Methods for isoelectric particle separation

Details Methods for isoelectric particle separation Methods for isoelectric particle separation

1999-05-24

2004-12-21

Nguyen, Nam (Department: 1753)

Chemistry: electrical and wave energy

Processes and products

Electrophoresis or electro-osmosis processes and electrolyte...

C204S450000, C204S459000, C204S600000, C204S610000, C204S644000

Reexamination Certificate

active

06833061

ABSTRACT:

The invention concerns methods and devices for isoelectric separation of particles whose charge characteristics depend on the pH value of a guiding fluid, especially for separating ampholytic suspended particles, colloids or biological cells. The invention concerns in particular the separation of such particles from a guiding fluid flow.
Numerous separation techniques are known from molecular biology; biochemistry, medicine and biotechnology whose function is based on charge differences of molecules within a substance that is to be separated. In the case of amphoteric ion compounds (socalled ampholytes or ampholytic molecules), the molecular charge depends on the pH value of the surrounding solution. The isoelectric point (hereafter referred to as the IP) of a compound is the pH value at which the net charge of the amphoteric molecules equals zero. The charge of the molecule is positive for a pH value smaller than the IP and negative in the opposite case.
In isoelectric focusing (hereafter referred to as IEF), proteins with different IPs are separated by making use of spatial pH gradients along a separation length (see R. A. M. Osher et al. in “The Dynamics of Electrophoresis”, published by B. J. Radola, VCH, Weinheim, 1992, pp 163-231). IEF is performed, for example, using porous gel matrices or, for analytical separation of the smallest samples, using thin capillaries as what is called cIEF (see F. Foret et al. “Capillary Electrophoresis” in “Advances in Electrophoresis”, vol. III, published by A. Chrambach et al., VCH, Weinheim, pp 273-347).
For the following reasons conventional IEF presents disadvantages and its use is restricted. Retrieval of the separated substances from a carrier material, especially for further processing like analysis, medication applications or the like, calls for elaborate procedures by which the separated substances themselves may be modified, or which lead to substance losses. The necessary use of additional substances to form a pH gradient that is as wide and linear as possible produces restrictions in terms of further use of the separated samples. The socalled carrier ampholytes used as additional substances are, in chemical terms, a highly diverse substance mix that is difficult to separate from the separated protein fractions. Furthermore, cIEF is restricted to minimal sample quantities that cannot be collected separately and are not separable from the carrier ampholytes.
The problem when socalled IPG membranes are used to form the pH gradient is that proteins, because of the necessity of passing through such a membrane, must pass through a milieu whose ionic concentration is very low. Consequently, sensitive proteins can denaturize or precipitate in this region. For this reason the strictest requirements are made in IEF for uniformity of the voltage gradients in the separation length. Finally, problems can occur in IEF in the form of electroosmosis in extreme pH ranges, the carrier medium (gel) heating up and altering (destruction of the gel) for instance.
To overcome such drawbacks, systems were developed in which ampholyte separation is produced by the effect of an external electric field and without modifying additional substances. One separating system is known, for example, that works by the method of socalled electric field flow fractionating (eFFF) (see K. D. Caldwell et al. in “Science”, vol. 176, 1972, pp 296-298). In this separating system a continuous fluid stream is conducted through a narrow duct between two ion flow permeable membranes, into which the sample to be separated is injected as a narrow band. External electrodes, in electrical contact with the duct through a surrounding fluid, produce different degrees of retardation, as a function of charge, of the protein molecules in the fluid stream. However, use of this method is restricted to protein molecules with IPs that are relatively far apart. Furthermore, no complete protein separation could be achieved. Control of the pH value of the fluid stream—and thus of the molecular charge state—is not a facet of this familiar separating system.
The eFFF system is not applicable in practice. Although the samples could be separated (incompletely), collection of the separated fractions was not implemented. Furthermore, the times for separation are unacceptably high. A follow-on development of the above mentioned eFFF system (see L. F. Kesner et al. in “Analytical Chemistry”, vol. 48, 1976, pp 1834-1839) produced analysis or separation times of several hours even on a laboratory scale for example.
A modified eFFF system described by Lightfoot et al. (see “Separation Science and Technology”, vol. 16, 1981, pp 619-656) makes use off cylindrical duct geometry. The separation performance of this system was also unacceptable in practical terms. Furthermore, protein retardation showed itself to be a complex function of a large number of parameters, eg the buffer ions that were used, the sample quantity, the sort of protein and properties of the duct wall. Nor is this familiar system intended for pH control or, say, separation of proteins with different IPs.
There is pronounced interest in substance separation for the production of high-purity substances, beyond the sphere of the laboratory, on an industrial scale. Because of the restrictions and disadvantages mentioned however, no continuous substance separation is known to date, using the named systems, that produces adequate separation performance and speed for the practical sphere.
The object of the invention is to indicate improved methods of isoelectric particle separation distinguished, in particular, by higher separation speed, greater reliability, and a broader range of application in terms of separable particles and the surrounding solutions. The object of the invention is also to indicate corresponding, continuous isoelectric purification methods. The object of the invention also consists in providing devices for implementing the methods of particle separation and further application possibilities.
The named objects are achieved by methods for isoelectric particle separation in which particles with a net charge or charge density that is a function of the pH value of the surroundings are exposed to electric field forces in a guiding fluid passing by collection means, whereby the pH value of the guiding fluid is set so that, through the effect of the electric field forces, at least one predetermined particle type undergoes a change in motion as a function of charge and is moved to the collection means, intended for soluble fixing of the charged particles or by devices for isolectric particle separation that include: electrode means for forming an electric field in a guiding fluid with particles whose net electric charge or charge density is a function of the pH value of the guiding fluid; pH setting means being adapted to set the pH value of the guiding fluid so that at least one predetermined particle type of the particles in the guiding fluid undergoes a change in motion as a function of charge through the effect of the electric field; and collection means arranged between the electrode means and the guiding fluid for soluble fixing of charged particles. Preferred embodiments of the invention are disclosed hereinafter.
The separation technique according to the invention is based on the idea of controlling or setting the pH value in a guiding fluid in which the particles to be separated are exposed to electric field forces so that all particle types with a net charge go through a motion dependent on charge, and the remaining, for the most part neutral particles show no change in their state of motion, whereby the particles moving as a function of charge are moved to collection means (collection device). The particles are retained at least temporarily on the collection means. This involves fixing on the surface or in the volume of the collection means. The duration of fixing is determined by the separation conditions, especially modulation with time of the electric field forces, alteration of the pH value, the structure of the col

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