CN1706536A - Electric chromatographic separation process of preparing biomass - Google Patents
Electric chromatographic separation process of preparing biomass Download PDFInfo
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- CN1706536A CN1706536A CN 200510025071 CN200510025071A CN1706536A CN 1706536 A CN1706536 A CN 1706536A CN 200510025071 CN200510025071 CN 200510025071 CN 200510025071 A CN200510025071 A CN 200510025071A CN 1706536 A CN1706536 A CN 1706536A
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Abstract
The electric chromatographic separation process for large scale preparation of biomass includes the following steps: 1. loading column of applying the processed fixing phase into electric chromatographic separating column; 2. balancing electric chromatographic separating column of injecting the column with electric chromatographic solution until obtaining the same pH value and composition of the buffering solution inflowing to and outflowing from the column; 3. applying the sample to be separated into the column; and 4. electric chromatographic separation through applying buffering solution to the column, turning on the high voltage power supply to the electrodes on ends of the column and collecting the effluent for analysis or further purification. The present invention has the advantages one high selectivity, high resolution, low cost, great capacity of sample processing and wide application range.
Description
Technical field
What the present invention relates to is a kind of separation method that is used for bio-science field, specifically is that a kind of electrochromatography separates the method for preparing biological substance.
Background technology
The capillary electrochromatography is to combine the advantage of high performance liquid chroma-tography and Capillary Electrophoresis and a kind of microanalysis technology of growing up, have efficient, fast, characteristics that amount of samples is few.The limitation of capillary electrochromatography is to be used for micro-analysis, can not be used for the preparation of sample.
At present, abroad some scholar is being prepared the research of type electrochromatography.Find that through literature search more typically preparation type electrochromatography method sees that Carlos M.Tellez and Kenneth D.Cole are published in the article Preparativeelectrochromatography of proteins in various types of porous media (the preparation type electrochromatography of protein in various types of porous medias) of 2000 the 21st volumes of Electrophoresis (electrophoresis) 1001-1009 page or leaf to prior art.The purpose of this document is from protein-based large biological molecule with preparation type electrochromatography technical point, used fixing is volume exclusion sephadex and Ago-Gel mutually, eluent is the buffer solution (pH8.3) that 3.9mmol/L Tris (trishydroxymethylaminomethane) and 47mmol/L glycine are formed, and separates α-breast and asks albumen, beta lactoglobulin, gamma globulin, bovine serum albumin(BSA).The deficiency of this method and defective are to be used for large biological molecules such as isolated protein, can not be used to separate small-molecule substance.
Summary of the invention
The present invention is directed to the deficiencies in the prior art and defective, propose a kind of electrochromatography and separate the method for preparing biological substance.By changing mutually fixing and buffer solution, separable as small-molecule substances such as antibiotic, organic acid, alkaloids, also separable as macromolecular substances such as protein, nucleic acid, applied widely.
The present invention is achieved by the following technical solutions: the present invention is with preparative scale electrochromatography method separating bio material, and concrete steps are as follows:
(1) dress post is with fixedly being added in the electrochromatography post of handling well;
(2) balance electrochromatography post injects buffer solution in the electrochromatography post, in entering post with flow out post outside buffer solution pH and form identical;
(3) application of sample, the sample that needs are separated adds in the electrochromatography post;
(4) electrochromatography adds buffer solution in the electrochromatography post, open the high voltage source that is connected with the electrode at post two ends, collects and flows out liquid, analyzes or is further purified.
Can load various fixedly phases in the described electrochromatography post.Fixedly the granular size of phase is 10-1000 μ m.Stationary phase volume is 1-4000ml.Described fixing be any of ion exchange resin, polymeric adsorbent, sephadex, Ago-Gel mutually.Used buffer solution is that inorganic salts or organic salt and acid, alkali are mixed with.Comprise phosphate, citrate, borate, acetate, Tris (trishydroxymethylaminomethane), amino acid.
Add ethanol, methyl alcohol, acetone water-miscible organic solvent in the buffer solution; The salt density scope is 0.01-0.50mol/L in the buffer solution, and the pH scope is 2-10, and organic solvent content is 0%-60%.
The described sample that needs to separate is water miscible ionic biologic, as antibiotic, alkaloid, organic acid, protein, nucleic acid etc.
Import department at the electrochromatography post adds the sample that needs separation, and the application of sample amount is 0.1-2000mg.
The present invention has the advantage that the chromatography selectivity is good, electrophoresis resolution ratio is high; Cost is low; Quantity of sample handling is big, can reach the milligram level to the gram level, can do preparation and use; Can adorn post by hand by the operator; By changing mutually fixing and buffer solution, can be used for separating macromolecular substances (as protein, nucleic acid), also can be used for separating small-molecule substance (as antibiotic, organic acid, alkaloid); Highly versatile, applied widely.The present invention uses for fields such as chemistry, chemical industry, biology, pharmacy, food, environmental protection, is used for the separation and purification and the sample preparation of laboratory biological substance.
The specific embodiment
Embodiment 1
Tetracycline and bleomycin use 0.06mol/L alanine-acetate buffer (pH3.0) to be formulated as 10mg/ml solution respectively, respectively get 25 μ l samples, mix the back sample introduction.Fixing is the MCI-Gel macroporous absorbent resin mutually, uses 0.06mol/L alanine-acetate buffer (pH3.0): and ethanol (V: V)=3: 1 wash-outs.Under the energising situation, tetracycline flows out earlier, flows out behind the bleomycin, and two kinds of materials obtain separating.If no power, by common chromatography operation, then tetracycline can not separate with bleomycin under the same conditions.
Embodiment 2
Cefazolin sodium, Cefotaxime Sodium, Ceftriaxone Sodium, cefotaxime, 5 kinds of antibiotic of Cefradine are mixed with the 50mg/ml aqueous solution respectively.5 kinds of antibiotic aqueous solution are respectively got 100 μ l, mix the back sample introduction.Fixing highly acid 1 * 2 type cationic ion-exchange resin, 0.1mol/L alanine-acetate buffer used mutually: ethanol (V: V)=40: 60 wash-outs, pH is elevated to 8.8 from 3.0 gradients.When electrochromatography, antibiotic flows out from post successively, obtains separating.
Embodiment 3
Purifies and separates ester catechin from the Tea Polyphenols crude extract (the L-Epigallo-catechin gallate (EGCG), L-EGCG).The Tea Polyphenols crude extract is mixed with the 40mg/ml aqueous solution, gets the 0.5ml sample introduction.Fixing is the MCI-Gel macroporous absorbent resin mutually, 0.02mol/L alanine-acetate buffer (pH 6.5): and ethanol (V: V)=80: 20 wash-outs.Electrochromatography is isolated two peaks, and the front is an ester catechin, and the back is an impurity.If no power, chromatography under similarity condition has only a peak to flow out, and ester catechin can not separate with impurity.
Embodiment 4
Sample is the mixture of 0.5ml bovine serum albumin(BSA) (50mg/ml) and 0.5ml BHb (20mg/ml).Fixing is DEAE-Sepahdex A-50 sephadex mutually, after 0.05mol/L Tris-HCl (pH8.3) balance, with 0.05mol/L Tris-HCl (pH8.3 contains 0.1mol/L NaCl) wash-out.Two kinds of albumen are separated into two peaks during energising.If no power, chromatography under similarity condition has only a peak to flow out, and two kinds of albumen are not separated.
Embodiment 5
Sample is the 20ml human serum.Fixing is DEAE-Sepahdex A-50 sephadex mutually, after 0.05mol/LTris-HCl (pH8.3 the contains 0.1mol/L NaCl) balance, with 0.05mol/L Tris-HCl (pH8.3 contains 0.2mol/L NaCl) wash-out.A peak surplus electrochromatography isolates 10.Detect albumen kind contained in each peak with polyacrylamide gel electrophoresis, each peak includes 1～3 kind of protein.If no power, make chromatography with same post, same fixedly phase and same sample size, buffer solution is 0.01mol/L Tris-HCl (pH8.3, NaCl concentration is increased to 0.7mol/L from the 0.2mol/L linearity), only isolate 5 peaks, each peak includes 1～5 kind of protein.
Claims (10)
1, a kind of electrochromatography separates the method for preparing biological substance, it is characterized in that with preparative scale electrochromatography method separating bio material, concrete steps are as follows:
(1) dress post is with fixedly being added in the electrochromatography post of handling well;
(2) balance electrochromatography post injects buffer solution in the electrochromatography post, in entering post with flow out post outside buffer solution pH and form identical;
(3) application of sample, the sample that needs are separated adds in the electrochromatography post;
(4) electrochromatography adds buffer solution in the electrochromatography post, open the high voltage source that is connected with the electrode at post two ends, collects and flows out liquid, analyzes or is further purified.
2, electrochromatography according to claim 1 separates the method for preparing biological substance, it is characterized in that the various fixedly phases of filling in the described electrochromatography post.
3, separate the method for preparing biological substance according to claim 1 or 2 described electrochromatographies, it is characterized in that the granular size of described fixedly phase is 10-1000 μ m, stationary phase volume is 1-4000ml.
4, separate the method that prepare biological substance according to claim 1 or 2 described electrochromatographies, it is characterized in that, described fixing is any of ion exchange resin, polymeric adsorbent, sephadex, Ago-Gel mutually.
5, electrochromatography according to claim 1 separates the method for preparing biological substance, it is characterized in that, described buffer solution is that inorganic salts or organic salt and acid, alkali are mixed with.
6, separate the method for preparing biological substance according to claim 1 or 5 described electrochromatographies, it is characterized in that described buffer solution comprises phosphate, citrate, borate, acetate, trishydroxymethylaminomethane, amino acid.
7, separate the method for preparing biological substance according to claim 1 or 5 described electrochromatographies, it is characterized in that described buffer solution adds ethanol, methyl alcohol, acetone water-miscible organic solvent in the buffer solution.
8, separate the method for preparing biological substance according to claim 1 or 5 described electrochromatographies, it is characterized in that, described buffer solution, the salt density scope is 0.01-0.50mol/L in the buffer solution, and the pH scope is 2-10, and organic solvent content is 0%-60%.
9, electrochromatography according to claim 1 separates the method for preparing biological substance, it is characterized in that, the described sample that needs separation is water miscible ionic biologic.
10, separate the method that prepare biological substance according to claim 1 or 9 described electrochromatographies, it is characterized in that, the described sample that separates of needing adds the sample that needs separation in the import department of electrochromatography post, and the application of sample amount is 0.1-2000mg.
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| CN 200510025071 CN1706536A (en) | 2005-04-14 | 2005-04-14 | Electric chromatographic separation process of preparing biomass |
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| CN 200510025071 CN1706536A (en) | 2005-04-14 | 2005-04-14 | Electric chromatographic separation process of preparing biomass |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101773521B (en) * | 2010-02-07 | 2012-05-30 | 董栗 | Method for purifying deer serum |
| CN104056468A (en) * | 2014-06-25 | 2014-09-24 | 上海交通大学 | Electrofocusing method for separating amphoteric matter without amphoteric electrolyte |
-
2005
- 2005-04-14 CN CN 200510025071 patent/CN1706536A/en active Pending
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101773521B (en) * | 2010-02-07 | 2012-05-30 | 董栗 | Method for purifying deer serum |
| CN104056468A (en) * | 2014-06-25 | 2014-09-24 | 上海交通大学 | Electrofocusing method for separating amphoteric matter without amphoteric electrolyte |
| CN104056468B (en) * | 2014-06-25 | 2016-01-13 | 上海交通大学 | Without the need to electrofocusing's method of the separation amphiprotic substance of ampholytes |
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