Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
1998-01-09
2001-10-02
Houtteman, Scott W. (Department: 1656)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C204S451000, C502S005000
Reexamination Certificate
active
06297009
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to a method and composition for conditioning a silica surface for use in separating chemical and biochemical analytes.
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BACKGROUND OF THE INVENTION
Silica surface play an important role in the purification and analysis of chemical and biochemical analytes. In chromatographic applications, silica matrices (e.g., comprising beads or gels) have been used for decades to separate organic compounds based on differences in binding affinities under selected solvent conditions. In recent years, applications of silica matrices have been expanded to include separating non-traditional materials, such as nucleic acids, for example.
Silica gels and beads have also been used as solid-phase supports for attaching, covalently or by adsorption, coating materials that impart unique and highly advantageous separation properties. For example, a vast number of derivatized silica gel materials have been developed for analytical and preparative standard and high-pressure liquid chromatography (HPLC) to provide high-resolution separations.
Silica surfaces have also been used in the form of glass plates, tubes, and channels, to define passageways in which sample materials migrate during chromatographic or electrophoretic separations. In many of these applications, including uses in chromatography, slab gel electrophoresis, and capillary electrophoresis, it is often desired that the silica surface be inert towards the analytes of interest so as not to interfere with the separation process. For example, glass plates and columns have been treated with blocking agents, such as dichlorodimethylsilane and other silylating agents, to block surface silanol groups which would otherwise adsorb analytes or interfere with the separation medium.
In electrophoretic techniques carried out in silica-lined channels, particularly with narrow channels, the physical condition of the silica surface can have a significant effect on analyte mobility as a consequence of electroosmotic flow. Electroosmotic flow (EOF) is the bulk flow of the liquid electrophoresis medium which arse due to the effect of the electric field on counterions adjacent to the negatively charged channel wall. Because the channel wall is negatively charged under most pH conditions, there is a build-up of positive counterions (cations) in the solution adjacent to the wall. In an electric field, this cylindrical shell of cations causes the bulk flow of the medium to assume the character of a positively charged column of fluid which migrates toward the cathodic electrode at an EOF rate dependent on the thickness of the shell.
The rate of EOF can provide an important variable that can be optimized to improve die separation of two or more closely migrating species. In particular, when electrophoresis is carried out under conditions in which EOF and the migration of species to be separated are in opposite directions, the effective column length for separation can be made extremely long by making the rate of EOF in one direction nearly equal to the electrophoretic migration rate of the analyte attracted most strongly in the opposite direction by the electric field. A significant problem with using such conditions in capillary electrophoresis (CE) applications has been that the rate of EOF is highly sensitive to the nature and composition of the selected electrophoretic medium, as well as to the chemical condition of the capillary wall. That is, it has been difficult to sustain consistent migration times from run to run and from capillary to capillary due to chemical changes at the surface of the capillary wall after successive runs, and due to variability in the condition of the capillary walls of different capillary tubes from the same or different suppliers.
SUMMARY OF THE INVENTION
The invention includes, in one aspect, a method for increasing the electroosmotic flow rate available for a silica surface. In the method, there is provided an electrophoretic channel which is defined by one or more silica surfaces. The surface(s) are contacted with an alkaline aqueous solution containing a solubilized silicate-monovalent metal complex in an amount effective to increase the acidity of the silica surface(s), as evidenced by a reduction in the average bulk pKa of the surface(s). The achieved increase in acidity is greater than would be obtained using an otherwise identical solution lacking said silicate. In one preferred embodiment, the monovalent metal used in the solution is Li
+
, Na
+
, or K
+
. Prior to treatment with silicate reagent, the silica surface(s) may be contacted with an aqueous solution of MOH having a pH greater than 11, where M is selected from the group consisting of Li
+
, Na
+
, and K
+
.
In one embodiment, the solution contains a SiO
2
concentration which is from 0.05 to 5.0 weight %, preferably from 0.05 to 1.0 weight %. In yet a more preferred embodiment, the concentration is from 0.2 to 0.5 weight %.
One advantage of the method is that the maximum possible electroosmotic flow of the capillary tube is increased, allowing improved separations of analytes of interest. The method is especially useful in association with counter-current separation methods, such as micellar electrokinetic capillary chromatography (MECC).
In a related aspect, the invention includes an electrophoresis method for analysis of one or more sample analytes. In the method, there is provided a silica surface which defines an electrophoretic channel having an inlet end and an outlet end. The surface is contacted with a silicate solution of the type above, in an amount effective to increase the acidity of the silica surface. After a selected time, the alkaline aqueous solution is replaced with running buffer, and the sample is loaded into the inlet end of the channel. The ends of the channel are immersed in anodic and cathodic reservoirs containing electrolyte solution, and an electric field is applied across the ends of the channel under conditions effective to induce the analyte(s) to migrate toward the outlet end of the tube for detection.
In a more general aspect, the invention includes a method for increasing the acidity of a silica surface, by contacting the surface with an alkaline aqueous solution of the type above, in an amount effective to increase the acidity of the silica sur
Chiesa Claudia
Demorest David M.
Moring Stephen E.
Houtteman Scott W.
Perkin-Elmer Corporation
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