Chemistry: electrical and wave energy – Processes and products – Electrophoresis or electro-osmosis processes and electrolyte...
Reexamination Certificate
1999-08-20
2003-06-03
Warden, Jill (Department: 1743)
Chemistry: electrical and wave energy
Processes and products
Electrophoresis or electro-osmosis processes and electrolyte...
C204S644000, C204S450000, C204S600000
Reexamination Certificate
active
06572751
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to purification, isolation, concentration and separation of organic or biological substances by a novel continuous flow isoelectric focusing method (contifocusing) and apparatus (contifocuser), a technique which employs an electrically low-conductive field for concentration or separation of charged substances. Centrifugation, filtration, absorption steps or carrier electrolyte or ampholyte buffers, to establish suitable pH gradients, are not used.
2. Description of the Related Art
Isoelectric focusing is a known technique for separating charged molecules such as proteins from a mixture containing them, using the principle that such charged amphoteric molecules have a zero net charge at a particular pH, the so-called isoelectric point (pI value). The compounds will migrate to the pH of their own isoelectric point if they are subjected to an electric field, and are separated accordingly.
Methods and apparatus for static isoelectric focusing are known, e.g. from WO 79/00942; EP-A-256552 and U.S. Pat. No. 5,256,269. A shortcoming of this type of arrangement is the small quantity to be purified, and its collection in a batch-wise operation. The technological background of the present invention is described in a continuous flow electrophoresis apparatus in U.S. Pat. No. 4,465,582 (1982) which employs a controlled fluid flow in the electrode chambers, which flow is running parallel but separate from the flow in the “working” chambers. U.S. Pat. No. 5,160,594 discloses an isoelectrofocusing apparatus having a forced recirculation in every cell, i.e. a recycling isoelectric focusing process. Recycling is not used in the present invention. The US patent employs a plurality of devices for preventing mixing of semi-purified and crude amphoteric substances. Additional cooling is required in every cell to reduce ohmic heating. Strong acids and bases, a catholyte or anolyte, are confined to the electrode chambers by ion-selective membranes to repel the negatively or positively charged ions and to establish suitable pH gradients. As opposed to U.S. Pat. No. 5,160,594, the present invention uses ion-selective membranes for different purposes, namely to selectively permit passing of low-molecular substances towards the electrode space for discarding from the electrode outflow port. All prior art devices require carrier ampholytes for the working chambers, which have a relatively low conductivity and a buffering capacity. U.S. Pat. No. 5,160,594 describes a maximum conductivity up to 600 &mgr;S/cm, which is far less than the conductivity up to 5000 &mgr;S/cm of the present invention. Like other prior art apparatus it is not designed for purification on an industrial scale and, in addition, U.S. Pat. No. 5,160,594 does not provide examples of substances to be purified.
A continuous flow isoelectric focusing method and apparatus, without using carrier ampholyte buffers but for use on an industrial scale, are disclosed in Hungarian patent 210,584 (1992). It describes an upward flow system through the chambers by gravitation into the separation chambers up to the level of the outflow port. The system includes two non-conductive solution chambers where the second is having two or more solution outflow ports and a passageway for a downwards flow of the solution from the first chamber into the bottom of the second chamber. A pair of electrodes are located in the second chamber. A plurality of adjacent partition members are located in the second chamber and extended there across in order to provide substantially convection-free solution and current flow pathways in the horizontal direction. A shortcoming of HU 210,584 is the fact that in the inflow solution chamber a gradient can be formed by itself. The inflow of the solution by dropping is difficult to control. The upward flow system of the Hungarian patent is less vulnerable to intrinsic gradient formation, alike in the present invention, but the two-chamber system is complicated and difficult to construct. In addition, separation of organic solutions occurs at both opposite sides of the second separating chamber close to the electrodes. Inorganic ions are easily mixing with the single fractions of inorganic or biological substances to be separated. A second separation step is frequently required.
Citation of any document herein is not intended as an admission that such document is pertinent prior art, or considered material to the patentability of any claim of the present application. Any statement as to content or a date of any document is based on the information available to applicant at the time of filing and does not constitute an admission as to the correctness of such a statement.
SUMMARY OF THE INVENTION
The present invention provides a contifocuser for separating substances in an electrically conductive solution, which apparatus is made from electrically non-conductive material and includes a brick-form separating chamber having one liquid inflow, two electrode space outflows and one or more working solution outflow ports and a passageway for upward flow of the solution from the bottom up to top outflows. The ratio of the height, the width and the depth of the separation chamber can be e.g. 3:2:0.2. A pair of electrodes are located in vertical position along both opposite most narrow walls of the separating chamber. They are adapted to be coupled to an external direct current (DC) power supply.
The apparatus is divided into three parts by two partitions made from ion-selective membranes. These ion-selective membrane partitions are situated in vertical position at both sides of the apparatus near the electrodes, so that they each separate about {fraction (1/25)} to {fraction (1/15)} parts from both ends. Both vertical ion-selective partitions have an opening at the bottom with a diameter of one half of the inflow pipe of the apparatus. In particular, the one enclosing the cathode space is a cation-selective membrane and the other is an anion-selective membrane. The ion-selective membranes have a specificity chosen to permit the transfer of either cations or anions in order to be eliminated in the waste outflow ports. At both ends, the waste outflow openings are located near the electrodes at {fraction (1/40)} to {fraction (1/10)} from the top rim of the apparatus. The working outflows, for collecting the substances to be purified, are situated at the same level in the middle. Advantageously, the contifocuser comprises at least three of said outflow ports. Between the outflow ports, except in the electrodes spaces, are dividing vertical partitions made from electrically non-conductive material, closed in their upper quarter and with small openings from bottom up to e.g. three quarters of their height.
As a further improvement, a plurality of adjacent alternating zigzag-shaped or U-shaped or multiple U form partition members may be located in the remaining space of the apparatus between the above-mentioned partitions, but not in the electrode spaces. These are also made of electrically non-conductive material and are perforated or made from porous material permeable for ions and molecules in both directions, and extend from the bottom up to the lowest level of the working outflow port and across the chamber. The partition members are placed freely over the entire width of the apparatus, and are the novel means to provide a substantially convection-free solution but provide an undisturbed flow of electrolytes or amphoteric particles in the horizontal direction. The perforated membranes are in a spaced-apart relationship in order to provide an undisturbed vertical flow. This invention has important advantages over prior art devices in that it can process up to 3,600 L/day of earthworm enzymes (prior art according to HU 210,584: 280-300 L) producing 80,000-170,000 U. of purified enzyme per L/day (HU 210,584: 60,000-70,000 U.).
Another advantage of this invention is that the solution or suspension may have a conductivity in the range of 50-5000 &mgr;S/cm, instead
De Boer Gerben Foppe
Sova Oto
Browdy and Neimark PLLC
Cerberus Enterprises B.V.
Starsiak Jr. John S.
Warden Jill
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