Amphoteric dissociation ion exchange medium and uses thereof and method for calibrating separation capacity thereof

Abstract

An amphoteric dissociation ion exchange separation medium, the surface of which is an amphoteric dissociation covalently-modified layer. When an environmental pH value is lower than the isoelectric point, pIm, of the covalently-modified layer, the type of net charges on the surface of the covalently-modified layer is positive and the separation medium has the properties of an anion exchanger; when the environmental pH value is higher than the pIm, the type of net charges on the covalently-modified layer surface is negative and the separation medium has the properties of a cation exchanger. The separation medium has the properties of an anion exchanger and a cation exchanger at both sides of the pIm, respectively. The pH of an eluent can be adjusted to allow the separation medium surface and the target substance to have the same type of net charges, so that the target substance can be released by electrostatic repulsion.

We claim: 
     
         1 . An amphoteric dissociation ion exchange separation medium, wherein a surface of the amphoteric dissociation ion exchange separation medium is an amphoteric dissociation covalently-modified layer; the amphoteric dissociation covalently-modified layer has an isoelectric point (pIm) that is an environmental pH value at which a net charge on the surface of the amphoteric dissociation ion exchange separation medium is zero; wherein when the environmental pH value is lower than the pIm, the net charge on a surface of the amphoteric dissociation covalently-modified layer is positive and the amphoteric dissociation ion exchange separation medium acts as an anion exchanger; when the environmental pH value is higher than the pIm, the net charge on the surface of the amphoteric dissociation covalently-modified layer is negative and the amphoteric dissociation ion exchange separation medium acts as a cation exchanger;
 the amphoteric dissociation covalently-modified layer on the surface of the amphoteric dissociation ion exchange separation medium comprises both a group which dissociates to generate positive charges only, and a group which dissociates to generate negative charges only; wherein the group which dissociates to generate negative charges only is an aliphatic carboxyl group, and the group which dissociates to generate positive charges only is one or more groups selected from the group consisting of an aliphatic primary amine group, an aliphatic secondary amine group, an aliphatic tertiary amine group and an imidazolyl group;   the amphoteric dissociation covalently-modified layer is derived from covalent modification of a surface of a separation medium substrate with an amphoteric dissociation group precursor;   the surface of the separation medium substrate does not contain a long linear chain having a length of more than 9 carbon atoms, but contains a reactive group for covalently linking with the amphoteric dissociation group precursor to form the amphoteric dissociation covalently-modified layer; the reactive group is any one group selected from the group consisting of an aliphatic primary amine group, an aliphatic secondary amine group, an aliphatic carboxyl group and a thiol reactive group; wherein the thiol reactive group contains a small-size group that is substituted by a thiol to leave from the surface of the separation medium substrate;   the amphoteric dissociation group precursor is a hydrophilic material having a molecular weight less than 500 Daltons and no hydrocarbon chains or hydrocarbon rings with more than 5 successive carbon atoms; wherein the amphoteric dissociation group precursor contains an alkylthiol group or an aliphatic primary amine group as a covalently-linking group and an aliphatic carboxyl group as the group which dissociates to generate negative charges only, and/or one or more groups selected from the group consisting of an aliphatic primary amine group, an aliphatic secondary amine group, an aliphatic tertiary amine group and an imidazolyl group as a group which dissociates to generate positive charges only; the amphoteric dissociation group precursor comprises a type A precursor, a type B precursor and a type C precursor, wherein the type A precursor is an amphoteric dissociation group precursor comprising only the group which dissociates to generate negative charge besides the covalently-linking group, the Type B precursor is an amphoteric dissociation group precursor comprising only the group which dissociates to generate positive charge besides the covalently-linking group, and the type C precursor is an amphoteric dissociation group precursor comprising the group which dissociates to generate negative charge and the group which dissociates to generate positive charge besides the covalently-linking group; wherein the type C precursor is selected from the group consisting of lysine, ornithine, histidine, N,N-dicarboxymethylethylenediamine, cysteine, 3-thiolhistidine, 3-thiol lysine and 3-thiol glutamic acid; the type A precursor is selected from the group consisting of glutamic acid, 3-thiol-1,5-glutaric acid, thioglycolic acid and tris-(carboxymethyl)-aminomethane; and the type B precursor is selected from the group consisting of mercaptoethylamine, 3-thiol-2-hydroxypropylamine, 2-mercaptoimidazole, diethylenetriamine, N,N-dimethylaminoethylenediamine and tetra-(aminomethyl)-methane.   
     
     
         2 . The amphoteric dissociation ion exchange separation medium of  claim 1 , wherein when the environmental pH value is lower than the pIm, the number of positive charges on the surface of the amphoteric dissociation covalently-modified layer increases as the difference between the pIm and the environmental pH value increases; when the environmental pH value is higher than the pIm, the number of negative charges on the surface of the amphoteric dissociation covalently-modified layer increases as the difference between the pIm and the environmental pH value increases; and the environmental pH value is within a pH range tolerated by both a target substance and the amphoteric dissociation ion exchange separation medium. 
     
     
         3 . The amphoteric dissociation ion exchange separation medium of  claim 1 , wherein the separation medium substrate is covalently modified with the amphoteric dissociation group precursor by one of the following methods:
 a) using one or more type C precursors; wherein, when two or more type C precursors are used in combination, the type C precursors are mixed in a given ratio for use, and the precursors for mixing contain the same covalently-linking group;   b) mixing one or more type A precursors with one or more type B precursors in a given ratio for use; wherein the precursors for mixing contain the same covalently-linking group;   c) mixing only one or more type A precursors with one or more type C precursors in a given ratio; wherein these precursors for mixing contain the same covalently-linking group; and   d) mixing only one or more type B precursors with one or more type C precursors in a given ratio; wherein the precursors for mixing contain the same covalently-linking group.   
     
     
         4 . The amphoteric dissociation ion exchange separation medium of  claim 3 , wherein when the amphoteric dissociation group precursor is used to covalently modify the separation medium substrate, a molar ratio of the total amount of aliphatic primary, secondary, tertiary amine groups and imidazolyl groups to the total amount of aliphatic carboxyl groups in the amphoteric dissociation group precursor is limited to 1:6 to 6:1. 
     
     
         5 . The amphoteric dissociation ion exchange separation medium of  claim 1 , wherein when an aliphatic secondary amine and/or an aliphatic tertiary amine are/is used in an amphoteric dissociation group precursor as the group which dissociates to generate positive charge, the total molar amount of the aliphatic secondary amine and the aliphatic tertiary amine does not exceed 30% of the total molar amount of the aliphatic primary amine and the imidazolyl group in the precursors used. 
     
     
         6 . The amphoteric dissociation ion exchange separation medium of  claim 4 , wherein the number of positive charge on the surface of the amphoteric dissociation ion exchange separation medium is not less than 90% of the maximum value at pH 3, and the number of negative charge on the surface of the amphoteric dissociation ion exchange separation medium is not less than 90% of the maximum value at pH 11. 
     
     
         7 . A use method of the amphoteric dissociation ion exchange separation medium of  claim 1 , comprising:
 applying the amphoteric dissociation ion exchange separation medium to separate a target substance; wherein a common logarithm of a dissociation constant obtained from the release of hydrogen ion from the target substance is pK or an isoelectric point of the target substance is pI; wherein the method further comprises:   a. selecting an amphoteric dissociation ion exchange separation medium; wherein difference between the pIm of the amphoteric dissociation ion exchange separation medium and the pK or pI of the target substance is not less than 1.0;   b. adsorbing the target substance; wherein an environment pH is selected to allow the type of net charges on the surface of the ion exchange separation medium to be opposite to the type of net charges of the target substance, thereby resulting in adsorption of the target substance by electrostatic attraction, wherein the environmental pH is between the pK or pI of the target substance and the pIm of the amphoteric dissociation ion exchange separation medium and the difference between the environmental pH and the pK or pI of the target substance and the difference between the environmental pH and the pIm of the amphoteric dissociation ion exchange separation medium are both greater than 0.3; and   c. eluting the target substance; wherein when pH of an eluent is lower than the pIm of the amphoteric dissociation ion exchange separation medium, the pH of the eluent is at least 0.3 lower than the lower one of the pK or pI of the target substance and the pIm of the amphoteric dissociation ion exchange separation medium; when the pH of the eluent is higher than the pIm of the amphoteric dissociation ion exchange separation medium, the pH of the eluent is at least 0.3 higher than the higher one of the pK or pI of the target substance and the pIm of the amphoteric dissociation ion exchange separation medium; in the use of the above eluent, the type of net charges of the surface of the amphoteric dissociation ion exchange separation medium is the same as that of the target substance, thereby resulting in elution of the target substance by electrostatic repulsion; and a water-soluble monovalent neutral inorganic salt is added to the eluent to promote the elution of the target substance.   
     
     
         8 . A method for calibrating separation capacity of the amphoteric dissociation ion exchange separation medium of  claim 1 , comprising:
 a) selecting an colored organic compound as a color-developing probe for the calibration of the separation capacity of the amphoteric dissociation ion exchange separation medium; wherein the colored organic compound has a dissociation constant of pK or an isoelectric point of pI, a molecular weight less than 600 Daltons, a visible light absorption coefficient greater than 14 mM −1 ·cm −1 , a solubility not less than 5.0 μmol/L at pH 3.0-11.0, and a positive or negative net charge after dissociation at pH 3.0-11.0;   b) calibrating the separation capacity of the amphoteric dissociation ion exchange separation medium; wherein step b comprises:   b1) when the amphoteric dissociation ion exchange separation medium has an isoelectric point pIm between 4.0 and 6.0, using a cationic probe with a dissociation constant of pK or an isoelectric point pI at least 2.0 greater than the pIm of the amphoteric dissociation ion exchange separation medium, or using an anionic probe with a dissociation constant pK or an isoelectric point pI at least 2.0 lower than the pIm of the amphoteric dissociation ion exchange separation medium; and   b2) when the amphoteric dissociation ion exchange separation medium has the pIm between 6.0 and 10.0, using the anionic probe with a dissociation constant of pK or an isoelectric point pI at least 2.0 lower than the pIm of the amphoteric dissociation ion exchange separation medium;   c) when calibrating the separation capacity of the amphoteric dissociation ion exchange separation medium, selecting a buffer solution corresponding to an environmental pH to allow the amphoteric dissociation ion exchange separation medium to adsorb a color-developing probe by electrostatic attraction, wherein the environmental pH is between the dissociation constant pK or the isoelectric point pI of the color-developing probe and the pIm of the amphoteric dissociation ion exchange separation medium, and differs from the pIm of the amphoteric dissociation ion exchange separation medium by not less than 1.3 and differs from the dissociation constant pK or the isoelectric point pI of the color-developing probe by not less than 0.5; selecting a buffer solution corresponding to an environmental pH to allow the type of net charges on the surface of the amphoteric dissociation ion exchange separation medium to be the same as that of the color-developing probe in the elution, thereby eluting a color-developing probe by electrostatic repulsion, wherein the environmental pH is at least 1.3 higher than the higher one or at least 1.3 lower than the lower one of the dissociation constant pK or the isoelectric point pI of the color-developing probe and the pIm of the amphoteric dissociation ion exchange separation medium; measuring the absorbance of the color-developing probe in an eluate followed by conversion into a separation capacity to the color-developing probe; and   determining pH effect on the separation capacity of the amphoteric dissociation ion exchange separation medium to a color-developing probe; wherein the minimum pH at which the separation capacity to the color-developing probe with negative net charge reaches zero, or the maximum pH at which the separation capacity to the color-developing probe with positive net charge reaches zero, is an approximation of the pIm of the amphoteric dissociation ion exchange separation medium.