CN110274972A - A method of series connection gel chromatography Dendrobium nobile polysaccharide molecular weight distribution - Google Patents
A method of series connection gel chromatography Dendrobium nobile polysaccharide molecular weight distribution Download PDFInfo
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- 229920001282 polysaccharide Polymers 0.000 title claims abstract description 87
- 239000005017 polysaccharide Substances 0.000 title claims abstract description 87
- 150000004676 glycans Chemical class 0.000 title claims abstract description 86
- 240000004638 Dendrobium nobile Species 0.000 title claims abstract description 56
- 238000000034 method Methods 0.000 title claims abstract description 35
- 238000005227 gel permeation chromatography Methods 0.000 title claims abstract description 11
- PXIPVTKHYLBLMZ-UHFFFAOYSA-N Sodium azide Chemical compound [Na+].[N-]=[N+]=[N-] PXIPVTKHYLBLMZ-UHFFFAOYSA-N 0.000 claims abstract description 47
- 239000007853 buffer solution Substances 0.000 claims abstract description 12
- 229910000162 sodium phosphate Inorganic materials 0.000 claims abstract description 12
- 239000001488 sodium phosphate Substances 0.000 claims abstract description 12
- RYFMWSXOAZQYPI-UHFFFAOYSA-K trisodium phosphate Chemical compound [Na+].[Na+].[Na+].[O-]P([O-])([O-])=O RYFMWSXOAZQYPI-UHFFFAOYSA-K 0.000 claims abstract description 12
- 239000000523 sample Substances 0.000 claims description 43
- 230000014759 maintenance of location Effects 0.000 claims description 14
- 239000012488 sample solution Substances 0.000 claims description 13
- 239000000243 solution Substances 0.000 claims description 11
- 229920001503 Glucan Polymers 0.000 claims description 10
- 238000004587 chromatography analysis Methods 0.000 claims description 8
- 239000012528 membrane Substances 0.000 claims description 7
- 239000012086 standard solution Substances 0.000 claims description 7
- 238000010790 dilution Methods 0.000 claims description 5
- 239000012895 dilution Substances 0.000 claims description 5
- 238000004090 dissolution Methods 0.000 claims description 5
- 238000004611 spectroscopical analysis Methods 0.000 claims description 3
- 239000004575 stone Substances 0.000 claims description 3
- -1 polysaccharide sodium azide Chemical class 0.000 claims 1
- 238000001514 detection method Methods 0.000 abstract description 12
- 238000000926 separation method Methods 0.000 abstract description 9
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 18
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 10
- 229910001868 water Inorganic materials 0.000 description 10
- 229920002307 Dextran Polymers 0.000 description 8
- 238000004458 analytical method Methods 0.000 description 8
- 235000011008 sodium phosphates Nutrition 0.000 description 8
- 239000007788 liquid Substances 0.000 description 7
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 6
- 230000000052 comparative effect Effects 0.000 description 6
- 229910000403 monosodium phosphate Inorganic materials 0.000 description 6
- 235000019799 monosodium phosphate Nutrition 0.000 description 6
- AJPJDKMHJJGVTQ-UHFFFAOYSA-M sodium dihydrogen phosphate Chemical compound [Na+].OP(O)([O-])=O AJPJDKMHJJGVTQ-UHFFFAOYSA-M 0.000 description 6
- 239000003153 chemical reaction reagent Substances 0.000 description 5
- 239000000843 powder Substances 0.000 description 5
- 238000002360 preparation method Methods 0.000 description 5
- 239000003814 drug Substances 0.000 description 4
- 238000002474 experimental method Methods 0.000 description 4
- 238000012360 testing method Methods 0.000 description 4
- 241001523681 Dendrobium Species 0.000 description 3
- 230000000694 effects Effects 0.000 description 3
- 235000019441 ethanol Nutrition 0.000 description 3
- 238000002270 exclusion chromatography Methods 0.000 description 3
- 239000000945 filler Substances 0.000 description 3
- 239000000463 material Substances 0.000 description 3
- 238000002156 mixing Methods 0.000 description 3
- 239000000047 product Substances 0.000 description 3
- 239000000126 substance Substances 0.000 description 3
- 229910021642 ultra pure water Inorganic materials 0.000 description 3
- 239000012498 ultrapure water Substances 0.000 description 3
- 239000003643 water by type Substances 0.000 description 3
- 239000008280 blood Substances 0.000 description 2
- 210000004369 blood Anatomy 0.000 description 2
- 238000009835 boiling Methods 0.000 description 2
- 238000002425 crystallisation Methods 0.000 description 2
- 230000008025 crystallization Effects 0.000 description 2
- 229940079593 drug Drugs 0.000 description 2
- 229960004756 ethanol Drugs 0.000 description 2
- 239000004744 fabric Substances 0.000 description 2
- 239000000706 filtrate Substances 0.000 description 2
- 238000005259 measurement Methods 0.000 description 2
- 229920000642 polymer Polymers 0.000 description 2
- 238000006116 polymerization reaction Methods 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 244000025254 Cannabis sativa Species 0.000 description 1
- RYAHJFGVOCZDEI-UFFNCVEVSA-N Dendrobine Chemical compound C([C@H]1CC[C@@H]2[C@@]31C)N(C)[C@@H]3[C@H]1[C@@H](C(C)C)[C@@H]2C(=O)O1 RYAHJFGVOCZDEI-UFFNCVEVSA-N 0.000 description 1
- DGAQECJNVWCQMB-PUAWFVPOSA-M Ilexoside XXIX Chemical compound C[C@@H]1CC[C@@]2(CC[C@@]3(C(=CC[C@H]4[C@]3(CC[C@@H]5[C@@]4(CC[C@@H](C5(C)C)OS(=O)(=O)[O-])C)C)[C@@H]2[C@]1(C)O)C)C(=O)O[C@H]6[C@@H]([C@H]([C@@H]([C@H](O6)CO)O)O)O.[Na+] DGAQECJNVWCQMB-PUAWFVPOSA-M 0.000 description 1
- 238000010521 absorption reaction Methods 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
- 230000033228 biological regulation Effects 0.000 description 1
- 238000011088 calibration curve Methods 0.000 description 1
- 239000003086 colorant Substances 0.000 description 1
- 238000012937 correction Methods 0.000 description 1
- 230000007812 deficiency Effects 0.000 description 1
- 229960000935 dehydrated alcohol Drugs 0.000 description 1
- RYAHJFGVOCZDEI-CZKZLRAZSA-N dendrobine Natural products O=C1O[C@@H]2[C@H](C(C)C)[C@H]1[C@H]1[C@@]3(C)[C@@H]2N(C)C[C@H]3CC1 RYAHJFGVOCZDEI-CZKZLRAZSA-N 0.000 description 1
- CBMPTFJVXNIWHP-UHFFFAOYSA-L disodium;hydrogen phosphate;2-hydroxypropane-1,2,3-tricarboxylic acid Chemical group [Na+].[Na+].OP([O-])([O-])=O.OC(=O)CC(O)(C(O)=O)CC(O)=O CBMPTFJVXNIWHP-UHFFFAOYSA-L 0.000 description 1
- 230000009881 electrostatic interaction Effects 0.000 description 1
- 230000007717 exclusion Effects 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 238000004108 freeze drying Methods 0.000 description 1
- 230000001900 immune effect Effects 0.000 description 1
- 238000011835 investigation Methods 0.000 description 1
- 229920002521 macromolecule Polymers 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 230000001151 other effect Effects 0.000 description 1
- 239000008055 phosphate buffer solution Substances 0.000 description 1
- 229920003229 poly(methyl methacrylate) Polymers 0.000 description 1
- 239000004926 polymethyl methacrylate Substances 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 238000010298 pulverizing process Methods 0.000 description 1
- 230000001603 reducing effect Effects 0.000 description 1
- 239000013049 sediment Substances 0.000 description 1
- 150000003384 small molecules Chemical class 0.000 description 1
- 229910052708 sodium Inorganic materials 0.000 description 1
- 239000011734 sodium Substances 0.000 description 1
- 230000003595 spectral effect Effects 0.000 description 1
- 238000001228 spectrum Methods 0.000 description 1
- 201000002282 venous insufficiency Diseases 0.000 description 1
Classifications
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- G—PHYSICS
- 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
-
- G—PHYSICS
- 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
- G01N30/06—Preparation
-
- G—PHYSICS
- 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
- G01N30/26—Conditioning of the fluid carrier; Flow patterns
- G01N30/28—Control of physical parameters of the fluid carrier
- G01N30/34—Control of physical parameters of the fluid carrier of fluid composition, e.g. gradient
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- G01N30/48—
-
- G—PHYSICS
- 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
- G01N30/60—Construction of the column
- G01N30/6034—Construction of the column joining multiple columns
- G01N30/6039—Construction of the column joining multiple columns in series
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- G—PHYSICS
- 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
- G01N30/62—Detectors specially adapted therefor
- G01N30/74—Optical detectors
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
- B01J20/281—Sorbents specially adapted for preparative, analytical or investigative chromatography
- B01J20/291—Gel sorbents
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- G—PHYSICS
- 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
- G01N2030/022—Column chromatography characterised by the kind of separation mechanism
- G01N2030/027—Liquid chromatography
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- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Immunology (AREA)
- Pathology (AREA)
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- Polysaccharides And Polysaccharide Derivatives (AREA)
Abstract
The invention discloses a kind of methods of gel chromatography Dendrobium nobile polysaccharide molecular weight distribution of connecting, using concatenated 500 column of Ultrahydrogel and 120 column of Ultrahydrogel as chromatographic column, using the buffer solution of sodium phosphate of 0.01 ~ 0.1mol/L of the sodium azide Han 0.01 ~ 0.05wt% as mobile phase.The present invention is capable of the separation of convenient, the quick Dendrobium nobile polysaccharide for realizing different molecular weight, improves the accuracy of Dendrobium nobile polysaccharide molecular weight distribution by the selection of chromatographic column and mobile phase.Verified, the method for the present invention is compared with using the method for single chromatographic column or other kinds of Coupled columns, and peak shape is good, result reproducibility is high, separating degree is good, accuracy is high, molecular weight detection range is wide, has general applicability.
Description
Technical field
The present invention relates to a kind of methods for measuring Dendrobium nobile polysaccharide molecular weight distribution, and in particular to a kind of series connection gel color
The method of spectrometry measurement Dendrobium nobile polysaccharide molecular weight distribution.
Background technique
HERBA DENDROBII is China's tradition rare Chinese medicine, is orchid family Dendrobium Sw also known as flat Jin Chai, golden hairpin stone, flat yellow grass
Deng, be traditional Chinese medicine dendrobium nobile one of source, recorded by Chinese Pharmacopoeia.The principle active component of HERBA DENDROBII be dendrobine and
Polysaccharide has nourishing Yin and clearing heat, immunological regulation, anti-oxidant, antitumor, reducing blood sugar and blood fat and other effects.Dendrobium nobile polysaccharide is with it
Unique effect is clinically with a wide range of applications.
However, in the research process of exploitation Dendrobium nobile polysaccharide, the molecular weight and molecular weight distribution of Dendrobium nobile polysaccharide
There is no the detection method of standard, so that the standard that Dendrobium nobile polysaccharide drug is not unified, makes the application and popularization of the drug
At greatly hindering, therefore, it is highly desirable to establish a set of accurate, stable Dendrobium nobile polysaccharide molecular weight and molecular weight point
Cloth detection method.
Summary of the invention
Polysaccharide is actually that a kind of chemical structure is similar and molecule mixture that extent of polymerization is different, molecular weight ranges across
More several orders of magnitude, exclusion chromatography are one of the polysaccharide molecular weight research methods being most widely used so far.For mesh
The preceding deficiency without Dendrobium nobile polysaccharide molecular weight and molecular weight distribution determination method, the present invention provides a kind of series connection gel colors
The method that spectrometry measures Dendrobium nobile polysaccharide molecular weight distribution, the method achieve efficiently dividing for Dendrobium nobile polysaccharide macromolecular
From, while effectively expanding molecular weight determination range, improve the detection of Dendrobium nobile polysaccharide molecular weight distribution accuracy and
Convenience.
The separating mechanism of exclusion chromatography be using volume exclude principle, polysaccharide during being eluted on gel column,
The polysaccharide and retention time of different molecular weight size have certain relationship, and the present invention first uses a series of glucan of known molecular amounts
For standard items, standard items and retention time correction are obtained into standard curve, then average molecular is calculated by the retention time of sample
Amount.Since the chemical structure of Dendrobium nobile polysaccharide is complicated, in the selection of gel chromatographic columns, the present invention is by two specific chromatographies
Column is together in series, and expands the detectable molecular weight ranges of Dendrobium nobile polysaccharide, to cover increasingly complex sample analysis, and color
Spectrum column series connection also improves the separation of the Dendrobium nobile polysaccharide of different molecular weight, improves detection effect.
Specific technical solution of the present invention is as follows:
A method of series connection gel chromatography Dendrobium nobile polysaccharide molecular weight distribution, using gel chromatography Jin Chai
The molecular weight of dendrobium polysaccharide, chromatographic condition are as follows:
Chromatographic column: concatenated 500 column of Ultrahydrogel and 120 column of Ultrahydrogel；
Detector: differential refraction detector；
Mobile phase: the buffer solution of sodium phosphate of 0.01 ~ 0.1mol/L of the sodium azide Han 0.01 ~ 0.05wt%；
Flow velocity: 0.6 ~ 1mL/min；
Column temperature: 40 ~ 60 DEG C；
Detector temperature: 30 ~ 45 DEG C；
Sample volume: 5 ~ 10 μ L.
Further, 500 column of Ultrahydrogel and 120 column of Ultrahydrogel are connected in series,
Sample and mobile phase first flow into 500 column of Ultrahydrogel, then flow into 120 column of Ultrahydrogel, Ultrahydrogel
500 columns first separate macromolecule component, and small molecule component then separates in 120 column of Ultrahydrogel.Wherein, chromatographic column
Specification are as follows: 300mm × 7.8mm, chromatographic column filler are hydroxylated polymethyl methacrylate.
Further, the pH of mobile phase is 6.5 ~ 7.5, preferably 7.
The present invention selects the buffer solution of sodium phosphate of 0.01 ~ 0.1mol/L of the sodium azide Han 0.01 ~ 0.05wt% as stream
Dynamic phase, when using the mobile phase, the peak shape of the Dendrobium nobile polysaccharide of different molecular weight is good, and separating degree is good, may be implemented different points
The appearance of the Dendrobium nobile polysaccharide of son amount distribution, accuracy in detection is high, this may be because phosphate buffer solution can be effectively
Inhibit absorption and electrostatic interaction of the chromatographic column filler to polysaccharide, to reach satisfactory separating effect.Mobile phase is preferably
The buffer solution of sodium phosphate of the 0.05mol/L of the sodium azide containing 0.02wt% of pH=7, when using the mobile phase, different molecular weight
Dendrobium nobile polysaccharide peak shape it is more preferable, separating degree more preferably, may be implemented the appearance of the Dendrobium nobile polysaccharide of more narrow molecular-weight, mention
High detection accuracy.
Further, it in the above method, using the glucan of different molecular weight as standard items, is first drawn by exclusion chromatography
Then the logarithm of molecular weight and the standard curve of retention time pass through the retention time meter of standard curve and Dendrobium nobile polysaccharide
Calculate the molecular weight of Dendrobium nobile polysaccharide.
Further, present invention preferably employs following detecting steps to be analyzed:
(1) solution is prepared
It is quiet after being sufficiently mixed uniformly then with flowing phase dilution by Dendrobium nobile polysaccharide aqueous sodium azide ultrasonic dissolution
2h is set, 0.45 μm of filter membrane is crossed, obtains sample solution；
By the glucan of different molecular weight flowing phased soln, 0.45 μm of filter membrane is crossed, standard solution is obtained.
(2) chromatographic condition
Chromatographic column: concatenated 500 column of Ultrahydrogel and 120 column of Ultrahydrogel, chromatographic column sequence are as follows: first connect
Ultrahydrogel 500, then meet Ultrahydrogel 120.
Detector: differential refraction detector；
Mobile phase: the buffer solution of sodium phosphate (pH=7) of the 0.05mol/L of the sodium azide containing 0.02wt%；
Flow velocity: 0.6 ~ 1mL/min；
Column temperature: 40 ~ 60 DEG C；
Detector temperature: 40 DEG C；
Sample volume: 10 μ L；
(3) standard curve is drawn
The standard solution of different molecular weight is subjected to chromatography, retention time is recorded, draws logarithm and the guarantor of molecular weight
Stay the standard curve of time；
(4) sample solution measures:
Sample solution is subjected to chromatography, obtains chromatogram, the golden hairpin stone under different retention times is calculated by standard curve
The molecular weight of dry measure used in former times polysaccharide obtains Dendrobium nobile polysaccharide molecular weight distribution.
Further, in above-mentioned steps (1), the concentration of glucan is 1.5 ~ 2.5mg/mL in standard solution.Sample is molten
The concentration of Dendrobium nobile polysaccharide is 1 ~ 3 mg/mL in liquid.When preparing sample solution, first using the folded of 0.01 ~ 0.05wt% of concentration
It nitrogenizes sodium solution and dissolves Dendrobium nobile polysaccharide, be then diluted to the sample solution of normal concentration with mobile phase again, it can also be direct
Sample is diluted to normal concentration with mobile phase.
Further, the group of the buffer solution of sodium phosphate becomes sodium dihydrogen phosphate, sodium hydroxide and water.
The present invention passes through 500 column of Ultrahydrogel and the series connection gel chromatographic columns of Ultrahydrogel 120 and stream
The selection of dynamic phase, realizes the measurement of Dendrobium nobile polysaccharide molecular weight and molecular weight distribution, under the chromatographic condition can it is convenient,
The separation for quickly realizing the Dendrobium nobile polysaccharide of different molecular weight realizes efficiently dividing for Dendrobium nobile polysaccharide different molecular weight
From improving the accuracy of Dendrobium nobile polysaccharide molecular weight distribution.Experiment proves that the method for the present invention and the single chromatographic column of use
Or the method for other kinds of Coupled columns is compared, peak shape is good, result reproducibility is high, separating degree is good, accuracy is high, molecular weight
Detection range is wide, has general applicability.
The Dendrobium nobile polysaccharide molecular weight and molecular weight distribution detection method simplicity, quick, data established in the present invention can
It leans on, can be produced for Dendrobium nobile polysaccharide from now on and the investigation of product stability provides foundation.
Detailed description of the invention
Fig. 1 is the dextran standards curve graph of the embodiment of the present invention 1.
Fig. 2 is the gel chromatography figure of the Dendrobium nobile polysaccharide sample of the embodiment of the present invention 1.
Fig. 3 is the gel chromatography figure of the branched polysaccharides sample of the wide molecular weight distribution of the embodiment of the present invention 2.
Fig. 4 is gel of the comparative example 1 of the present invention using the branched polysaccharides sample of the wide molecular weight distribution of other chromatogram column analysis
Chromatogram.
Specific embodiment
Below in conjunction with specific embodiment, technical scheme is described further.In following embodiments, such as without spy
Do not mentionlet alone bright, each concentration is mass percentage concentration.
Embodiment 1
1. instrument and equipment and reagent:
2695 high performance liquid chromatograph of Waters matches 2414 differential refraction detectors, 3 work station of Empower, gel chromatography
Column: Ultrahydrogel 120(300mm × 7.8mm) and Ultrahydrogel 500(300mm × 7.8mm) series connection.
Reagent: sodium dihydrogen phosphate；Sodium hydroxide；Sodium azide (analysis is pure)；Ultrapure water；Glucan serial standards
(Dextrans)；HERBA DENDROBII medicinal material.
2. experimental method
2.1 chromatographic condition
Chromatographic column are as follows: Ultrahydrogel 500(300mm × 7.8mm) and Ultrahydrogel 120(300mm × 7.8mm)
Series connection；
Mobile phase: the buffer solution of sodium phosphate (pH=7) of the 0.05mol/L of the sodium azide containing 0.02wt%, the mobile phase preparation side
Method are as follows: weigh sodium dihydrogen phosphate (without the crystallization water) 6g, add water 500ml to make to dissolve, separately take sodium azide 0.2g, add appropriate amount of water
Make to dissolve, after two liquid are mixed, 0.1mol/L sodium hydroxide solution added to adjust pH value to 7, be diluted with water to 1000ml to get.
Flow velocity: 0.6mL/min;
Column temperature: 40 DEG C；
Detector temperature: 40 DEG C；
Sample volume: 10 μ L.
The preparation of 2.2 Dendrobium nobile polysaccharide powder
It takes dry HERBA DENDROBII pulverizing medicinal materials to 10 mesh or so, weighs 2g, impregnate 2 h, ultrasonic wave with 30mL95wt% ethyl alcohol
20 min are extracted, are filtered, filtrate is collected；Residue adds 30 mL of 95wt% ethyl alcohol, 20 min of ultrasonic wave extraction again, filtering；Merge
Filtrate twice is concentrated under reduced pressure into 10 mL, is cooled to room temperature, and 30 mL boilings boiling is added to extract 3 times, and combined extract depressurizes dense
It is reduced to alcohol-free taste；The dehydrated alcohol of 3 times of volumes is added, 12h is precipitated, is centrifuged to obtain sediment, adds water sufficiently molten alcohol hypostasis
Powder is made in Xie Hou, freeze-drying.
The drafting of 2.3 standard curves
Take serial dextran standard, molecular weight Mp are as follows: 960,1460,7130,12900,43500,196000,401000；Point
Standard solution is not made with flowing phase dilution, concentration is controlled in 2mg/mL or so.By 2.1 chromatographic condition sample introductions, when sample introduction, sample
Product firstly flow through 500 column of Ultrahydrogel, again pass through 120 column of Ultrahydrogel, record chromatogram, soft in Empower3
The narrow ditribution molecular weight Mp that each standard items are inputted in part, using the logarithm of standard items narrow ditribution molecular weight as ordinate, corresponding color
The retention time of spectral peak is abscissa, is handled by software and draws standard curve, calibration curve equation logM = 11.639
Fig. 1 is seen in -0.307t, r=0.993.The retention time and logM of each molecular weight dextran standard items are as shown in table 1 below.
The preparation and analysis of 2.4 Dendrobium nobile polysaccharide test samples
Dendrobium nobile polysaccharide powder 10mg is weighed to be diluted to after 0.02wt% sodium azide solution ultrasonic dissolution with mobile phase
10mL stands 2h after mixing well, cross 0.45 μm of filter membrane, Dendrobium nobile polysaccharide test sample is obtained, by 2.1 chromatographic condition sample introductions, sample
Product firstly flow through 500 column of Ultrahydrogel, again pass through 120 column of Ultrahydrogel, obtain chromatogram.Pass through in software
Standard curve obtains the molecular weight and molecular weight distribution of dendrobium polysaccharide, as a result sees Fig. 2 and table 2.
Note: Mn is number-average molecular weight, and Mw is weight average molecular weight, and Mp is peak molecular weight, and Mz is viscosity average molecular weigh, Mw/
Mn is molecular weight polydispersity coefficient.
Average molecular weight and molecular weight distribution are that polysaccharide is most basic, are also one of most important parameter.To low molecule chemical combination
For object, molecular weight is a determining value, but for the polymer such as polysaccharide, due to degree of polymerization difference, strand length
Different, molecular size range is just different, and the ratio distribution of different molecular weight polysaccharide is also critically important, and polysaccharide may be because of its molecular weight point
Cloth is different and the difference of property occurs.Molecular weight distribution is indicated with polydispersity coefficient, the system of narrow molecular weight distribution
It (1.01 ~ 1.1) is monodisperse system, molecular weight distribution > 1.6 or 1 remoter system of deviation are polydisperse system.Calculated result
It has been shown that, the weight average molecular weight Mw of Dendrobium nobile polysaccharide are 1.43 × 105, molecular weight polydispersity coefficient is 2.146, is distributed band
It is wider.
The examination of 2.5 precision
According to above-mentioned 2.4 6 parts of method parallel processing same Dendrobium nobile polysaccharide samples, gained molecular weight and molecualr weight distribution
It is shown in Table 3.
As can be seen from Table 3, the RSD of the number-average molecular weight of parallel 6 tests gained sample is 1.20%, Weight-average molecular
The RSD of amount is 3.04%, and the RSD of molecular weight polydispersity coefficient is 1.20%, illustrate HERBA DENDROBII molecular weight that this method measures and
Molecular weight distribution precision is preferable, and accuracy is high.
Embodiment 2
1. instrument and equipment and reagent:
2695 high performance liquid chromatograph of Waters matches 2414 differential refraction detectors, 3 work station of Empower, gel chromatography
Column: Ultrahydrogel 120(300mm × 7.8mm) and Ultrahydrogel 500(300mm × 7.8mm) series connection.
Reagent: sodium dihydrogen phosphate；Sodium hydroxide；Sodium azide (analysis is pure)；Ultrapure water；Glucan serial standards
(Dextran company)；The branched polysaccharides sample (Dextran company) of wide molecular weight distribution.
2. experimental method:
2.1 chromatographic conditions:
Chromatographic column are as follows: Ultrahydrogel 500(300mm × 7.8mm) and Ultrahydrogel 120(300mm × 7.8mm)
Series connection；
Mobile phase: the buffer solution of sodium phosphate (pH=7) of the 0.05mol/L of the sodium azide containing 0.02wt%, the mobile phase preparation side
Method are as follows: weigh sodium dihydrogen phosphate (without the crystallization water) 6g, add water 500ml to make to dissolve, separately take sodium azide 0.2g, add appropriate amount of water
Make to dissolve, after two liquid are mixed, 0.1mol/L sodium hydroxide solution added to adjust pH value to 7, be diluted with water to 1000ml to get.
Flow velocity: 0.6mL/min;
Column temperature: 40 DEG C；
Detector temperature: 40 DEG C；
Sample volume: 10 μ L.
2.2 weigh branched polysaccharides sample 10mg, respectively after 0.02wt% sodium azide solution ultrasonic dissolution, with flowing
Phase dilution stands 2h, crosses 0.45 μm of filter membrane, obtain sample solution to 10mL after mixing well.
2.3 take branched polysaccharides sample solution, and by 2.1 chromatographic condition sample introductions, sample firstly flows through Ultrahydrogel 500
Column again passes through 120 column of Ultrahydrogel, obtains chromatogram, as shown in Figure 3.Pass through the standard in embodiment 1 in software
Curve obtains the molecular weight and molecular weight distribution of polysaccharide, the results are shown in Table 4.
Note: Mn is number-average molecular weight, and Mw is weight average molecular weight, and Mp is peak molecular weight, and Mz is viscosity average molecular weigh, Mw/
Mn is polydispersity coefficient.
As seen from Figure 3, the molecular weight distribution of branched polysaccharides sample is wider, may be implemented under the chromatographic condition compared with
Good separation.This is because the separating ranges of 500 column of Ultrahydrogel are 5000 ~ 500,000, Ultrahydrogel, 120 column
Separating ranges be 400 ~ 5000, the series connection of this two root chromatogram column can detect the molecular weight of ten thousand range of 400-50, simultaneously
Enable the polysaccharide of different molecular weight ranges appearance under different retention times by the selection of mobile phase, peak shape is good, separates
Degree is high, realizes efficient complex sample analysis.
Embodiment 3
1. instrument and equipment and material:
2695 high performance liquid chromatograph of Waters matches 2414 differential refraction detectors, 3 work station of Empower, gel chromatography
Column: Ultrahydrogel 120(300mm × 7.8mm) and Ultrahydrogel 500(300mm × 7.8mm) series connection.
Reagent: sodium dihydrogen phosphate；Sodium hydroxide；Sodium azide (analysis is pure)；Ultrapure water；Glucan serial standards；Certain
The Dendrobium nobile polysaccharide powder sample that drugmaker provides.
2. experimental method:
2.1 chromatographic conditions:
Chromatographic column are as follows: Ultrahydrogel 500(300mm × 7.8mm) and Ultrahydrogel 120(300mm × 7.8mm)
Series connection；
Mobile phase: the buffer solution of sodium phosphate (pH=7) of the 0.05mol/L of the sodium azide containing 0.02wt%, preparation method is the same as implementation
Example 1；
Flow velocity: 0.8mL/min or 0.6mL/min;
Column temperature: 40 DEG C；
Detector temperature: 40 DEG C；
Sample volume: 10 μ L.
2.2 weigh Dendrobium nobile polysaccharide powder sample 20mg, after 0.02wt% sodium azide solution ultrasonic dissolution, use
Mobile phase is diluted to 10mL, and 2h is stood after mixing well, and crosses 0.45 μm of filter membrane, obtains sample solution.
The drafting of 2.3 standard curves: serial dextran standard, narrow ditribution molecular weight Mp are taken are as follows: 960,146,7130,
12900,43500,196000,401000；The standard items that 2mg/mL is made with flowing phase dilution respectively in dextran standard are molten
Then liquid carries out chromatography according to 2.1 chromatographic condition.When sample introduction, sample firstly flows through 500 column of Ultrahydrogel, then
120 column of Ultrahydrogel is flowed through, chromatogram is recorded, the narrow ditribution molecular weight of each standard items is inputted in Empower3 software
Mp is handled by software and is drawn standard curve.
2.4 take Dendrobium nobile polysaccharide sample solution, carry out chromatography, when sample introduction, sample according to 2.1 chromatographic condition
500 column of Ultrahydrogel is firstly flowed through, 120 column of Ultrahydrogel is again passed through, obtains chromatogram, pass through mark in software
Directrix curve obtains the molecular weight and molecular weight distribution of lower Dendrobium nobile polysaccharide different in flow rate, the results are shown in Table 5.
As can be seen from Table 5, in the case where other chromatographic conditions are constant, flow velocity is 0.8 mL/min and 0.6 mL/min
When the molecular weight analyzed and molecular weight distribution result it is undifferentiated, so the change of flow velocity will not cause analysis result
It influences.
Comparative example 1
The molecular weight and its molecular weight distribution of the branched polysaccharides sample of wide molecular weight distribution are detected according to the method for embodiment 2, no
With: chromatographic column used is TSK-G4000PWxl chromatographic column.The results show that TSK-G4000PWxl chromatographic column is only capable of
To a chromatographic peak, the separation of polysaccharides of four kinds of different molecular weights can not be come, accuracy in detection is low, sees Fig. 4.
Comparative example 2
The molecular weight and its molecular weight distribution of the branched polysaccharides sample of wide molecular weight distribution are detected according to the method for embodiment 2, no
It is concatenated TSK-G4000PWxl chromatographic column and Ultrahydrogel 500 with: chromatographic column used, when sample introduction, sample
TSK-G4000PWxl chromatographic column is firstly flowed through, 500 column of Ultrahydrogel is again passed through.The results show that being also only capable of obtaining one
Chromatographic peak can not come the separation of polysaccharides of four kinds of different molecular weights, and accuracy in detection is low.
Comparative example 3
The molecular weight and its molecular weight distribution of the branched polysaccharides sample of wide molecular weight distribution are detected according to the method for embodiment 2, no
With: chromatographic column used is concatenated Ultrahydrogel 2000 and Ultrahydrogel 500.Ultrahydrogel
The filler aperture of 2000 chromatographic columns is larger, its exclusion limit and permeation limit range is 50,000 ~ 5,000,000, is suitble to separation molecular weight
Bigger polysaccharide or polymer, the series connection of the two pillars are only capable of coming out the biggish several separation of polysaccharides of molecular weight, Wu Faquan
Part from, illustrate pillar aperture selection it is improper, cannot achieve the separation of relatively small molecular weight.
Comparative example 4
The molecular weight and its molecular weight distribution of the branched polysaccharides sample of wide molecular weight distribution are detected according to the method for embodiment 2, no
With: mobile phase is the aqueous sodium azide of 0.02wt%.The results show that these four polysaccharide also can appearance, but peak type
It is poor, and have the phenomenon that overlap of peaks and trailing phenomenon, so that testing result, there are biggish deviation, be unsatisfactory for accurately analyzing wants
It asks.
Comparative example 5
According to the molecular weight and its molecular weight distribution of the polysaccharide sample of the method detection different molecular weight of embodiment 2, the difference is that:
Mobile phase is disodium hydrogen phosphate-citric acid solution (pH=7) of the sodium azide containing 0.02wt%.The results show that these four are more
Sugar also can appearance, but peak type is poor, there is trailing phenomenon, is unsatisfactory for the requirement accurately analyzed.
Claims (9)
1. a kind of method for gel chromatography Dendrobium nobile polysaccharide molecular weight distribution of connecting, it is characterized in that: using gel color
Spectrometry measures the molecular weight of Dendrobium nobile polysaccharide, and chromatographic condition is as follows:
Chromatographic column: concatenated 500 column of Ultrahydrogel and 120 column of Ultrahydrogel；
Detector: differential refraction detector；
Mobile phase: the buffer solution of sodium phosphate of 0.01 ~ 0.1mol/L of the sodium azide Han 0.01 ~ 0.05wt%；
Flow velocity: 0.6 ~ 1mL/min；
Column temperature: 40 ~ 60 DEG C；
Detector temperature: 30 ~ 45 DEG C；
Sample volume: 5 ~ 10 μ L.
2. according to the method described in claim 1, it is characterized in that: 500 column of Ultrahydrogel and Ultrahydrogel 120
Column is connected in series, and sample and mobile phase first flow into 500 column of Ultrahydrogel, then flow into 120 column of Ultrahydrogel.
3. according to the method described in claim 1, it is characterized in that: the pH of mobile phase is 6.5 ~ 7.5, preferably 7.
4. method according to claim 1,2 or 3, it is characterized in that: mobile phase is the sodium azide containing 0.02wt%
The buffer solution of sodium phosphate of 0.05mol/L.
5. according to the method described in claim 1, it is characterized in that: passing through gel using the glucan of different molecular weight as standard items
Chromatography draws the logarithm of molecular weight and the standard curve of retention time, then passes through standard curve and Dendrobium nobile polysaccharide
The molecular weight of retention time calculating Dendrobium nobile polysaccharide.
6. method according to claim 1,2 or 3, it is characterized in that specifically includes the following steps:
(1) solution is prepared
By Dendrobium nobile polysaccharide sodium azide solution ultrasonic dissolution, then with flowing phase dilution, stood after being sufficiently mixed uniformly
2h crosses 0.45 μm of filter membrane, obtains sample solution；
By the glucan of different molecular weight flowing phased soln, 0.45 μm of filter membrane is crossed, standard solution is obtained；
(2) chromatographic condition
Chromatographic column: concatenated 500 column of Ultrahydrogel and 120 column of Ultrahydrogel, chromatographic column sequence are as follows: first connect
Ultrahydrogel 500, then meet Ultrahydrogel 120；
Detector: differential refraction detector；
Mobile phase: the buffer solution of sodium phosphate of the 0.05mol/L for the sodium azide containing 0.02wt% that pH is 7；
Flow velocity: 0.6 ~ 1mL/min；
Column temperature: 40 ~ 60 DEG C；
Detector temperature: 40 DEG C；
Sample volume: 10 μ L；
(3) standard curve is drawn
The standard solution of different molecular weight is subjected to chromatography, retention time is recorded, draws logarithm and the guarantor of molecular weight
Stay the standard curve of time；
(4) sample solution measures:
Sample solution is subjected to chromatography, obtains chromatogram, the golden hairpin stone under different retention times is calculated by standard curve
The molecular weight of dry measure used in former times polysaccharide obtains Dendrobium nobile polysaccharide molecular weight distribution.
7. according to the method described in claim 6, it is characterized in that: the concentration of glucan is 1.5 ~ 2.5mg/ in standard solution
mL。
8. according to the method described in claim 6, it is characterized in that: the concentration of Dendrobium nobile polysaccharide is 1 ~ 3 mg/ in sample solution
mL。
9. according to the method described in claim 6, it is characterized in that: molten using the aqueous sodium azide of 0.01 ~ 0.05wt% of concentration
Solve Dendrobium nobile polysaccharide sample.
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