CN104056468A - Electrofocusing method for separating amphoteric matter without amphoteric electrolyte - Google Patents
Electrofocusing method for separating amphoteric matter without amphoteric electrolyte Download PDFInfo
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Abstract
The invention discloses an electrofocusing method for separating amphoteric matter without amphoteric electrolyte. The method comprises the following steps: filling anti-convection media into a chromatographic column; filling the chromatographic column longitudinally to prepare a buffer solution with gradually changing pH, so that the pH value changes gradually from an inlet to an outlet in the chromatographic column; adding a sample to the inlet, externally adding an electric field parallel to the flowing direction of a fluid in the chromatographic column, and electrofocusing; continuously adding a buffer solution with pH value termination, so that the pH gradient transfers gradually along with the buffer solution towards the outlet of the column, and eluting the sample focused at different pH parts out of the chromatographic column; collecting the buffer solution which flows out of the chromatographic column and enters an online detection and collecting device online. According to the invention, different pH buffer solutions are prepared by utilizing micromolecule organic acid and organic alkaline, and a pH gradient buffer solution band is prepared in the chromatographic column; an amphoteric matter is electrofocused by utilizing the externally added electric field, and a semi-preparation technique for separating and concentrating is provided for amphoteric polarity micromolecule matters.
Description
Technical field
The present invention relates to the high efficient separation technology of amphiprotic substance, be specifically related to a kind of electrofocusing's method of the separation amphiprotic substance without ampholytes.
Background technology
IEF is the electrophoretic techniques of the protein that a kind of pH of utilization gradient media separated PI of middle 1960s appearance is different.Due to 0.01 pH unit of its resolution, be particularly suitable for the close and protein component that pI is different of isolated molecule amount.IEF need to add special ampholytes in certain anti-convective media (as gel), sucks acid solution at positive terminal simultaneously, as sulfuric acid, phosphoric acid or acetic acid etc., introduces alkali lye, as NaOH solution, ammoniacal liquor etc. at negative pole end.In the time of extra electric field, electrolyte and both sexes sample start mobile, the ampholytes molecule that in mixture, pH is minimum, electronegative maximum, the fastest to anodal translational speed, in the time moving near anodal acid solution interface, pH declines suddenly, and this part no longer moves forward and rests in this region.The essential ampholytes that uses of these class methods because ampholytes has certain buffer capacity, make its around the pH in certain region remain on its isoelectric point scope.The second ampholytes that pH is slightly high, also shifts to positive pole, after being positioned the first ampholytes.Like this, after certain hour, the ampholytes with different pI is arranged in order by pI separately, has formed the gradient progressively rising to negative pole pH value from positive pole, and both sexes sample is also enriched in the region equating with its pI, thereby separates according to pI.The resolution ratio of this technology is high, and has concentrated effect, for purity analysis or the separation and purification of numerous protein sample.But can not carry out the online collection of sample, need to be by wash-out again after the gel band cutting that contains sample, complex operation, length consuming time, and ampholytes price is more expensive, therefore this method is not used widely, and approaches and be worth at present higher protein example mainly for separating of pI.Find by the retrieval to existing document, Chinese invention patent CN103235026A discloses a kind of method and device thereof of protein isoelectric focusing electrophoresis; That the method needs ampholytes to build district's band that pH changes, thereby realizes isoelectric focusing about the isoelectric focusing apparatus and method of point subsample greatly such as protein, nucleic acid; Sample finally focuses in anti-convective media, can not realize equally the online collection of sample.
Exploitation is efficient, stable, the preparative isolation technics of automation, sample wide coverage, is the key of biomedical product low cost, efficient industrialization.Increasing active small molecular polar substances is found, for example, and active small peptide, oligosaccharides, micromolecule nucleotide, organic acid.But, the preparation of these materials separates and has technological difficulties, these materials can not be by conventional liquid chromatogram separation and purification [Strege MA.Hydrophilic interaction chromatography-electrospray mass spectrometryanalysis of polar compounds for natural product drug discovery[J] .Anal Chem1998,70 (13): 2439-2445. because can not effectively retaining on reverse-phase chromatographic column]; [Jiang Z, Smith NW, Ferguson PD, et al.Hydrophilicinteraction chromatography using methacrylate-based monolithic capillarycolumn for the separation of polar analytes[J] .Anal Chem2007,79:1243-1250.]; [Strege MA, Stevenson S, Lawrence SM.Mixed-mode anion-cat ion exchange/hydrophilic interaction liquid chromatography-electrospray mass spectrometryas an alternative to reversed phase for smal l molecule drug discovery[J] .AnalChem2000,72:4629-4633].Although ion-exchange chromatography can meet the separation of ionic compound, but can not separate non-polar compound simultaneously, high salt eluent also increases desalination burden [JonesIL, Carta G.Ionexchange of amino acids and dipeptides on cation resins with varying degree ofcrosslinking.1.Equilibrium[J] .Ind Eng Chem Res1993,32:107-117].
Conventionally, very complicated sample, especially biological sample must be through separating and concentrating before analyzing.IEF can realize the separation of sample simultaneously and concentrate, and is very effective separation means, is mainly used at present in the separation of protein.But the essential ampholytes of introducing of IEF of existing maturation is made pH gradient, thereby is mixed in separated sample collection tube, causes matrix interference effect to analyzing.IEF technology is generally used for the separation preparation of protein, polypeptide sample, but, a lot of polar micromolecules medicines, for example, cephalosporins also has the feature of both sexes, if IEF is used for to relevant new drug development field, significant.
Summary of the invention
The object of the invention is to overcome above-mentioned the deficiencies in the prior art, a kind of electrofocusing's method of the separation amphiprotic substance without ampholytes is provided.The present invention utilizes simple small molecular organic acid, organic base to make different pH buffer solutions, and in conventional little molecule preparative chromatography post manual manufacture pH gradient buffering liquid band, the focusing that realizes amphiprotic substance by extra electric field separates, and provides a kind of simultaneously realization to separate and half concentrated technology of preparing for having the polar micromolecules material of both sexes; Especially can refining for this class sample.
The object of the invention is to be achieved through the following technical solutions:
A kind of electrofocusing's method that the present invention relates to separation amphiprotic substance without ampholytes, described method comprises the steps:
A, in chromatographic column, fill anti-convective media;
B, the buffer solution that longitudinally packing chromatography column production pH gradually changes, make to form pH value from the inlet to the outlet in chromatographic column and gradually change;
C, sample is added to chromatographic column porch, the additional electric field parallel with chromatographic column inner fluid flow direction, makes sample electrofocusing in post;
D, constantly add pH value continue change buffer solution, make pH gradient along with buffer solution flow move to column outlet gradually; The sample that focuses on different pH place is eluted to chromatographic column;
The buffer solution of E, outflow chromatographic column enters online detection and collector, realizes online and collecting.
Preferably, in steps A, described anti-convective media and sample room are faint without suction-operated or suction-operated.Thereby make sample mainly be subject to electric field force effect.
Preferably, described anti-convective media is chromatograph packing material; Described chromatograph packing material comprises sephadex particle or reverse-phase chromatography filler.
Preferably, in step B, it is to change continuously or phase change that described pH gradually changes; Described pH scope is chosen according to the isoelectric point scope of separated sample.
Preferably, the buffer solution that described pH gradually changes be by little molecule weak acid and weak base according to different proportionings, make different pH buffer, and add successively chromatographic column to form.
Preferably, described little molecule weak acid is acetic acid; Described little molecule weak base is ammoniacal liquor.
Preferably, in step C, extra electric field direction is by determining to the pH change direction of outlet from column inlet; If the pH value of column inlet is higher than column outlet, porch connects high voltage source negative pole, and exit connects high-voltage power cathode.Vice versa.Thereby in sample is slowly moved with buffer solution in chromatographic column, can focus on the buffer solution banded zone that pH is suitable with pI by hard to bear electric field action.
Preferably, in step D, if the pH of cushioning fluid of column inlet higher than column outlet, the pH value that described pH value continues the buffer solution changing is equal to or higher than the pH of cushioning fluid of column inlet; If the pH of cushioning fluid of column inlet is lower than column outlet, the pH value of the buffer solution that described pH value continuation changes is equal to or less than the pH of cushioning fluid of column inlet.The buffer solution that this pH value continues to change can be the buffer solution that pH gradually changes, and can be also single stop buffer.
Compared with prior art, the present invention has following beneficial effect:
(1) can use the buffer solution (for example, monobasic weak acid and weak monoacidic base solution) of the pre-configured a series of pH graded of simple soda acid of low concentration, replace ampholytes mixture essential in traditional isoelectric focussing.Although increased the operation of preparing in advance pH gradient, cost reduces greatly, also can reduce the operation of later separation ampholytes and sample, isoelectric focussing can be popularized.
(2) this method can realize online detection and collection, do not need separate finish after again cutting and elution samples, operating process is simple, consuming time short.
(3) this method can be used for Separation of Hydrophilic both sexes small-molecule substance, and general separation for macromolecular substances such as protein of conventional method.
Brief description of the drawings
By reading the detailed description of non-limiting example being done with reference to the following drawings, it is more obvious that other features, objects and advantages of the present invention will become:
Fig. 1 is isoelectric focusing electrophoresis separation principle schematic diagram;
Fig. 2 separates cynnematin mixture chromatogram for carry out isoelectric focusing electrophoresis by pH gradient.
Detailed description of the invention
Below in conjunction with specific embodiment, the present invention is described in detail.Following examples will contribute to those skilled in the art further to understand the present invention, but not limit in any form the present invention.It should be pointed out that to those skilled in the art, without departing from the inventive concept of the premise, can also make some distortion and improvement.These all belong to protection scope of the present invention.
In the present invention, amphiprotic substance: both containing acidic-group, containing the material of basic group, have isoelectric point again, in the buffer solution equating with isoelectric point in pH value, sample institute static electrification lotus is zero.Such as protein, amino acid, both sexes cynnematin etc.
Isoelectric focusing (IEF): be the high efficient separation technology of amphiprotic substance, its principle is to utilize ampholytes to create a pH gradient in gel utilizes protein to have that both sexes are dissociated and the feature of isoelectric point simultaneously, and sample is carried out to compartment analysis.IEF has the advantage such as enrichment that separative efficiency is high, realize sample in separating.The existence of pH gradient is the prerequisite of isoelectric focusing.
Operation principle of the present invention: first longitudinally chromatographic column (filling common anti-convective media) is made the buffer solution that pH gradually changes.Utilize separated material (both containing acidic-group, contain again the amphiprotic substance of basic group) opposite direction of suffered electric field force under the environment of pH < pI and pH > pI, separated material is drawn repeatedly, finally focus on the region of pH=pI, the separated material of different pI is separated and concentrated.Complete because focusing is separated on common chromatogram arrangement, can realize online detection and collect each component, complete quick preparation, and there is no high concentration ampholytes in the sample of collecting, be beneficial to follow-up Sample Purification on Single.
In chromatographic column before and after loading, the schematic diagram of pH variation and separation principle as shown in Figure 1.
Just (Figure 1A) when loading, sample is at bed face, and it is electronegative that pI is less than the sample of environment pH of living in, under electric field force effect, moves fast to post bottom; In the time moving to the pH band identical with pI (Figure 1B), mobile slowing down gradually; Move to if continue while being with lower than the pH of pI, positively charged, be subject to electric field force upwards, move to column inlet direction.And so forth, finally this sample is brought focusing on the pH identical with its pI.It is positively charged that when loading, (Figure 1A) pI is greater than the sample of environment pH of living in, is retained in chromatographic column bed face by electric field; Along with adding pH of cushioning fluid to increase, sample is reduced by electric field force gradually, until while adding pH of buffer to be equal to or greater than its pI (Figure 1B), enter chromatographic column with mobile phase.Concrete Application Example is as follows:
embodiment
Preparation cynnematin sample mixture, is dissolved in 0.2mL pure water by 2mg brizolina, 2mg cefotaxime, 3mg Cefotiam and 4mg Cefradine, centrifugal.Pack Ago-Gel Sepharose6B in electric chromatographic column (40cm × 1.2cm I.D.).Be mixed with the buffer solution of following different pH values with the acetic acid of 0.01mol/L and ammoniacal liquor, be pH=3.4, 4.4, 5.4, 6.4, 7.4, 8.4, 9.4 buffer solution, and with pH3.4 as initial buffer solution balance chromatographic column, the buffer solution that the pH that adds in turn each 7.5mL to prepare increases gradually, be pH4.4, 5.4, 6.4, 7.4, 8.4, then loading, add again the stop buffer of pH9.4, (column inlet arranges negative pole to add reversed electric field, outlet arranges positive pole) 60V/cm carries out electrofocusing's experiment, flow rate of mobile phase is controlled at 0.5mL/min, online detection wavelength is 254nm, the every 5mL of efflux collects one and manages and carry out HPLC and detect analysis.
Analysis result as shown in Figure 2.Result shows, the order separation that can increase successively according to isoelectric point the online both sexes cynnematin of collecting.Result can be with the concentration loading 11mg cynnematin blend sample of 1.1mg/mL in the chromatographic column of 40cm × 1.2cm I.D., in 200min, complete the separation of sample, the HPLC purity of the various cynnematins that obtain is all more than 92%, and yield is all more than 75%.Therefore, this buffer system can be used for analyzing the material that isoelectric point differs greatly.
Above specific embodiments of the invention are described.It will be appreciated that, the present invention is not limited to above-mentioned specific implementations, and those skilled in the art can make various distortion or amendment within the scope of the claims, and this does not affect flesh and blood of the present invention.
Claims (8)
1. without electrofocusing's method of the separation amphiprotic substance of ampholytes, it is characterized in that, described method comprises the steps:
A, in chromatographic column, fill anti-convective media;
B, the buffer solution that longitudinally packing chromatography column production pH gradually changes, make to form pH value from the inlet to the outlet in chromatographic column and gradually change;
C, sample is added to chromatographic column porch, the additional electric field parallel with chromatographic column inner fluid flow direction, makes sample electrofocusing in post;
D, constantly add pH value continue change buffer solution, make pH gradient along with buffer solution flow move to column outlet gradually; The sample that focuses on different pH place is eluted to chromatographic column;
The buffer solution of E, outflow chromatographic column enters online detection and collector, realizes online and collecting.
2. electrofocusing's method of the separation amphiprotic substance without ampholytes as claimed in claim 1, is characterized in that, in steps A, described anti-convective media and sample room are faint without suction-operated or suction-operated.
3. electrofocusing's method of the separation amphiprotic substance without ampholytes as claimed in claim 2, is characterized in that, described anti-convective media is chromatograph packing material; Described chromatograph packing material comprises sephadex particle or reverse-phase chromatography filler.
4. electrofocusing's method of the separation amphiprotic substance without ampholytes as claimed in claim 1, is characterized in that, in step B, it is to change continuously or phase change that described pH gradually changes; Described pH scope is chosen according to the isoelectric point scope of separated sample.
5. electrofocusing's method of the separation amphiprotic substance without ampholytes as claimed in claim 4, it is characterized in that, the buffer solution that described pH gradually changes be by little molecule weak acid and weak base according to different proportionings, make different pH buffer, and add successively chromatographic column to form.
6. electrofocusing's method of the separation amphiprotic substance without ampholytes as claimed in claim 5, is characterized in that, described little molecule weak acid is acetic acid; Described little molecule weak base is ammoniacal liquor.
7. electrofocusing's method of the separation amphiprotic substance without ampholytes as claimed in claim 1, is characterized in that, in step C, extra electric field direction is by determining to the pH change direction of outlet from column inlet; If the pH value of column inlet is higher than column outlet, porch connects high voltage source negative pole, and exit connects high-voltage power cathode; If the pH value of column inlet is lower than column outlet, porch connects high-voltage power cathode, and exit connects high voltage source negative pole.
8. electrofocusing's method of the separation amphiprotic substance without ampholytes as claimed in claim 1, it is characterized in that, in step D, if the pH of cushioning fluid of column inlet higher than column outlet, the pH value that described pH value continues the buffer solution changing is equal to or higher than the pH of cushioning fluid of column inlet; If the pH of cushioning fluid of column inlet is lower than column outlet, the pH value of the buffer solution that described pH value continuation changes is equal to or less than the pH of cushioning fluid of column inlet.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109270153A (en) * | 2018-11-28 | 2019-01-25 | 华东理工大学 | A kind of no ampholytes free flow isoelectric focusing electrophoresis separation method |
| CN113563412A (en) * | 2021-06-23 | 2021-10-29 | 上海蓝析生物技术有限公司 | Free-current isoelectric focusing electrophoresis buffer system |
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| CN1634642A (en) * | 2004-11-25 | 2005-07-06 | 上海交通大学 | Preparative electro-chromatography separation instrument for chemical and biological substance |
| CN1706536A (en) * | 2005-04-14 | 2005-12-14 | 上海交通大学 | Electric chromatographic separation process of preparing biomass |
| CN101294930A (en) * | 2007-04-27 | 2008-10-29 | 杨春 | Integral immobilization pH gradient production method and application thereof |
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Patent Citations (4)
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|---|---|---|---|---|
| US20030226752A1 (en) * | 2002-06-05 | 2003-12-11 | Gradipore Limited | Method for pH-biased isoelectric trapping separation |
| CN1634642A (en) * | 2004-11-25 | 2005-07-06 | 上海交通大学 | Preparative electro-chromatography separation instrument for chemical and biological substance |
| CN1706536A (en) * | 2005-04-14 | 2005-12-14 | 上海交通大学 | Electric chromatographic separation process of preparing biomass |
| CN101294930A (en) * | 2007-04-27 | 2008-10-29 | 杨春 | Integral immobilization pH gradient production method and application thereof |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109270153A (en) * | 2018-11-28 | 2019-01-25 | 华东理工大学 | A kind of no ampholytes free flow isoelectric focusing electrophoresis separation method |
| CN109270153B (en) * | 2018-11-28 | 2020-12-15 | 华东理工大学 | Non-amphoteric electrolyte free-flow isoelectric focusing electrophoresis separation method |
| CN113563412A (en) * | 2021-06-23 | 2021-10-29 | 上海蓝析生物技术有限公司 | Free-current isoelectric focusing electrophoresis buffer system |
| CN113563412B (en) * | 2021-06-23 | 2023-12-19 | 上海蓝析生物技术有限公司 | Free-flow isoelectric focusing electrophoresis buffer system |
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