CN109459504A - A method of measurement guluronic acid sulfuric acid weight average molecular weight and content - Google Patents
A method of measurement guluronic acid sulfuric acid weight average molecular weight and content Download PDFInfo
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Abstract
The invention belongs to Natural Medicine Chemistry and field of quality control, it is related to a kind of method of weight average molecular weight and content for measuring guluronic acid sulfuric acid, including using the metal ion content in guluronic acid sulfuric acid to be corrected the weight average molecular weight and content results of molecular sieve chromatography combination multiple angle laser light scattering detector SEC-MALS measurement guluronic acid sulfuric acid.It can quick, Accurate Determining guluronic acid sulfuric acid weight average molecular weight and content using the method for the present invention.
Description
Technical field
The invention belongs to Natural Medicine Chemistry and its field of quality control fields, are related to a kind of measurement guluronic acid
The method of sulfuric acid weight average molecular weight and content.
Background technique
Guluronic acid is prepared after acidolysis is classified in sodium alginate, and guluronic acid through sulphation, obtains again
Guluronic acid sulfuric ester, structure are the L- guluronic acid (L-Guluronic acid, G) of 2-OH/3-OH sulphation
Pass through α-Isosorbide-5-Nitrae-glucosides key connection composition linear compound.Currently, there is document report guluronic acid sulfuric ester both at home and abroad
It can be used for preventing and treating lithangiuria, also there is antitumor biological activity.
Guluronic acid sulfuric acid structural formula
The weight average molecular weight and content of guluronic acid sulfuric acid are to develop guluronic acid sulfuric acid ester drug
Important location parameter.Currently, in Chinese Pharmacopoeia, high performance gel permeation chromatography (Gel Permeation
Chromatography, GPC) it is the Normal practice for measuring molecular weight, it is the principle according to exclusion chromatography, different molecular weight
Polysaccharide successively flows out chromatographic column according to its size order, related to retention time by a series of different molecular weight standard polysaccharide
Property the calibration curve that the measures and relative molecular weight for calculating corresponding polysaccharide to be measured.But the conventional method must have corresponding more
Saccharide and have certain limitation.
In addition, the measurement common method of polysaccharide and oligosaccharide content is sulfuric acid carbazole method, this method is first with concentration and monosaccharide
Standard curve is done in the colour developing of standard items, then the content of sample to be tested is read on standard curve.But the monosaccharide of acid sugar is not
There is standard items sale, it, cannot be right if being had differences using glucuronic acid as standard items with the structure and colour developing efficiency of acid sugar
Acid sugar carries out accurate quantification.
The size exclusion chromatography combination multiple angle laser light scattering (Size-Exclusion occurred in recent years
Chromatography/Multi-Angle Laser Light Scattering, SEC-MALS) measurement sample absolute weight average point
Son amount, molecular weight distribution and content in the industry cycle cause extensive concern.18 multi-angle laser light scattering apparatus (MALS) working principles
Be: laser beam can generate light scattering by solution, and scattered light intensity is directly proportional to molecular weight analyte and solution concentration, difference scattering
The light intensity and molecular dimension of angle are related, therefore different location sets up the (survey of molecular weight of multiple detection probes around sample cell
Accuracy of measurement is directly proportional to angle number purpose square root) the scattered light intensity signal of different angle is received simultaneously.According to light scattering side
Journey K*c/R (θ)=1/MWP(θ)+2A2C makees Zimm figure, and the equal absolute molecular weight Mw of weight of macromolecular can be obtained, side's rotation half
Diameter rg and second ties up sharp coefficient A2.MALS and size exclusion chromatography (SEC) combination, it is not necessary to rely on the flow velocity of pump, calibration curve and
Other mistake it is assumed that the data such as the molecular weight that can directly ask and molecular weight distribution.The sample that MALS is isolated using chromatographic column
Product obtain eluate concentration by differential refraction (RI) detector and matter are calculated in dn/dc value in the amount of light scatter of different angle
Amount, both comprehensive molecular weight that can calculate each slice.This method is not required to standards calibration, overcomes sample and standard items
Chemical composition, molecular structure and bring relative molecular weight of different sizes measurement error.Existing research person uses this side
Method determine chitosan, hyaluronic acid average molecular weight (Monica Fee, Neil Errington, Kornelia Jumel,
et al.Correlation of SEC/MALLS with ultracentrifμge and viscometric data for
Chitosans.Eur Biophys J (2003) 32:457~464；Sanya Hokputsa,Kornelia Jumel,
Catherine Alexander,et al.Comparison of molecular mass determination of
Hyaluronic acid using SEC/MALLS and sedimentation equilibrium.Eur Biophys J
(2003) 32:450~456), there are also researchers to quantify natural neutral polysaccharide using this method, and predicts that this method will
A kind of routine techniques (Kit-Leong Cheong, Ding tao Wu, Jing Zhao, the et al.A quantitative as polysaccharide
rapid and accurate method for the quantitative estimation of natural
polysaccharides and their fractions using high performance size exclusion
chromatography coupled with multi-angle laser light scattering and refractive
Index detector.Journal of Chromatography A (2015) 1400:98~106).European Pharmacopoeia has been at present
Through the measuring method (European using the method as hydroxyethyl starch weight average molecular weight and molecular weight distribution
pharmacopoeia 8.0,p3307-3308,starches hydroxyethyl)。
To sum up, SEC-MALS can successfully measure neutral polysaccharide weight average molecular weight and content.
Summary of the invention
The purpose of the present invention is to provide a kind of detection guluronic acid sulfuric acid that can be accurate, efficient, simple, stable
Ester salt weight average molecular weight, the method for content.
One aspect of the present invention provides a kind of weight average molecular weight and content for measuring guluronic acid sulfuric acid
Method, which is characterized in that the measuring method comprises the steps of:
(1) the weight percentage A of guluronic acid sulfuric acid Gold Samples category ion is measured；
(2) weight average molecular weight Mw1 and content X is measured using SEC-MALS；
(3) it is calculated, respectively obtain the weight average molecular weight of guluronic acid sulfuric acid and is contained using following formula
Amount:
In above formula (1): Mw1 is the Weight-average molecular for the guluronic acid sulfuric acid that SEC-MALS method directly obtains
It measures (Da)；A is the content of metal ion in guluronic acid sulfuric acid；
In above formula (2): X is the quality that map processing software is calculated；Y is the quality of sample introduction；A is poly- gulose
The content of metal ion in aldehydic acid sulfuric acid.
Using measuring method provided by the invention, the weight average molecular weight of guluronic acid sulfuric acid can be accurately measured
And content, successfully solve the problems, such as that guluronic acid sulfuric acid weight average molecular weight measurement deviation is big, content quantitative is inaccurate.
Detailed description of the invention
Fig. 1 shows the SEC-ICP-MS spectrogram of aqueous solvent (A) and guluronic acid sulfuric ester sodium salt (B), Yi Jiju
The SEC-MALS difference spectrum figure (C) of guluronic acid sulfuric ester sodium salt
Specific embodiment
Definition
Unless otherwise stated, otherwise all percentage, ratio, ratio or number be by weight.
Unless otherwise defined, known to all professional and scientific terms as used herein and those skilled in the art
Known meaning it is identical.In addition, any method similar to or equal to what is recorded and material all can be applied to the present invention
In method.The preferred methods and materials described herein are for illustrative purposes only.
As used herein, term " dn/dc " refers to refractive index increment, and method commonly used in the art can be used to be measured
(absolute molecular weight of the Medical sodium hyaluronate gel such as Liu Lili and its measuring method [J] Pharmaceutical Analysis magazine of distribution
2013,33 (8), 1435-1438)；It can also inquire and obtain from relevant technical literature disclosed in this field.
As used herein, term " SEC-MALS " refers to that size exclusion chromatography is combined multiple angle laser light scattering (Size-
Exclusion Chromatography/Multi-Angle Laser Light Scattering), it is widely used in recent years
Method well known in the art can be used in measurement sample absolute weight average molecular, molecular weight distribution and content, specific measuring method
(Monica Fee,Neil Errington,Kornelia Jumel,et al.Correlation of SEC/MALLS with
ultracentrifuge and viscometric data for chitosans.Eur Biophys J(2003)32:457
~464；Sanya Hokputsa,Kornelia Jumel,Catherine Alexander,et al.Comparison of
molecular mass determination of Hyaluronic acid using SEC/MALLS and
Sedimentation equilibrium.Eur Biophys J (2003) 32:450~456；Kit-Leong Cheong,
Ding tao Wu,Jing Zhao,et al.A rapid and accurate method for the quantitative
estimation of natural polysaccharides and their fractions using high
performance size exclusion chromatography coupled with multi-angle laser
light scattering and refractive index detector.Journal of Chromatography A
(2015) 1400:98~106).
As used herein, term " ICP-MS " refers to inductively coupled plasma mass spectrometry (Inductively coupled
Plasma mass spectrometry) it is significant development in contemporary element composition measuring technology, there is high sensitivity, interference
Less, many advantages, such as ultra trace detection limit, Simultaneous multi element analysis, method well known in the art is can be used in specific measuring method,
Such as the inorganization credit of Feng Xianjin, Qu Taiyuan inductively coupled plasma mass spectrometry (ICP-MS) more recent application progress China
Analyse method documented by (2011) 1:46~52.
As used herein, term " ICP-OES " refers to inductively coupled plasma emission spectrography (Inductively
Coupled Plasma Optical Emission Spectrometer), it is this field for measuring metal ion content
Method well known in the art can be used in common method, specific measuring method.
The discovery of technical problem
The inventors of the present application found that when measuring guluronic acid sulfuric acid sample using existing SEC-MALS,
There is unknown peak after sample peak, and the content that guluronic acid sulfuric acid is calculated is unreasonable, MALS is on this content basis
On obtained weight average molecular weight also have deviation.For above-mentioned phenomenon, it is generally recognized that absorption occurs on pillar for sample causes to contain
Amount reduces, but inventors have found that such case is not also resolved after using pillar separating for several times.To explore the problem,
Unknown peak when inventor measures guluronic acid sulfuric acid to SEC-MALS parses, as a result, it has been found that the unknown peak pair
In obtaining the accurate weight average molecular weight of guluronic acid sulfuric acid and content is most important.
It was found by the inventors of the present invention that unknown peak belongs to the metal ion in guluronic acid sulfuric acid structure,
Therefore, the weight average molecular weight and content of guluronic acid sulfuric acid are also contemplated that the influence of metal ion content, carry out school
Just, i.e. the weight average molecular weight and content of SEC-MALS measurement are divided by matter shared by sugar chain portion in guluronic acid sulfuric acid
Weight average molecular weight and content that score (1- metal ion content) obtains guluronic acid sulfuric acid are measured, that is, uses following calculating
Formula 1, formula 2 are corrected:
In above formula (1): Mw1 is that SEC-MALS method directly obtains weight average molecular weight (Da)；A is guluronic acid sulphur
The content of metal ion in acid esters salt；
In above formula (2): X is the quality (μ g) that map processing software is calculated；Y is the quality (μ g) of sample introduction；A is
The content of metal ion in guluronic acid sulfuric acid.
Using measuring method provided by the invention, the weight average molecular weight of guluronic acid sulfuric acid can be accurately measured
And content, successfully solve the problems, such as that guluronic acid sulfuric acid weight average molecular weight measurement deviation is big, content quantitative is inaccurate.
In some embodiments of the present invention, the guluronic acid sulfuric acid is alkali metal salt.In the present invention
Other embodiments in, the guluronic acid sulfuric acid be sodium salt, sylvite, calcium salt or magnesium salts.Of the invention
In other embodiments, the guluronic acid sulfuric acid is sodium salt.
The measurement of metal ion content
In embodiments of the invention, in the guluronic acid sulfuric acid metal ion content measuring method
It is selected from: ICP-MS, ICP-OES, Atomic absorption or the chromatography of ions.It is preferred that ICP-MS, the chromatography of ions, more preferable ion chromatography
Method.These measuring methods are as described previously.They are usually used together with SEC.
Inductively coupled plasma mass spectrometry (Inductively coupled plasma mass spectrometry,
ICP-MS) be significant development in contemporary element composition measuring technology, there is high sensitivity, interference less, it is ultra trace detection limit, more
Many advantages, such as element is analyzed simultaneously (Feng Xianjin, Qu Taiyuan inductively coupled plasma mass spectrometry (ICP-MS) more recent application into
Open up China inorganic chemical analysis (2011) 1:46~52).Inventor attempts for SEC and ICP-MS to be combined for the first time, successfully confirms
Unknown peak after SEC-MALS measurement guluronic acid sulfuric acid sample peak is metal ion, while can obtain metal
The related data of the type and content of ion.
On this basis, by the type and its content of measurement ion come the Weight-average molecular to be measured to SEC-MALS method
Amount and content are corrected: by by the weight average molecular weight measured and content respectively divided by sugared in guluronic acid sulfuric acid
Mass fraction shared by chain part, and then respectively obtain the weight average molecular weight and content of guluronic acid sulfuric acid.
SEC-MALS method measures molecular weight and content
In the present invention, SEC-MALS method is used to measure the molecular weight and content of acid carbohydrate.Inventor has found poly-
Guluronic acid sulfuric acid sample dissociates when entering the analysis of SEC column, it follows that SEC-MALS method obtains being dissociation
The not weight average molecular weight and content of the acid sugar of metal ion afterwards, therefore the weight average molecular weight of guluronic acid sulfuric acid
With content it is also contemplated that the influence of metal ion content, there is not yet the report of this respect in current SEC-MALS method.
In the SEC-MALS method that the present invention uses, alcohol+salt solution can be used as mobile phase；Its flow velocity may
For 0.05-0.2mL/min；Wherein, the content of alcohol is 0-20% in the mobile phase.The alcohol can for methanol, ethyl alcohol, propyl alcohol,
Or mixtures thereof cyclohexanol, the salt are selected from NH4Ac, NaCl or Na2SO4, the concentration of salting liquid is 0.05-0.15mol/L.
In another preferred embodiment of the invention, SEC-MALS measurement uses 20% methanol+80%80mmol/L
NH4Ac is as mobile phase；Its flow velocity is 0.1mL/min.
In another preferred embodiment of the invention, SEC-MALS measurement uses being capable of good separation 2k-30kDa polysaccharide
Chromatographic column；Preferably include, but is not limited to following chromatographic column: Waters ACQUITYSEC
1.7um 4.6 × 150mm column, TSK G4000PWXL 7.8 × 300mm (TOSOH) Shodex OHpak SB-803HQ 7.8 ×
300mm(Shodex)AdvanceBio SEC2.7 μm of 4.6 × 150mm columns (Agilent).
In some embodiments of the present invention, the measuring method further includes measuring poly- guluronic acid sulfate folding
The step of optical index increment (dn/dc).
In some embodiments of the present invention, the poly- guluronic acid sulfate refractive index increment (dn/dc)
Measurement comprise the steps of:
(1) using the poly- guluronic acid sulfate sample of flowing phased soln of SEC-MALS measurement, and sample is diluted, is obtained
To the serial solution of 5-6 concentration point in 0.1mg/ml-2mg/ml concentration ranges；
(2) with differential refraction detector (Wyatt, Optilab T-rEX (WTREX-08) respectively successively sample introduction mobile phase,
The sample solution prepared in step 2, and acquire signal；
(3) dn/dc is calculated by Astra software.
In one embodiment of the invention, the weight average molecular weight of guluronic acid sulfuric acid and assay side
Method is as follows:
Step 1: with gold in ICP-MS, ICP-OES, Atomic absorption or ion chromatography guluronic acid sulfuric acid
Belong to the weight percentage A of ion.
Step 2: the dn/dc of measurement guluronic acid sulfuric acid
(1) SEC mobile phase (such as mobile phase used in following steps 3) dissolution, configuration is added in the sample of weighed constant weight
At 0.1-5mg/ml, the preferably serial solution of 0.2-2mg/ml, the concentration of the serial solution can for such as 0.2mg/ml,
0.4mg/ml、0.8mg/ml、1.2mg/ml、1.8mg/ml、2mg/ml。
(2) with differential refraction detector (such as Wyatt, Optilab T-rEX (WTREX-08) respectively successively sample introduction flow
The serial solution prepared in phase, back, and signal is acquired,
(3) dn/dc is calculated by Astra software in dn/dc.
Step 3:SEC-MALS measures guluronic acid sulfuric acid molecular weight and content
(1) according to the molecular weight ranges for estimating guluronic acid sulfuric acid to be measured, suitable chromatographic column is selected；
(2) gradient solution for preparing guluronic acid sulfuric acid divides 3-10 ladder in the range of 2-500mg/ml
Degree；The solution of e.g., about 2,10,50,100,300mg/mL (concentration=weight/volume).
(3) use alcohol+salt solution as mobile phase；Its flow velocity may be 0.05-0.2mL/min；Wherein, the flowing
The content of alcohol is 0-20% in phase.The alcohol can be or mixtures thereof methanol, ethyl alcohol, propyl alcohol, cyclohexanol, and the salt is selected from
NH4Ac, NaCl or Na2SO4, the concentration of salting liquid is 0.05-0.15mol/L, and detector is using ten octagonal laser detectors and shows
Poor refraction detector；
(4) the guluronic acid sulfuric acid weight average molecular weight Mw1 directly obtained using SEC-MALS method；
(5) guluronic acid sulfuric acid quality X in the sample being calculated using map processing software；
(6) it is calculated, respectively obtain the weight average molecular weight of guluronic acid sulfuric acid and is contained using following formula
Amount:
In above formula (1): Mw1 is the Weight-average molecular for the manna oligosacchride diacid soluble-salt that SEC-MALS method directly obtains
It measures (Da)；A is the content of metal ion in guluronic acid sulfuric acid；
In above formula (2): X is the quality that map processing software is calculated；Y is the quality of sample introduction；A is poly- gulose
The content of metal ion in aldehydic acid sulfuric acid.
SEC-MALS method measure weight average molecular weight and content concrete operations condition can according to conventional experimental program into
Row.
Compared with the prior art, the present invention has the following advantages:
(1) the unknown peak in guluronic acid sulfuric acid sample SEC-MALS analysis is explained.
(2) guluronic acid sulfuric acid weight average molecular weight and content can be accurately measured, it is reproducible.
(3) weight average molecular weight and content can obtain in primary analysis, easy to operate.
(4) accuracy for improving measurement guluronic acid sulfuric acid facilitates point for more accurately studying polysaccharide
Minor structure.
Embodiment
Present invention will be further explained below with reference to specific examples.It should be understood that these embodiments are merely to illustrate the present invention
Rather than it limits the scope of the invention.In the following examples, the experimental methods for specific conditions are not specified, usually according to conventional strip
Part or according to the normal condition proposed by manufacturer.
Instrument used in embodiment part includes:
1260 liquid chromatograph of LC:Agilent
18 multi-angle laser detectors: Wyatt DAWN HELEOS II
Differential refraction detector: Wyatt Optilab T-rEX
ICP-MS:Agilent 7900ICP-MS
Ion chromatography: Switzerland ten thousand is logical
ICP-OES: Perkin-Elmer company, U.S. Optima7000DV Inductively coupled plasma optical emission spectrometer
Flame Atomic Absorption Spectrometry: Beijing Rayleigh WFX-120B type flame atomic absorption spectrophotometer
1 SEC of embodiment and ICP-MS discloses acid sugar in the separating mechanism of size exclusion chromatography, and measures poly- gulose
The weight average molecular weight and content of aldehydic acid sulfuric ester sodium salt
Step 1:SEC-ICP-MS measurement metal ion goes out peak position and content
(1) chromatographic column Waters ACQUITY is usedSEC 1.7um 4.6×150mm
Column, 0.08mol/L NH4Ac are mobile phase, flow velocity 0.1mL/min.
(2) SEC and ICP-MS is combined: sample is soluble in water, concentration 0.02mg/ml, sample introduction 20ul, acquisition23Na,43Ca,44The signal of Ca.The concentration being respectively configured is the Na ion mark of 0.1ppm, 0.5ppm, 1.0ppm, 5.0ppm and 10.0ppm
Quasi- solution, and with the content (A value) of external standard method calculating Gold Samples category ion.A value the results are shown in Table 1.
Step 2:SEC-MALS measures weight average molecular weight and content
(1) chromatographic column Waters ACQUITY is usedSEC 1.7um 4.6 × 150mm column
0.1mol/L NH4Ac is mobile phase, flow velocity 0.1mL/min.
(2) SEC and MALS is combined, sample solution flows in phase, concentration 10mg/ml, sample volume 20ul, i.e., in formula
Y value is 200ug.Experimental result is shown in attached drawings 1 and table 1.
Fig. 1 is the SEC-ICP-MS spectrogram of aqueous solvent (Figure 1A) and guluronic acid sulfuric ester sodium salt (Figure 1B), and
The SEC-MALS difference spectrum figure (Fig. 1 C) of guluronic acid sulfuric ester sodium salt.The results show that in SEC-ICP-MS spectrogram, phase
Than in negative control, guluronic acid sulfuric ester sodium salt has obvious Na salt peak at a, compare Figure 1B and Fig. 1 C it is found that poly- Gu
Na salt peak of sieve uronic acid sulfuric ester sodium salt at SEC-ICP-MS spectrogram a and the peak appearance position consistency at difference spectrum figure b, say
Expressing the unknown peak at poor spectrogram b is sodium salt peak, it means that hair when guluronic acid sulfuric ester sodium salt enters the analysis of SEC column
Raw dissociation, SEC-MALS method obtain be after dissociation not the weight average molecular weight of the guluronic acid sulfuric ester of metal ion and
Content prompts in order to accurately calculate the weight average molecular weight and content of guluronic acid sulfuric ester sodium salt, using SEC-MALS
When method, it is necessary to consider that the metal ion in guluronic acid sulfuric ester sodium salt is corrected, specific calculate sees formula 1, public affairs
Formula 2.
In above formula (1): Mw1The weight average molecular weight (Da) directly obtained for SEC-MALS method；A is containing for metal ion
Amount.
In above formula (2): X is the quality (μ g) that map processing software is calculated；Y is the quality (μ g) of sample introduction；A is
The content of metal ion.
Table 1: the metal ion content of guluronic acid sulfuric ester sodium salt and the weight average molecular weight and content of correction front and back
Y value is 200ug；*: using formula correction；A is Na%
As known from Table 1, after corrected, SEC-MALS can accurately calculate the weight of guluronic acid sulfuric ester sodium salt
Average molecular weight and content.
The weight average molecular weight and content of 2 SEC-MALS of embodiment measurement guluronic acid sulfuric ester sylvite
Step 1: the ratio of ion chromatography metal ion
K ion standard solution is configured to 25,10,12.5,6.25,3.125ppm.Sample concentration is 2mg/ml.
Ion chromatography condition: pre-column: Metrosep C4Guard/4.0；Splitter: Metrosep
C5-150/4.0；Mobile phase: the ultra-pure water solution of 5mM nitric acid；Flow velocity: 0.9ml/min；Sample volume 20ul.In addition
The measurement of mark method progress metal ion content.
Step 2: measurement dn/dc
(1) sample of weighed constant weight is 0.2mg/ml, 0.4mg/ml, 0.8mg/ml, 1.2mg/ with mobile phase compound concentration
A series of solution of ml, 1.8mg/ml, 2mg/ml.
(2) with differential refraction detector, (successively hand sampling flows Wyatt, Optilab T-rEX (WTREX-08) respectively
Phase, 0.2mg/ml, 0.4mg/ml, 0.8mg/ml, 1.2mg/ml, 1.8mg/ml, 2mg/ml sample solution, and signal is acquired,
(3) dn/dc is calculated by Astra software, is shown in Table 2.
Step 3:SEC-MALS measures weight average molecular weight and content
(1) chromatographic column AdvanceBio SEC is used2.7 μm of 4.6 × 150mm column (Agilent) 0.3mol/L
Na2SO4For mobile phase, flow velocity 0.2mL/min.
(2) SEC and MALS is combined, sample solution flows in phase, concentration 10mg/ml, sample volume 20ul, i.e., in formula
Y value is 200ug.Experimental result is as shown in table 2.
The metal ion content of 2 guluronic acid sulfuric ester sylvite of table and the weight average molecular weight and content of correction front and back
Y value is 200ug；*: using the formula correction in embodiment 1；A is K%
From table 2 it can be seen that corrected, SEC-MALS can accurately calculate guluronic acid sulfuric ester sylvite
Weight average molecular weight and content.
The weight average molecular weight and content of 3 SEC-MALS of embodiment measurement guluronic acid sulfuric ester calcium salt
Step 1: flame atomic absorption spectrometry metal ion content:
The spectral line of Ca is acquired, the Ca standard solution of 1mg/L, 10mg/L, 50mg/L, 75mg/L and 100mg/L are prepared；Sample
Solution concentration preparation is 200mg/L.
Step 2: measurement dn/dc
With 2 step 2 of embodiment.Measured value is shown in Table 3.
Step 3:SEC-MALS measures weight average molecular weight and content
(1) 7.8 × 300mm of chromatographic column Shodex OHpak SB-803HQ (Shodex), 0.15mol/L NaCl are used
For mobile phase, flow velocity 0.5mL/min.
(2) SEC and MALS is combined, sample solution flows in phase, concentration 10mg/ml, sample volume 20ul, i.e., in formula
Y value is 200ug.Experimental result is as shown in table 3.
The metal ion content of 3 guluronic acid sulfuric ester calcium salt of table and the weight average molecular weight and content of correction front and back
Y value is 200ug；*: using the formula correction in embodiment 1；A is Ca%
From table 3 it can be seen that corrected, SEC-MALS can accurately calculate guluronic acid sulfuric ester calcium salt
Weight average molecular weight and content.
The weight average molecular weight and content of 4 SEC-MALS of embodiment measurement guluronic acid sulfuric ester magnesium salts
Step 1:ICP-OES measures metal ion content:
The analysis spectral line of Mg is acquired, the Na standard solution of 1mg/L, 5mg/L, 10mg/L are prepared；
Sample solution concentration is formulated as 20mg/L, and thus obtained A value is as shown in table 4.Thus step 2: measurement dn/dc
With 2 step 2 of embodiment.Measured value is shown in Table 4.
Step 3:SEC-MALS measures weight average molecular weight and content
(1) 7.8 × 300mm of chromatographic column TSK G4000PWXL (TOSOH), 0.5mol/L NaNO are used3For mobile phase,
Flow velocity 0.5mL/min.
(2) SEC and MALS is combined, sample solution flows in phase, concentration 10mg/ml, sample volume 20ul, i.e., in formula
Y value is 200ug.Experimental result is shown in Table 4.
The metal ion content of 4 guluronic acid sulfuric ester magnesium salts of table and the weight average molecular weight and content of correction front and back
Y value is 200ug；*: using the formula correction in embodiment 1；A is Mg%
From table 4, it can be seen that corrected, SEC-MALS can accurately calculate guluronic acid sulfuric ester magnesium salts
Weight average molecular weight and content.
Claims (4)
1. a kind of method for the weight average molecular weight and content for measuring guluronic acid sulfuric acid, which is characterized in that the survey
The method of determining comprises the steps of:
(1) the weight percentage A of guluronic acid sulfuric acid Gold Samples category ion is measured；
(2) weight average molecular weight Mw1 and content X is measured using SEC-MALS；
(3) it is calculated using following formula, respectively obtains the weight average molecular weight and content of guluronic acid sulfuric acid:
Weight average molecular weight
In above formula (1): Mw1 is the weight average molecular weight for the guluronic acid sulfuric acid that SEC-MALS method directly obtains
(Da)；A is the content of metal ion in guluronic acid sulfuric acid；
In above formula (2): X is the quality that map processing software is calculated；Y is the quality of sample introduction；A is guluronic acid
The content of metal ion in sulfuric acid.
2. measuring method as described in claim 1, which is characterized in that the guluronic acid sulfuric acid is alkali metal
Salt；Particular certain cancers, sylvite, calcium salt or magnesium salts；More preferable sodium salt.
3. measuring method described in claim 1, which is characterized in that SEC-MALS measurement uses alcohol+salt solution in step (2)
As mobile phase；Its flow velocity can be 0.1-0.5mL/min；Wherein, the content of alcohol is 0-20% in the mobile phase；The alcohol
It can be or mixtures thereof methanol, ethyl alcohol, propyl alcohol, cyclohexanol；The salt is selected from NH4Ac、NaCl、NaNO3Or Na2SO4, salting liquid
Concentration be 0.05-0.5mol/L；The chromatographic column is to be capable of the chromatographic column of good separation 2k-30kDa polysaccharide.
4. measuring method as claimed in claim 1 or 2, which is characterized in that the measuring method of the metal ion content is selected from:
ICP-MS, ICP-OES, Atomic absorption or the chromatography of ions.
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