CN114441684B - A method for qualitative and quantitative analysis of high molecular sugar compounds - Google Patents
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- 238000000034 method Methods 0.000 title claims abstract description 27
- 238000004445 quantitative analysis Methods 0.000 title claims abstract description 22
- 238000004451 qualitative analysis Methods 0.000 title claims abstract description 7
- 150000001875 compounds Chemical class 0.000 title claims description 22
- 238000004128 high performance liquid chromatography Methods 0.000 claims abstract description 24
- 230000014759 maintenance of location Effects 0.000 claims abstract description 20
- 230000004044 response Effects 0.000 claims abstract description 19
- 238000001514 detection method Methods 0.000 claims abstract description 9
- 229920001218 Pullulan Polymers 0.000 claims abstract description 6
- 239000004373 Pullulan Substances 0.000 claims abstract description 6
- 229920000642 polymer Polymers 0.000 claims abstract description 6
- 235000019423 pullulan Nutrition 0.000 claims abstract description 6
- -1 saccharide compounds Chemical class 0.000 claims abstract description 6
- 239000000126 substance Substances 0.000 claims abstract 7
- 239000000523 sample Substances 0.000 claims description 36
- 229920001282 polysaccharide Polymers 0.000 claims description 31
- 239000005017 polysaccharide Substances 0.000 claims description 31
- 150000004676 glycans Chemical class 0.000 claims description 30
- 241000196252 Ulva Species 0.000 claims description 8
- 239000012086 standard solution Substances 0.000 claims description 8
- 238000005227 gel permeation chromatography Methods 0.000 claims description 7
- 238000000926 separation method Methods 0.000 claims description 6
- 229910021642 ultra pure water Inorganic materials 0.000 claims description 5
- 239000012498 ultrapure water Substances 0.000 claims description 5
- 235000014633 carbohydrates Nutrition 0.000 claims description 4
- 238000002347 injection Methods 0.000 claims description 4
- 239000007924 injection Substances 0.000 claims description 4
- 239000012488 sample solution Substances 0.000 claims description 4
- 239000012528 membrane Substances 0.000 claims description 2
- 238000012360 testing method Methods 0.000 claims description 2
- 241000908178 Tremella fuciformis Species 0.000 claims 1
- 238000000611 regression analysis Methods 0.000 abstract description 2
- 150000001720 carbohydrates Chemical class 0.000 abstract 1
- 238000001914 filtration Methods 0.000 abstract 1
- 239000012982 microporous membrane Substances 0.000 abstract 1
- 241001506047 Tremella Species 0.000 description 8
- 238000011002 quantification Methods 0.000 description 3
- 238000004458 analytical method Methods 0.000 description 2
- 238000004364 calculation method Methods 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- CTYRPMDGLDAWRQ-UHFFFAOYSA-N phenyl hydrogen sulfate Chemical compound OS(=O)(=O)OC1=CC=CC=C1 CTYRPMDGLDAWRQ-UHFFFAOYSA-N 0.000 description 2
- 230000008569 process Effects 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 239000000243 solution Substances 0.000 description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 2
- 229920002307 Dextran Polymers 0.000 description 1
- QAOWNCQODCNURD-UHFFFAOYSA-L Sulfate Chemical compound [O-]S([O-])(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-L 0.000 description 1
- 238000004164 analytical calibration Methods 0.000 description 1
- RJGDLRCDCYRQOQ-UHFFFAOYSA-N anthrone Chemical compound C1=CC=C2C(=O)C3=CC=CC=C3CC2=C1 RJGDLRCDCYRQOQ-UHFFFAOYSA-N 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 230000007717 exclusion Effects 0.000 description 1
- 238000013365 molecular weight analysis method Methods 0.000 description 1
- 238000010606 normalization Methods 0.000 description 1
- 230000009965 odorless effect Effects 0.000 description 1
- 230000000144 pharmacologic effect Effects 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
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- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
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Abstract
Description
技术领域Technical field
本发明属于糖类化合物的分析技术领域，具体涉及一种用于分析高分子糖类化合物定性和定量的方法。The invention belongs to the technical field of analysis of sugar compounds, and specifically relates to a method for qualitative and quantitative analysis of high molecular sugar compounds.
背景技术Background technique
公开该背景技术部分的信息仅仅旨在增加对本发明的总体背景的理解，而不必然被视为承认或以任何形式暗示该信息构成已经成为本领域一般技术人员所公知的现有技术。The information in this Background section is disclosed solely for the purpose of increasing understanding of the general background of the invention and is not necessarily considered to be an admission or in any way implying that the information constitutes prior art that is already known to a person of ordinary skill in the art.
多糖的药理活性与其相对分子质量(Mw)及其分布密切相关，高分子糖类化合物由于其分子量大、结构复杂，而且缺少易于检测的发光基团，相关的定性和定量方法尚未完全建立，限制了人们对其进一步的研究和应用，如何快速准确地鉴别及定性定量分析糖类化合物成为糖类产业化过程中的关键问题。The pharmacological activity of polysaccharides is closely related to its relative molecular mass (Mw) and its distribution. Due to their large molecular weight, complex structure, and lack of easy-to-detect luminescent groups, the relevant qualitative and quantitative methods of high-molecular sugar compounds have not yet been fully established, limiting their This has led to further research and application, and how to quickly and accurately identify and qualitatively and quantitatively analyze sugar compounds has become a key issue in the process of sugar industrialization.
目前，硫酸苯酚法、硫酸蒽酮法等常作为多糖的测定方法，虽然可以较为迅速地进行测定，但其只能测定样品的总糖含量，无法准确地针对一些特定分子量的高分子糖类化合物进行定量分析。高效液相色谱技术(HPLC)是目前应用比较广泛的多糖的分子量测定和定量的分析方法，可以对高分子和低分子的多糖化合物进行分离、定性及定量。但目前该方法只能建立对应多糖标准品下的定量曲线，因而只能对有限的和分子量确定或接近标准品的目标化合物进行定量分析，无法对其他未知或更高分子量的糖类化合物进行定量分析。现有的一种方法中，为了较为准确地测定样品中多糖的含量和相对分子质量分布，通过选用分子量与样品相近的葡聚糖为标准品，采用面积归一法计算各组分含量。虽然该方法一定程度上可以实现相近分子量样品的定量，但是当样品与标准品分子量差距过大时，用于定量分析是极为不准确的。At present, the phenol sulfate method and the anthrone sulfate method are often used as methods for the determination of polysaccharides. Although they can be measured relatively quickly, they can only measure the total sugar content of the sample and cannot accurately target some high molecular sugar compounds with specific molecular weights. Perform quantitative analysis. High-performance liquid chromatography (HPLC) is currently a widely used analytical method for the molecular weight determination and quantification of polysaccharides. It can separate, characterize and quantify high-molecular and low-molecular polysaccharide compounds. However, at present, this method can only establish a quantitative curve corresponding to the polysaccharide standard. Therefore, it can only quantitatively analyze a limited number of target compounds with a certain molecular weight or close to the standard, and cannot quantify other unknown or higher molecular weight carbohydrate compounds. analyze. In an existing method, in order to more accurately measure the content and relative molecular mass distribution of polysaccharides in a sample, dextran with a molecular weight similar to that of the sample is selected as a standard, and the area normalization method is used to calculate the content of each component. Although this method can achieve quantification of samples with similar molecular weights to a certain extent, when the molecular weight difference between the sample and the standard is too large, it is extremely inaccurate for quantitative analysis.
发明内容Contents of the invention
针对上述现有技术中存在的问题，本发明的目的是提供一种用于分析高分子糖类化合物定性和定量的方法。In view of the problems existing in the above-mentioned prior art, the purpose of the present invention is to provide a qualitative and quantitative method for analyzing high molecular sugar compounds.
为了解决以上技术问题，本发明的技术方案为：In order to solve the above technical problems, the technical solution of the present invention is:
一种用于分析高分子糖类化合物定性和定量的方法，所述方法为：A method for qualitative and quantitative analysis of high molecular sugar compounds, the method is:
1)选用普鲁兰多糖(Pullulan KIT)用流动相溶解为标准品溶液，将待测样品用流动相溶解为样品溶液；1) Use Pullulan KIT to dissolve it into a standard solution using mobile phase, and dissolve the sample to be tested into a sample solution using mobile phase;
2)将步骤1)的待测样品溶液和标准品溶液经过微孔滤膜进行过滤后利用高效液相色谱和示差折光检测器进行检测；2) Filter the sample solution to be tested and the standard solution in step 1) through a microporous filter membrane and then detect using high performance liquid chromatography and a differential refractive index detector;
3)取不同分子量的标准品溶液利用步骤2)的方法进行检测后得到标准品的色谱图和保留时间(RT)，以标准品分子量的对数logMw为纵坐标、色谱峰的保留时间为横坐标进行回归分析，得出回归方程和计算分子量的标准曲线；3) Take standard solutions of different molecular weights and use the method in step 2) to detect them and obtain the chromatogram and retention time (RT) of the standard. Take the logarithm of the molecular weight of the standard, logMw, as the ordinate and the retention time of the chromatographic peak as the abscissa. Perform regression analysis on the coordinates to obtain the regression equation and the standard curve for calculating molecular weight;
4)取不同分子量的标准品进行色谱检测，得到标准品的色谱图和响应值(Mv)，根据步骤3)的标准曲线以及不同标准品的浓度，拟合得到不同浓度下的拟合曲线；4) Take standards of different molecular weights for chromatographic detection to obtain the chromatogram and response value (Mv) of the standard. According to the standard curve in step 3) and the concentrations of different standards, fit the fitting curves at different concentrations;
5)将待测样品利用步骤2)的方法进行检测后，得到色谱图的响应值(Mv)和保留时间(RT)，利用步骤3)的标准曲线和步骤4)的拟合曲线得到HPLC定量曲线计算待测样品的重均相对分子质量和浓度。5) After testing the sample to be tested using the method of step 2), obtain the response value (Mv) and retention time (RT) of the chromatogram, and use the standard curve of step 3) and the fitting curve of step 4) to obtain HPLC quantification The curve calculates the weight average relative molecular mass and concentration of the sample to be tested.
响应值为色谱图的Mv，保留时间能够通过色谱图的峰对应的横坐标得到。通过色谱图得到出现的峰的保留时间，通过步骤3)的分子量标准曲线得到分子量对数值logMw，进而得到分子量。进一步通过步骤4)的拟合曲线，得到HPLC定量曲线，通过分子量和响应值，得到对应的浓度。所以能够对高分子糖类化合物进行定性和定量。The response value is the Mv of the chromatogram, and the retention time can be obtained from the abscissa corresponding to the peak of the chromatogram. Obtain the retention time of the peak that appears from the chromatogram, obtain the logarithmic value of the molecular weight logMw through the molecular weight standard curve in step 3), and then obtain the molecular weight. Further, the HPLC quantitative curve is obtained through the fitting curve in step 4), and the corresponding concentration is obtained through the molecular weight and response value. Therefore, it is possible to characterize and quantify high molecular sugar compounds.
选择普鲁兰多糖相比于其它多糖具有溶解度较大、可塑性和粘性较强，具有易溶于水、无色无味等优良特性，非常适合于仪器的校准。Compared with other polysaccharides, pullulan polysaccharide has greater solubility, stronger plasticity and viscosity. It has excellent properties such as being easily soluble in water, colorless and odorless, and is very suitable for instrument calibration.
在本发明的一些实施方式中，步骤2)中检测条件为分离柱为Shodex SB-804和806串联，流动相为超纯水或0.1M NaNO3，流速为0.5-1ml/min，柱温为35-40℃，进样量为10-20μL。本发明中选择分离柱为Shodex SB-804和806串联，Shodex SB-806的排阻极限是2*107，可以满足更高分子量糖类化合物或多糖的分子量分析。使用0.1M NaNO3可以满足相近分子量的多糖的分离。In some embodiments of the present invention, the detection conditions in step 2) are that the separation column is Shodex SB-804 and 806 in series, the mobile phase is ultrapure water or 0.1M NaNO 3 , the flow rate is 0.5-1ml/min, and the column temperature is 35-40℃, injection volume is 10-20μL. In the present invention, the selected separation column is Shodex SB-804 and 806 connected in series. The exclusion limit of Shodex SB-806 is 2*10 7 , which can meet the molecular weight analysis of higher molecular weight carbohydrate compounds or polysaccharides. The use of 0.1M NaNO 3 can satisfy the separation of polysaccharides with similar molecular weights.
在本发明的一些实施方式中，步骤2)中普鲁兰多糖分子量Mw为180Da、504Da、991Da、6600Da、9900Da、23000Da、50600Da、115000Da、202000Da、343000Da、805000Da、1330000Da。In some embodiments of the present invention, the molecular weight Mw of pullulan in step 2) is 180Da, 504Da, 991Da, 6600Da, 9900Da, 23000Da, 50600Da, 115000Da, 202000Da, 343000Da, 805000Da, 1330000Da.
在本发明的一些实施方式中，步骤1)中得到的标准品溶液的浓度为1.0mg/ml、0.8mg/ml、0.5mg/ml、0.2mg/ml、0.1mg/ml。In some embodiments of the present invention, the concentration of the standard solution obtained in step 1) is 1.0 mg/ml, 0.8 mg/ml, 0.5 mg/ml, 0.2 mg/ml, or 0.1 mg/ml.
在本发明的一些实施方式中，色谱图为横坐标为时间和纵坐标为电压的色谱图。In some embodiments of the invention, the chromatogram is a chromatogram with time as the abscissa and voltage as the ordinate.
在本发明的一些实施方式中，步骤4)中拟合曲线的方法为：通过步骤3)的分子量标准曲线，得到色谱图的保留时间与标准品的分子量相对应，然后通过分子量与标准样品的浓度进行对应，将分子量浓度与色谱图的响应值进行拟合，得到拟合曲线。可进一步得到任意分子量下的HPLC定量曲线。In some embodiments of the present invention, the method of fitting the curve in step 4) is: using the molecular weight standard curve of step 3) to obtain the retention time of the chromatogram corresponding to the molecular weight of the standard sample, and then using the molecular weight and the molecular weight of the standard sample to obtain Correspond to the concentration, and fit the molecular weight concentration with the response value of the chromatogram to obtain a fitting curve. HPLC quantitative curves at any molecular weight can be further obtained.
在本发明的一些实施方式中，步骤4)得到的拟合曲线的回归方程为y＝Ae-Bx，R2＝0.9236-0.9698。进一步，浓度为1.0mg/ml，A＝363.07，B＝0.349；浓度为0.8mg/ml，A＝295.18，B＝0.361；浓度为0.5mg/ml，A＝143.32，B＝0.334；浓度为0.2mg/ml，A＝65.347，B＝0.372；浓度为0.1mg/ml，A＝54.458，B＝0.485。In some embodiments of the present invention, the regression equation of the fitting curve obtained in step 4) is y=Ae -Bx , R 2 =0.9236-0.9698. Further, the concentration is 1.0mg/ml, A=363.07, B=0.349; the concentration is 0.8mg/ml, A=295.18, B=0.361; the concentration is 0.5mg/ml, A=143.32, B=0.334; the concentration is 0.2 mg/ml, A=65.347, B=0.372; the concentration is 0.1mg/ml, A=54.458, B=0.485.
在本发明的一些实施方式中，步骤5)的具体步骤为：通过检测待测样品得到色谱图的保留时间和响应值，然后利用步骤3)得到的分子量标准曲线计算样品分子量，得到分子量的对数值logMw，然后利用步骤4)中不同浓度的拟合曲线得到其分子量下对应的HPLC定量曲线，通过代入响应值，得到待测样品的浓度。In some embodiments of the present invention, the specific steps of step 5) are: obtaining the retention time and response value of the chromatogram by detecting the sample to be tested, and then using the molecular weight standard curve obtained in step 3) to calculate the molecular weight of the sample to obtain a correlation of the molecular weight. Value logMw, and then use the fitting curves of different concentrations in step 4) to obtain the corresponding HPLC quantitative curve at its molecular weight. By substituting the response value, the concentration of the sample to be tested is obtained.
在本发明的一些实施方式中，步骤3)和5)中色谱图的保留时间(RT)以及响应值等通过GPC凝胶色谱软件进行计算。In some embodiments of the present invention, the retention time (RT) and response value of the chromatograms in steps 3) and 5) are calculated by GPC gel chromatography software.
在本发明的一些实施方式中，所述待测样品可以为银耳多糖、浒苔多糖等高分子多糖。In some embodiments of the present invention, the sample to be tested may be high molecular polysaccharides such as Tremella polysaccharide and Enteromorpha polysaccharide.
在本发明的一些实施方式中，HPLC定量曲线的方程为y＝Ax-B，R2＝0.9958-0.9959，，x为浓度，y为响应值；其中A和B的值根据不同的糖类化合物的分子量确定的。In some embodiments of the invention, the equation of the HPLC quantitative curve is y=Ax-B, R 2 =0.9958-0.9959,, x is the concentration, and y is the response value; where the values of A and B are based on different carbohydrate compounds The molecular weight is determined.
本发明一个或多个技术方案具有以下有益效果：One or more technical solutions of the present invention have the following beneficial effects:
(1)本发明运用高效凝胶色谱法建立了一种新型的用于高分子糖类化合物定性及定量分析方法，摆脱了当前高分子糖类化合物HPLC定量受标准品的限制，该方法具有精密准确、重现性好、高效快速等优势，实现了任意分子量范围下的定性定量分析。(1) The present invention uses high-efficiency gel chromatography to establish a new method for qualitative and quantitative analysis of polymer sugar compounds. It gets rid of the current limitations of HPLC quantitative standards for polymer sugar compounds. This method has precise With the advantages of accuracy, good reproducibility, high efficiency and rapidity, it can achieve qualitative and quantitative analysis in any molecular weight range.
(2)相较于传统的经典方法如硫酸苯酚法等，本发明重在可用于评估不同方法制备糖化合物时的分子量差异以及精确计算某一分子量或分子量区间样品的浓度，解决了当样品与标准品分子量差距过大时用于定量分析不准确的问题，弥补了目前国标中多糖检测方法的不足，推动高分子糖类化合物在化学、生物、医药等领域的研究进展。(2) Compared with traditional classic methods such as the phenol sulfate method, the present invention focuses on evaluating the difference in molecular weight when preparing sugar compounds by different methods and accurately calculating the concentration of a sample with a certain molecular weight or molecular weight range. It solves the problem when the sample is different from the sample. The problem of inaccurate quantitative analysis when the molecular weight gap of the standard is too large makes up for the shortcomings of polysaccharide detection methods in the current national standards and promotes the research progress of polymer sugar compounds in the fields of chemistry, biology, medicine and other fields.
附图说明Description of the drawings
构成本发明的一部分的说明书附图用来提供对本申请的进一步理解，本发明的示意性实施例及其说明用于解释本发明，并不构成对本发明的不当限定。The description and drawings that constitute a part of the present invention are used to provide a further understanding of the present application. The illustrative embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute an improper limitation of the present invention.
图1为不同浓度下拟合曲线；Figure 1 shows the fitting curves at different concentrations;
图2为银耳多糖GPC色谱图；Figure 2 is the GPC chromatogram of Tremella polysaccharide;
图3为银耳多糖样品峰1的HPLC定量曲线；Figure 3 is the HPLC quantitative curve of Tremella polysaccharide sample peak 1;
图4为银耳多糖样品峰2的HPLC定量曲线；Figure 4 is the HPLC quantitative curve of peak 2 of Tremella polysaccharide sample;
图5为浒苔多糖GPC色谱图；Figure 5 is the GPC chromatogram of Enteromorpha Enteromorpha polysaccharide;
图6为浒苔多糖样品峰1的HPLC定量曲线。Figure 6 is the HPLC quantitative curve of Peak 1 of Enteromorpha polysaccharide sample.
具体实施方式Detailed ways
应该指出，以下详细说明都是例示性的，旨在对本发明提供进一步的说明。除非另有指明，本文使用的所有技术和科学术语具有与本发明所属技术领域的普通技术人员通常理解的相同含义。It should be noted that the following detailed description is illustrative and is intended to provide further explanation of the present invention. 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.
需要注意的是，这里所使用的术语仅是为了描述具体实施方式，而非意图限制根据本申请的示例性实施方式。如在这里所使用的，除非上下文另外明确指出，否则单数形式也意图包括复数形式，此外，还应当理解的是，当在本说明书中使用术语“包含”和/或“包括”时，其指明存在特征、步骤、操作、器件、组件和/或它们的组合。下面结合实施例对本发明进一步说明It should be noted that the terms used herein are only for describing specific embodiments and are not intended to limit the exemplary embodiments according to the present application. As used herein, the singular forms are also intended to include the plural forms unless the context clearly indicates otherwise. Furthermore, it will be understood that when the terms "comprises" and/or "includes" are used in this specification, they indicate There are features, steps, operations, means, components and/or combinations thereof. The present invention will be further described below in conjunction with the examples.
实施例1Example 1
此实施例详细为HPLC拟合曲线的建立过程。This embodiment details the establishment process of HPLC fitting curve.
将不同浓度下的12个不同分子量的标准品的色谱结果进行处理，借助分子量标准曲线得到logMw，后与响应值(Mv)进行特殊拟合，构建1.0mg/ml、0.8mg/ml、0.5mg/ml、0.2mg/ml、0.1mg/ml 5个不同浓度下的拟合曲线，用于不同分子量下定量曲线的建立(见图1)。The chromatographic results of 12 standards of different molecular weights at different concentrations were processed, logMw was obtained with the help of the molecular weight standard curve, and then specially fitted with the response value (Mv) to construct 1.0mg/ml, 0.8mg/ml, 0.5mg /ml, 0.2mg/ml, 0.1mg/ml 5 fitting curves at different concentrations, used to establish quantitative curves at different molecular weights (see Figure 1).
得到的拟合曲线的回归方程为y＝Ae-Bx，R2＝0.9236-0.9698。进一步，浓度为1.0mg/ml，A＝363.07，B＝0.349；浓度为0.8mg/ml，A＝295.18，B＝0.361；浓度为0.5mg/ml，A＝143.32，B＝0.334；浓度为0.2mg/ml，A＝65.347，B＝0.372；浓度为0.1mg/ml，A＝54.458，B＝0.485。The regression equation of the obtained fitting curve is y=Ae -Bx , R 2 =0.9236-0.9698. Further, the concentration is 1.0mg/ml, A=363.07, B=0.349; the concentration is 0.8mg/ml, A=295.18, B=0.361; the concentration is 0.5mg/ml, A=143.32, B=0.334; the concentration is 0.2 mg/ml, A=65.347, B=0.372; the concentration is 0.1mg/ml, A=54.458, B=0.485.
实施例2Example 2
此实施例详细以银耳多糖为例通过HPLC定量曲线的构建，进行样品的定量分析。In this example, Tremella polysaccharide is used as an example to construct a HPLC quantitative curve to conduct quantitative analysis of the sample.
银耳多糖样品用流动相超纯水溶解，测定银耳多糖样品的分子量分布及在各分子量下的具体浓度，采用HPLC-RID进行检测，分离柱为Shodex SB-804和806串联，流动相为超纯水,流速1ml/min，柱温为35℃，进样量为10μL；时间为30min。The Tremella polysaccharide sample was dissolved in ultrapure water in the mobile phase. The molecular weight distribution of the Tremella polysaccharide sample and the specific concentration at each molecular weight were measured. HPLC-RID was used for detection. The separation column was Shodex SB-804 and 806 in series, and the mobile phase was ultrapure. Water, flow rate 1ml/min, column temperature 35°C, injection volume 10μL; time 30min.
银耳多糖GPC色谱图如图2所示，以计算峰1和峰2为例，峰1的保留时间RT为8.768min，峰2的保留时间RT为13.439min，利用分子量标准曲线计算，峰1的logMw为7.63，计算Mw为42657951Da，峰2的logMw为6.15，计算Mw为1412537Da；通过实施例1中的不同浓度的拟合曲线分别构建峰1和峰2的对应分子量下的HPLC定量曲线(见图3和图4)，计算峰1的HPLC浓度为0.393mg/ml，峰2的HPLC浓度为0.133mg/ml。The GPC chromatogram of Tremella polysaccharide is shown in Figure 2. Taking the calculation of peak 1 and peak 2 as an example, the retention time RT of peak 1 is 8.768min, and the retention time RT of peak 2 is 13.439min. Calculated using the molecular weight standard curve, the retention time RT of peak 1 The logMw is 7.63, the calculated Mw is 42657951Da, the logMw of peak 2 is 6.15, and the calculated Mw is 1412537Da; the HPLC quantitative curves at the corresponding molecular weights of peak 1 and peak 2 are constructed through the fitting curves of different concentrations in Example 1 (see Figure 3 and Figure 4), the calculated HPLC concentration of peak 1 is 0.393 mg/ml, and the HPLC concentration of peak 2 is 0.133 mg/ml.
实施例3Example 3
此实施例浒苔多糖样品为例通过HPLC定量曲线的构建，进行样品的定量分析。In this embodiment, the Enteromorpha polysaccharide sample is taken as an example to perform quantitative analysis of the sample by constructing an HPLC quantitative curve.
浒苔多糖样品用流动相超纯水溶解，测定浒苔多糖样品的分子量分布及在各分子量下的具体浓度，采用HPLC-RID进行检测，分离柱为Shodex SB-804和806串联，流动相为超纯水，流速1ml/min，柱温为35℃，进样量为10μL；时间为50min。The Enteromorpha polysaccharide sample was dissolved in mobile phase ultrapure water, and the molecular weight distribution and specific concentration at each molecular weight of the Enteromorpha polysaccharide sample were determined. HPLC-RID was used for detection. The separation column was Shodex SB-804 and 806 in series, and the mobile phase was Ultrapure water, flow rate 1ml/min, column temperature 35°C, injection volume 10μL; time 50min.
浒苔多糖GPC色谱图如图5所示，以计算峰1为例，峰1的保留时间RT为9.1min，利用分子量标准曲线计算，峰1的logMw为7.51，Mw为32359365Da；通过实施例1中的不同浓度的拟合曲线构建峰1对应分子量下的HPLC定量曲线(见图6)，计算峰1的HPLC浓度为0.267mg/ml。The GPC chromatogram of Enteromorpha polysaccharide is shown in Figure 5. Taking the calculation of peak 1 as an example, the retention time RT of peak 1 is 9.1min. Calculated using the molecular weight standard curve, the logMw of peak 1 is 7.51 and the Mw is 32359365Da; through Example 1 The fitting curves of different concentrations in were used to construct the HPLC quantitative curve at the corresponding molecular weight of peak 1 (see Figure 6), and the HPLC concentration of peak 1 was calculated to be 0.267mg/ml.
以上所述仅为本发明的优选实施例而已，并不用于限制本发明，对于本领域的技术人员来说，本发明可以有各种更改和变化。凡在本发明的精神和原则之内，所作的任何修改、等同替换、改进等，均应包含在本发明的保护范围之内。The above descriptions are only preferred embodiments of the present invention and are not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and changes. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention shall be included in the protection scope of the present invention.
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