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Amphoteric dissociation ion exchange medium, application method and separation capacity calibration method

An ion exchange and separation medium technology, applied in the field of amphoteric dissociation ion exchange medium, to achieve the effect of high separation capacity, avoiding subsequent problems and high separation capacity

Active Publication Date: 2019-02-26
CHONGQING FARSIGHTED BLUE DRAGON FBD BIOTECH CO LTD CHINA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there is only one type of dissociation groups on the surface of classical ion exchangers, changing the pH cannot make the net charge on the surface of the separation medium and the net charge of the target substance change from opposite to the same, resulting in electrostatic repulsion

Method used

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  • Amphoteric dissociation ion exchange medium, application method and separation capacity calibration method
  • Amphoteric dissociation ion exchange medium, application method and separation capacity calibration method
  • Amphoteric dissociation ion exchange medium, application method and separation capacity calibration method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0112] Example 1: Preparation of representative structural forms of amphoteric dissociative ion exchange separation media

[0113] In the first step, prepare the following solution (filter with a 0.22um filter membrane to sterilize at the same time):

[0114] (1) Lysine solution: 100mM lysine was adjusted to pH 6.0 with 50mM hydrochloric acid, diluted with water to 30mM, and filtered for later use;

[0115] (2) Histidine solution: 50mM histidine solution, adjusted to pH 6.0 with 10mM hydrochloric acid, diluted to 30mM;

[0116] (3) Glycine solution: 100mM glycine solution was adjusted to pH 6.0 with dilute sodium bicarbonate, diluted with water to 30mM, and filtered for later use;

[0117] (4) N,N-dimethylethylenediamine solution: 10mM N,N-dimethylethylenediamine was adjusted to pH 6.0 with 0.10m hydrochloric acid, diluted to 10mM;

[0118] (5) Cysteine ​​solution: 50mM cysteine ​​aqueous solution, adjusted to pH 6.0 with 10mM sodium bicarbonate, diluted to 10mM, filtered fo...

Embodiment 2

[0137] Example 2: The operation process for measuring the adsorption and separation capacity of amphoteric dissociation ion exchange medium for acid red 13

[0138] The first step is to measure the influence of pH on the extinction coefficient of acid red 13; the results are shown in Figure 5 . The extinction coefficient of acid red 13 has no change between pH 4.0 and 8.9, both are 18.0 (mM) -1 .cm -1 ; The following acid red 13 all use this extinction coefficient.

[0139] In the second step, the amphoteric dissociation ion exchange separation medium is resuspended in the buffer solution with pH 3.6 for 3 times;

[0140] The third step is to dilute Acid Red 13 with adsorption buffer to not less than 5.0mM;

[0141] Step 4: Take the required amount of ion-exchange separation medium, remove the water phase, resuspend in 0.80ml of adsorption buffer, add diluted acid red 13 solution not exceeding 50μl to the required final concentration, and continue to mix gently at room te...

Embodiment 3

[0145] Example 3. The influence of adsorption and elution conditions on the separation capacity of the amphoteric dissociation separation medium (operate according to Example 2).

[0146] The first step is to adsorb at pH 3.6, and Acid Red 13 is 60M. See Table 1 for the pH effect of the eluent.

[0147] The pH of the eluent from 8.0 to 8.9 only has a strong impact on the capacity of FBD-MSP-ZEWC and FBD-MSP-ZEW3 to separate Acid Red 13, but it does not increase significantly after pH>8.9; if not stated below, For adsorbed acid red 13 or nucleic acid, use pH 8.9 eluent. The amount of acid red 13 before adsorption is taken as the total amount; under the washing conditions used, the proportion of acid red 13 in the washing liquid and the washing loss rate are lower than 2%. The reduction of Acid Red 13 in the above supernatant is the total amount of adsorption, and the elution rate of Acid Red 13 adsorbed on the representative amphoteric dissociation ion-exchange magnetic beads ...

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Abstract

The invention provides an amphoteric dissociation ion exchange separation medium, the surface of which is an amphoteric dissociation covalent modification layer; when the environmental pH value is lower than the isoelectric point of the amphoteric dissociation covalent modification layer, the surface of the amphoteric dissociation covalent modification layer The net charge is positive and has the property of an anion exchanger; when the environmental pH value is higher than the isoelectric point of the amphoteric dissociation covalent modification layer, the surface net charge of the amphoteric dissociation covalent modification layer is negative and has the property of a cation exchanger; the present invention The separation medium has the properties of anion exchanger and cation exchanger on both sides of the isoelectric point respectively. Adjusting the pH of the eluent makes the surface of the separation medium and the net charge of the target substance the same to generate electrostatic repulsion to release the target substance, which not only enhances the elution The efficiency also avoids the follow-up problems of using high-concentration inorganic ions. It has high separation capacity, high elution efficiency of adsorbed ions under mild conditions, accurate calibration of separation capacity, low non-specific adsorption of hydrophobic small molecules, and high regeneration efficiency under mild conditions. new ion exchange media.

Description

technical field [0001] The invention relates to an ion exchange separation medium for separating biologically active substances, in particular to an amphoteric dissociation ion exchange medium, an application method thereof, and a separation capacity calibration method thereof. Background technique [0002] Ion exchange separation medium is suitable for rapid separation and reanalysis of biomolecules such as protein / nucleic acid, purification and preparation of protein / nucleic acid, chromatographic quantitative analysis of dissociated charged substances; these applications require ion exchange separation medium to have high adsorption capacity and be absorbed under mild conditions. It has the characteristics of high elution efficiency of adsorbed substances, accurate calibration of separation capacity, low non-specific adsorption of interfering substances, and high regeneration efficiency under mild conditions. The existing ion exchange separation medium cannot meet the abov...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): B01J43/00B01J47/014G01N21/31
CPCG01N21/31B01J43/00B01J47/014C07K1/18C12N15/101C12Q1/6806G01N1/4005G01N21/80G01N2001/4011
Inventor 廖飞龙高波谢燕玲黄铭通谢万军
Owner CHONGQING FARSIGHTED BLUE DRAGON FBD BIOTECH CO LTD CHINA
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