Size exclusion chromatography method for separating biomacromolecule of preparation type transverse electric field

A technology of biological macromolecules and transverse electric field, applied in the field of biological product processing and separation, can solve the problems of large cooling load, long distance, product loss, etc., and achieve the effect of avoiding high-voltage power supply, broad application prospects and reducing energy consumption

Inactive Publication Date: 2006-02-22
TIANJIN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
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AI Technical Summary

Problems solved by technology

The common disadvantages of this type of axial electric field electrochromatography are: (1) The distance between the two electrodes of axial electric field electrochromatography is long, and the applied voltage is high, resulting in serious Joule heating effect in the column and large cooling load
(2) During the operation, the continuous directional migration of positive and negative ions will cause the increase of opposite charges at the electrodes, which will lead to a decrease in the electric field strength in the column
(3) During the operation, a large amount of protein will be adsorbed on the separator of the opposite electrode, resulting in product loss

Method used

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  • Size exclusion chromatography method for separating biomacromolecule of preparation type transverse electric field
  • Size exclusion chromatography method for separating biomacromolecule of preparation type transverse electric field
  • Size exclusion chromatography method for separating biomacromolecule of preparation type transverse electric field

Examples

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Effect test

Embodiment 1

[0022] The size of the electrochromatographic gel chamber for separating bovine serum albumin and immunoglobulin G mixture is (length×width×depth) 20.0×0.5×1.2 cm, and the size of the electrode chamber is 20.1×0.8×0.8 cm. The mobile phase buffer was 3.9mmol / L Tris-47mmol / L Gly buffer (pH8.2) containing 5.0mmol / L sodium chloride. The protein mixture with a total concentration of 1.0mg / mL is prepared by mixing equal volumes of bovine serum albumin and immunoglobulin sample solutions with a concentration of 1.0mg / ml. All buffers were filtered through a 0.45 μm microporous membrane and degassed by ultrasonication for 15 minutes. After the gel chamber and the electrode chambers on both sides were equilibrated with buffer solution for 10 minutes, the Sephadex G-75 gel was filled into the gel chamber of the electrochromatographic column by gravity filling. Then, the medium in the gel chamber was equilibrated with the mobile phase buffer until the UV absorbance value (280 nm) reached...

Embodiment 2

[0024] The size of the electrochromatographic gel chamber for separating myoglobin and lysozyme mixture is (length×width×depth) 20.0×0.5×1.2 cm, and the size of the electrode chamber is 20.1×0.8×0.8 cm. The mobile phase buffer is 8.0mmol / L acetate buffer (pH4.9). A protein mixture with a total concentration of 1.0 mg / mL was prepared by mixing the same volume of the above single protein sample solutions. All buffers were filtered through a 0.45 μm microporous membrane and degassed by ultrasonication for 15 minutes. After the gel chamber and the electrode chambers on both sides were equilibrated with buffer solution for 10 minutes, the Sephadex G-75 gel was filled into the gel chamber of the electrochromatographic column by gravity filling. Then, the medium in the gel chamber was equilibrated with the mobile phase buffer until the UV absorbance value (280 nm) reached the baseline and stabilized around the baseline. The buffer flow rate was 0.2ml / min. On both sides of the gel ...

Embodiment 3

[0026] The size of the electrochromatographic gel chamber for separating bovine serum albumin and myoglobin mixture is (length×width×depth) 20.0×0.5×1.2 cm, and the size of the electrode chamber is 20.1×0.8×0.8 cm. The mobile phase buffer is 8.0mmol / L acetate buffer (pH4.9). A protein mixture with a total concentration of 1.0 mg / mL was prepared by mixing the same volume of the above single protein sample solutions. All buffers were filtered through a 0.45 μm microporous membrane and degassed by ultrasonication for 15 minutes. After the gel chamber and the electrode chambers on both sides were equilibrated with buffer solution for 10 minutes, the Sephadex G-75 gel was filled into the gel chamber of the electrochromatographic column by gravity filling. Then, the medium in the gel chamber was equilibrated with the mobile phase buffer until the UV absorbance value (280 nm) reached the baseline and stabilized around the baseline. The buffer flow rate was 0.2ml / min. On both sides...

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Abstract

The invention discloses a method for chromatographic separation of biomacromolecule such as protein by volume exclusion electrochromatogram and with an alternating electric field. Said preparative electrochromatogram separation device comprises of a gel chamber and the electrode chambers at double- side, the method for separation of biomacromolecule with this device being characterized in that: acetate, phosphonate, hydrogen carbonate, or trishydroxymethylaminomethane- hydrochloric acid buffer liquid is prepared as a mobile phase; gel fills the gel chamber by gravity filling method after the balance of mobile phase; the flow velocity of the mobile phase is 10- 200cm / h and the temperature is 2- 25Deg. C; iso- period alternating electric field with a voltage of 1- 600V and a current alternating period of 2- 200 seconds is stressed at the two sides of gel chamber, the flow velocity of electrode liquid is 20- 400cm / h and the temperature is 4- 25 Deg. C; and the specimen is added by means of impulse. Invented is characterized in that the separating power is strong, the handling capacity is large, the applicability extensive, and it has an extensive application prospect in separating and purifying the biomacromolecule such as protein and nucleic acid.

Description

technical field [0001] The invention relates to a chromatographic separation method for separating biological macromolecules such as proteins by using size exclusion electrochromatography under the action of an alternating electric field, and belongs to the technical field of biological product processing and separation. Background technique [0002] Among the various methods of biochemical analysis of biological macromolecules such as proteins and nucleic acids, electrophoresis is considered to be an analytical method with the highest resolution. However, electrophoresis is also the least amplifying technique. So far, the improvement from microgram-level analysis to gram-level preparation has not been realized. Chromatography is considered a technique second only to electrophoresis in its separation power, while still being a method of scale-up that works well. Among them, size exclusion chromatography is often used for the separation of...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): B01D15/08C07K1/16
Inventor 孙彦谭国民史清洪董晓燕白姝
Owner TIANJIN UNIV
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