Separation of micromolecules

Abstract

This invention is directed to an apparatus and method for the separation of molecules, particularly micromolecules having a molecular mass of less than 5000 Dalton. The present invention is directed to an apparatus for separating micromolecules by electrophoretic separation, the apparatus comprising: (a) an anode; (b) a cathode disposed relative to the anode so as to be adapted to generate an electric field in an electric field area therebetween upon application of a voltage potential between the anode and the cathode; (c) a separation membrane disposed in the electric field area; (d) a first restriction membrane disposed between the anode and the separation membrane so as to define a first interstitial volume therebetween; (e) a second restriction membrane disposed between the cathode and the separation membrane so as to define a second interstitial volume therebetween; and (f) means adapted to provide a sample constituent in a selected one of the first and second interstitial volumes; wherein upon application of the voltage potential, a selected separation product is removed from the sample constituent, thorough the separation membrane, and provided to the other of the first and second interstitial volumes, wherein a micromolecule is capable of being retained in at least one of the interstitial volumes, and wherein a micromolecule is capable of being retained in at least one of the interstitial volumes.

1 . An electrophoretic separation apparatus for separating micromolecules, the apparatus comprising: 
 (a) an anode;    (b) a cathode disposed relative to the anode so as to be adapted to generate an electric field in an electric field area therebetween upon application of a voltage potential between the anode and the cathode;    (c) a separation membrane disposed in the electric field area;    (d) a first restriction membrane disposed between the anode and the separation membrane so as to define a first interstitial volume therebetween;    (e) a second restriction membrane disposed between the cathode and the separation membrane so as to define a second interstitial volume therebetween; and    (f) means adapted to provide a sample constituent in a selected one of the first and second interstitial volumes; 
 wherein upon application of the voltage potential, a selected separation product is removed from the sample constituent, thorough the separation membrane, and provided to the other of the first and second interstitial volumes and wherein a micromolecule is capable of being retained in at least one of the interstitial volumes.  
   
     
     
         2 . The apparatus according to  claim 1  wherein at least one restriction membrane is formed as a composite arrangement with at least two materials.  
     
     
         3 . The electrophoretic separation apparatus of  claim 1  wherein at least one of the restriction membranes is comprised of at least two membranes having distinctive pores sizes.  
     
     
         4 . An apparatus for electrophoretic separation of micromolecules, the apparatus comprising: 
 (a) an anode buffer compartment and a cathode buffer compartment;    (b) electrodes positioned in the buffer compartments;    (c) a first chamber and a second chamber positioned on either side of an ion-permeable separation membrane having a defined molecular mass cut-off, the first chamber and the second chamber being positioned between the anode and the cathode buffer compartments and separated by an ion-permeable restriction membrane positioned on at least one side of the separation membrane, the restriction membrane allowing flow of ions into and out of the compartments and chambers under the influence of an electric field but substantially restrict movement of at least one micromolecule type from the second chamber into the buffer compartment.    
     
     
         5 . The apparatus according to  claim 4  wherein the ion-permeable separation membrane has a molecular mass cut-off greater than the molecular mass of the micromolecule to be separated.  
     
     
         6 . The apparatus according to  claim 4  wherein at least one buffer compartment, sample chamber or product chamber is configured to allow flow of the respective buffer, sample or product solution to form a stream.  
     
     
         7 . The apparatus according to  claim 4  wherein at least one restriction membrane is formed as a composite arrangement with at least materials.  
     
     
         8 . The apparatus according to  claim 4  wherein at least one restriction barrier is formed as a sandwich arrangement with at least two layers of material.  
     
     
         9 . The apparatus according to  claim 8  wherein the sandwich arrangement includes an inner layer comprising a membrane having a pore size with a molecular mass cut-off less than the about 5000 Da and an outer layer comprising a membrane having a molecular mass cut-off of greater than about 5000 Da.  
     
     
         10 . The apparatus according to  claim 9  wherein the inner layer is made from an ultrafiltration, electrodialysis or haemodialysis membrane material and the outer layer is a membrane material made from polyacrylamide.  
     
     
         11 . The apparatus according to  claim 10  wherein the ultrafiltration membrane has a molecular mass cut-off between 100 Da and 5000 Da.  
     
     
         12 . The apparatus according to  claim 11  wherein the ultrafiltration membrane has a molecular mass cut-off of about 1 kDa.  
     
     
         13 . The apparatus according to  claim 4  wherein the ion-permeable separation membrane is made from polyacrylamide and having a molecular mass cut-off from 5 to 1000 kDa.  
     
     
         14 . A separation cartridge suitable for use in an electrophoretic separation apparatus for separating micromolecules, the cartridge comprising: 
 (a) a housing;    (b) an ion-permeable separation membrane having a defined molecular mass cut-off positioned in the housing;    (c) an ion-permeable restriction membrane positioned either side of the separation membrane in the housing and spaced to form a first chamber and second chamber on either side of the separation membrane, wherein the restriction membrane is adapted to allow flow of ions into and out of the compartments and chambers under the influence of an electric field but substantially restrict movement of at least one micromolecule type from the second chamber.    
     
     
         15 . The cartridge according to  claim 14  further including: 
 (d) electrodes positioned in the housing on the outer sides of the restriction barriers.  
 
     
     
         16 . The apparatus according to  claim 14  wherein the ion-permeable separation membrane has a molecular mass cut-off greater than the molecular mass of a micromolecule to be separated.  
     
     
         17 . The cartridge according to  claim 14  wherein the separation membrane is composed of polyacrylamide and having a molecular mass cut-off from about 5 to 1000 kDa.  
     
     
         18 . The cartridge according to  claim 14  wherein at least one restriction membrane is formed as a composite arrangement with at least two materials.  
     
     
         19 . The cartridge according to  claim 14  wherein at least one restriction membrane is formed as a sandwich arrangement of membranes with at least two layers of material.  
     
     
         20 . The cartridge according to  claim 19  wherein the sandwich arrangement includes an inner layer comprising a membrane having a pore size with a molecular mass cut-off less than the about 5000 Da and an outer layer comprising a membrane having a molecular mass cut-off of greater than about 5000 Da.  
     
     
         21 . The cartridge according to  claim 19  wherein the inner layer is made from an ultrafiltration, electrodialysis or haemodialysis membrane material and the outer layer is a membrane material made from polyacrylamide.  
     
     
         22 . The cartridge according to  claim 20  wherein the ultrafiltration membrane has a molecular mass cut-off between 100 Da and 5000 Da.  
     
     
         23 . The cartridge according to  claim 22  wherein the ultrafiltration membrane has a molecular mass cut-off of about 1 kDa.  
     
     
         24 . The cartridge according to  claim 23  wherein the ion-permeable separation barrier is a membrane made from polyacrylamide and having a molecular mass cut-off from 5 to 1000 kDa.  
     
     
         25 . A method of separating a micromolecule from a liquid sample, the method comprising: 
 (a) providing an electrophoresis apparatus according to clam  4 ;    (b) placing the sample in the first chamber of the apparatus;    (c) selecting a solvent for the first chamber having a pH such that the micromolecule to be separated is charged;    (d) applying an electric potential between the first and second chambers causing movement of micromolecules in the first stream through the separation membrane into the second chamber while unwanted molecules are substantially prevented from entering the second chamber;    (e) optionally, periodically stopping and reversing the electric potential to cause movement of molecules having entered the separation membrane to move back into the first chamber, while substantially not causing any micromolecules that have entered the second chamber to re-enter first chamber; and    (f) maintaining steps (d) and optionally (e) until the desired amount of micromolecules are moved to the second chamber.    
     
     
         26 . The method according to  claim 25  wherein the micromolecule is selected from the group consisting of biotin, Brilliant Blue FCF (BB FCF), azorubine, phytoestrogen, digoxigenin, hormones, cytokines, dyes, vitamins, chemicals, neutraceuticals, pharmaceuticals food diet supplements, and combinations thereof.  
     
     
         27 . The method according to  claim 25  wherein the sample is selected from the group consisting of crude extracts, microbial cultures, cell lysates, cellular products, chemical processing mixtures, cell culture media, plant products or extracts.  
     
     
         28 . The method according to  claim 25  wherein the solvent is Tris Borate buffer around pH 9.  
     
     
         29 . The method according to  claim 28  wherein buffer has a concentration of 10 mM to 200 mM.  
     
     
         30 . The method according to  claim 29  wherein the buffer has a concentration of 20 mM to 80 mM.  
     
     
         31 . A micromolecule purified or separated by the method according to  claim 25 .  
     
     
         32 . The micromolecule according to  claim 31  selected from the group consisting of biotin, Brilliant Blue FCF (BB FCF), azorubine, phytoestrogen, digoxigenin, hormones, cytokines, dyes, vitamins, chemicals, neutraceuticals, pharmaceuticals, food supplements, and combinations thereof.