CN109991067A - A kind of accurate method for detecting the gastrodia elata polysaccharide radius of gyration and molecular weight distribution - Google Patents
A kind of accurate method for detecting the gastrodia elata polysaccharide radius of gyration and molecular weight distribution Download PDFInfo
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- 239000005017 polysaccharide Substances 0.000 title claims abstract description 120
- 150000004676 glycans Chemical class 0.000 title claims abstract description 118
- 229920001282 polysaccharide Polymers 0.000 title claims abstract description 117
- 241000305491 Gastrodia elata Species 0.000 title claims abstract description 102
- 238000009826 distribution Methods 0.000 title claims abstract description 60
- 238000000034 method Methods 0.000 title claims abstract description 39
- 238000000926 separation method Methods 0.000 claims abstract description 32
- 238000001514 detection method Methods 0.000 claims abstract description 27
- 238000005516 engineering process Methods 0.000 claims abstract description 25
- 238000000569 multi-angle light scattering Methods 0.000 claims abstract description 23
- 238000012512 characterization method Methods 0.000 claims abstract description 8
- 239000007864 aqueous solution Substances 0.000 claims description 23
- 238000013016 damping Methods 0.000 claims description 16
- 125000006850 spacer group Chemical group 0.000 claims description 16
- 238000010828 elution Methods 0.000 claims description 14
- 239000012530 fluid Substances 0.000 claims description 4
- PXIPVTKHYLBLMZ-UHFFFAOYSA-N sodium azide Substances [Na+].[N-]=[N+]=[N-] PXIPVTKHYLBLMZ-UHFFFAOYSA-N 0.000 claims description 4
- 238000004458 analytical method Methods 0.000 abstract description 5
- 230000002349 favourable effect Effects 0.000 abstract description 2
- 238000011065 in-situ storage Methods 0.000 abstract description 2
- 208000034628 Celiac artery compression syndrome Diseases 0.000 description 22
- 230000000052 comparative effect Effects 0.000 description 21
- 239000000243 solution Substances 0.000 description 12
- 238000001914 filtration Methods 0.000 description 6
- 238000005227 gel permeation chromatography Methods 0.000 description 5
- 239000012528 membrane Substances 0.000 description 5
- 238000012360 testing method Methods 0.000 description 5
- 229920002678 cellulose Polymers 0.000 description 3
- 239000001913 cellulose Substances 0.000 description 3
- 230000007423 decrease Effects 0.000 description 3
- 230000014759 maintenance of location Effects 0.000 description 3
- 239000004627 regenerated cellulose Substances 0.000 description 3
- 230000000694 effects Effects 0.000 description 2
- 239000007788 liquid Substances 0.000 description 2
- 238000005259 measurement Methods 0.000 description 2
- 238000003756 stirring Methods 0.000 description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 2
- 230000003712 anti-aging effect Effects 0.000 description 1
- 230000000259 anti-tumor effect Effects 0.000 description 1
- 239000003963 antioxidant agent Substances 0.000 description 1
- 230000003078 antioxidant effect Effects 0.000 description 1
- 235000006708 antioxidants Nutrition 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 238000001816 cooling Methods 0.000 description 1
- 238000004132 cross linking Methods 0.000 description 1
- 230000007717 exclusion Effects 0.000 description 1
- 239000004744 fabric Substances 0.000 description 1
- 239000002808 molecular sieve Substances 0.000 description 1
- 239000002245 particle Substances 0.000 description 1
- BULVZWIRKLYCBC-UHFFFAOYSA-N phorate Chemical compound CCOP(=S)(OCC)SCSCC BULVZWIRKLYCBC-UHFFFAOYSA-N 0.000 description 1
- 150000004804 polysaccharides Polymers 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 238000002203 pretreatment Methods 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 238000010008 shearing Methods 0.000 description 1
- URGAHOPLAPQHLN-UHFFFAOYSA-N sodium aluminosilicate Chemical compound [Na+].[Al+3].[O-][Si]([O-])=O.[O-][Si]([O-])=O URGAHOPLAPQHLN-UHFFFAOYSA-N 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
- G01N1/38—Diluting, dispersing or mixing samples
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
- G01N21/41—Refractivity; Phase-affecting properties, e.g. optical path length
- G01N21/4133—Refractometers, e.g. differential
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
- G01N21/47—Scattering, i.e. diffuse reflection
- G01N21/49—Scattering, i.e. diffuse reflection within a body or fluid
- G01N21/51—Scattering, i.e. diffuse reflection within a body or fluid inside a container, e.g. in an ampoule
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Abstract
The present invention provides a kind of accurate detection gastrodia elata polysaccharide radius of gyration and the methods of molecular weight distribution, it is using asymmetric field flow separation technology combination multi-angle laser light scattering detector and the information such as differential refraction detector separation characterization gastrodia elata polysaccharide and the radius of gyration and the molecular weight distribution that provide sample, separation characterization is being carried out to sample close under in-situ condition, it was found that and overcome the influence of existing steric hindrance during using asymmetric field flow separation technology detection gastrodia elata polysaccharide, obtain the more accurate radius of gyration and its molecular weight distribution, and result favorable reproducibility, reduce analysis time, improve detection efficiency, it is with a wide range of applications.
Description
Technical field
The present invention relates to a kind of detection methods of polysaccharide, concretely relate to a kind of accurate detection gastrodia elata polysaccharide revolution half
The method of diameter and molecular weight distribution.
Background technique
Gastrodia elata polysaccharide is one of Rhizoma Gastrodiae important component, has the bioactivity such as immune, anti-oxidant, anti-aging, antitumor.It grinds
Study carefully and shows gastrodia elata polysaccharide generally and have super high molecular weight and wider particle diameter distribution, the bioactivity of polysaccharide and its radius of gyration,
Molecular weight distribution etc. is closely related.Therefore, using a kind of point for efficiently detecting the gastrodia elata polysaccharide radius of gyration and molecular weight distribution
Analysis method is necessary the activity research of gastrodia elata polysaccharide.
Currently, the common method of characterization polysaccharide molecular weight distribution is gel permeation chromatography (Gel permeation
Chromatography, GPC), the separating mechanism based on volume exclusion passes through the fixation relative sample with molecular sieve property
It is separated.GPC applies in general to molecular weight ranges less than 107 The sample of g/mol (divides super high molecular weight polysaccharide sample
Son amount is greater than 107 G/mol), GPC need to carry out pre-treatment to sample, and shearing force present in separation process destroys the original knot of polysaccharide
Structure leads to not the molecular weight distribution for accurately measuring gastrodia elata polysaccharide.Therefore, develop a kind of super suitable for separation characterization polydispersity
The analysis method of the polysaccharide sample of high molecular weight is particularly significant.
Summary of the invention
It is an object of the invention to provide a kind of accurate detection gastrodia elata polysaccharide radius of gyration and the method for molecular weight distribution, with
It solves existing gastrodia elata polysaccharide detection method and destroys its original structure, can not accurately measure the gastrodia elata polysaccharide radius of gyration and molecular weight point
The problem of cloth.
The object of the present invention is achieved like this:
A kind of accurate method for detecting the gastrodia elata polysaccharide radius of gyration and molecular weight distribution, includes the following steps:
(a) gastrodia elata polysaccharide aqueous solution is prepared；
(b) using asymmetric field flow separation technology combination multi-angle laser light scattering detector and differential refraction detector (AF4-
MALS-dRI) come separate characterization gastrodia elata polysaccharide aqueous solution in the gastrodia elata polysaccharide radius of gyration and molecular weight distribution, wherein elution stream
Speed uses exponential damping cross-current flow velocity and initial flow rate for 0.8 ~ 1 mL/min, and half-life period is 1.5 ~ 2.5 min.
In step (a), the concentration of the gastrodia elata polysaccharide aqueous solution is 1 ~ 2 mg/mL；Preferably, the gastrodia elata polysaccharide is water-soluble
The concentration of liquid is 1 mg/mL.
Gastrodia elata polysaccharide aqueous solution is prepared using following methods: the gastrodia elata polysaccharide of specific quantity is mixed with pure water, needed for being made
The gastrodia elata polysaccharide aqueous solution of concentration；Preferably, it places it in 50 DEG C of water-baths, continuously stirs 2 h, natural cooling with 200 rpm
To room temperature, 1 mL is taken to be put into spare in 1.5 mL EP pipe.
In step (b), carrier fluid used includes NaNO when separation characterization3And NaN3Mixed aqueous solution；Specifically, the load
Liquid is the NaNO that concentration is 10 mM3Aqueous solution and 3 mM NaN3The mixed liquor of aqueous solution, pH 7.00.
Asymmetric field flow separation technology and multi-angle laser light scattering detector and differential refraction detector (AF4-MALS-
When dRI) being combined, spacer height used is 490 μm.
Asymmetric field flow separation technology and multi-angle laser light scattering detector and differential refraction detector (AF4-MALS-
When dRI) being combined, detector flow velocity is 1 mL/min.
Preferably, exponential damping cross-current flow velocity is down to 0.05 mL/min by 0.8 ~ 1 mL/min,；It is highly preferred that described
Initial flow rate is 0.9 mL/min, i.e., exponential damping cross-current flow velocity is to be down to 0.05 mL/min by 0.9 mL/min.
Preferably, the half-life period is 2 min；It is highly preferred that the cross-current flow rate detection program is as shown in table 1 below:
1 gastrodia elata polysaccharide field flow of table separates elution program
The present invention is using asymmetric field flow separation technology combination multi-angle laser light scattering detector and differential refraction detector point
From information such as the radius of gyration and the molecular weight distributions for characterizing gastrodia elata polysaccharide and providing sample, sample structure is avoided damage to, close
Separation characterization is carried out to sample under in-situ condition, while finding and overcoming detecting Rhizoma Gastrodiae using asymmetric field flow separation technology
The influence of existing steric hindrance, obtains the more accurate radius of gyration and its molecular weight distribution, and result during polysaccharide
Favorable reproducibility reduces analysis time, improves detection efficiency, is that one kind is mild, fast and efficiently gastrodia elata polysaccharide separation characterizes
Technology is with a wide range of applications.
Detailed description of the invention
Fig. 1 is using comparative example 1, the 2 methods detection gastrodia elata polysaccharide radius of gyrationR gDistribution map.
Fig. 2 is using comparative example 1,2 methods detection gastrodia elata polysaccharide molecular weightM wDistribution map.
Fig. 3 detects the gastrodia elata polysaccharide radius of gyration using comparative example 3,4 methodsR gLocal distribution figure, wherein embedded figure is pair
The complete AF4-dRI elution profile of ratio 3,4 methods detection gastrodia elata polysaccharide.
Fig. 4 detects gastrodia elata polysaccharide molecular weight using comparative example 3,4 methodsM wLocal distribution figure, wherein embedded figure is comparison
Example 3,4 methods detect the complete AF4-MALS elution profile of gastrodia elata polysaccharide.
Fig. 5 is using 5 ~ 6 method of comparative example and 1 ~ 3 method of the embodiment detection gastrodia elata polysaccharide radius of gyrationR gDistribution map.
Fig. 6 is using 5 ~ 6 method of comparative example and 1 ~ 3 method of embodiment detection gastrodia elata polysaccharide molecular weightM wDistribution map.
Fig. 7 is using 7 ~ 8 method of comparative example and embodiment 1,4 ~ 5 methods detection gastrodia elata polysaccharide radius of gyrationR gDistribution map.
Fig. 8 is using 7 ~ 8 method of comparative example and embodiment 1,4 ~ 5 methods detection gastrodia elata polysaccharide molecular weightM wDistribution map.
Fig. 9 is using embodiment 1, the 6 methods detection gastrodia elata polysaccharide radius of gyrationR gDistribution map.
Figure 10 is using embodiment 1,6 methods detection gastrodia elata polysaccharide molecular weightM wDistribution map.
Figure 11 is the gastrodia elata polysaccharide radius of gyration using the detection of 1 method of embodimentR gDistribution reappears figure.
Figure 12 is the gastrodia elata polysaccharide molecular weight using the detection of 1 method of embodimentM wDistribution reappears figure.
Specific embodiment
Below with reference to embodiment, the present invention is further elaborated, and following embodiments are only as explanation, not with any
Mode limits the scope of the invention.
The process and method being not described in detail in the following embodiments are conventional method well known in the art, institute in embodiment
It is to analyze pure or chemical pure and commercially available or prepared by method well known within the skill of those ordinarily skilled with reagent.Under
Stating embodiment realizes the purpose of the present invention.
Use following manner preparation concentration for 1 mg/mL gastrodia elata polysaccharide aqueous solution in following embodiments, comparative example: accurate
It weighs 3 mg of gastrodia elata polysaccharide to be placed in 20 mL sample bottles, 3 mL pure water is added, the gastrodia elata polysaccharide solution of 1 mg/mL is made；By its
It is placed in 50 DEG C of water-baths, continuously stirs 2 h with 200 rpm；Sample bottle is taken out from 50 DEG C of water-baths, cooled to room temperature.
1 mL is taken to be put into spare in 1.5 mL EP pipe.
Multi-angle laser light scattering detector and differential refraction detector (AF4- are combined using asymmetric field flow separation technology
When MALS-dRI), carrier fluid used is the NaNO that concentration is 10 mM3The NaN of aqueous solution and 3 mM3The mixed liquor of aqueous solution, pH
It is 7.00.
Comparative example 1
Multi-angle laser light scattering detector and differential refraction detector (AF4- are combined using asymmetric field flow separation technology
MALS-dRI the radius of gyration and molecular weight distribution of gastrodia elata polysaccharide) are measured, wherein spacer height is 350 μm, 1 mg/mL Rhizoma Gastrodiae
The sample volume of polysaccharide solution is 50 μ L, and detector flow velocity is 1 mL/min；Using constant cross-current flow velocity, 0.5 mL/min.
Comparative example 2
It is after 1 mg/mL gastrodia elata polysaccharide aqueous solution is cooled to room temperature, to pass through 0.45 μm of regenerated cellulose film syringe-type mistake by concentration
Filter filtering is collected into spare in 1.5 mL EP pipe.
Multi-angle laser light scattering detector and differential refraction detector (AF4- are combined using asymmetric field flow separation technology
MALS-dRI the radius of gyration and molecular weight distribution of gastrodia elata polysaccharide) are measured, wherein spacer height is 350 μm, again through 0.45 μm
The sample volume of the raw filtered 1 mg/mL gastrodia elata polysaccharide aqueous solution of cellulose membrane syringe filter disk is 50 μ L, detector flow velocity
For 1 mL/min；Using constant cross-current flow velocity, 0.5 mL/min.
1 ~ 2 gained testing result of comparative example is as shown in Figure 1 and 2.It can be seen from figure 1 that the radius of gyration of gastrodia elata polysaccharide is distributed model
It encloses: 95 ~ 143 nm；The range of molecular weight distributions of gastrodia elata polysaccharide: 4.9 × 107~2.5×108g/mol.It can be seen in fig. 2 that through
The radius of gyration distribution of 0.45 μm of filtered gastrodia elata polysaccharide: 85 ~ 150 nm；The molecular weight of filtered gastrodia elata polysaccharide
Distribution: 4.5 × 107~2.5×108g/mol.Gastrodia elata polysaccharide retention time before filteringt r Have at=20 ~ 23 min bright out
Aobvious eluting peak, and existed by the eluting peak of 0.45 μm of filtered gastrodia elata polysaccharidet r =25 min occur, unfiltered Rhizoma Gastrodiae
The gastrodia elata polysaccharide that polysaccharide is filtered than 0.45 μm is eluted out in advance, illustrates there is " space during AF4 separates gastrodia elata polysaccharide
Steric hindrance " phenomenon.
Comparative example 3
Multi-angle laser light scattering detector and differential refraction detector (AF4- are combined using asymmetric field flow separation technology
MALS-dRI the radius of gyration and molecular weight distribution of gastrodia elata polysaccharide) are measured, wherein spacer height is 350 μm, 1 mg/mL Rhizoma Gastrodiae
The sample volume of polysaccharide solution is 50 μ L, and detector flow velocity is 1 mL/min；Using linear attenuation cross-current flow velocity, originate with 2
ML/min runs 5 min, is then down to after 1 mL/min runs 8 min and is gradually decrease to 0 mL/min, total elution time 50
min。
Comparative example 4
It is after 1 mg/mL gastrodia elata polysaccharide aqueous solution is cooled to room temperature, to pass through 0.45 μm of regenerated cellulose film syringe-type mistake by concentration
Filter filtering is collected into spare in 1.5 mL EP pipe.
Multi-angle laser light scattering detector and differential refraction detector (AF4- are combined using asymmetric field flow separation technology
MALS-dRI the radius of gyration and molecular weight distribution of gastrodia elata polysaccharide) are measured, wherein spacer height is 350 μm, again through 0.45 μm
The sample volume of the raw filtered 1 mg/mL gastrodia elata polysaccharide aqueous solution of cellulose membrane syringe filter disk is 50 μ L, detector flow velocity
For 1 mL/min；Using linear attenuation cross-current flow velocity, starting runs 5 min with 2 mL/min, is then down to 1 mL/min fortune
0 mL/min, total 50 min of elution time are gradually decrease to after 8 min of row.
3 ~ 4 gained testing result of comparative example is as shown in Fig. 3 ~ 4.By Fig. 3 ~ 4 as it can be seen that with this elution mode, elution time
Too long, eluting peak is rearward；Also, the gastrodia elata polysaccharide retention time before filteringt r There is apparent eluting peak out at=40.4 min, and
Existed by the eluting peak of 0.45 μm of filtered gastrodia elata polysaccharidet r =42 min occur, and unfiltered gastrodia elata polysaccharide is than 0.45 μm
The gastrodia elata polysaccharide of filtering is eluted out in advance.In addition, the radius of gyration and molecular weight distribution of the gastrodia elata polysaccharide of measurement are presented obviously
" V-type ", illustrate there is apparent " steric hindrance " phenomenon under this elution mode.
Embodiment 1
Multi-angle laser light scattering detector and differential refraction detector (AF4- are combined using asymmetric field flow separation technology
MALS-dRI the radius of gyration and molecular weight distribution of gastrodia elata polysaccharide) are measured, wherein spacer height is 490 μm, 1 mg/mL Rhizoma Gastrodiae
The sample volume of polysaccharide solution is 50 μ L, and detector flow velocity is 1 mL/min；Using exponential damping cross-current flow velocity, by 0.9
ML/min is down to 0.05 mL/min, and half-life period is 2 min, and specific cross-current flow rate detection program is shown in Table 1.
1 gastrodia elata polysaccharide field flow of table separates elution program
Embodiment 2
Multi-angle laser light scattering detector and differential refraction detector (AF4- are combined using asymmetric field flow separation technology
MALS-dRI the radius of gyration and molecular weight distribution of gastrodia elata polysaccharide) are measured, wherein spacer height is 490 μm, 1 mg/mL Rhizoma Gastrodiae
The sample volume of polysaccharide solution is 50 μ L, and detector flow velocity is 1 mL/min；Using exponential damping cross-current flow velocity, by 0.8
ML/min is down to 0.05 mL/min, and half-life period is 2 min.
Comparative example 5
Multi-angle laser light scattering detector and differential refraction detector (AF4- are combined using asymmetric field flow separation technology
MALS-dRI the radius of gyration and molecular weight distribution of gastrodia elata polysaccharide) are measured, wherein spacer height is 490 μm, 1 mg/mL Rhizoma Gastrodiae
The sample volume of polysaccharide solution is 50 μ L, and detector flow velocity is 1 mL/min；Using exponential damping cross-current flow velocity, by 0.5
ML/min is down to 0.05 mL/min, and half-life period is 2 min.
Comparative example 6
Multi-angle laser light scattering detector and differential refraction detector (AF4- are combined using asymmetric field flow separation technology
MALS-dRI the radius of gyration and molecular weight distribution of gastrodia elata polysaccharide) are measured, wherein spacer height is 490 μm, 1 mg/mL Rhizoma Gastrodiae
The sample volume of polysaccharide solution is 50 μ L, and detector flow velocity is 1 mL/min；Using exponential damping cross-current flow velocity, by 1.2
ML/min is down to 0.05 mL/min, and half-life period is 2 min.
Embodiment 3
Multi-angle laser light scattering detector and differential refraction detector (AF4- are combined using asymmetric field flow separation technology
MALS-dRI the radius of gyration and molecular weight distribution of gastrodia elata polysaccharide) are measured, wherein spacer height is 490 μm, 1 mg/mL Rhizoma Gastrodiae
The sample volume of polysaccharide solution is 50 μ L, and detector flow velocity is 1 mL/min；Using exponential damping cross-current flow velocity, by 1 mL/
Min is down to 0.05 mL/min, and half-life period is 2 min.
Embodiment 1 ~ 3 and 5 ~ 6 gained testing result of comparative example are as shown in Fig. 5 ~ 6.By Fig. 5 ~ 6 as it can be seen that AF4-MALS-dRI
When initial flow rate is 0.8 ~ 1 mL/min under exponential damping intersects flow velocity, detection gastrodia elata polysaccharide can effectively shorten elution time
And reach good separating effect.When intersecting flow velocity is 0.5 mL/min, dRI signal is shown, gap peak and gastrodia elata polysaccharide
Main eluting peak separating degree is low；When intersecting flow velocity is 1.2 mL/min, the rate of recovery is reduced.This is because intersection flow velocity is larger,
Sample is increased by downward external force, makes gastrodia elata polysaccharide sample closer to AF4 filter membrane, it is caused to increase with filter membrane crosslinking probability
Greatly.
Embodiment 4
Multi-angle laser light scattering detector and differential refraction detector (AF4- are combined using asymmetric field flow separation technology
MALS-dRI the radius of gyration and molecular weight distribution of gastrodia elata polysaccharide) are measured, wherein spacer height is 490 μm, 1 mg/mL Rhizoma Gastrodiae
The sample volume of polysaccharide solution is 50 μ L, and detector flow velocity is 1 mL/min；Using exponential damping cross-current flow velocity, by 0.9
ML/min is down to 0.05 mL/min, and half-life period is 1.5 min.
Comparative example 7
Multi-angle laser light scattering detector and differential refraction detector (AF4- are combined using asymmetric field flow separation technology
MALS-dRI the radius of gyration and molecular weight distribution of gastrodia elata polysaccharide) are measured, wherein spacer height is 490 μm, 1 mg/mL Rhizoma Gastrodiae
The sample volume of polysaccharide solution is 50 μ L, and detector flow velocity is 1 mL/min；Using exponential damping cross-current flow velocity, by 0.9
ML/min is down to 0.05 mL/min, and half-life period is 1 min.
Comparative example 8
Multi-angle laser light scattering detector and differential refraction detector (AF4- are combined using asymmetric field flow separation technology
MALS-dRI the radius of gyration and molecular weight distribution of gastrodia elata polysaccharide) are measured, wherein spacer height is 490 μm, 1 mg/mL Rhizoma Gastrodiae
The sample volume of polysaccharide solution is 50 μ L, and detector flow velocity is 1 mL/min；Using exponential damping cross-current flow velocity, by 0.9
ML/min is down to 0.05 mL/min, and half-life period is 3 min.
Embodiment 5
Multi-angle laser light scattering detector and differential refraction detector (AF4- are combined using asymmetric field flow separation technology
MALS-dRI the radius of gyration and molecular weight distribution of gastrodia elata polysaccharide) are measured, wherein spacer height is 490 μm, 1 mg/mL Rhizoma Gastrodiae
The sample volume of polysaccharide solution is 50 μ L, and detector flow velocity is 1 mL/min；Using exponential damping cross-current flow velocity, by 0.9
ML/min is down to 0.05 mL/min, and half-life period is 2.5 min.
Embodiment 1,4 ~ 5 and 7 ~ 8 gained testing result of comparative example are as shown in Fig. 7 ~ 8.By Fig. 7 ~ 8 as it can be seen that when half-life period is 1
When min, gap peak decreases compared with other eluting peaks with the separating degree between the main eluting peak of gastrodia elata polysaccharide, when partly declining
When phase is 3 min, signal strength is reduced, and retention time increases.
Embodiment 6
It is after 1 mg/mL gastrodia elata polysaccharide aqueous solution is cooled to room temperature, to pass through 0.45 μm of regenerated cellulose film syringe-type mistake by concentration
Filter filtering is collected into spare in 1.5 mL EP pipe.
Multi-angle laser light scattering detector and differential refraction detector (AF4- are combined using asymmetric field flow separation technology
MALS-dRI the radius of gyration and molecular weight distribution of gastrodia elata polysaccharide) are measured, wherein spacer height is 490 μm, again through 0.45 μm
The sample volume of the raw filtered 1 mg/mL gastrodia elata polysaccharide aqueous solution of cellulose membrane syringe filter disk is 50 μ L, detector flow velocity
For 1 mL/min；Using exponential damping cross-current flow velocity, 0.05 mL/min is down to by 0.9 mL/min, half-life period is 2 min.
Embodiment 1,6 gained testing results are as shown in Fig. 9 ~ 10.It can be seen from the figure that unfiltered gastrodia elata polysaccharide retains
Timet r There is apparent eluting peak at=11 min, is also existed by the eluting peak of 0.45 μm of filtered gastrodia elata polysaccharidet r =11
Min occurs, and elution time is almost the same, and elution mode is the same, shows that gradient elution can solve analysis gastrodia elata polysaccharide process and go out
The problem of existing steric hindrance.
The radius of gyration distribution of lower the surveyed gastrodia elata polysaccharide of gradient cross stream flow conditions are as follows: 71 ~ 106 nm；Rhizoma Gastrodiae is more
The range of molecular weight distributions of sugar are as follows: 3.4 × 107~3.1×108g/mol；Time through 0.45 μm of filtered surveyed gastrodia elata polysaccharide
Turn radius distribution range are as follows: 71 ~ 101 nm；The range of molecular weight distributions of filtered gastrodia elata polysaccharide are as follows: 2.2 × 107~2.9×
108g/mol.The radius of gyration measured under gradient cross stream flow conditions is lower than constant intersection flow velocity, it may be possible to due to constant friendship
Distributary flow velocity elution time is longer, signal peak is lower and there is " steric hindrance " phenomenon, measurement result inaccuracy.
Embodiment 7
The gastrodia elata polysaccharide sample of embodiment 1 is analyzed through AF4-MALS-dRI, is resolved the reproducibility map of steric hindrance, institute
Result is obtained as shown in Figure 11 ~ 12.
From Figure 11 ~ 12 as can be seen that the radius of gyration distribution of gastrodia elata polysaccharide are as follows: 71 ~ 106 nm, point of gastrodia elata polysaccharide
Son amount distribution are as follows: 3.4 × 107~3.1×108Therefore g/mol, relative standard deviation 1.66% are based on AF4-MALS-
DRI, which detects gastrodia elata polysaccharide with this method, has preferable reproducibility.
Claims (7)
1. a kind of method of accurate detection gastrodia elata polysaccharide radius of gyration and molecular weight distribution, which comprises the steps of:
(a) gastrodia elata polysaccharide aqueous solution is prepared；
(b) multi-angle laser light scattering detector and differential refraction detector are combined using asymmetric field flow separation technology to separate
Characterize the gastrodia elata polysaccharide radius of gyration and molecular weight distribution in gastrodia elata polysaccharide aqueous solution, wherein elution flow rate uses exponential damping
Cross-current flow velocity and initial flow rate are 0.8 ~ 1 mL/min, and half-life period is 1.5 ~ 2.5 min.
2. the method for the accurate detection gastrodia elata polysaccharide radius of gyration according to claim 1 and molecular weight distribution, feature exist
In the concentration of the gastrodia elata polysaccharide aqueous solution is 1 ~ 2 mg/mL.
3. the method for the accurate detection gastrodia elata polysaccharide radius of gyration according to claim 1 and molecular weight distribution, feature exist
In in step (b), carrier fluid used includes NaNO when separation characterization3And NaN3Mixed aqueous solution.
4. the method for the accurate detection gastrodia elata polysaccharide radius of gyration according to claim 3 and molecular weight distribution, feature exist
In the carrier fluid is the NaNO that concentration is 10 mM3Aqueous solution and 3 mM NaN3The mixed liquor of aqueous solution, pH 7.00.
5. the method for the accurate detection gastrodia elata polysaccharide radius of gyration according to claim 1 and molecular weight distribution, feature exist
In in step (b), asymmetric field flow separation technology and multi-angle laser light scattering detector and differential refraction detector are combined
When, spacer height used is 490 μm.
6. the method for the accurate detection gastrodia elata polysaccharide radius of gyration according to claim 1 and molecular weight distribution, feature exist
In in step (b), detector flow velocity is 1 mL/min.
7. the method for the accurate detection gastrodia elata polysaccharide radius of gyration according to claim 1 and molecular weight distribution, feature exist
In in step (b), exponential damping cross-current flow velocity is down to 0.05 mL/min by 0.8 ~ 1 mL/min.
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