CN109115907A - A kind of levan molecular weight determination and the method for separation preparation - Google Patents
A kind of levan molecular weight determination and the method for separation preparation Download PDFInfo
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- CN109115907A CN109115907A CN201810955625.1A CN201810955625A CN109115907A CN 109115907 A CN109115907 A CN 109115907A CN 201810955625 A CN201810955625 A CN 201810955625A CN 109115907 A CN109115907 A CN 109115907A
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- G—PHYSICS
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- 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
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- G01N30/02—Column chromatography
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Abstract
The invention belongs to polysaccharose substance molecular weight detection technical fields, and in particular to a kind of method for being levan (Levan) molecular weight determination and separation preparation.A kind of levan molecular weight determination and the method for separation preparation, the following steps are included: self-chambering GPC osmogels chromatographic column molecular weight detection optimization of parameter choice, isolating and purifying optimization of parameter choice, enzyme linked immunosorbent assay high throughput detection sugared content, the measurement of glucide molecular weight and separation preparation process.This method can quickly and easily isolate and purify the levan of different molecular weight, be especially suitable for the quick analysis measurement of levan molecular weight and prepared according to the separation that different molecular weight carries out levan.The present invention has the advantages that at low cost, easy to operate, accuracy is high, has good production application and researching value.
Description
Technical field
The invention belongs to polysaccharose substance molecular weight detection technical fields, and in particular to be a kind of levan molecule measuring
Fixed and separation preparation method.
Background technique
The molecular weight determination of polysaccharide is a more important job for studying polysaccharide properties, no matter grinding in polysaccharide properties
Study carefully, or it in terms of, the problem of being directed in terms of molecular weight, generally require the molecular weight for measuring it, thus
Measurement molecular weight becomes research and prepares one regular work of polysaccharide.
The measuring method for being commonly used for polysaccharide material molecular weight has: osmometry, Steam soak meter method, end group method, viscosity
Method, light scattering method, exclusion chromatography and be more than rate method etc..
Wherein (GPC) exclusion chromatography is also known as size exclusion chromatography, and measuring principle is point based on volume exclusion
It disembarks reason, by the stationary phase with molecular sieve property, for separating the substance of different molecular weight, and can be with analyzing molecules
Volume difference, the macromolecule homologue with identical chemical property.Without phase between sample molecule and stationary phase gel in the method
Mutual fixed function is separated fully according to molecular sieve principle.
(HPGPC) High Performance Gel Permeation Chromatography of present use for laboratory, chromatographic column used in the experiment cannot oneself dress
It fills out, is commodity column.Common commodity column has μ-Bondagel column system and TSK column system.It generally requires to increase resolution ratio by two
The chromatographic column combination of a difference separating ranges, this just increases more laboratory instrument expense.Efficient liquid is needed in addition
The instruments such as chromatography and Composition distribution, a whole set of instrument get off costly.
Another method that oneself can load pillar is tested for (HPGPC) High Performance Gel Permeation Chromatography relatively
Instrument expense is reduced, but filler used is mostly import, such as: the series filler such as Sephacyl, Sephadex, but price is equal
It is costly.
In addition, Phenol sulfuric acid procedure is more common in the polyoses content detection of self-chambering column method.Phenol Sulphuric acid colorimetry is surveyed
The principle for determining polysaccharide is that polysaccharide is first hydrolyzed into monosaccharide to be quickly generated alditol derivative and then and phenol again under concentrated sulfuric acid effect
Chromogenic reaction occurs.Most reaction systems are that 1ml phenol solution and 5ml phenol solution are reacted in test tube, then with taking
It is detected in cuvette with spectrophotometer in right amount.The method reaction agents useful for same has certain toxicity and risk, anti-adding
Answer reagent, in reaction process, reagent is relatively large to the toxic action of people in the cleaning that is finished after test tube.And point to have reached
It often collects many pipes from effect to collect, with spectrophotometer, test experience process is slower one by one, and error is larger.
And for most of factories and small-size laboratory, there is no enough hardware conditions to reach both the above inspection
The requirement of survey method.Therefore, it finds a kind of gel column packing of economical and efficient and develops a kind of high-throughput detection of safety and precise
System tool has very important significance.
The information disclosed in the background technology section is intended only to increase the understanding to general background of the invention, without answering
When being considered as recognizing or imply that the information constitutes the prior art already known to those of ordinary skill in the art in any form.
Summary of the invention
The purpose of the present invention is to solve the above problem, a kind of levan molecular weight determination and separation preparation are provided
Method.
Technical solution provided by the invention is as follows:
A kind of levan molecular weight determination and the method for separation preparation, include the following steps:
(1) running parameter and experimental implementation: 1) chromatographic column: self-chambering osmogels column is used；2) column temperature: 4-25 DEG C；3) it flows
Dynamic phase；4) flow velocity；5) sampling volume；6) it capture range: is collected using automatic collector with 1-2min/ pipe；
(2) it detects: being loaded manually based on phend-sulphuric acid, microplate reader OD490nmThe high-throughput detection sugar read
Content method, steps are as follows: take 200 μ l sample prepare liquids in EP pipe → be added 100 μ L6% phenol solutions after be rapidly added
500 μ L concentrated sulfuric acid solutions → cover EP pipe lid, gently concussion shakes up rear boiling water bath 6min → be placed in ice-water bath and cools off, and takes
Solution after 200 μ l reaction is in ELISA Plate microplate reader OD490nmIt is measured；
(3) formulation of glucose standard curve: the formulation of glucose standard curve is using method described in step (2)；
(4) molecular weight determination operates: being that guidance carries out molecular weight detection and separation with the characteristic parameter in above-mentioned steps (1)
Preparation: dress column → processing sample → loading → elution → collection → reagent adding detects → handles data and analyzes；
(5) isolate and purify process: dress column → processing sample → loading → elution → collection → freeze-drying → weighing calculates
The rate of recovery → molecular weight detection verifying, if not single symmetrical peak, then need to repeat loading separation.
Preferably, the filler of the self-chambering osmogels column in step (1) be high flow rate 6FF agarose microbeads, it is described from
Dress its exclusion limit range of osmogels column is 10-400kDa, and the column diameter of the self-chambering osmogels column is 1:15-1 than range:
20。
Preferably, the mobile phase in step (1) is the Na of distilled water, deionized water, 0.1%-2%2SO4Buffer or
The BP buffer of 0.1M.
Preferably, the sampling volume in step (1) is the 1-2% of column volume；Elution flow rate is 0.2-1mL/min.
Compared with prior art, the beneficial effects of the present invention are:
(1) this method can quickly and easily isolate and purify the levan of different molecular weight, be especially suitable for levan molecule
The quick analysis of amount is measured and is prepared according to the separation that different molecular weight carries out levan.
(2) the method for the present invention does not need expensive liquid chromatograph, the high and recyclable multiplicating benefit using cost performance
Particular fillers preparative separation column, process are easy, advantage of lower cost, good separating effect, filler long service life, both can be with
Qualitative and quantitative analysis micro-example can also have at low cost, operation side for separating preparation, the present invention by amplification system
Just, the high advantage of accuracy has good production application and researching value.
Detailed description of the invention
Fig. 1 is glucose standard curve；
Fig. 2 is quantitation curves；
Fig. 3 is levan (levan) molecular weight determination；
Fig. 4 is 22min molecular weight detection；
Fig. 5 is 46min molecular weight detection.
Specific embodiment
With reference to the accompanying drawing, specific embodiments of the present invention will be described in detail, it is to be understood that guarantor of the invention
Shield range is not limited by the specific implementation.
Unless otherwise explicitly stated, otherwise in entire disclosure and claims, term " includes " or its change
Changing such as "comprising" or " including " etc. will be understood to comprise stated element or component, and not exclude other members
Part or other component parts.
Embodiment 1:
A kind of method of phenolsulfuric acid colour developing enzyme linked immunosorbent assay high throughput detection sugared content, the formulation including standard curve, step
It is as follows:
(1) reagent: 6% phenol solution (weighs phenol, deionized water stirring is added, to fixed with volumetric flask after completely dissolution
Hold, be configured to 6% phenol solution), the concentrated sulfuric acid (reagent can not be placed for a long time)
(2) operating procedure: take 200 μ l sample prepare liquids in EP pipe → be added 100 μ L6% phenol solutions after be rapidly added
500 μ L concentrated sulfuric acid solutions → cover EP pipe lid, gently concussion shakes up rear boiling water bath 6min → be placed in ice-water bath and cools off, and takes
Solution after 200 μ l reaction is in ELISA Plate microplate reader OD490nmIt is measured.
(3) production of glucose standard curve
1. the preparation of glucose standards solution: accurately weighing the 0.1g glucose mark product that drying to constant weight in beaker, add
Enter buffer and stir to polysaccharide to be completely dissolved, is settled to 1000mL, obtains glucose standards solution.
2. taking a series of glucose standards solution (being shown in Table 1) of different volumes respectively, deionized water buffer is added and supplies
To 200 μ L, a series of prepare liquid of different glucose final concentrations is obtained.Using phenolsulfuric acid colour developing enzyme linked immunosorbent assay high throughput detection
Operating procedure in sugared content carries out carry out experimental implementation, obtains OD490nmDegree, then using absorbance as ordinate, with grape
Sugared mark product concentration is abscissa, draws standard curve.
1 glucose standards solution of table
As shown in Figure 1, obtaining calibration curve equation is y=0.0498x-0.005, R2=0.9993, it is linear good, it says
Bright the method for the present invention can carry out quantitative analysis.
Embodiment 2:The formulation of polysaccharide molecular weight mark song
1, column is filled
(1) all material and reagent is allowed to reach room temperature.Prepare buffer.Gel chromatography loading, balance and elution are only used
A kind of buffer of low salt concn.
(2) pillar for selecting a root long internal diameter 1cm long 40cm, takes the desired amount of gel (about column bed according to pillar size
1.15 times of volume), 20% ethyl alcohol is washed, is drained, is made into homogenate with buffer (in gel: buffer=3:1 ratio).
Homogenate supersonic wave cleaning machine makees 20min degassing process.
(3) fixed pillar, it is perpendicular to the ground.
(4) pillar upper end is opened, closing pillar lower end will be in column and pillar bottom end water or buffer soak and keep one
Segment liquid level (liquid level is slightly above filter membrane), must make bottom end bubble-free.
(5) it is disposably poured into column with glass bar guidance homogenate along column wall, pays attention to not making to generate bubble.Open outlet
Clip is held, efflux receives waste liquid cylinder.
(6) as the outflow of liquid constantly supplements glue into pillar, until glue bed deposits to ideal height, and shape
At horizontal glue plane.Glue bed 20cm is formed by sedimentation in test.
(7) lower outlet is closed, eluent 5cm is supplemented in column, pillar upper end is closed and is connect with constant flow pump and eluent.
(8) peristaltic pump is opened, 1.33 times of flow velocity of flow velocity flows through when buffer being allowed to use, and stablizes column bed.With slow
Fliud flushing balances pillar, stablizes to column bed.
2, it balances
Buffer is allowed to flow through pillar with certain flow rate, it is constant to efflux conductance and pH.It balances pillar and elutes 2-3 cylinder
Product.
3, loading
(1) sample solution prepares (2ml buffer is added in EP pipe and sufficiently dissolves for precise 0.02g sample), muddy
Sample to be centrifuged and filter after loading.
(2) medium to the separation of sample component is carried out by component molecular amount size, and molecular weight is big first to be flowed out.
(3) loading volume is about 1% (157 μ l) of column volume, and smaller separation is better.It carefully is loaded onto column bed surface, is protected
It is smooth to hold glue surface.
With a small amount of eluent rinse vial wall 3 times, chromatographic column outlet end is opened.
(4) chromatographic column top connection is connected, outlet is opened, passes through peristaltic pump (SHENCHEN Lab2015) coutroi velocity
0.5mL/min collects efflux with 2min/ pipe automatic collector (BSZ-100), samples one column volume of range.
(5) sample will sufficiently dissolve.By being centrifuged or filtering removal particulate matter.
(6) in chromatography process, using the eluent of degassing, stationary temperature is maintained, avoids introducing bubble in pillar.
(7) sample introduction process center pillar pressure cannot excessive (flow velocity is too fast), column bed is easy to appear slight crack tomography.
(8) before using a new sample, pillar is rebalanced with the eluent of a column volume.
(9) standard items sample introduction: T-20, T-200, T-500, T-1000, T-2000 distinguish 157 μ l of loading；
(10) automatic collector takes 0.2ml collection liquid after collecting, with phenolsulfuric acid colour developing enzyme linked immunosorbent assay high throughput detection
Polyoses content in each collecting pipe.
(11) qualitative analysis standard can be determined according to appearance time after standard items detection, or according to peak area out come really
Determine quantitative analysis standard
4, it elutes
It is eluted with buffer, keeps flow velocity, buffer composition constant in elution.
Sample detection is carried out to following mark product respectively, calculated through glucose mark song and MATLAB Software Integration is used to calculate peak face
Product obtains result such as table 2.
Table 2
As shown in Fig. 2, obtaining calibration curve equation is that y=-1224x+9.928, R2=0.9969 are linearly well said
Bright this method can carry out quantitative analysis.
Because of the macromolecular glucan mark product of not no very high purity, so quantitative and fixed being large molecular weight polysaccharides mark Qu Shiqi
Property analysis will appear deviation, if being only tentatively to judge polysaccharide molecular weight size and analysis, judge i.e. according to qualitative analysis mark song
It can.
Embodiment 3:Molecular weight detection is carried out by taking levan polysaccharide as an example
1, process: dress column → processing sample → loading → elution → collection → reagent adding detects → handles data and analyzes；
2, it weighs the dry polysaccharide 0.02g to constant weight and takes 157 μ l samples (loading 1%) after completely dissolution in 2mlEP pipe
Loading elutes (flow velocity 0.5ml/min, eluent ultrapure water), collects (2min/ pipe), reagent adding phenolsulfuric acid colour developing enzyme mark
Method high throughput detects polyoses content (every pipe take 200 μ l detect) in each collecting pipe, analysis data.
3, result
As shown in figure 3, calculating peak area qualitative analysis with MATLAB Software Integration, peak there are two levan levans is tested,
First appearance time is 2000kDa or so in 22min, molecular weight, accounts for about measured levan 54.17%, second appearance
Time is 20kDa or so in 46min, molecular weight, accounts for about measured levan 45.83%.
Embodiment 4:Isolating and purifying for polysaccharide is carried out by taking levan polysaccharide as an example
There is the not single situation of polysaccharide molecular weight of product for example 3, we isolate and purify polysaccharide.
1, process is as follows:
(1) isolate and purify: dress column → processing sample → loading → elution → collection → freeze-drying → weighing calculates recycling
Rate；
(2) molecular weight detection is verified: taking 22min and 46min to collect and the separation component being freeze-dried carries out
Molecular weight detection verifies separating effect, if not single symmetrical peak then needs to repeat loading separation.
2, experiment parameter:
(1) it isolates and purifies: at this time by the amplification of cylinder system (i.e. 1.6 × 32cm filling material), weighing the dry levan to constant weight
Polysaccharide 0.8g (i.e. 40% initial levan polysaccharide solution) in 2mlEP pipe is taken after completely dissolution on 2% (i.e. 1287 μ l) sample
Sample elutes (flow velocity 1ml/min, eluent ultrapure water), collects (1min/ pipe)；
(2) molecular weight detection is verified: weighing the dry levan polysaccharide (the 22nd pipe and 46 pipes) to after the isolating and purifying of constant weight
0.02g takes 157 μ l sample loadings after completely dissolution in 2mlEP pipe, elutes (flow velocity 0.5ml/min, eluent ultrapure water), receives
Collect (2min/ pipe), reagent adding detection (every pipe takes 200 μ l to detect), analysis data.
3, result is as follows:
(1) total collection of weighing to obtain after obtained every pipe sample is freeze-dried, total collection/separation will the rate of recovery: be collected
Applied sample amount=rate of recovery before purification.Calculated result is 93.08%.
(2) separating effect: 22min and 46min polysaccharide after taking separation respectively carry out molecular weight detection, such as Fig. 4-5
It is shown, it can obviously be observed by figure, Levan polysaccharide elution curve peak shape is single after isolating and purifying, and form is more symmetrical, explanation
The polysaccharide component of two different molecular weights is separated, and can reach the effect isolated and purified.
The aforementioned description to specific exemplary embodiment of the invention is in order to illustrate and illustration purpose.These descriptions
It is not wishing to limit the invention to disclosed precise forms, and it will be apparent that according to the above instruction, can much be changed
And variation.The purpose of selecting and describing the exemplary embodiment is that explaining specific principle of the invention and its actually answering
With so that those skilled in the art can be realized and utilize a variety of different exemplary implementation schemes of the invention and
Various chooses and changes.The scope of the present invention is intended to be limited by claims and its equivalents.
Claims (4)
1. a kind of levan molecular weight determination and the method for separation preparation, which comprises the steps of:
(1) running parameter and experimental implementation: 1) chromatographic column: self-chambering osmogels column is used；2) column temperature: 4-25 DEG C；3) mobile phase；
4) flow velocity；5) sampling volume；6) it capture range: is collected using automatic collector with 1-2min/ pipe；
(2) it detects: being loaded manually based on phend-sulphuric acid, microplate reader OD490nmThe high-throughput detection sugared content read
Method, steps are as follows: take 200 μ l sample prepare liquids in EP pipe → be added 100 μ L6% phenol solutions after be rapidly added 500 μ L
Concentrated sulfuric acid solution → cover EP pipe lid, gently concussion shakes up rear boiling water bath 6min → be placed in ice-water bath and cools off, and takes 200 μ l anti-
Solution after answering is in ELISA Plate microplate reader OD490nmIt is measured；
(3) formulation of glucose standard curve: the formulation of glucose standard curve is using method described in step (2)；
(4) molecular weight determination operates: being that guidance carries out molecular weight detection and separation system with the characteristic parameter in above-mentioned steps (1)
Standby: dress column → processing sample → loading → elution → collection → reagent adding detects → handles data and analyzes；
(5) isolate and purify process: dress column → processing sample → loading → elution → collection → freeze-drying → weighing calculates recycling
Rate → molecular weight detection verifying, if not single symmetrical peak, then need to repeat loading separation.
2. levan molecular weight determination according to claim 1 and the method for separation preparation, which is characterized in that step (1)
In self-chambering osmogels column filler be high flow rate 6FF agarose microbeads, described its exclusion limit model of self-chambering osmogels column
It encloses for 10-4000kDa, the column diameter of the self-chambering osmogels column is 1:15-1:20 than range.
3. levan molecular weight determination according to claim 1 and the method for separation preparation, which is characterized in that step (1)
In mobile phase be distilled water, deionized water, 0.1%-2% Na2SO4The BP buffer of buffer or 0.1M.
4. levan molecular weight determination according to claim 1 and the method for separation preparation, which is characterized in that step (1)
In sampling volume be column volume 1-2%；Elution flow rate is 0.2-1mL/min.
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