuring polyguluronic acid sulfate is improved, and the molecular structure of polysaccharide can be researched more accurately.
Examples
The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. The experimental procedures, in which specific conditions are not noted in the following examples, are generally carried out according to conventional conditions or according to conditions recommended by the manufacturers.
The apparatus used in the examples section included:
LC: agilent 1260 liquid chromatograph
18-angle laser detector: wyatt DAWN HELEOS II
Refractive index detector: wyatt Optilab T-rEX
ICP-MS：Agilent 7900ICP-MS
Ion chromatography: swiss Wantong
ICP-OES: optima7000DV inductively coupled plasma emission spectrometer (Perkin-Elmer, USA)
Flame atomic absorption: beijing Rayleigh WFX-120B type flame atomic absorption spectrometer
Example 1 SEC and ICP-MS reveal the separation mechanism of acidic sugars by size exclusion chromatography and determine the weight average molecular weight and content of Polyguluronic acid sulfate sodium salt
Step 1: SEC-ICP-MS (SEC-inductively coupled plasma-mass spectrometry) for determining peak position and content of metal ions
(1) Using a chromatographic column Waters ACQUITY
SEC 1.7um 4.6X 150mm column, 0.08mol/L NH4Ac as mobile phase, flow rate 0.1 mL/min.
(2) SEC in combination with ICP-MS: dissolving the sample in water at a concentration of 0.02mg/ml, injecting 20ul, and collecting23Na,43Ca,44Signal of Ca. Na ion standard solutions with concentrations of 0.1ppm,0.5ppm,1.0ppm,5.0ppm and 10.0ppm were prepared, and the content of metal ions in the sample (A value) was calculated by the external standard method. The A values results are shown in Table 1.
Step 2: determination of weight average molecular weight and content by SEC-MALS
(1) Using a chromatographic column Waters ACQUITY
SEC 1.7um 4.6X 150mm column 0.1mol/L NH4Ac as mobile phase, flow rate 0.1 mL/min.
(2) SEC and MALS are combined, the concentration of the sample solution in the mobile phase is 10mg/ml, the sample amount is 20ul, and the Y value in the formula is 200 ug. The results are shown in FIG. 1 and Table 1.
FIG. 1 is a SEC-ICP-MS spectrum of water (FIG. 1A) as solvent and polyguluronic acid sulfate sodium salt (FIG. 1B), and a SEC-MALS difference spectrum of polyguluronic acid sulfate sodium salt (FIG. 1C). The results show that in the SEC-ICP-MS spectrogram, compared with a negative control, the polyguluronic acid sulfate sodium salt has a distinct Na salt peak at a, and as can be seen by comparing FIG. 1B with FIG. 1C, the Na salt peak of the polyguluronic acid sulfate sodium salt at SEC-ICP-MS spectrogram a is consistent with the peak position at a differential spectrogram B, which indicates that the unknown peak at the differential spectrogram B is a sodium salt peak, which means that the polyguluronic acid sulfate sodium salt is dissociated when entering SEC column analysis, the SEC-MALS method obtains the weight average molecular weight and content of the polyguluronic acid sulfate which does not contain metal ions after dissociation, and the SEC-MALS method indicates that in order to accurately calculate the weight average molecular weight and content of the polyguluronic acid sulfate sodium salt, the metal ions in the polyguluronic acid sulfate sodium salt must be considered for correction, and the specific calculation is shown in formula 1, Equation 2.
In the above equation (1): mw1The weight average molecular weight (Da) directly obtained by an SEC-MALS method; a is the content of metal ions.
In the above equation (2): x is the mass (mug) calculated by the map processing software; y is the mass (mug) of the sample; a is the content of metal ions.
Table 1: the content of metal ions in polyguluronic acid sulfate sodium salt, and the weight average molecular weight and content before and after correction
The Y value is 200 ug; correcting by adopting a formula; a is Na%
As can be seen from Table 1, the weight average molecular weight and content of polyguluronic acid sulfate sodium salt can be accurately calculated by SEC-MALS after correction.
EXAMPLE 2 SEC-MALS determination of the weight-average molecular weight and content of Polyguluronic acid sulfate Potassium salt
Step 1: ion chromatography determination of the proportion of metal ions
The K ion standard solution is configured to be 25, 10, 12.5, 6.25 and 3.125 ppm. The sample concentration was 2 mg/ml.
Ion chromatography conditions: pre-column: metrosep C4 Guard/4.0; separating the column: metrosep
C5-150/4.0; mobile phase: 5mM nitric acid in ultrapure water; flow rate: 0.9 ml/min; the sample size is 20 ul. And measuring the content of the metal ions by an external standard method.
Step 2: measuring dn/dc
(1) The samples were weighed to constant weight and a series of solutions at concentrations of 0.2mg/ml, 0.4mg/ml, 0.8mg/ml, 1.2mg/ml, 1.8mg/ml, 2mg/ml were prepared using mobile phase.
(2) The mobile phase, 0.2mg/ml, 0.4mg/ml, 0.8mg/ml, 1.2mg/ml, 1.8mg/ml, 2mg/ml sample solutions were each manually injected in this order with a differential refractometer (Wyatt, Optilab T-rEX (WTREX-08), and signals were collected,
(3) dn/dc was calculated by Astra software, see Table 2.
And step 3: determination of weight average molecular weight and content by SEC-MALS
(1) Advance Bio SEC Using chromatography column
2.7 μm 4.6X 150mm column (Agilent)0.3mol/L Na2SO4As a mobile phase, the flow rate was 0.2 mL/min.
(2) SEC and MALS are combined, the concentration of the sample solution in the mobile phase is 10mg/ml, the sample amount is 20ul, and the Y value in the formula is 200 ug. The results of the experiment are shown in table 2.
TABLE 2 Metal ion content of Polyguluronic acid sulfate Potassium salt and weight average molecular weight and content before and after correction
The Y value is 200 ug; corrected using the formula in example 1; a is K%
As can be seen from Table 2, SEC-MALS can accurately calculate the weight average molecular weight and content of polyguluronic acid sulfate potassium salt through correction.
EXAMPLE 3 SEC-MALS determination of the weight-average molecular weight and the content of Polyguluronic acid sulfate calcium salt
Step 1: and (3) determining the content of metal ions by a flame atomic absorption method:
collecting the spectral line of Ca, and preparing Ca standard solutions of 1mg/L, 10mg/L, 50mg/L, 75mg/L and 100 mg/L; the concentration of the sample solution was set to 200 mg/L.
Step 2: measuring dn/dc
Same as example 2, step 2. The measured values are shown in Table 3.
And step 3: determination of weight average molecular weight and content by SEC-MALS
(1) A chromatographic column Shodex Ohpak SB-803HQ 7.8X 300mm (Shodex) was used, with 0.15mol/L NaCl as mobile phase and a flow rate of 0.5 mL/min.
(2) SEC and MALS are combined, the concentration of the sample solution in the mobile phase is 10mg/ml, the sample amount is 20ul, and the Y value in the formula is 200 ug. The results of the experiment are shown in table 3.
TABLE 3 content of metal ion of Polyguluronic acid sulfate calcium salt and weight average molecular weight and content before and after calibration
The Y value is 200 ug; corrected using the formula in example 1; a is Ca%
As can be seen from Table 3, SEC-MALS can accurately calculate the weight average molecular weight and content of polyguluronic acid sulfate calcium salt by calibration.
EXAMPLE 4 SEC-MALS determination of the weight-average molecular weight and the content of the Polyguluronic acid sulfate magnesium salt
Step 1: ICP-OES determination of metal ion content:
collecting an analysis spectral line of Mg, and preparing 1Mg/L, 5Mg/L and 10Mg/L Na standard solutions;
the sample solution was prepared at a concentration of 20mg/L, and the A value thus obtained is shown in Table 4. Thereby step 2: measuring dn/dc
Same as example 2, step 2. The measured values are shown in Table 4.
And step 3: determination of weight average molecular weight and content by SEC-MALS
(1) The chromatographic column TSK G4000PWXL 7.8X 300mm (TOSOH), 0.5mol/L NaNO was used3As a mobile phase, the flow rate was 0.5 mL/min.
(2) SEC was used in combination with MALS, and the sample solution was in the mobile phase at a concentration of 10mg/ml and a sample size of 20ul, i.e. Y value of 200ug in the formula. The results are shown in Table 4.
TABLE 4 Metal ion content of Polyguluronic acid sulfate magnesium salt, weight average molecular weight before and after calibration, and content
The Y value is 200 ug; corrected using the formula in example 1; a is Mg%
As can be seen from Table 4, SEC-MALS can accurately calculate the weight average molecular weight and content of the polyguluronic acid sulfate magnesium salt by calibration.
Claims (5)
1. A method for determining the weight average molecular weight and content of polyguluronic acid sulfate, said method comprising the steps of:
(1) determining the weight percentage content A of metal ions in the polyguluronic acid sulfate sample;
(2) determination of the weight average molecular weight Mw1 and the mass X using size exclusion chromatography in combination with multi-angle laser light scattering;
(3) the weight average molecular weight and the content of polyguluronic acid sulfate were calculated using the following equations:
in the above equation (1): mw1 is the weight average molecular weight (Da) of polyguluronic acid sulfate directly obtained by size exclusion chromatography combined with multi-angle laser light scattering; a is the content of metal ions in polyguluronic acid sulfate;
in the above equation (2): x is the mass calculated by the map processing software; y is the quality of sample introduction; a is the content of metal ions in polyguluronic acid sulfate.
2. The method according to claim 1, wherein the polyguluronic acid sulfate salt is an alkali metal salt.
3. The method according to claim 1, wherein the polyguluronic acid sulfate salt is a sodium salt, a potassium salt, a calcium salt, or a magnesium salt.
4. The method of claim 1, wherein the polyguluronic acid sulfate salt is a sodium salt.
5. The method of claim 1A method, characterized in that in step (2) size exclusion chromatography is combined with multi-angle laser light scattering method to determine the use of alcohol + salt solution as mobile phase; the flow rate is 0.1-0.5 mL/min; wherein, the content of alcohol in the mobile phase is 0-20%; the alcohol is methanol, ethanol, propanol, cyclohexanol or a mixture thereof; the salt is selected from NH4Ac、NaCl、NaNO3Or Na2SO4The concentration of the salt solution is 0.05-0.5 mol/L; the chromatographic column is a chromatographic column capable of well separating 2k-30kDa polysaccharide.
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