CN104345102A - Method for measuring chitosan oligosaccharide content in specific polymerization degree range - Google Patents
Method for measuring chitosan oligosaccharide content in specific polymerization degree range Download PDFInfo
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- CN104345102A CN104345102A CN201410605992.0A CN201410605992A CN104345102A CN 104345102 A CN104345102 A CN 104345102A CN 201410605992 A CN201410605992 A CN 201410605992A CN 104345102 A CN104345102 A CN 104345102A
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Abstract
The invention belongs to an ocean chemical engineering technology, and particularly relates to a method for measuring the chitosan oligosaccharide content in a specific polymerization degree range. According to the method, a dextran standard product is subjected to chromatographic analysis, a standard curve is drawn, and a linear regression equation is obtained; chitosan oligosaccharide molecular weights M1 and M2 corresponding to two end values n1 and n2 of the specific polymerization degree range to be measured are substituted into the linear regression equation, and the chitosan oligosaccharide chromatographic retention time t1 and t2 of the two end values of the specific polymerization degree range to be measured; a chitosan oligosaccharide solution to be measured is analyzed by measuring the chromatographic analysis condition of the standard sample, the chromatographic peak total area A corresponding to the sample to be measured and the chromatographic area A<t1-t2> in the chitosan oligosaccharide chromatographic retention time t1 to t2 of the two end values of the specific polymerization degree range to be measured are obtained through a chromatogram of the sample to be measured, and the chitosan oligosaccharide content of the specific polymerization degree range n1 to n2 is obtained according to a chromatographic area formula. The method has the advantages that the chitosan oligosaccharide content of the specific polymerization degree is indirectly measured by using the molecular weight as a calibration measure through gel exclusion chromatography, the analysis steps are few, and the operation is simple and convenient.
Description
Technical field
The invention belongs to thalassochemistry engineering, be specifically related to a kind of method measuring specific aggregation degree scope chitosan oligosaccharide content.
Background technology
Chitosan oligosaccharide, also known as glucose oligosaccharide amine, oligo-glucosamine, is a kind of to be formed by connecting linear oligosaccharides by β-Isosorbide-5-Nitrae glycosidic bond by D-Glucosamine and N-acetyl-D Glucosamine.Chitosan oligosaccharide is a kind of important living marine resources, is found to have multiple physiologically active, as antitumor, antibacterial, and anti-inflammatory is anti-oxidant, regulates blood pressure and blood lipoid, develop immunitypty, activation gut flora etc.In May, 2014, national health committee has ratified chitosan oligosaccharide as new raw-food material, and this is significant for chitosan oligosaccharide industry development.At present, chitosan oligosaccharide mainly through the acidolysis of shitosan, oxidative degradation, or enzymolysis three kinds of technology obtain.Chitosan oligosaccharide product prepared by these technology is a very complicated potpourri, the chitosan oligosaccharide wherein containing each degree of polymerization.The distribution of polymerization degree of chitosan oligosaccharide has material impact for its physiologically active.The new raw-food material regulation of chitosan oligosaccharide, in chitosan oligosaccharide, the oligomerization Glucosamine content of 2-10 the degree of polymerization must not lower than 80%.Therefore, how the chitosan oligosaccharide content of Accurate Determining specific aggregation degree scope is significant.
At present, the detection for oligosaccharides distribution of polymerization degree mainly adopts thin layer chromatography (TLC), high performance liquid chromatography (HPLC) and mass spectroscopy.Thin-layer chromatography method does not need large-scale instrument and equipment, easy and simple to handle, it is the method for the inspection oligosaccharides distribution of polymerization degree that laboratory is commonly used at first, the chitosan oligosaccharide that general polymerization degree is less than 6 can realize well being separated on thin layer plate, but its colour band skewness, long with conditions of streaking, and revision test differs greatly, and is rarely used in the quantitative test of oligosaccharides distribution of polymerization degree.Mass spectrum is a kind of very strong Oligosaccharide Analysis technology, it directly can provide the accurate molecular masses distribution of each composition in oligosaccharides, be widely used the analysis in oligosaccharide mixture, wherein Matrix Assisted Laser Desorption ionization time of flight mass spectrometry (MALDI-TOF/MS) and electrospray ionization mass spectrometry (ESI/MS) the most common.Although there is certain associating with the content of corresponding component in mass signal abundance, but do not have quantitative relationship accurately, therefore mass spectroscopy generally can only be used for the qualitative analysis of oligosaccharide compositions, about mass spectroscopy only has a small amount of bibliographical information at present in the quantitative test of each component of oligosaccharides, be in development, technological means is not yet ripe.
For low-molecular-weight chitosan oligosaccharide, specific high phase liquid chromatography (HPLC) system of usual employing can determine distribution of polymerization degree concrete in chitosan oligosaccharide and the content of each chitooligose monomer, comprise amino positive/reverse-phase chromatography, ion-exchange chromatography, hydrophilic chromatographic etc.But; calibrate owing to lacking single degree of polymerization chitosan oligosaccharide standard items; especially for the degree of polymerization be greater than 6 chitooligose monomer and have particular sequence arrangement N-acetylated chitooligose monomer at present be also difficult to obtain standard items, this give method practical application bring difficulty.Therefore, a kind of method researching and developing Accurate Determining specific aggregation degree scope chitosan oligosaccharide content is very necessary, and this production for the new raw-food material of chitosan oligosaccharide, supervision and whole industry development are significant.
Summary of the invention
The object of the present invention is to provide a kind of method measuring specific aggregation degree scope chitosan oligosaccharide content.
For achieving the above object, the technical solution used in the present invention is:
Measure a method for specific aggregation degree scope chitosan oligosaccharide content,
Stratographic analysis is carried out to dextran standard items, with the retention time (t) of the standard items measured and its molecular weight logarithm drawing standard curve, obtains the equation of linear regression of molecular weight and retention time;
By the endpoints thereof n of specific aggregation degree scope to be measured
1and n
2the molecular weight M of corresponding chitosan oligosaccharide
1and M
2bring the chromatographic retention t that above-mentioned equation of linear regression draws the endpoints thereof chitosan oligosaccharide of specific aggregation degree scope to be measured into
1and t
2;
With the chromatographiccondition of bioassay standard sample, chitosan oligosaccharide solution to be measured is analyzed, obtain the chromatographic retention t of chromatographic peak total area A corresponding to testing sample and the endpoints thereof chitosan oligosaccharide in specific aggregation degree scope to be measured with testing sample chromatogram
1~ t
2interior chromatogram area A
t1-t2, obtain specific aggregation degree scope n according to chromatogram area formula
1~ n
2chitosan oligosaccharide content;
Wherein, chromatogram area formula=A
t1-t2× 100%/A.
Described stratographic analysis adopts gel exclusion chromatography.Described gel exclusion chromatography is TSK G3000-PW
xLor the chromatography packed column of same nature.
Described gel exclusion chromatography analytic system analysis condition is: applied sample amount is 2-50 μ L, and mobile phase is: the NaAc_HAc buffer solution of 0.02-0.2mol/L, and flow velocity is: 0.5-1.0mL/min, and detecting device is: differential refraction detector.
The dextran standard items molecular weight of described known molecular amount is 180,1000,2700,5250,9750,13050,36800Da or the dextran molecule amount standard series with same nature.
The advantage that the present invention has:
1. the present invention starts with from macroscopic perspective; with molecular weight, chitosan oligosaccharide sample is characterized; by defining the molecular weight ranges of specific aggregation degree chitosan oligosaccharide; chromatographic peak area is adopted to carry out quantitative measurement; avoid the use of the single degree of polymerization, specific N-acetylated chitooligose standard items, workable.
2. the present invention adopts gel exclusion chromatography to be calibrate the content of indirect determination specific aggregation degree chitosan oligosaccharide with molecular weight, and analytical procedure is few, easy and simple to handle, is a kind of simple and effective mensuration chitosan oligosaccharide distribution of polymerization degree method.
Accompanying drawing explanation
The calibration curve of the dextran standard items molecular weight that Fig. 1 provides for the embodiment of the present invention and chromatographic retention.
The chromatographic integration scope schematic diagram of degree of polymerization 2-10 chitosan oligosaccharide in the testing sample that Fig. 2 provides for the embodiment of the present invention.
The chromatographic integration scope schematic diagram of degree of polymerization 3-8 chitosan oligosaccharide in the testing sample that Fig. 3 provides for the embodiment of the present invention.
Specific embodiment
Below in conjunction with Figure of description, the invention will be further described, and protection scope of the present invention is not only confined to following examples.
Embodiment 1
Measuring the degree of polymerization in chitosan oligosaccharide sample is the oligosaccharide content of 2-10:
Take molecular weight and be respectively 180,1000,2500, the each 4mg of dextran standard items of 5000,7100,12000, (in buffer solution, acetate concentration is 0.2mol/L to be dissolved in 1ml NaAc_HAc buffer solution, sodium acetate concentration is 0.1mol/L) in, adopt high performance size exclusion chromatography to measure each dextran standard items chromatographic retention respectively, chromatographic condition is:
Chromatographic column: TSK G3000-PW
xL(250 × 4.6mm)
Mobile phase: 0.2M acetic acid/0.1M sodium-acetate buffer
Flow velocity: 0.8mL/min.
Detecting device: Composition distribution.
Each dextran standard items to obtain corresponding molecular weight through stratographic analysis be 180,1000, the chromatographic retention of the dextran standard items of 2500,5000,7100,12000 is respectively 11.938,10.872,10477,10.015,9.676,9.433min.With molecular weight logarithm (log Mp) the drawing standard curve (see Fig. 1) of retention time (t) with corresponding dextran standard items, obtain equation of linear regression, log Mp=-0.7254t+10.926; (R
2=0.9949).
By the molecular weight M of complete deacetylated chitobiose
2(340Da) and the molecular weight M of complete deacetylated shell ten sugar
1(1628Da) bring above-mentioned regression equation respectively into, calculate chitobiose and the theoretical retention t of shell ten sugar in gel exclusion chromatography
2and t
1be respectively t
2=11.572min and t
1=10.635min.
With under the chromatographiccondition of above-mentioned bioassay standard sample, testing sample is injected chromatographic system analysis, obtain the gel exclusion chromatography analysis of spectra of chitosan oligosaccharide sample to be measured, adopt gel chromatography analysis software (chromatograph carries software) respectively to chromatographic peak and the testing sample specific aggregation topology degree retention time t of testing sample
2(11.572min) to t
1(10.635min) peak area in scope carries out integration (see Fig. 2), calculates chromatographic peak total area A (830641) corresponding to testing sample and at t
1~ t
2chromatogram area A in time
t1-t2(709456), in chitosan oligosaccharide sample so to be measured, the chitosan oligosaccharide content of the degree of polymerization 2 ~ 10 is: 709456 × 100%/830641=85.41%.
Embodiment 2
Measure the oligosaccharide content of degree of polymerization 3-8 in chitosan oligosaccharide sample
Take molecular weight and be respectively 180,1000,2500,5000, the each 4mg of dextran standard items of 7100,12000 is dissolved in 1ml NaAc_HAc buffer solution (in buffer solution, acetate concentration is 0.2mol/L, and sodium acetate concentration is 0.1mol/L), adopt high performance size exclusion chromatography to measure each dextran standard items chromatographic retention respectively, chromatographic condition is:
Chromatographic column: TSK G3000-PW
xL(250 × 4.6mm)
Mobile phase: 0.2M acetic acid/0.1M sodium-acetate buffer
Flow velocity: 0.8mL/min.
Detecting device: Composition distribution.
Each dextran standard items to obtain corresponding molecular weight through stratographic analysis be 180,1000, the chromatographic retention of the dextran standard items of 2500,5000,7100,12000 is respectively 11.938,10.872,10477,10.015,9.676,9.433min.With molecular weight logarithm (log Mp) the drawing standard curve (see Fig. 1) of retention time (t) with corresponding dextran standard items, obtain equation of linear regression, log Mp=-0.7254t+10.926; (R
2=0.9949).
By the molecular weight M of complete deacetylated chitotriose
2(501Da) and the molecular weight M of complete deacetylated shell eight sugar
1(1306Da) bring above-mentioned regression equation respectively into, calculate chitotriose and the theoretical retention t of shell eight sugar in gel exclusion chromatography
2and t
1be respectively t
2=11.340min and t
1=10.767min.
With under the chromatographiccondition of above-mentioned bioassay standard sample, testing sample is injected chromatographic system analysis, obtain the gel exclusion chromatography analysis of spectra of chitosan oligosaccharide sample to be measured, adopt gel chromatography analysis software respectively to chromatographic peak and the testing sample specific aggregation topology degree retention time t of testing sample
2(11.340min) to t
1(10.767min) peak area in scope carries out integration (see Fig. 3), calculates chromatographic peak total area A (830641) corresponding to testing sample and at t
1~ t
2chromatogram area A in time
t1-t2(563545), in chitosan oligosaccharide sample so to be measured, the chitosan oligosaccharide content of the degree of polymerization 3 ~ 8 is: 563545 × 100%/830641=67.84%.
Claims (3)
1. measure a method for specific aggregation degree scope chitosan oligosaccharide content, it is characterized in that:
Stratographic analysis is carried out to dextran standard items, with the retention time (t) of the standard items measured and its molecular weight logarithm drawing standard curve, obtains the equation of linear regression of molecular weight and retention time;
By the endpoints thereof n of specific aggregation degree scope to be measured
1and n
2the molecular weight M of corresponding chitosan oligosaccharide
1and M
2bring the chromatographic retention t that above-mentioned equation of linear regression draws the endpoints thereof chitosan oligosaccharide of specific aggregation degree scope to be measured into
1and t
2;
With the chromatographiccondition of bioassay standard sample, chitosan oligosaccharide solution to be measured is analyzed, obtain the chromatographic retention t of chromatographic peak total area A corresponding to testing sample and the endpoints thereof chitosan oligosaccharide in specific aggregation degree scope to be measured with testing sample chromatogram
1~ t
2interior chromatogram area A
t1-t2, obtain specific aggregation degree scope n according to chromatogram area formula
1~ n
2chitosan oligosaccharide content;
Wherein, chromatogram area formula=A
t1-t2× 100%/A.
2. by the method for mensuration specific aggregation degree scope chitosan oligosaccharide content according to claim 1, it is characterized in that: described stratographic analysis adopts gel exclusion chromatography.
3. by the method for mensuration specific aggregation degree scope chitosan oligosaccharide content according to claim 1, it is characterized in that: described gel exclusion chromatography analytic system analysis condition is: applied sample amount is 2-50 μ L, mobile phase is: the NaAc_HAc buffer solution of 0.02-0.2mol/L, flow velocity is: 0.5-1.0mL/min, and detecting device is: differential refraction detector.
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Cited By (5)
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| CN105461761A (en) * | 2015-12-16 | 2016-04-06 | 中国科学院理化技术研究所 | Preparation method of monomers with different polymerization degrees of fully deacetylated chitosan oligosaccharide capable of being detected on line |
| CN106153764A (en) * | 2016-06-17 | 2016-11-23 | 青岛科海生物有限公司 | The detection method of 2～6 degree of polymerization oligochitosan total contents |
| CN108548875A (en) * | 2018-04-02 | 2018-09-18 | 中国药科大学 | A kind of method of chitobiose in measurement chitosan oligosaccharide, chitotriose content |
| WO2020048269A1 (en) * | 2018-09-06 | 2020-03-12 | 广东药科大学 | Method for measuring content of chitosan oligosaccharide |
| CN116297996A (en) * | 2023-05-23 | 2023-06-23 | 中国人民解放军军事科学院军事医学研究院 | UPLC-MS/MS method for accurately measuring chitosan content in aqueous solution |
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105461761A (en) * | 2015-12-16 | 2016-04-06 | 中国科学院理化技术研究所 | Preparation method of monomers with different polymerization degrees of fully deacetylated chitosan oligosaccharide capable of being detected on line |
| CN105461761B (en) * | 2015-12-16 | 2018-09-14 | 中国科学院理化技术研究所 | Preparation method of monomers with different polymerization degrees of fully deacetylated chitosan oligosaccharide capable of being detected on line |
| CN106153764A (en) * | 2016-06-17 | 2016-11-23 | 青岛科海生物有限公司 | The detection method of 2～6 degree of polymerization oligochitosan total contents |
| CN108548875A (en) * | 2018-04-02 | 2018-09-18 | 中国药科大学 | A kind of method of chitobiose in measurement chitosan oligosaccharide, chitotriose content |
| WO2020048269A1 (en) * | 2018-09-06 | 2020-03-12 | 广东药科大学 | Method for measuring content of chitosan oligosaccharide |
| CN116297996A (en) * | 2023-05-23 | 2023-06-23 | 中国人民解放军军事科学院军事医学研究院 | UPLC-MS/MS method for accurately measuring chitosan content in aqueous solution |
| CN116297996B (en) * | 2023-05-23 | 2023-08-15 | 中国人民解放军军事科学院军事医学研究院 | UPLC-MS/MS method for accurately measuring chitosan content in aqueous solution |
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Application publication date: 20150211 |