METHOD AND APPARATUS OF SEGREGATING AT LEAST ONE SPECIES OF DESIRED IONS

Abstract: Novel method and apparatus for separating, purifying and/or concentrating ions in a mixture of different molecular species using a method of electrophoresis in the presence of a counterflowing carrier fluid are provided. By selecting the appropriate carrier fluid flow rate, magnitude of the current and varying separation characteristics in a separation chamber, one or more similarly charged species of ions can be segregated into separate or discrete equilibrium zones within the chamber. The ionic species include isotopes, proteins, antibiotics, nucleic acids and the like.

Description: Method and Apparatus of Segregating at least one Species of Desired Ions This invention relates to methods and apparatuses for segregating, i.e. separating, purifying and/or concentrating ions in a mixture of different molecular species; and,according to one embo diment;Lo a novel method of isotope purification and/or enrichment. More particularly, this invention relates to a combination of electrophoresis and a flowing carrier fluid. The principles of electrophoresis are known whereby separation of a mixture of ions can be effected by an applied voltage field since different species will migrate toward an oppositely charged electrode at different rates proportional to their charge to mass ratio. It is also known that partic ular hydrated supports or matrices can have an influence on electrophoresis rates and can be used in conjunction with electrophoresis to effect the separation of ions according to physical properties other than their charge to mass ratio. In addition, a specialized technique is known, in which an electric potential forces complex polyionic molecules to their isoelectric point in a pH gradient. Electrophoresis is typically used as an analytical tool in bischemical research. When used in this manner, electrophoresis separates components of a mixture and allows certain measurements, e.g. quantification, of the separated components. Usually analytical electrophoresis methods do not include the recovery of the separated components, i.e. the species are both separated and measured in the separation medium and the separated components are recovered only as a mixture or not at all. Several electrophoretic techniques have been developed for preparat ve use, i.e. to recover the separated components. The simplest of the preparative electrophoresis systems operates on the same principles as the analytical methods, wherein a zone containing a mixture of ion species is applied at the origin and the ions are electrophoresed into or through a supporting matrix whose major importance is to prevent turbulent mixing, i.e. as an anticonvection medium. These techniques include a method for recovering the different ions which are separated by virtue of differing rates of electrophoresis through the supporting matrix. U.S. Patent No. 3,847,785 is illustrative of such techniques. These techniques are extremely limited in that they have a very low capacity. Further, because electrophoresis cannot be done at high concentrations of ionic components and because electric heating limits the size of the apparatus, such techniques cannot advantageously be operated on a large scale basis. Moreover, the sample must be in a concentrated form, while recovered isolated components are generally very dilute. Finally, such techniques have the added disadvantage of very limited resolution, i.e. poor capacity to separate two or more very similar components. Methods of continuous electrophoresis are known in which electrically charged species are continuously fed into an apparatus and are segregated by application of a direct current perpendicular to the flow of a carrier solution. Each charged component moves in a particular direction defined by its rate of electrophoresis in one direction and the rate at which it is carried by the bulk solution flow in the perpendicular direction. Components can thus be collected continuously from the position of zones on the downstream end of the apparatus (see e.g. Ravoo, Gellings and Vermculen, Anal. Chim. Acta 38 (1967) 219-232). By virtue of allowing continuous operation, these techniques have increased the capacity of electrophoretic techniques. However, despite this increase, the capacity is still low, while the other disadvantages of electrophoresis, e.g. poor resolution, remain. Additionally, these continuous electrophoresis processes cannot be adapted to very large scale operation because of inadequate heat dissipation. Also known is a mode of electrophoresis referred to as isotachophoresis or displacement electrophoresis which was developed following the principles described by Ornstein in U.S. 3,384,564. According to these principles, under particular conditions, an ion of intermediate electrophoretic mobility can be "sandwiched" between an ion with higher electrophoretic mobility and one of lower electrophoretic mobility. Isotachophoresis has the advantage of providing a force which counteracts diffusion and also can concentrate the separated components. However, several problems have been encountered in attempts to apply the method of isotachophoresis. In the absence of spacer ions, the different separated zones of a mixture border on each other and are thus difficult to recover without contamination from adjacent components. Spacer ions must possess very particular properties and thus are not always available. To obtain adequate separation the components must be electrophoresed a considerable distance which demands a high voltage. Also isotachophoresis has a limited capacity. A method has been described (Preetz and Pfeifer, Anal. Chim. Acta 38 (1967) 255-260) in which bulk fluid flow counteracts isotachophoresis. The purpose of this counter flow is that isotachophoresis can continue for a long time in a small chamber or column. This decreases the power requirements as disclosed in U.S. 3,705,845. Furthermore, bulk fluid flow perpendicular to direction of isotachophoresis has been used to give a continuous method analogous to that described for electrophoresis above. In a special field, viz. in radiochemical industry, methods of purifying radioactive isotopes (also stable isotopes with unusual mass) from the more abundant chemically identical isotopic forms are required. Most importantly, a large industry is involved in the enrichment of the isotope uranium-235 from natural uranium. To be used for powering nuclear reactors, the uranium-235 content is typically increased from 0.71 percent to 3 percent. Similarly, large scale enrichment plants exist for the isolation of the stable heavy isotope of hydrogen, deuterium, which is used in heavy water-cooled nuclear reactors. Known processes for enrichment of uranium in the fissionable isotope uranium-235 include gaseous diffusion processes and gas centrifuge processes. Typically, such processes are multi-stage and involve significant capital outlay for equipment. Also, gaseous diffusion suffers from the disadvantage that large energy expenditures are required to achieve enrichment. Furthermore, because the enrichment achieved in any one step is small, a large multiunit plant is required. Additionally, the low enrichment levels make it impractical to recover more than a fraction of the uranium-235. Gas centrifugation requires the design of a centrifuge having the fastest and longest rotor possible, since separation increases with speed and is proportional to length. However, the level of materials technology and engineering, in general, impose practical limits on the size and speed of the rotors. The limit of the length of the machine is determined in part by the difficulty of controlling the straightness of the rotor, the uniformity of the wall in mass production manufacture, and by the durability of the bottom bearing, which must support the weight of the rotor. Another disadvantage of the centrifuge is that those with large length to diameter ratios have a problem getting up to operational speed, since they may pass through critical speeds at which large scale vibrations can occur. These problems are compounded by the fact that typically, three or more centrifuges are utilized in series as a "cascade". Thus, although the gas centrifuge requires substantially less energy than gaseous diffusion, establishment of plants requires a large capital outlay and their maintenance involves high costs. Another more recently developed method of isotope separation is laser separation which utilizes the differences in the electronic structure, i.e. in the electron clouds that surround the nuclei, of isotopes. The differences, though small, affect the wavelengths of light absorbed by the isotopes. Because a laser emits light of a very pure color, it can thus be utilized to "tag", i.e. excite the electrons ofssone isotope but not the other. Once the various isotopes are distinguished a variety of methods are then available for actually sorting them into different "bins". For example, application of an electric or magnetic field may be used to deflect the excited isotope along a different path from that followed by the unexcited one. Alternatively, the excited isotopes may be converted into ions which are more easily manipulated. Still other classes of laser separation decompose molecules containing one isotope into stable products that can be simply removed from the mixture or utilize chemical scavengers which will react only with a molecule in its excited state. In addition to the similar disadvantage of the other laser techniques, i.e. requiring specialized and costly equipment, these techniques have the added difficulty of finding compounds which decompose into stable components or of finding appropriate scavenger molecules. The laser separation techniques have considerable potential but these have yet not been fully developed. These laser techniques all suffer from the disadvantage of retrieving only a small proportion of the available isotope. Thus, these tec#iriiques will require multiple stages with the accompanying reduction in efficiency and furthermore are unlikely to achieve com plete recovery of the rarer isotope. Accordingly, there is a need for a technique for isotope separation which has a high capacity and high degree of enrichment per stage, which recovers the large majority of the desired isotope, and which does not require large sophisticated equipment. The invention comprises a novel method and apparatus for isolating, purifying and/or concentrating any charged species, i.e., any atom, molecule, compound or particle which possesses ionic character (ions). The processes of the present invention comprise a combination of electrophoreis, a counter flowing carrier fluid and a separation chamber having longitudinally varying separation characteristicst#o produce equilibrium positions to which particular ions move According to the present inven tion by selecting the appropriate carrier fluid flow rate, the magnitude of the applied electric field, i.e. the cur rent, and the varying separation characteristics in the separation chamber, one or more similarly charged species of ions can be segregated into separate or discrete equilibrium zones within the chamber. The process of the present invention is based on the simple principle that the movement of a given ion can be in the direction of flow or in the direction of electro phoresis and that due to the varying separation character istics movement due to flow can dominate in one compartment of the separation chamber and movement due to electrophoresis can dominate in a second compartment; Applying this principle to isotopes, the desired isotopes will electrophorese slightly faster than the desired isotopes will flow in the opposite direction, and a contiguous compartment of the separation chamber contains a second matrix in which the desired iso topes flow slightly faster than they electrophorese. Thus, conditions can be selected such that a given ion will move toward the boundary of two compartments or to the interface of the matrices, and will be concentrated there while impurities will be washed through the system. By compartments is meant a portion or region of the separation chamber posses sing a unique combination of separation characteristics. The longitudinal variation in separation characteristics can be achieved by several methods. In one type of embodiment each compartment is defined by a unique matrix or particular combination of matrices (granular or porous materials through which a solvent can flow) which by virtue of interactions with ions within the separation chamber establish particular separation characteristics. In a second type of embodiment the electrical potential (voltage) across different compartments of the separation chamber differs. In a third type of embodiment the rate of fluid flow through different compartments differs. The first of the above methods (that using one or more matrices) is particularly practical for large scale applications and is described in detail below. The method of the present invention proceeds by introducing a S.sample of a mixture of different molecular p i a mlxture a separation chamber having longitudinally varying separation characteristics in the form of at least two compartments, flowing a carrier fluid through the chamber and applying an electric field across the chamber such that the ions to be separated or collected electrophorese in a direction counter to the flow of the carrier fluid. Typically, one compartment will comprise a uniform support matrix or a volume of free flowing carrier fluid. Alternatively, a non-uniform or gradient support matrix may be utilized, such matrix providing a multiplicity of compartments. The flow rate and the electric field strength are selected so that the species to be separated have a net mobility of substantially zero at some position in the chamber, e.g. in two contiguous compartments the ions move toward the common boundary and have a net mobility of zero in an equilibrium zone at and adjacent the boundary. In one embodiment, one compartment of the separation system comprises a matrix of a type in which, at suitable flow rates and applied voltages, i.e., current, the movement of the species of desired ions due to electrophoresis is slightly faster than their movement due to flow in the opposite direction, i.e. the ions migrate slightly faster in the direction of electrophoresis than in the opposite carrier fluid flow direction. A compartment of the separation system contiguous to the first in the direction of electrophoresis comprises a second matrix in which the desired ions move due to flow slightly faster than they move due to electrophoresis. Under the appropriate flow rate and applied voltage, the result of the imbalances in the movements due to electrophoresis and due to flow is that the desired ions migrate to the common boundary of the two compartments, i.e. of the two matrices, and are concentrated there in a discrete equilibrium zone while impurities are washed through the system. The separation system of the present invention provides a high resolution method for purifying desired ions and, importantly, can be used for large scale amounts and/or on a continuous basis, while providing relatively comparably high resolution coupled with high capacity and sufficient flexibility to be generally applicable for a wide range of separation requirements. The present invention is further illustrated by the follo wing drawings in which: FIG. 1 is a graph depicting the direction and rate of net movement (Ri) of an ion as a function of the current (a) and flow rate (dv/dt). FIG. 1A is a graph depicting the direction of net movement of an ion as a function of the ratio a:dv/dt on a particular matrix. FIG. 2 is a fragmentary, elevational, sectional view, taken along a vertical axis of an electrophoresis apparatus that includes an embodiment of the invention which utilizes two uniform support matrices; FIG. 3 is a fragmentary, elevational, sectional view, taken along a vertical axis of an electrophoresis apparatus that includes another embodiment of the invention which utilizes a gradient support matrix; FIG. 4 is an elevational, sectional view taken along a vertical axis of an apparatus according to another embodiment of the present invention which utilizes a single uniform support matrix; FIG. 5 is an elevational, sectional view, taken along a vertical axis of an apparatus according to another embodiment of the present invention; FIG. 6 is a schematic representation of another embodiment; FIG. 7 is a perspective view of an embodiment of the Fig. 6; ; FIG. 8 is a cross-sectional view of the embodiment depicted in Fig. 7 taken along line 8-8; FIG. 9 is a fragmentary, elevational, sectional view, taken along a vertical axis of an electrophoresis apparatus that includes one embodiment of the invention; FIG. 10 is a fragmentary, elevational, sectional view, taken along a vertical axis of an electrophoresis apparatus that includes one embodiment of the invention. Fig. 11 is a fragmentary,elevational sectional view taken along a vertical axis of an apparatus similar to that of Fig. 2 and useful in a method of separating uranium-235 from uranium238; Fig. 12 is a fragmentary, elevational, sectional view taken along a vertical axis of an apparatus useful in a method of separating uranium-235 from uranium-238 and Fig. 13 is a fragmentary, elevational, sectional view taken along a vertical axis of an apparatus useful in a method of separating uranium-235 from uranium-238. The electrophoretic mobility of an ion is proportional to the charge to mass ratio of that ion and by the method of the present invention it is possible to separate different ions or isotopes on the basis of their differing mass. For example, an ionic form of uranium, the uranyl ion, would have about a 1 percent higher electrophoretic mobility if made from the lighter isotope, uranium-235, than if made from the heavier form, uranium-238. This difference in mobility allows separation. The present invention utilizes the influence of the applied voltage and carrier fluid flow on an ion to effect separation. Ions, such as uranium ions, in solution or suspension will migrate in an electric field at a rate Re (distance traversed per unit time) determined by (a) the electrical current, a,(amperes); (b) the electrical resistance of the carrier fluid, r; (c) the reciprocal of the cross sectional area of the separation chamber, 1/A; (d) the influence of the separation medium on electrical resistance, Ce; (e) the electrophoretic mobility Me of the ion (proportional to the total charge of the ion and inversely proportional to its total molecular weight); and (f) by physical characteristics of the medium through which the ions must migrate, usually expressed as ke (the factor by which a particular medium influences the elec ¯mo ec The trophoretic mobility of a particuarylon. The rate of movement due to electrophoresis Re is represented by the e formula: (1) Re = a ¯ r ¯ 1/A ¯ Ce ¯ Me s ke Similarly, the ions in solution or suspension will move, when the carrier fluid is made to flow, at a rate, Rf (distance traversed per unit time), determined by (a) the rate of flow of the entire solution, dv/dt ¯ 1/A (the volume of solution flowing past a point over the time interval dt divided by the cross sectional area, A,of flow) and (b) the physical characteristics of the medium through which the ions must move, kf (the degree to which a particular medium influences the movement due to flow of a particular ion). The movement due to flow Rf is represented by the formula: (2) Rf = dv/dt ¯ l/A ¯ kf In the process of the present invention, the movement of a given ion due to electrophoresis is counterbalanced by its movement due to flow in the opposite directions In addition, a heterogeneous medium which has varying separation characteristics, results in an equilibrium force which counteracts diffusion and concentrates the desired ion in an equilibrium zone. The terms r, 1/A, Ce, Me, ke, and k are referred to as separation characteristics. The terms Ce, k and k are also referred to as medium characte e f ristics. In addition, in accordance with the principles described below, the present invention provides a separation chamber with varying separation characteristics, i.e. divides the separation chamber into different compartments or regions, and produces an equilibrium force by taking advantage of the fact that differing separation characteristics, can have different influences on the rate of electrophoresis (Re) and on the rate of flow (Rf), e.g. by utilizing two matrices which have different values for ke, Ce and k. As can be seen from formula (1) the rate of movement due to electrophoresis, Re, can be varied independently by varying the applied voltage, i.e. regulating the amount of electrical current, a, put through the separation chamber. Similarly, in formula (2) the rate of movement due to flow, Rf, can be independently varied by varying the rate of carrier fluid flow, dv/dt. The dependence of Re on the current, a, is linear if other parameters are constant, i.e. the ion species and the separation characteristics. Similarly, the dependence of Rf on the rate of flow of the carrier fluid is linear when other parameters are constant, i.e. the separation characteristics, the chamber cross-sectional area, the ion species and the carrier fluid. In each compartment n = i, ii, iii, etc. a given ion will have a net mobility Rn equal to the sum of its rate of movement due to electrophoresis, Re (defined as in the positive direction) and its rate of movement due to flow, Rf. For example, in compartment i the net mobility Ri is equal to Re-Rf (minus because flow and electrophoresis are counterdirectional). If movement due to electrophoresis dominates then R. is positive while if movement due to flow dominates Ri is negative. Since both Re and Rf can be adjusted independently, Ri can be made to be positive, zero or negative (Fig. 1). An alternate way of expressing this is, if Ri is positive, then Re :Rf > 1, if Ri is negative, is zero then Re:Rf=l This absolute ratio EMI12.1 nd if Ri varies in proportion to the ratio of a:dv/dt, as depicted in Fig. 1A. The direction of net movement of an ion is depen dent on the separation characteristics, as well as on the ratio of a:dv/dt. Where two matrices are selected which do not have the same k , Ce and kf, a given ion will have a e different net mobility, Rnl in the different matrices in the separation chamber despite the same values of a and dv/dt. Thus in Fig. 1A two curves are given: one for a matrix i and one for a matrix ii. Typically, when two matrices are used in one separation chamber, the same electric current and the flowing solvent pass through both matrices. (Note that in some embodiments, such as that depicted in Fig. 9, the same electric current and same carrier fluid do not pass through all compartments of the separation chamber) Thus, although a:dv/dt can be varied, it typically will only have one value at a time for one separation chamber and thus is the same for all compartments of the chamber, e.g. matrix i and matrix ii. It can be seen from Fig. 1A, at all values of a:dv/dt between V1 and V2, the net mobility of the given ion will be in opposite directions in matrix i and matrix ii. Thus, if the two matrices are positioned such that the matrix in which the net mobility is negative is placed contiguous to and in the direction of electrophoresis with respect to the matrix in which the net mobility is positive, then the ion will migrate toward the common boundary of the two matrices. The ion will concentrate adjacent the boundary of these matrices and will form an equilibrium zone. An equilibrium zone is defined as that region of the separation chamber in which a particular species is concentrated and has a net longitudinal movement of substantially zero. The details of the conditions within the equilibrium zone are described below (section entitled: Equilibrium Zones). Fig. 2 depicts one embodiment of the present invention in which two compartments or regions comprising two different matrices 116, 118 are utilized to produce longitudinally varying medium characteristics in a separation chamber 100. The medium characteristics, applied voltage and rate of carrier fluid flow produce dynamic equilibrium conditions for given preselected Y ions 120. By dynamic equilib rium conditions for a species of ion is meant conditions in a separation system the effect of which is that a particular species of ion remains stationary at the position in the system where those conditions exist, i.e. the ion concentrates in an equilibrium zone. Any pump 114 capable of maintaining a constant output may be utilized to pump the carrier fluid through the separation chamber 100. The pumping rate chosen is as dictated by the separation requirements. That is, the movement due to flow (equation 2) is proportional to the pumping rate which can be selected to give dynamic equilibrium conditions for the desired ions. The separation chamber 100 can be of almost any form as demanded by the particular separation technique. Although simple changes in the shape of a separation chamber can produce gradients in the voltage field and fluid flow rates, it should be noted that these trivial changes cause parallel changes in both the rates of movement due to flow and movement due to electrophoresis and do not produce the variation in the ratio of these rates required to implement the present invention. The material contacting the solution 110 is typically a non-conductor of electricity. The chamber 100 is usually closed, except for exit ports which may be disposed at any point along the chamber 100 for convenient removal of desired ions at their position of dynamic equilibrium, i.e. at the position of about zero net mobility. The chamber 100 is impermeable to the solution 110 so that constant fluid flow can be maintained. A preferred separation chamber 100 is a vertical cylindrical chamber (column), typically of glass or suitable plastic. A transverse disc or mesh 138 at the bottom of the column 100 supports the matrices 116, 118 which longitudinally fill the cavity of the column 100. This disc 138 allows the carrier fluid to flow and ions to move. Preferably, the electrodes 130, 132 are isolated from the separation chamber 100 by having the electrodes 130, 132 contact the solution 11Q in reservoirs 134, 136 which are connected to the separation chamber 100 by a liquid contact 139, 141 such as a membrane barrier or matrix (different from the support matrices discussed above) which resists fluid flow but does not 'resist the electric current flow, i.e. the flow of ions, through the solution. A carrier fluid 110 containing A ions 112 and X ions 113 pumped by a pump 114 flows down through a first support matrix 116 of type i, then through a second support matrix 118 of type ii. Under the configuration depicted in Fig. 2 matrix i 116 is positioned above matrix ii 118, the movement of Y ions 120 due to flow is downward, and the movement of Y ions 120 due to electrophoresis is upward. In this embodiment the matrices i, ii are chosen such that Ce ke/kf for matrix i 116 differs from Ce ke/kf for matrix ii 118. As an example, in a matrix i having Cei k . equal to 1 ei Ce. keii of matrix ii, and having a kfi equal to twice ii eii that for matrix ii (kfii), the electrophoresis rate of Y ion 120 is the same in matrix i 116 and matrix ii 118 (Rei = a . r. 1/A . Cei .Me ¯kei = Reii = a . r ¯1/A ¯ Ceii ¯ Me . keii). However, the flow mobility of Y ion 120 in matrix i 116 (Rfi) is twice the flow mobility (Rfii) in matrix ii 118 (dv/dt ¯ 1/A ¯ kfi = dv/dt ¯ 1/A ¯ 2 x kfii). That is, even though the carrier fluid 110 is flowing at the same rate (dv/dt ¯ 1/A) in matrix i 116 and ii 118, the Y ion 120 flows at twice the rate in matrix i 116 (Rfi) as it does in matrix ii 118 (Rfii). Thus, when a voltage applied by a power source 122 is chosen so that the electrophoresis rate for Y ion 120 in both matrices (since Rei = Reii) is less than Rfi but greater than Rfii (Rfi > a ¯ r ¯ 1/A ¯ Ce ¯ Me ke > Rfii), then the movements of Y ions 120 due to electrophoresis and due to flow are imbalanced in both support matrix i 116 and ii 118. In matrix i 116 the movement due to flow (Rfi) dominates, Rfi:Rei is greater than 1, and the net downward movement is Rfi -a . r ¯ 1/A ¯ Ce ¯ Me ¯ ke. In matrix ii 118 the movement due to electrophoresis dominates, i.e. Rfii :Reiiis less than 1, and the net upward movement is a ¯ r ¯ 1/A ¯ Ce ¯ Me ¯ ke - Rfii As a result, the Y ions 120 concentrate at the boundary 124 of the two matrices 116, 118 and are prevented from diffusing away because the described forces return the Y ions 120 to the boundary 124. The only ions which will concentrate at the boundary are those for which Ri (the net mobility in matrix i) is negative and Rii (the net mobility in matrix ii) is positive. Thus, Y ions i20 are separated from impurities by choosing conditions such that Y ions 120 satisfy these conditions and the impurities do not. When the segregation of Y ions 120 is completed, the purified Y ions are removed from the chamber by interrupting the applied voltage, i.e. current through the chamber. As carrier fluid 110 continues to flow the zone of Y ions 120 will move down the column and may be collected by any conventional collection means known in the art, e.g. a fraction collector which collects successive volumes of the eluting fluid in different test tubes. Fig. 3 illustrates an alternative embodiment of the invention in which equilibrium points for a plurality of ions are established using a non-uniform or gradient support matrix 217, e.g. two or more different support media combined in different proportions to produce a continuously varying gradient of their separation medium characteristics. For example, two support matrices having kf = a and 2a may be combined to form a multiplicity of contiguous compartments producing a gradient of kf from a to 2a. The carrier fluid is then made to flow through the gradient and a sample 208 containing a mixture of different ions is added to the system. In the instant example, only those ions with electrophoresis rates (Re) between dv/dt - 1/A ¯ a and dv/dt 1/A ¯ 2a achieve equilibrium. Different ions which satisfy these boundary conditions will have equilibrium points at different locations, i.e. positions of the varying medium characteristics, in the gradient, (in different compartments), and therefore the ions separate out in different discrete zones, each at its particular equilibrium position in the gradient, i.e. in the compartment at which the dynamic equilibrium conditions for that species of ions exists. In Fig. 3, as in Fig. 2, flow is downward and electrophoresis movement of the desired ions is upward. A sample 208 containing S ions 212, T ions 214, V ions 216, U ions 218, Z ions 220, and Y ions 222 is introduced into the separation chamber 100. A carrier solution 110 comprised of ions A+ 112 and X 113, is pumped by pump 114 through the sptio chamber 10. The chamber 100 contains a gradient support matrix 217 whose characteristics are such that S , T , V , and U ions 212, 214, 216, 218, each have equilibrium points at different locations on the gradient, while Z and Y ions 220, 222 do not. Thus, S , T , V and U ions 212, 214, 216, 218 can be collected from their respective equilibrium zones 232, 234, 236, 238, by removal through exit ports 227, 228, 229, 230. For this purpose, a plurality of pumps 252, 254, 256, 258 and the associated conduits are provided, each pump being coupled to the respective exit port. The location of the exit ports relative to each other is not important and thus they may be aligned as depicted in Fig. 2 or may be in a spiral pattern about the exterior of the column. Also the chamber may be pre-equipped with a series of potential ports. Only those ports adjacent equilibrium zones formed are opened, either simultaneously or sequentially. The outlet of each pump 252, 254, 256, 258 is coupled to appropriate downstream equipment, such as a reservoir. In each of the embodiments of the present invention, it is frequently advantageous to re-circulate, as depicted in Fig. 3, the electrolyte solutions 135, 137 present in reservoirs 134, 136 by means of a pump 204 in order to reduce any effects due to electrolysis. As depicted in Fig. 3, the process is a continuous one in that sample 208 is continuously fed into the system while concentrated S ions 212, T ions 214, V ions 216, and U ions 218 are continuously withdrawn. As will be understood by those skilled in the art, for continuous operation the rate of withdrawal of a given ion must be about equal to the rate of accumulation of that ion and each rate of withdrawal must be such as not to interfere with the equilibrium positions. In order to attain rates of withdrawal of ions which approximate the rates of accumulation, sensing means 207, 208, 209, 210, such as thermocouples, spectrophotometers, geiger counters or the like, may be utilized to detect the boundaries of the equilibrium zones 232, 234, 236, 238 and optionally to con trol pumps 252, 254, 256, 258, and thus the rates of withdrawal. For example, the presence of the ions in each equilibrium zone will influence the electrical resistance of the solution in the equilibrium zone. Since the rate of heat generation, will increase as the resistance increases (W = a2R or wattage is equal to the square of the electrical current times the resistance), the temperature of the equilibrium zone will be somewhat different than that at adjacent parts of the separation chamber. Sensing means, such as thermocouples (not depicted) placed on the exterior of the column may be used to detect the temperature discontinuities in order to monitor the positions of the equilibrium zones. As the zone expands in size, the rate of withdrawal is increased and as the zone decreases in size the rate of withdrawal is decreased. Fig. 4 depicts an embodiment of the present invention wherein a single matrix is utilized. In Fig. 4 flow is upward and electrophoretic migration is downward. Carrier fluid 110 is pumped by a pump 302 through a separation column 100 having a single matrix 310 of uniform medium characteristics. In this embodiment the matrix 310 defines compartment i and compartment ii is a free solution. In this embodiment the crude material 300 is fed into the separation chamber 100 by pump 300 via port 304. As has been described the rate of movement due to electrophoresis and movement due to flow are given by equations 1 and 2, respectively. Where there is no matrix, i.e. in a compartment or volume of free flowing carrier fluid, equation 1 reduces to R = a r 1/A Me and equation 2 reduces to Rf = dv/dt 1/A This simplification amounts to no more than a special case in which the separation medium characteristics, Ce, ke and kf, are unity. Compartment ii can be treated exactly as if it contained a matrix with these properties. Thus, in Fig. 4 of the matrix 310 is one in which at a particular ratio of a:dv/dt, the net mobility of the Y ions will be negative within the matrix while it will be positive in free solution. Under these circumstances the Y ions will migrate toward the boursucL-y of trie two compartments and, as described below, will accumulate in an equilibrium zone with one of its two boundaries coinciding with the boundary of the two compartments. Where crude material 200 containing Y is continuously fed into the system, Y ion 120 in a concentrated or purified form may be continuously removed from port 305 by the action of pump 307. Referring again to Fig. 1, it will be understood that the imbalances in movement due to electrophoresis and movement due to flow will cause the desired ion, Y , to move toward the boundary of the two compartments. Since the ion Y is not normally at equilibrium in either compartment i or compartment ii, an additional factor must be involved in the formation of the equilibrium zone. The additional factor is the concentration of Y . (The movements of ions within a compartment, Re and R have been described without consideration of the effects of the concentrations of the ions being separated. Although this represents a simplification of the real situation, throughout most of each compartment the concentrations of the ions being separated are typically too low to have a substantial impact on Re and Rf.) Since the ion being separated, Y , reaches a high concentration within its equilibrium zone, the rate of movement due to electrophoresis within an equilibrium zone is R = Re ¯ fl(c) and the rate of movement due to fluid flow within an equilibrium zone is Rf' = Rf - f2(c) where f (c) and f2(c) are correction factors which vary as a function of the concentration, c, of the ion forming the equilibrium zone. The correction factors f1(c) and f (e) differ for different ions and for different combinations of separation conditions. As will be understood from the following descriptions fl(c) and f2(c) need not be known. They simply serve here as an attempt to explain the behavior of the equilibrium zone. Consider first the following specific case. Increasing concentrations of Y ions cause a decrease in Ret e.g., f1(c) = 1/c and Ret = Re 1/c. Furthermore, the concentration of Y ions has no influence on Rf. That is, f2(c) = 1 and Pbf' = Rf. Consider that for Y ions, fin = Re - Rf is positive in compartment i (Ri > 0) and negative in compartment ii (Rii < O) and that compartment ii is on the electrophoresis side of compartment i, i.e. contiguous to compartment i in the direction of electrophoresis. Under these conditions the equilibrium zone is thought to form and behave as follows. Ion Y accumulates at the boundary of the two compartments and the increasing concentration of Y results in a decreased movement due to electrophoresis. When Rel is reduced to the point where it equals Rf in compartment i, the equilibrium zone has been established. If the concentration increases further the movement due to flow dominates and Y moves into compartment i, thereby decreasing the concentration of Y , increasing the size of the zone and reestablishing the equilibrium situation. If the concentration of Y were to decrease, Re would increase and thus movement due to electrophoresis would dominate which would cause the Y ions to move toward the boundary of compartments i and ii, and once again increase the concentration of Y ions and reestablish the equilibrium situation. Thus, it is clear that the equilibrium zone is self-regulating and the ion, Y , composing that zone is maintained at just that concentration which causes Re' to counterbalance Rf'. When additional Y ions are added to the zone, the zone will increase in size and similarly the removal of Y ions from the zone will cause it to decrease in size. In this particular case the equilibrium zone will form in compartment i. One boundary of the equilibrium zone will correspond to the boundary of compartments i and ii, and the other boundary of the zone will be within compartment i (its position being determined by the total amount of Y which has accumulated in the equilibrium zone). It should be emphasized that these conditions establish themselves automatically and that an equilibrium zone will be established irrespective of the values of f1(c) and f2(c). However, the position of the equilibrium zone will depend on the values of fl(a) and ¯2(c). Although for the instant example one of the boundaries of the equilibrium zone will always correspond with the boundary of compartments i and ii, the other boundary of the equilibrium zone may lie in either compartment i or ii depending on the values of f (c) and f2(c). Consider the following cases wherein for ion Y- Rn = Re - Rf is positive in compartment i (Ri > 0) and negative in compartment ii (Rii < 0) and that compartment ii is on the electrophoresis side of compartment i. Case 1: If increasing concentration of Causes Re' to increase, then when a certain concentration is reached Rii will become zero. That is, normally Rii = Reii - Rfii, whereas within an equilibrium zone lying in compartment ii Rii' = Re' - Rf' and in this case Rf' = Rfii. Thus, if Re' = Rfii then within the equilibrium zone Rii' = Re' - Rfii = O. This condition develops because Reii < Rfii and the increasing concentration of Y ions cause Ret to increase until it equals Rfii. In contrast, in this particular case Ri cannot become zero because Rei > Rfi and increasing the concentration of Y ions would only increase Ri. Thus, under these conditions the equilibrium zone forms in compartment ii and one boundary corresponds to the common boundary of compartment i and compartment ii. Case 2: If increasing concentration of Y causes Rf' to increase, then when a certain concentration is reached R. will become zero. Note that in this case Rii cannot become zero but rather becomes more negative. Thus the equilibrium zone will form adjacent to compartment ii but within compartment i. Case 3: If increasing concentration causes Rf' to decrease, then when a certain concentration is reached Rii will become zero and Ri cannot become zero. The equilibrium zone will form adjacent to compartment i but within compartment ii. More complicated cases which can exist are those wherein both Re and R are affected, Rei and Reii are affected differently, Rfi and Rfii are affected differently, or combinations thereof. As will be apparent to those skilled in the art, a quasi equilibrium can be reached in all cases and the concentration of a species in the equilibrium zone will be determined by the separation system characteristics. As will be apparent to one skilled in the art, a number of different factors could be involved in the influ ence of concentration on R and Rf. For example, the ion, e Y , forming an equilibrium zone may alter the electrical conductivity of the equilibrium zone, and thereby influence its own mobility. Alternately, Y ions might interact with biospecific sites on one of the matrices, and the high concentration of Y ions in the equilibrium zone might saturate these sites and alter both Re' and Rf'. When more than one species of ions satisfies the boundary conditions, Ri > 0 and Rii < 0i (as depicted in Fig. 5), all such ions are forced toward the boundary of the two matrices. However, each of the different ion species will typically form a separate or discrete equilibrium zone. This follows from the above described considerations and can be understood as follows. Consider a system in which one ion, Y , has formed an equilibrium zone where one boundary of the zone coincides with the boundaries of matrices i and ii which form the compartments of the separation system (e.g. Y ions in Fig. 5). Furthermore, in the instant example, matrix ii (416 in Fig. 5) is on the electrophoresis side of matrix i (418 in Fig. 5) and according to principles described Ri > O and Rii < O for ion Y whose equilibrium zone lies in matrix i. If a second ion V is introduced into this separation system and if this ion also satisfies the boundary conditions, Ri > 0 and Rii < 0, then it will be forced toward the boundaries of compartments i and ii. However, the net rate of movement of ion V will typically be affected by the high concentration of ion y in the existing equilibrium zone. Thus, assume the net rate of movement of ion V within the equilibrium zone of y (designated Rn') is less than 0. Thus, Ri > O while Rnl and Rii are less than zero and V would concentrate at the boundary of the equilibrium zone for y ions and the remainder of compartment i. Following the principles described ab#ove, ion V would then form its own equilibrium zone in this position (Fig. 5). Similarly, if a third ion species, ZI, were intro- duced into the separation system, it would typically form a third discrete equilibrium zone if it satisfies the boundary conditions, wi > O and Rii < O. Additionally, if the net rate of movement of Z ions within the equilibrium zone of Y , Rnl, is less than zero and similarly the net rate of movement of Z ions within the equilibrium zone of V , Rn ", is also less than zero, then Z ions will form an equilibrium zone between the equilibrium zone of V ions and the remainder of compart ment i (Fig. 5). That is, since Rii, Rnt and Rn'' < 0, V ions will migrate in the direction of flow whereas since Ri > 0 it will migrate in the direction of electrophoresis in compartment i and thus be forced to this equilibrium position. To one skilled in the art it will be obvious that this condition of multiple discrete boundaries will form automatically and does not require that the different ions species be introduced sequentially. Referring again to Fig. 5, crude material 400 containing Y ions 420 to be purified is fed into the separation chamber 410 with the flowing carrier fluid 440. Port(s) 402 are attached to the separation column just below the boundary 424 of the two matrices 416, 418 (below assuming that the equilibrium zone will form in the lower compartment; under other conditions the port would be positioned above the boundary) and both fluid 440 and the purified Y ions 420 can be withdrawn from the equilibrium zone 450, provided only that the following requirements are satisfied: (a) either the Y ion 420 of interest must be the only component which comes to equilibrium in the separation system or if several ions, i.e. Y ions 420, V ions 419, and Z ions 431, come to equilibrium, y ions 420 must form the first equilibrium zone (the one bordering on the boundary 424 of the two matrices 416, 418) so that the position of this zone will not change with time or if the ion of interest forms an equilibrium zone whose boundary is defined by another equilibrium zone, i.e. V ions 419, X ions must be withdrawn at a rate corresponding to their rate of accumulation so that the position of the equilibrium zone of the desired ion remains constant; and (b) the rate of carrier fluid 440 withdrawal from the port 402 must not perturb the fluid flow 440 through the separation chamber 100 to an extent which would destroy the e riwn force. Furthermore,- the rate of withdrawal of material at the port 402 must substantially match the rate of accumulation of the desired Y ion 420 in the equilibrium zone 450 if maximum purification and recovery are required (this can easily be accomplished since the zone 450 can be detected by one or more sensing means 480 which regulates the withdrawal from the ports by control of the withdrawing pump 482). Another embodiment of the present invention depicted in Figs. 6, 7 and 8 provides for, and is particularly useful in those applications which require high purification factors. In this design, the flow force (and thus the movement of the desired ions due to flow) and the electrophoretic force (and thus the movement due to electrophoresis) do not directly oppose each other but are at an oblique angle relative to each other. This is shown schematically in Fig. 6, where the direction of the electric field 502 is at an angle, 6, to the direction of fluid flow 500. The electric field of magnitude V can be considered to be composed of two vectors. One of these would have a magnitude of cos o V and would directly oppose the movement due to fluid flow and the other vector would have a magnitude of sin e V and would act in the direction at right angles to the direction of fluid flow. If the rates of net movement, Ri and Riit in the two compartments 521, 522 are only considered in the direction parallel to the direction of fluid flow, then the only relevant voltage field is that which is in parallel with this direction of flow. An appropriate electric force, V, would generate a value of cos o V such that for a particular ion species, Y , Ri < 0 and Rii > 0. Thus, Y ions will be forced toward the boundary of the two compartments 521, 522 and will form an equilibrium zone 506. Although y ions in the equilibrium zone will have no substantial movement in the direction defined by the direction of fluid flow, these ions will be influenced by the other vector of the electric field, sin e.V, and will migrate laterally from the position of the inlet port 504 to the position of the outlet port 508. In order to avoid distortions in the electric field or the pattern of fluid flow, this design incorporates a wedge 510 on each side of the separation chamber composed of a third matrix which resists fluid flow but which allows ions to move under the influence of a voltage gradient. Figs. 7 and 8 illustrate one embodiment of an apparatus for combining lateral movement and dynamic equilibrium electrophoresis. Carrier fluid enters through an entry trough 705 and flows through a separation chamber having longitudinally varying medium characteristics exiting through an exit trough 707. The crude material is fed in on one side of the separation chamber 701 at the entry port 703 and an equilibrium zone 706 is formed at the boundary 724 of the two matrices, matrix i 721 and matrix ii 722. However, the ions move laterally along the equilibrium zone 706 because the movements of the ion due to electrophoresis and flow cancel out only along the longitudinal direction or axis and a residual lateral force remains along the other lateral axis. Thus, the purified material can be collected from an exit port 708 at the boundary 724 of the two matrices but positioned on the side of separation chamber opposite the input part 703. Typically a third matrix iii 710 or other material which resists fluid flow but which allows ions to move under the influence of a voltage gradient will be positioned along the lateral sides of the separation chamber. In this embodiment electrodes 731, 732 are placed in long electrode reservoirs 733, 734 which are parallel with each other but at an angle to the entry and exit troughs 705, 707. The electrode reservoirs 733, 734 are connected to the separation chamber 701 by membranes 735, 736 which resist fluid flow but allow electric current to pass. Fig. 8 is a cross-sectional view of the apparatus of Fig. 7 taken along line 8-8. In all of the embodiments described above, the longitudinal variation in separation characteristics is produced by the presence of one or more matrices. In an alternate embodiment described here, the concentration of the carrier fluid ions varies longitudinally resulting in a longitudinal variation in the resistance of the fluid to electrical current and thus producing a longitudinal variation in electric force affecting the ions. As can be seen from equation 1, the rate of movement due to electrophoresis, Ret varies in direct proportion to a change in the electrical resistance of the carrier fluid, r. Since Rf is unaffected by r (equation 2) the production of a continuous gradient of r along the longitudinal dimension of a separation chamber will produce a multiplicity of compartments with differing ratios of Re/Rf. As described above, these are the conditions needed for dynamic equilibrium electrophoresis. An increase in the concentration of ions causes a decrease in the electrical resistance of a fluid. The embodiment shown in Fig. 9 is illustrative of methods which can be used to generate such a gradient of ion concentration. A second chamber 901 whose walls are constructed of a semipermeable membrane, i.e., a material which resists bulk fluid flow and restricts the passage of the desired Y ion, but allows the carrier ions A+ and X to freely diffuse across (e.g. cellulose dialysis membrane) is enclosed within the separation chamber 900. Pump 902 pumps a dilute solution of A+X through the inner chamber 901 and pump 903 pumps a concentrated solution of A X through the separation chamber such that the direction of flow in the separation chamber 900 is counter to the direction of flow in the inner chamber 901. Under these conditions A X will diffuse from the separation chamber 900 into the inner chamber 901 so that the concentration of A+X in the carrier fluid will increase as the carrier fluid passes through the inner chamber 901. Of course the concentration of A X in the carrier fluid will decrease as it passes through the separation chamber 900. Because of the high concentration-of A+X and thus the low electrical resistance of the carrier fluid when it enters the separation chamber, Re for ion Y will be low and Rf will dominate so that net movement will be in the direction of flow. However, as Y moves up the separation chamber, r increases as does Re and under the appropriate conditions Re will reach a value which counterbalances Rf and an equilibrium zone 910 will form. Note that the electrode reservoir 136 connected to the end of the separation chamber containing a high concentration of A X will be filled with a carrier solution containing the high concentration of A X while the electrode reservoir 134 attached to the end of the separation chamber containing the low concentration of A X will be filled with the carrier solution containing the low concentration of A+X . It should be further noted that this embodiment requires that rates of flow are sufficiently low to allow the concentration of A X to approach equilibrium across the walls of the inner chamber 901. The rate of diffusion of A X across this wall is likely to limit the practicality of large scale apparati of this particular type. It should be possible to circumvent this limitation by substituting the inner chamber 901 with a multitude of small chambers. The construction of chambers of this type is a known art, e.g. the Diaflo hollow fiber units made by Amicon. In another embodiment of the present invention, unequal flow rates in different compartments of the separation chamber provide the longitudinal variation in separation characteristics. Fig. 10 illustrates one such embodiment. A carrier fluid containing carrier ions 112, 113 as well as the desired ion species, Y , is pumped through the separation chamber which can be filled with a matrix whose only importance is anticonvection, e.g. the influence of this matrix on Re and Rf is not relevant to operation in this mode. Pump 1082 pumps carrier fluid containing the desired ion out of the separation chamber and returns it to the inlet of the separation chamber. Because of the operation of pump 1082 the rate of carrier fluid flow in compartment 1012 is lower than the rate of fluid flow in compartment 1011. Under the appropriate operating conditions Re/Rf > l in compartment 1012 and Re/Rf < l in compartment 1011. Thus, the desired ion would collect at an equilibrium zone which typically will form adjacent to the exit port 1020 serving the pump 1082. The desired ion y can be collected by another exit port 1021 by the action of pump 1083 or alternate methods described above could be usea for collection. Because the action of pump 1082 tends to distort the path of fluid flow, this embodiment works most effectively with a layered matrix which partly resists fluid flow in the longitudinal dimension but gives little resistance to lateral fluid flow (i.e. where a large pressure differential is required to drive the fluid longitudinally through the separation chamber). Fig 11 depicts one embodiment of the present invention in which two different matriceslll6,11lS are utilized in -the separation chamber 100 to produce equilibrium. A carrier solution 110 pumped by a pump 114 flows down through a first support matrix4ll6 of type i then through a second support matrixll8 of type ii. The matrices i, ii are selected for their differing influence on either the electrophoresis rate or flow rate of uranium ions20, i.e. ke/kf for matrix i is not the same as ke/kf for matrix ii. For example, if ke and Ce are the same for matrix i and ii, and kf for matrix i (Kfi) was twice kf for matrix ii (kfii), then the electro phoresis rate (Re) of 235U+ ion @20 will be the same in matrix i 1116 and matrix ii1118 (a ¯ r . 1/A ¯Cei ¯ Me k i ei mobil- Ceii. Me .keii). However, the flow mobil- as in matrix ii (dv/dt e 1/A $ kfi = dv/dt ¯ 1/A ¯ 2 x kf That is, even though the carrier fluid 110 is flowing at the same rate (dv/dt o I/A) in matrix j1116 and matrix ii@118, the 235U+ ion 120 flows at twice the rate in matrix i@ 116 as it does in matrix ii 1118 (Rfi = 2 . Rfii) Thus, when a voltage of a particular magnitude is applied by a power source 122 such that the electrophoresis rate (Rf) for 235U+ ion 1120 is less than Efi but greater than Rfii (Rfi > a r 1/A ¯ Ce e Me ' ke > Rii) then the electrophoresis rate of the 235U+ ion@120 and the flow rate of the 235U+ ionsll20 are imbalanced in both support matrix i1116 and matrix ii1118. In matrix i1116 the flow rate dominates, i.e., the ratio of the magnitudes of Rfi to Rei is greater than 1, and the net rate of movement in compartment i, Ri, will be Rei-Rfi where movement in the direction of electrophoresis is defined as positive. In matrix ii 1118 the electrophoresis rate dominates, i.e. the ratio of Rfii to Reii is less than 1, and the net rate of movement in compartment ii, Rii, will be Reii - Rfii. Thus, the 235U+ ions 1120 concentrate at the boundary 224 of the two matrices1116,1118 and are not able to diffuse away because the described forces return the 235U+ ions 1120 to the boundary 1124. The only ions which will concentrate at the boundary are those which satisfy the inequality, Rfi > Re > Rfii Typically, because their electrophoretic mobilities are so similar, 235U+ and 238U+ ions will satisfy the inequality under very similar conditions. Thus, it would be difficult to select conditions where only 235U+ ions would concentrate at the boundary of the two matrices. Nonetheless, these two isotopic forms can be separated according to the present invention because they will form distinct equilibrium zones adjacent the boundary of the two compartments. By compartment is meant a portion or region of the separation chamber in which a particular species of ion has a unique ratio of its movement due to flow and movement due to electrophoresis under particular conditions, e.g. when the electric field, rate of carrier fluid flow and composition of the carrier fluid are constant and where ions other than those of the carrier fluid are at an insignificant concentration. In this example the compartments correspond to the regions of the separation chamber occupied by matrix i and matrix ii. The formation of distinct equilibrium zones is explained as follows. Since the ions under consideration, U ions, are not at equilibrium in either compartment i or compartment ii, an additional factor must be involved in the formation of the equilibrium zone. The additional factor is the concentration of the U ions. The U ions will migrate toward the boundary of compartments i and ii. First, considering only 235U ions, as the concentration of 235U ions increases at the boundary, Re and Rf will be affected such that Re' = Re fl(c) and Rf' Rf f2(c) where Re' is the rate of movement due to electrophoresis when influenced by the concentration of 235U+ ions, Rf' is the rate of movement due to flow when influenced by the concentration of 235U ions, and fl(c) and f2(c) are correction factors which vary as a function of the concentration, c, of the ion forming the equilibrium zone, 235U+. Consider the following specific case: Increasing concentrations of 235U+ ions cause Rel to decrease but have no effect on Rf', e.g. Re' = Re ' I/e and Rf' = Rf ¯ 1. Thus, as 235U+ ions accumulate at the boundary of the two compartments Re' will decrease until it equals Rfii and will thereby establish an equilibrium zone where one of the boundaries of the zone correspond to the boundary of compartments i and ii and the other boundary lies within compartment ii. The position of the boundary within compartment ii, and thus the size of the zone, depends on the amount of 235U ions in the equilibrium zone. That is, if the amount of 235U ions were to decrease, then the concentration, c, would decrease and Re' would increase which would force the 235U ions toward the boundary of compartments i and ii, thereby concen trating the 235U+ ions and reestablishing an equilibrium zone. Similarly an increase in 235u ions would case Rue 2 to decrease so the movement due to flow (Rfii) would dominate and 235U ions would move further into compartment ii to establish a larger equilibrium zone where, once again, Re' = Rfii. Thus, it is clear that the equilibrium zone is selfregulating and the concentration of 23so+ ions is maintained at just that level which causes Re' to counterbalance Rfii It will be understood that the values of f1(c) and f2(c) will differ for different separation conditions and different ions. However, the values of fl(c) and f2(c) do not have to be known; an equilibrium zone will form in all cases except for the unlikely case where f1(c) - f2(c). It will be apparent to one skilled in the art that an equilibrium zone abutting the boundary of compartment i or ii can lie in either compartment i or ii and that this will be determined by the values of fl(c) and f(c) and the particular conditions. Returning now to the explanation of why 235U+ ions and 238U+ ions will form distinct equilibrium zones, consider the behavior of 238U+ ions in the above described example where 235u+ ions are trapped in an equilibrium zone bordering compartments i and ii but lying in compartment ii. For the instant example, 238U+ ions are also assumed to satisfy the inequality Rfi > Re > Rfii and thus should form an equilib 238 + rium zone. However, note that since R for U ions is less than Re for 235U ions, the 238U ions will not be at equilibrium within the equilibrium zone for 235U ions. Within the equilibrium zone of 235U+ ions, the rate of move- ment of 238U+ ions due to electrophoresis will be about 0.99 Re' and thus the 238U+ ions will be forced to a position between the equilibrium zone of 235U ions and the remainder of compartment ii. Furthermore, as described for the equilibrium zone for 235U+ ions, 238U+ ions will form a second equilibrium zone in which the concentration of 238U+ ions will be automatically regulated so that the rate of movement due to electrophoresis precisely counterbalances their movement due to flow. Since the 238U+ ions are the more abundant ions, continued addition of uranium salts to the separation system will lead to an expansion of the equilibrium zone of 238U+ ions until this equilibrium zone fills that part of compartment ii not occupied by the equilibrium zone of 235U+ ions (Fig.41) at which point additional 238U+ ions will be eluted with the fluid flowing from the separation system. Thus, in effect, 235U ions will be the only ions concentrating adjacent the boundary of compartments i and ii and the 235U+ enriched material can be collected from this position of the separation chamber. Although the present invention is described in terms of the separation of uranium isotopes it will be known and understood by those skilled in the art that the descriptions herein provided are equally applicable to all other isotopes, e.g. hydrogen and deuterium, isotopes of carbon, sulfur, chlorine, oxygen, and the like. For purposes ot any of the#embodiments of the present invention any one of a large number of commercially -available electrical power sources 122 having a stable direct current output can be used. The voltage V applied to the system is chosen as dictated by separation requirements. That is, the voltage gradient dV/dD is directly proportional to the applied voltage or applied current. Thus, by equation 1, the movement due to electrophoresis is proportional to the input voltage or input current. Means for applying the voltage are those known in the art, such as those described in U.S. Pat. No. 3.737.758. The electrodes 130, 132 are typically isolated from the separation chamber 100 by having the electrodes contact the solution in reservoirs 134, 136 which are connected to the separation chamber 100 by liquid contacts 139, 141, e.g. a membrane barrier or matrix (different from the support matrices discussed above) which resists fluid flow but does not resist the electric current flow through the solution. Similarly any pump 114 capable of maintaining a constant output may be utilized to pump the fluid 110 through the separation chamber 100. The pumping rate chosen is as dictated by the separation requirements. That is, the flow rate (equation 2) is proportional to the pumping rate which can be selected to give the desired equilibrium. The separation chamber 100 can be of almost any form as demanded by the particular separation technique. The material contacting the solution is typically a non-conductor of electricity. The chamber is usually closed, except for exit and input ports 102, and impermeable to the solution so that constant fluid flow can be maintained. A preferred separation chamber is a vertical cylindrical chamber (column), typically glass or suitable plastic, in which the fluid flows in the downward direction and electrophoresis drives the desired molecules in the upward direction. A mesh on the bottom 138 of such a column supports the matrices which fill the cavity of the column. Port 102 can be included to permit withdrawal of the purified 235U+ ions 120 from the equilibrium point. A wide range of support matrices have been developed for use in liquid chromatographic procedures. These same matrices can be used in the procedures of the present invention. Some of these matrices may have a different effect on the mobility of ions# of interest driven by flow than they have on the electrophoretic mobility of these molecules and theoretical considerations indicate that virtue ally all such matrices will have the desired differential influence on mobilities. Because of the variety of such matrices, and the variety of their modes of interaction with particular molecules, it is possible, as will be known and understood by those skilled in the art, to separate ions on the basis of virtually any chemi'cal/physical property (e.g., molecular size, ionic character, hydrophobicity, and electro phoretic mobility) by selecting the right matrices. preferred embodiment of the present invention for large scale applications is a continuous flow process. By continuous flow process is meant a process wherein the impure raw material is fed into the system continuously and the purified product removed continuously. Accordingly, the present invention provides a simple means of operating separation as a continuous flow process. As shown in Fig.11, the crude material containing uranium salt 200 is fed into the separation chamber 100 with the flowing solvent 110. Port 1102 is disposed on the separation column just below the common boundary1124 of the two matrices1116,1118 (below if flow is down through matrix i first and then matrix ii, electrophoresis is up, and the Rf:Re is greater than 1 in matrix i and less than 1 in matrix ii) and both carrier fluid 110 and the purified 235U+ ions 1120 can be withdrawn from the equilibrium zone 1150, provided only that the following requirements be satisfied: a) either the 235U+ ions1120 of interest must be the only component which comes to equilibrium in the separation system or if several ions come to equilibrium, it must form the first equilibrium zone (the one bordering on the common boundary 1124 of the two matrices)116, 1118) so that its position will not change with time, b) the rate of fluid 110 withdrawal from the port t02 must not perturb the flow through the separation chamber 100 to an extent which would destroy the equilibrium force. This design offers high capacity and efficient use of power. The embodiment shown in Fig. 11 may be modified to include a) the incorporation of sensors to regulate the rate of input of crude material and withdrawal of purified product; b) a method of inputting the crude material which would avoid any loss to the electrode reservoirs, and c) elimination of carrier ions from the separation chamber so that the efficiency of utilization of electric power would be increased. Fig. 12 shows a preferred embodiment of an apparatus for 235U+ / 238U+ separation. A carrier solution containing A+X is pumped into the separation chamber by pump 114. The positive ions, A ions, in the carrier solution have a higher electrophoretic mobility than the ions to be separated, e.g. U ions. The crude material, e.g. a solution of a salt of natural uranium such as uranylacetate, is pumped into the separation chamber by pump1225. Following principles described by Orstein in U.S. 3,384,564, when a voltage is imposed, the rapidly migrating A ions will not mix with the more slowly moving U+ ions and a distinct boundary 1201 will form between the solution containing U ions and the solution containing A+ ions. Although this boundary 1201 between A+ and U+ ions would normally move toward the negative electrode, in the method of the present invention this movement is counterbalanced by the flowing solution. To ensure that the boundary 901 is maintained in a stable position, pump 114 is regulated by two sensors?227,1228. Typically, sensors1227,1228 are radiation detectors, e.g. Geiger counters, which detect the presence of 235U. As the amount of radioactivity increases and is detected by sensorS227, the pumping rate of pump 114 increases and the increased flow moves the U ions further into the separation chamber 100. Similarly, as the level of radioactivity detected by sensor 1228 decreases, the pumping rate of pump 114 is decreased. The additional flow due to the action of pump;225 causes the U ions to move through compartment i 4116 toward the interface of compartment i and compartment it 4124. To those skilled in the art, it will be apparent that within compartment i, 116, the U+ ions will electrophorese at the same rate as the A+ ions and as the boundary between these ions and thus in the absence of the flow from pump 1225 the movement of U+ ions due to electrophoresis according to the instant invention will be precisely counterbalanced by the flow induced by pump 114. If Rii > b forboth 235U+ and 238U ions, then both these uranium ions will form equilibrium zones adjacent the boundary of compartment i and ii 124. However, as described above, the equilibrium zone-composed of 238U+ ions will quickly fill up compartment it 1118 and therefore only the equilibrium zone for 235U ions 1150 is shown in Fig.42. The 235U+ ions are collected from the equilibrium zone by the action of pump 1226. The sensors1224,4230 are used to regulate pump4225 to obtain maximum throughput. Because of diffusion, the 235U+ ion concentration in compartment i will be somewhat elevated adjacent to the boundary1124 of compartments i and ii. This elevated level of 235U+ ions will be detected by sensor 4229 which will increase the pumping rate of pump 225. Additionally, sensor1230 can monitor the concentration of 235U+ ions within the equilibrium zone to ensure that the flow rate does not exceed the range in which the equilibrium zone can be maintained. Similarly, sensors 231 and 232 can regulate the pumping rate of pump ". 226 so that the rate of collection can match the rate of accumulation. Ion exchange matrices can be used to provide the required differential affect on the flow mobility and elec tòphoretic mobility of the uranyl-235 ion'(120. For example, a cationic exchange resin used as matrix i in compartment i 1116 provides the desired effect: Although this resin will not interact strongly with the uranyl-235 ion, it will bind the counter ion (e.g., acetate), and as a result the salt concentration and the electrical conductivity will increase in this zone. Consequently, the voltage gradient will decrease, reducing the electrophoretic mobility in this matrix. As a second matrix ii in compartment ii1118 either an inert matrix could be used or, in fact, this region of the separation chamber can be left as free carrier fluid (viz. ke in the free solvent will differ from ke in matrix i). Fig. 12 depicts a preferred embodiment wherein compartment ii of the separation chamber comprises free carrier fluid rather than a second matrix. In the embodiment of Fig./2, the electrodes and thus the direction of flow have been reversed to illustrate such a possibility. Otherwise operation is similar to that described with regard to Fig.41. Fig.13 depicts another embodiment which utilizes a gradient matrix in the separation chamber to produce multiple compartments each differing in separation characteristics, e.g. Re/Rf for a particular ion would vary through the separation chamber. Typically a gradient matrix would be produced by mixing in different proportions two matrices with differing characteristics. In this process two different ions might satisfy the boundary conditions and form equilibrium zones, but these equilibrium zones could occupy different regions of the separation chamber and are unlikely to be contiguous. In Fig.13 a-carrier fluid 110 containing carrier ions A+ and X is pumped by pump 114 into the separation chamber 100 which is filled with a gradient matrix4ll9. The power supply 122 imposes a voltage across the separation chamber 100 such that the positively charged (in this instant example) ions electrophorese in the direction counter to the fluid flow. The ions to be separated 235U and 238U+ are fed into the separation chamber 100 by pump 1225. In those areas of the column where electrophoresis dominates the particular ions will move in the direction of electrophoresis while in those regions where flow dominates they will move in the direction of flow and for those ions which satisfy the boun dary conditions the ions will concentrate in that region of the separation chamber where the rates of movement due to flow and electrophoresis are balanced. Because a multitude of compartments exist in the gradient matrix even ions with very similar mobilities can form separate equilibrium zones 1150, 1351. If desired the different ions can be removed from their respective equilibrium zones. Accordingly, pump1226 can remove + can remove a solution rich in U ions via port1102 from equilibrium zone#l50. Similarly, pump 1327 can remove a solution rich in 238U ions via port1303 from equilibrium zone1351, This process could be useful when high purifica tion ratios are needed. A wide range of support matrices has been developed for use in liquid chromatographic procedures. These same matrices can be used in the procedures of the present invention. Virtually all of these matrices may have a different effect on the movement of ions due to flow than they have on the movement due to electrophoresis of the same ions and will therefore be useful in the process of the present invention. Because of the variety of such matrices, and the variety of their modes of interacttDn with particular ions, it is possible, as will be known and understood by those skilled in the art, to separate ions on the basis of virtually any chemical/physical property (e.g., molecular size, ionic character, hydrophobicity, and electrophoretic mobility), by selecting the right matrices. Typical of such matrices are polyacrylamide gels, cellulose, agarose; copolymers of acrylic and methacrylic acids and the like, with or without charged functionalities such as diethyl amino ethyl (DEAE) and carboxy methyl (CM). Other embodiments of the present invention relate to continuous purification or enrichment processes suited to industrial scale applications. By a continuous flow process is meant a process in which the impure raw material is fed into the system continuously and the purified product is removed continuously. In carrying out the present method, a number of alternative configurations and processes are available. General designs by which the process of the present invention can be used as a continuous flow process are as shown in FIGS. 3-8 and 10-13. The process of the present invention, in addition to isotope separation, was utilized for the following specific applications: 1) Protein purification: The process of the present invention was utilized for its capacity to resolve proteins. The high resolution of the system allowed for separation of proteins which differ by less than 1% in their electrophoretic mobility or degree of interaction with the gel permeation matrices used in the separation chamber. Other matrices such as ion exchange resins, hydrophobic affinity resins, absorption resins, and specific affinity resins, can also be used. The number of adjustable parameters are sufficient to allow the procedure to be used for the purification of nucleic acids, proteins, antibodies, antigens, haptens, and hormones, e.g. the purification of interferon, useful for treatment of viral diseases and the isolation of peptide hormones. 2) Purification of antibiotics: Most antibiotics contain a number of amino acids in their structures and can be anticipated to behave somewhat like very small proteins in the described separation procedure. Thus, the process is useful for drug purification and provides an economical method for purifying antibiotics which are made in low amounts and/or are difficult to separate from contaminants. Separation of colored protein, ferritin, was carried out utilizing a carrier buffer solution of 10 mM Tris Acetate, pH 7.4, a 50 cm long vertical glass column with an internal diameter of 7 mm as a separation chamber and commercial gel permeation matrices (agarose A 50 m and Biogel P-10 available from Bio-Rad Laboratories, Richmond, California). Matrix i, the Biogel P-10, was layered above a zone of matrix ii, the agarose A 50 m. The flow direction was downward while the electrophoresis direction was upward (since the protein, ferritin, is negatively charged, the positive electrode was at the top of the column in order to impart an upward electrophoretic motion to the protein). Equilibrium was obtained at an applied voltage of 600 V and a flow rate of 40 ml/hr. Under these conditions, 10 mg of added ferritin was concentrated into a narrow band about 2 mm wide. The protein zone remained stable in this position, but as expected, small adjustments of the applied voltage or flow caused the protein to be displaced from the equilibrium position. Resolution was tested by first determining whether the ferritin could be separated from other colored proteins. Secondly, the smallest change in applied voltage which would shift the position of myoglobin in the separation chamber was determined. A high resolution of separation between ferritin and other proteins was observed. In fact, hemoglobin, myoglobin, and cytochrome c, would not achieve equilibrium under the same conditions as those which forced ferritin to an equilibrium position. As a result, the other proteins ran off the column. However, using other voltage and flow settings, equilibrium conditions for these other proteins were defined. To give a measure of degree of resolution of these proteins, the equilibrium conditions for each protein are compared. Since both flow and applied voltage are adjusted to achieve these equilibrium conditions, and it is their ratio which defines the equilibrium conditions, the ratios for different proteins provide a comparison. In arbitrary units, the equilibrium ratios were ferritin 6.7, hemoglobin 32, and myoglobin 56 (the cytochrome c required an alteration in electrical polarity). Ferritin was brought to equilibrium between a boundary between two matrices (matrix i, Biogel P-300; matrix ii, mixture consisting of 80% Biogel P-300 and 20% Agarose A 5 m) and the applied voltage was changed until the protein shifted from equilibrium. The minimal voltage change required to shift the ferritin corresponded to a change in the equilibrium ratio of 3%. On the basis of these measurements, it can be seen that the process of the present invention equals or surpasses other known separation procedures in terms of resolution of proteins. Furthermore, even the small scale apparatus utilized herein has a capacity approaching 1 gm of purified protein, which equals or exceeds the capacity of other known methods of separation. While various embodiments of the present invention have been illustrated in detail, it is apparent that modifications and adaptations of those embodiments will occur to those skilled in the art. However, it is to be expressly understood that such modifications and adaptations are within the spirit and scope of the present invention, as set forth in the following claims.

Claims: CLAIMS 1. A method of segregating at least one species of desired ions in a separation chamber having longitudinally varying separation characteristics comprising the steps of: introducing said ions into said chamber; imparting said ions with a movement in a first direction by flowing a carrier fluid through said chamber; and applying an electric field across said chamber to electrophorese said ions in a second direction counter to said first direction to segregate each of said species in an equilibrium zone within the chamber. 2. A method according to Claim 1 further comprising the step of removing one or more of said species from the equilibrium zone of said species. 3. A method according to Claim 1 further comprising providing in said chamber a gradient matrix having continuously varying medium characteristics. 4. A method according to claims 1 or 3 further comprising providing in said chamber at least two contiguous matrices whereby at least one of said equilibrium zones will abut the common boundary of said matrices. 5. A method according to Claim 1 or 2 further comprising providing in said chamber at least one matrix of uniform medium characteristics contiguous to a volume of free flowing carrier fluid whereby at least one of said equilib rium zones will abut the common boundary of said matrix and said volume. 6. A method according to anyone of claims 1-5 further comprising providing in said chamber compartments having carrier fluid of differing electrical resistances. 7. A method according to anyone of claims 1 to 5 further comprising providing in said chamber carrier fluid having a gradient of electrical resistance. 8. A method according to anyone of claims 1 - 6, further comprising providing in said chamber compartments with differing rates of movement of said flowing carrier fluid. 9. A method according to anyone of claims 1-8, wherein said steps of removing and/or of introducing are continuous. 10. A method according to anyone of claims 1 - 8 further comprising the steps of terminating said step of applying after each of said species is segregated; allowing said species to move through said chamber due to said flowing carrier fluid; and collecting said species by collecting means. 11. A method according to anyone of claims 1-10 further comprising the step of detecting said zones by detection means. 12. A method according to anyone of claims 1-11 wherein said second direction is at an acute angle to said first direction whereby said equilibrium zones have lateral movement. 13. A method according to claim 12 further comprising the step of removing said species from a port disposed adjacent said at least one equilibrium zone in the direction of said lateral movement. 14. A method according to anyone of claims 1-8 and 10-13, wherein said steps of introducing and removing are inter mittent. 15. A method according to anyone of claims 1-14 wherein said matrix retards ions in relation to their size and/or has ionic interactions with ions 16. A method according to anyone of claims 1-15, wherein said matrix is a specific affinity resin, especially an adsorption resin. 17. A method according to anyone of claims 6-16, wherein said differences in electrical resistance are due to differences in concentrations of ions produced by diffusion of ions across a semipermeable membrane. 18. A method according to anyone of claims 1-17, wherein said species of desired ions is a mixture of isotopic species. 19. A method according to anyone of claims 1-18, wherein at least one of the isotopes is uranyl-235 or deuterium. 20. A method according to anyone of claims 1-17 for 235 concentrating positively charged U ions from a mixture of 235 238 positively charged U and U ions in a separation chamber provided with positive and negative electrodes and having first and second contiguous compartments between said electrodes, said first compartment being positioned between said negative electrode and said second compartment, said method comprising the steps of: flowing a carrier fluid comprising 235U and 238U ions through flowing a carrier fluid comprising 235U and 238U ions through said chamber, whereby said 235U and 238U ions are iNzted with a movement due to said flow in the direction of said positive electrode; and applying an electric field across said electrodes whereby said 235U and 238U ions are imparted with a movement due to electrophoresis in the direction of said negative electrode, said electric field having a magnitude such that 235U ions when in said first compartment have a net movement in the direction of siad positive electrode and when in said com partment have a net movement in the direction of said negative electrode, whereby said 235U ions accumulate in said first compartment in a region adjacent the common boundary of said first and second compartments. 21. A method according to claim 20 further comprising the step of removing said concentrated 235U ions from a port disposed adjacent said region. 22. A method according to claim 21 wherein said step of removing further comprises removing at a rate substantially 235 equal or less than the rate of accumulation of said 235U ions. 23. A method according to anyone of claims 1 to 17 wherein said species is a protein or a plurality of proteins, especially an antibody, antigen and/or hapten. 24. A method according to anyone of claims 1 to 17 wherein said at least one species is an antibiotic or a nucleic acid. 25. An apparatus for segregating at least one species of ions, said apparatus comprising: means for producing longitudinally varying separation characteristics within a separation chamber; means for imparting movement to said ions in a first longitudinal direction within the chamber; and means for electrophoresing said ions in a second direction counter to said first direction to segregate said species into equilibrium zones within the chamber. 26. An apparatus according to claim 25 further comprising means for removing said species from said chamber. 27. An apparatus according to claims 25 and 26 further comprising means for detecting said equilibrium zones. 28. An apparatus according to claims 26 or 27, wherein said removing means further comprises means for continuously removing said species. 29. An apparatus according to claims 27 and 28, wherein said removing means further comprises means for continuously removing said species and said detecting means further comprises means for controlling said removing means. 30. An apparatus according to anyone of claims 25-29, wherein said means for electrophoresing further comprises means for moving said ions in a lateral direction perpendicular to said first direction and said apparatus further comprising means for removing said species disposed adjacent said equilibrium zone in said lateral direction.

Inventors: O'FARRELL, PATRICK H.
Applicants: THE REGENTS OF THE  UNIVERSITY OF CALIFORNIA
Application year: 1980
Main classification: B01D59
IPC codes: B01D59, G01N27