US4431544A
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High pressure liquid affinity chromatography
Assignee: PBLIC HEALTH LABORATORY SERVICE BOARD
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Filing: 1982-04-26
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Granted: 1984-02-14
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Expires: 2002-04-26
Abstract
Abstract A process for the high pressure liquid affinity chromatographic separation of at least one biological or related substance from a mixture in which the contact, washing and eluting phases are performed on a binding material made from a ligand, containing at least one of the groups anthraquinone, phthalocyanine or aromatic azo, coupled to a matrix through a spacer arm, the binding material being so constructed that at least one biological or related substance is retained on the binding material during the contact and washing phases. In one preferred embodiment the ligand is a reactive dye, especially a triazinyl dye, the matrix is agarose or silica and the spacer arm is a substituted aminohexyl group. The chromatographic procedure is preferably performed at 100-3500 psi, at a flow rate of 0.5-2.0 ml/min. The choice of washing and eluting solutions depends on the material to be separated. However buffer solution to wash the column and a desorbing agent to elute the material are preferred. When the biological substance is an enzyme the desorbing agent is preferably an enzyme substrate, cofactor, inhibitor or analogue thereof. A binding material for use in the above HPLAC procedure is also provided. This is made from a ligand containing at least one of the groups anthraquinone, phthalocyanine or aromatic azo, coupled to a matrix through a spacer arm.
Inventors
- 👤 Anthony Atkinson
- 👤 Christopher R. Lowe
- 👤 Klaus Mosbach
- 👤 David A. P. Small
Bibliographic Details
Application Number
—
Jurisdiction
US
Data Source
google_patents
Last Synchronized
2026-10-25 23:50
CPC Classifications
B; B01; B01D; B01D15/00; B01D15/08; B01D15/26; B01D15/38; B01D15/3804; B01D15/3828; B01J; B01J20/00; B01J20/22; B01J20/26; B01J20/265; B01J20/267; B01J20/281; B01J20/286; B01J20/289; B01J20/30; B01J20/3092; B01J20/32; B01J20/3202; B01J20/3204; B01J20/3206; B01J20/3208; B01J20/321; B01J20/3212; B01J20/3214; B01J20/3217; B01J20/3219; B01J20/3231; B01J20/3242; B01J20/3244; B01J20/3246; B01J20/3257; B01J20/3259; B01J20/3261; B01J20/3263; C; C12; C12N; C12N9/00; B01J2220/00; B01J2220/40; B01J2220/49; B01J2220/50; B01J2220/54; B01J2220/58
Claims Analysis
Total Claims: 14
Claim 1
Independent Claim
287 words
A process for the high pressure liquid affinity chromatographic separation of at least one biological substance from a mixture containing same comprising the steps of (a) contacting the mixture containing the at least one biological substance with a binding material retained on a chromatographic column to bind the biological substance to the binding material, (b) passing a washing solution through the binding material to remove non-binding species from the column, and (c) passing an eluting solution through the binding material to recover the at least one biological substance from the column, the improvement which comprises performing at least the steps (a) and (b) at a pressure of between 10 and 5000 psi on a binding material of general formula ##STR5## wherein the matrix is an affinity chromatographic support, compatible with a pressure of between 10 and 5000 psi, selected from alumina, titania, zirconia, silica and glass beads, X--Y--R is a spacer arm wherein X is ##STR6## R 1 is selected from H and alkyl, R 2 is at least one group selected from alkyl, substituted alkyl, diol, ether, amido and amino, R is at least one group selected from alkyl, alkene, -aminoalkyl, -oxyalkyl, -thioalkyl, alkyl alcohol, diol, carboxylate, ether, thioether, amido, amino, amidino, imino carboxylate, isourea, carbamate, quanidino, hydrazino, aromatic and heterocyclic groups, Y is selected from alkyl, ether, thioether, amido, amino, --NH--, --O-- and --S--, A is an organic substituent containing a group selected from anthraquinone, aromatic azo and phthalocyanine and B is selected from an organic substituent, a halogen atom, an amino group and a substituted amino group said binding material being so constructed that the at least one biological substance is retained on the binding material during steps (a) and (b) of the process.
Claim 2
Dependent Claim
287 words
A process for the high pressure liquid affinity chromatographic separation of at least one biological substance from a mixture containing same comprising the steps of (a) contacting the mixture containing the at least one biological substance with a binding material retained on a chromatographic column to bind the biological substance to the binding material, (b) passing a washing solution through the binding material to remove non-binding species from the column, and (c) passing an eluting solution through the binding material to recover the at least one biological substance from the column, the improvement which comprises performing at least the steps (a) and (b) at a pressure of between 10 and 5000 psi on a binding material of general formula ##STR5## wherein the matrix is an affinity chromatographic support, compatible with a pressure of between 10 and 5000 psi, selected from alumina, titania, zirconia, silica and glass beads, X--Y--R is a spacer arm wherein X is ##STR6## R 1 is selected from H and alkyl, R 2 is at least one group selected from alkyl, substituted alkyl, diol, ether, amido and amino, R is at least one group selected from alkyl, alkene, -aminoalkyl, -oxyalkyl, -thioalkyl, alkyl alcohol, diol, carboxylate, ether, thioether, amido, amino, amidino, imino carboxylate, isourea, carbamate, quanidino, hydrazino, aromatic and heterocyclic groups, Y is selected from alkyl, ether, thioether, amido, amino, --NH--, --O-- and --S--, A is an organic substituent containing a group selected from anthraquinone, aromatic azo and phthalocyanine and B is selected from an organic substituent, a halogen atom, an amino group and a substituted amino group said binding material being so constructed that the at least one biological substance is retained on the binding material during steps (a) and (b) of the process.
Claim 3
Dependent Claim
12 words
A process according to claim 1 wherein A is a sulphonated anthraquinone.
Claim 4
Dependent Claim
13 words
A process according to claim 1 wherein B is a sulphonated aromatic group.
Claim 5
Dependent Claim
12 words
A process according to claim 1 wherein B is a chlorine atom.
Claim 6
Dependent Claim
102 words
A process according to claim 1 wherein the matrix is silica, X is ##STR7## wherein R 1 is selected from the group consisting of H and alkyl, R 2 is at least one group selected from the group consisting of alkyl, substituted alkyl, diol, ether, amido and amino, R is at least one group selected from the group consisting of alkyl, alkene, α-aminoalkyl, α-oxyalkyl, α-thioalkyl, alkyl alcohol, diol, carboxylate, ether, thioether, amido, amino, amidino, imino carboxylate, isourea, carbamate, guanidino, hydrazino, aromatic and heterocyclic groups and Y is selected from the group consisting of alkyl, ether, thioether, amido, amino, --NH--, --O-- and --S--.
Claim 7
Dependent Claim
83 words
A process according to claim 5 wherein R 2 is at least one group selected from the group consisting of alkyl, substituted alkyl and alkyl ether, R is selected from the group consisting of --(CH 2 ) n , --NH(CH 2 ) n , --O(CH 2 ) n , --S(CH 2 ) n wherein n is from 1 to 12, polyethyleneimine, polylysine, polyarginine, polyvinylpyrrolidone, polyornithine, polyvinylamine and a phenyl derivative and Y is selected from the group consisting of --NH--, --O-- and --S--.
Claim 8
Dependent Claim
43 words
A process according to claim 6 wherein R is selected from the group consisting of --(CH 2 ) n , --NH(CH 2 ) n , --O(CH 2 ) n , and --S(CH 2 ) n , wherein n is from 1 to 12.
Claim 9
Dependent Claim
59 words
A process according to claim 7 wherein --X--R--Y-- is selected from the group consisting of --OSi(OR 1 ) 2 (CH 2 ) 3 O CH 2 CH(OH)CH 2 NH(CH 2 ) 6 NH--, --OSi(OR 1 ) 2 CH 2 CH 2 CH 2 S-- and --OSi(OR 1 ) 2 (CH 2 ) 3 O CH 2 CH(OH)CH 2 O--.
Claim 10
Dependent Claim
27 words
A processs according to claim 1 wherein the at least one biological substance is selected from the group consisting of an enzyme, a polypeptide and a protein.
Claim 11
Dependent Claim
45 words
A process according to claim 9 wherein the at least one biological substance is selected from the group consisting of an albumin, a peptidase, a phosphatase, a kinase, a nuclease, a dehydrogenase, an esterase, a synthetase, a DNA binding protein and a RNA binding protein.
Claim 12
Dependent Claim
277 words
A binding material for use in a process according to claim 1 for the high pressure liquid affinity chromatographic separation of at least one biological substance from a mixture containing same, said process comprising the steps of (a) contacting the mixture containing the at least one biological substance with the binding material retained on a chromatographic column to bind the biological substance to the binding material, (b) passing a washing solution through the binding material to remove non-binding species from the column, and (c) passing an eluting solution through the binding material to recover the at least one biological substance from the column, wherein the binding material is of general formula ##STR8## wherein the matrix is an affinity chromatographic support, compatible with a pressure of between 10 and 5000 psi, selected from alumina, titania, zirconia, silica and glass beads, X--Y--R is a spacer arm wherein X is ##STR9## R 1 is selected from H and alkyl, R 2 is at least one group selected from alkyl, substituted alkyl, diol, ether, amido and amino, R is at least one group selected from alkyl, alkene, -aminoalkyl, -oxyalkyl, -thioalkyl, alkyl alcohol, diol, carboxylate, ether, thioether, amido, amino, amidino, imino carboxylate, isourea, carbamate, guanidino, hydrazino, aromatic and heterocyclic groups, Y is selected from alkyl, ether, thioether, amido, amino, --NH--, --O-- and --S--, A is an organic substituent containing a group selected from anthraquinone, aromatic azo and phthalocyanine and B is selected from an organic substituent, a halogen atom, an amino group and a substituted amino group and is so constructed that the at least one biological substance is retained on the binding material during steps (a) and (b) of the process.
Claim 13
Dependent Claim
96 words
A binding material according to claim 11 wherein the matrix is silica, X is ##STR10## wherein R 1 is selected from the group consisting of H and alkyl, and R 2 is at least one group selected from the group consisting of alkyl, substituted alkyl and alkyl ether, R is selected from the group consisting of --(CH 2 )-- n , --NH(CH 2 )-- n , --O(CH 2 )-- n and --S(CH 2 )-- n , wherein n is from 1 to 12 and Y is selected from the group consisting of --NH--, --O-- and --S--.
Claim 14
Dependent Claim
69 words
A process for the preparation of a binding material according to claim 12 wherein R is --NH (CH 2 ) n -- and Y is --NH-- comprising: (a) reacting a silica matrix with a w-glycidoxyalkyltrialkoxysilane to form a modified matrix, (b) reacting a monochloro triazine dye with an, w-diaminoalkane to form a modified dye, and (c) reacting the modified matrix with the modified dye to form the binding material.
References Cited (Backward Citations)
USPTO Annuity & Maintenance Windows
| Annuity Stage | Window Opens | Payment Deadline | Surcharge Period Until | Estimated Fee | Status |
|---|---|---|---|---|---|
| 3.5 Years (Year 4) | 1987-02-13 | 1987-08-15 | 1988-02-14 | $2000.00 | Window Closed / Lapsed |
| 7.5 Years (Year 8) | 1991-02-13 | 1991-08-15 | 1992-02-14 | $3760.00 | Window Closed / Lapsed |
| 11.5 Years (Year 12) | 1995-02-13 | 1995-08-15 | 1996-02-14 | $7700.00 | Window Closed / Lapsed |