Chemistry: electrical and wave energy – Processes and products – Electrophoresis or electro-osmosis processes and electrolyte...
Reexamination Certificate
1999-09-17
2001-12-11
Warden, Jill (Department: 1743)
Chemistry: electrical and wave energy
Processes and products
Electrophoresis or electro-osmosis processes and electrolyte...
Reexamination Certificate
active
06328868
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The invention relates to carrier-free deflection electrophoresis.
2. Description of the Related Art
Carrier-free deflection electrophoresis has previously been used exclusively in continuous processes. This process in fact yields outstanding results in a continuous operation if the negative influence of the laminar flow profile on the bandwidth of the substances to be separated can be minimized or not even come into play. This applies in particular to the process of continuous isoelectric focusing.
In all electromigration processes of deflection electrophoresis, the widening of the band caused by the laminar flow profile of the separating medium (media) triggers a serious deterioration in separation capacity.
The basics of carrier-free deflection electrophoresis in continuous processes were already described in the literature over 30 years ago, including in the 1968 yearbook of the Max-Planck company on pp. 117-137. This process is also described under the term FFE (free flow electrophoresis) or CFE (continuous flow electrophoresis). (K. Hannig: Carrier-free continuous electrophoresis and its application. Anal. Chem. 181, 233 (1961); M. C. Roman and P. R. Brown: Anal. Chem. Free Flow Electrophoresis. 66(N2), 86-94, (1994); R. Braun, H. Wagner and G. Weber: Preparative Free Flow Electrophoresis—a powerful procedure for separating natural substances, GIT Fachzeitschrift für das Laboratorium 39 (1995), 317-322).
FFE separation procedures are used to separate ions of any molecular weight up to bioparticles. It is here irrelevant whether the sample to be separated is charged itself, or whether the charge came about via the addition or sorption of ions.
SUMMARY OF THE INVENTION
This process is used for both preparative and analytical separations.
The applicant has improved essential aspects of the method for continuous deflection electrophoresis (German Patent DE 41 39 472 C1).
The order of magnitude of these improvements varied for the different FFE separation techniques:
In all FFE separation techniques, a distinct improvement in long-term stability was achieved for the separation.
The separation capacity was improved by a factor of 10-50 for continuous isoelectric focusing, while only an improvement by a factor of 2-4 was reached in the electromigration process, and this improvement was exclusively reached via the novel “dead volume-free” fractionation using the counterflow as described in the patent. The negative influence of the laminar flow profile on the separation capacity of the electromigration processes was not influenced by the aforementioned improvements.
A method for continuous deflection electrophoresis will be described below based on the “typical” electromigration process, the zone electrophoresis.
FIG. 1
presents a schematic view of a separation chamber
1
. Located at the lower end of separation chamber
1
are at least three inlet holes
2
,
3
,
4
, which are connected with the conveyance channels of a multi-channel pump
5
by means of three lines. The separation medium is introduced into the separating space via central inlet hole
3
, while the stabilization media are introduced via the two outside inlets
2
,
4
.
These media flow through the separating space under laminar flow conditions. Situated parallel to the side edges of the separation chamber are electrodes
6
,
7
, which are rinsed in circulation by an additional pump with a high flow rate. Membranes
8
,
9
, which conduct electrical current, separate the electrode spaces from the separating space, and prevent any exchange of media via the hydrodynamic flow.
If a sample mixture is now introduced into the flowing separation medium using an independent metering pump
10
at the optimal metering rate, this sample is transported with the separation medium as a fine jet.
If a high voltage is applied to the electrodes, all charged constituents in the media and sample are deflected out of the original direction, with this deflection increasing as do the number of charges possessed by these ionic constituents.
Constituents of varying charge hence migrate along separate paths (see
FIG. 1
) through the separating space, and reach a row of hose openings
11
arranged transverse to the direction of flow at different locations, after which they are routed to separate collecting basins
12
with the media. This series of hose openings with the smallest possible distance between the openings is referred to as a fractionating device, and the number of these hoses arranged in parallel ranges between 30 and a maximum of 180.
An additional medium (counterflow medium) flows from the top end of the separation chamber in an opposite direction via an inlet
13
to the separation media, and also exits the separating space via the hose openings of the fractionating device.
A device conceived in accordance with the above description yields separations that exhibit an outstanding long-term stability for the separation profiles in all separation techniques of continuous deflection electrophoresis, but the quality of separation (resolution) can be compared with the level attained in the analogous analytical separation technique only in the case of continuous isoelectric focusing.
By contrast, in all continuous electromigration processes, the separation capacity is modest in comparison with the analogous analytical separation techniques.
Numerous publications describe the physical or electrochemical effects that contribute to the so-called band widening of the analytes during separation in continuous deflection electrophoresis (J. A. Giannovario, R. Griffin, E. L. Gray: A mathematical model of free-flow electrophoresis. Journal of Chromatography, 153, 329-352 (1978); F. G. Boese: Contribution to a mathematical theory of free flow electrophoresis, J. Chromat. 483, 145-170 (1988); K. Hannig and H. G. Heidrich: Free-Flow Electrophoresis, 1990 by GIT Verlag Darmstadt ISBN 3-921956-88-9).
The most important of these effects are:
widening due to the laminar flow profile,
widening due to thermal convection,
widening due to electrical osmosis,
widening due to electrokinetic effects.
The negative influence of all electrokinetic effects described thus far can be minimized or eliminated by using separation media with suitable ionic constituents with sufficiently high ionic strength, and at the same time not excessively increasing the concentration of the sample.
There are numerous ways to minimize the negative influence of electrical osmosis, e.g., through the selection of a suitable wall material (plastics instead of glass or quartz), or most preferably by adding surface-active chemicals to the separation media that preclude electrical osmosis. This method is referred to as “dynamic coating” in the literature.
The negative influence of thermal convection can be reduced very easily by arranging the separation chamber horizontally instead of vertically.
The negative influence of the laminar flow profile does not exist for continuous isoelectric focussing as long as a sufficiently long separation time is selected that also enables the focussing of the analytes, which are transported at the highest linear velocity in the center of the separation chamber gap.
By contrast, the negative influence is very significant in the case of the electromigration processes. Analytes that migrate near or in the boundary surface to the walls of the separation chamber pass through the separation chamber in a considerably longer time than analytes at the center of the separation chamber gap (see
FIGS. 2.1
and
2
.
2
), and are hence deflected to a clearly greater extent. This effect results in a band widening detectable as a tailing in the direction of electromigration.
FIG. 3.1
depicts this effect of band widening for
2
low-molecular analytes with opposite charges, while
FIG. 3.2
illustrates the same for high-molecular particles.
Given a continuously executed electromigration process under the boundary conditions of carrier-free electrophoresis, then, the negative influence of the laminar flow
Nixon & Peabody LLP
Noguerola Alex
Safran David S.
Warden Jill
LandOfFree
Method for carrier-free deflection electrophoresis does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with Method for carrier-free deflection electrophoresis, we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Method for carrier-free deflection electrophoresis will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-2597925