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Description  |
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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.
REFERENCES
Anderson, D. J., Anal. Chem. 67:475R (1995).
Atamna, K. D., et al., J. Liq. Chrom. 13:2517-2528 (1990).
Boom, R., et al., J. Clin. Microbiol. 28:495-503 (1990).
Falcone, J. S., et al., "Silicates" in Encyclopedia of Polymer Science and
Engineering, Vol. 15, 2nd Edition, Wiley Publishing, NY, pp. 178-204
(1985).
Grob, K., Making and Manipulating Capillary Columns for Gas Chromatography,
Alfred Huthig Verlag, New York, N.Y.(1986).
Grossman, P. D., and Colburn, J. C., Eds., Capillary Electrophoresis,
Academic Press, Inc., San Diego, Calif. (1992).
Hearn, M. T. W., Ed., HPLC of Proteins, Peptides, and Polynucleotides, VCH,
New York, N.Y. (1991).
Henry, M. P., J. Chrom. 544:413 (1991).
Jacobson, S. C., et al., Anal. Chem. 66:4127-4132 (1994a).
Jacobson, S. C., et al., Anal. Chem. 66:1107-1113 (1994b) .
Majors, R. E., LC.GC 12:203 (1994).
Manz, A., et al. J. Micromech. Microeng. 4:257-265 (1994a).
McCormick, R., Anal. Chem. 60:2322-2328 (1988).
Otsuka, K., J. Microcol. Sep. 1:150-154 (1989).
Snyder, L. R., and Kirkland J. J., Introduction to Modern Liquid
Chromatography, 2nd Ed., John Wiley & Sons, Incl, New York, N.Y. (1979).
Unger, K. K., Ed., Packings and Stationary Phases in Chromatographic
Techniques, Marcel Dekker, New York, N.Y. (1990) .
Unger, K. K., and Trudinger, U., Chap. 3 in High Performance Liquid
Chromatography, Brown, P. R. and Hartwick, R. A., Eds, John Wiley, New
York, N.Y. (1989).
Vogelstein, B., et al., Proc. Natl. Acad. Sci. USA 76:615-619 (1979).
Wiktorowicz, J. E., and Colburn, J. D., Electrophoresis 1990 11:769-773
(1990).
Wiktorowicz, J. E., U.S. Pat. No. 5,015,350 (1991).
Yang, R. C. A., et al., Meth. Enzymol. 65:176-182 (1979).
BACKGROUND OF THE INVENTION
Silica surfaces 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 techiques 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 arises 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 the 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
extemely 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.sup.+, Na.sup.+, or K.sup.+. 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.sup.+, Na.sup.+, and K.sup.+.
In one embodiment, the solution contains a SiO.sub.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 surface(s), as evidenced by a reduction
in the average bulk pKa of the surface(s). Again, the achieved increase in
acidity is greater than would be obtained using an otherwise identical
solution lacking said silicate. The method can be used to enhance the
physical properties of a variety of silica surfaces, including those of
capillary tubes, microchannels formed on microchips, glass plates, silica
beads used in liquid chromatography, and capillary surfaces used in gas
chromatography. 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.sup.+, Na.sup.+, and K.sup.+.
In one embodiment, the method is useful for preparing a silica surface
which is to be subsequently derivatized with a covalently or
non-covalently attached chemical coating. In the method, an underivatized
silica surface is contacted with an alkaline aqueous silicate solution of
the type above, in an amount effective to increase the acidity of the
silica surface. After the alkaline solution is removed, the surface is
contacted with a derivating agent under conditions effective to allow the
derivatizing agent to bind to the surface. In a preferred embodiment, the
derivatizing agent is covalently bound to the silica surface.
The invention also includes fused silica capillary tubes,
microchannel-containing microchips, and silica beads produced using the
silicate solution.
In another aspect, the invention includes an improvement in a method for
capturing a nucleic acid on a silica particle. The improvement resides in
the step of, prior to nucleic acid capture, contacting the silica particle
with an alkaline aqueous solution containing a soluble silicate-monovalent
metal complex in an amount effective to increase the acidity of the silica
particle, whereby the binding capacity of the silica particle for the
nucleic acid is increased. In one embodiment, the silica particle may be
contacted with an aqueous solution of MOH of the type above, prior to
contact of the particle with silicate solution.
Also included are kits for use in carrying out the methods above. The kits
include a silicate reagent solution of the type described herein, together
with other reagents as appropriate for the selected application. In one
embodiment, for capillary electrophoresis applications, a kit will include
a stock solution of silicate reagent in a carbon dioxide impermeable
container, and selected buffer reagents and additives appropriate for
electrophoretic sepration.
These and other objects and features of the invention will become more
fully apparent when the following detailed description of the invention is
read in conjunction with the accompanying drawings.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 shows a plot of EOF mobility values (.mu..sub.EO) measured by
capillary electrophoresis (CE) of DMSO in 20 mM sodium acetate buffer, pH
4.5, as a function of concentration of silicate reagent (0.03 to 0.8%
SiO.sub.2 /100 mM NaOH) used in conditioning and pre-run cycles;
FIG. 2 shows a plot of EOF mobilities obtained using silicate reagent
solutions formed from a sodium silicate stock solution (27% SiO.sub.2 /14%
NaOH) (upper curve) and a solution prepared from solid anhydrous sodium
metasilicate (lower curve);
FIG. 3 shows a plot of three EOF mobility curves obtained in a conditioning
study with three different silicate concentrations over several NaOH
concentrations (.about.25-200 mM NaOH): left-hand curve, 0.2% SiO.sub.2 ;
middle curve, 0.4% SiO.sub.2, right-hand curve, 0.6% SiO.sub.2 ;
FIGS. 4A-4B show plots of EOF mobilities measured in a Tris/phosphate
running buffer, pH 7.4, additionally containing either no SDS (FIG. 4A) or
50 mM SDS (FIG. 4B) following conditioning with silicate reagent
containing 100 mM NaOH and SiO.sub.2 concentrations ranging from 0 to 0.8%
SiO.sub.2 ;
FIGS. 5A-5B show EOF mobility plots obtained under conditions similar to
those for FIGS. 4A-4B, except that the silicate concentration for silicate
reagent in the pre-run cycles was kept constant at 0.2% SiO.sub.2 (5A) or
0.4% SiO.sub.2 (5B) while the NaOH concentration was varied (.about.35-200
mM); the Tris/phosphate running buffer contained 50 mM SDS;
FIG. 6 shows a plot of EOF mobility measured in 20 mM sodium acetate
running buffer, pH 4.5, by CE as a function of time of exposure of the
capillary tube to silicate reagent (0.25% SiO.sub.2 /100 mM NaOH); lower
trace, 10 minute exposure; middle trace, 20 minute exposure; upper trace,
30 minute exposure;
FIG. 7 shows a plot of EOF mobilities obtained for 20 consecutive
electrophoretic separations of a neutral marker and three charged
standards in 20 mM sodium acetate running buffer, pH 4.5, where the
capillary tube had been preconditioned either with NaOH solution alone or
with a silicate/NaOH mixture (0.2% SiO.sub.2 /100 mM NaOH, designated by
"/Si" in legend);
FIG. 8 shows pH profiles of EOF mobilities measured by CE as a function of
silicate concentration in the prerun cycle; the silicate concentrations
were 0, 0.05, 0.1, 0.3, and 0.3% SiO.sub.2 (lowest to highest curve), the
latter two curves differing in the time of exposure to silicate reagent
(Example 7);
FIGS. 9A-9F shows plots of EOF mobility and electrophoretic mobilities
obtained with DMSO and a mixture of 3 chiral compounds, as obtained with
otherwise identical capillary tubes obtained from 6 different suppliers,
where the capillary tube was washed with 0.25% SiO.sub.2 /100 mM NaOH
prior to each run;
FIG. 10 shows a plot of EOF mobilities for DMSO and the chiral mixture used
in FIGS. 9A-9F, as measured in 10 consecutive runs under the same
conditions;
FIGS. 11A-11B shows electropherograms of a mixture of derivatized
monosaccharides separated in a capillary tube which was conditioned first
only with NaOH solutions (11A), and in the same capillary tube after
pre-conditioning with silicate reagent (0.34% SiO.sub.2 /100 mM NaOH);
FIG. 12 shows a plot of migration times of the monosaccharide mixture from
FIGS. 11A-11B over the course of 17 consecutive runs following exposure of
the capillary tube to silicate reagent (0.34% SiO.sub.2 /100 mM NaOH);
FIG. 13 shows a schematic view of a capillary electrophoresis system which
may be used in practicing the invention; and
FIG. 14 shows a schematic view of a miniature capillary electrophoresis
system formed in a microchip.
DETAILED DESCRIPTION OF THE INVENTION
The present invention provides, in its broadest embodiment, a method for
increasing the acidity of a silica surface by contacting the surface with
an alkaline aqueous silicate solution of the type detailed below. In one
aspect, the method is used to increase the maximum possible electroosmotic
flow (EOF) of electrophoretic channel defined by one or more silica
surfaces. This feature is especially useful in countercurrent
electrophoresis formats. In a second aspect, the method can be used to
increase the acidity of a silica surface, as a means of activating the
surface for derivatization with a coating agent. In this aspect, the
method can be used to prepare derivatized silica surfaces for
electrophoresis, as well as derivatized solid supports for chromatographic
applications such as liquid chromatography and gas chromatography.
As used herein, the term "silica" refers to a solid material consisting
predominantly of SiO.sub.2 and/or silicic acid groups (H.sub.2 SiO.sub.3).
"Silica surface" refers to a surface consisting predominantly or entirely
of SiO.sub.2 and/or silicic acid groups. The term as used herein encompass
such surfaces present on pure forms of SiO.sub.2 (such as quartz,
cristobalits, or fused silica), as wells as those of silicate glasses.
"Soluble silicate" or "soluble silicate-monovalent metal complex" refers to
a silicate composition of the general formula M.sub.2
O.multidot.(SiO.sub.2).sub.m .multidot.(H.sub.2 O).sub.n, where m and n
are integers and M is an alkali metal, i.e., lithium, sodium, potassium,
rubidium, cesium, or francium, most commonly lithium (Li), sodium (Na), or
potassium (K). The term encompasses all soluble monomeric, linear,
branched, and cyclic silicate structures in equilibrium in an alkaline
aqueous solution.
"Alkaline" as used herein, refers to a solution having a pH.gtoreq.10.
I. Silicate Reagent
The soluble silicate reagent of the invention is generally an alkaline
aqueous solution containing a soluble silicate in an amount effective to
reduce the average bulk pKa of the silica surface. The soluble silicate
has the general formula M.sub.2 O.multidot.(SiO.sub.2).sub.m
.multidot.(H.sub.2 O).sub.n, where m and n are integers and M is an alkali
metal, most usually lithium, sodium, potassium, or a mixture thereof. The
silicates form a number of structures in alkaline solution, including
orthosilicate (SiO.sub.4.sup.4-), pyrosilicate (Si.sub.2 O.sub.7.sup.6-)
and longer linear structures, and cyclic and branched structures, all of
which are in dynamic equilibrium under alkaline conditions, particularly
with pH.gtoreq.10. The distribution among possible structures depends on
several factors including the concentration of SiO.sub.2, the ratio of
SiO.sub.2 to alkali metal, and temperature.
The silicate reagent is prepared by any suitable means known in the art.
Suitable solutions may be prepared by solubilization of crystalline,
powdered, or glass-state SiO.sub.2 in aqueous LiOH, NaOH, or KOH, for
example, optionally under elevated temperature and pressure to accelerate
dissolution. Alternatively, silicate salts in dry form, such as Na.sub.2
SiO.sub.3, Na.sub.6 Si.sub.2 O.sub.7, Na.sub.2 Si.sub.3 O.sub.7, K.sub.2
Si.sub.2 O.sub.5, K.sub.2 Si.sub.3 O.sub.7, and Li.sub.2 SiO.sub.3, may be
dissolved in water or aqueous hydroxide to prepare a solution of desired
concentration. The concentration of alkali metal M may be adjusted by
suitable titration using concentrated LiOH, NaOH, or KOH, as appropriate.
Silicate stock solutions are also available from commercial suppliers. For
example, aqueous lithium silicate (Li.sub.2 SiO.sub.3, 20 weight %) is
available from Aldrich Chem. Co. (Milwaukee, Wisc.), and sodium silicate
(27% SiO.sub.2 /14% NaOH) is available from Aldrich and Fluka (Ronkonkoma,
N.Y.). The pH of the silicate reagent of the invention is usually greater
than 10, preferably greater than 11, and more preferably greater than 12.
Most commonly, the monovalent metal is sodium.
It will be appreciated that, given the high concentration of hydroxide ion
in the silicate reagent, the reagent readily captures carbon dioxide
(CO.sub.2) from the surrounding atmosphere, forming carbonate ions in the
reagent. Although the presence of carbonate ions in the reagent can be
tolerated to some degree without significantly diminishing the reagent's
advantageous effects, exposure to carbon dioxide should be avoided. To
minimize carbonate formation, the reagent should be kept in a sealed
container, preferably in an inert atmosphere (e.g., argon or nitrogen).
Further advantages in this regard may be achieved by preparing the
silicate reagent in a glove-box, dry-box, or other suitably controlled
atmosphere which is substantially devoid of water vapor and CO.sub.2.
In a preferred embodiment, the silicate reagent is prepared as a
concentrated stock solution in one or more sealable containers containing
a selected amount of reagent, e.g., a volume sufficient for one-day or
one-month use. For example, a 10.times. silicate stock consisting of
.about.3% SiO.sub.2 in 1N NaOH (for preparing working solutions containing
0.3% SiO.sub.2 in 0.1N NaOH can be used for a month or more when stored in
an air-tight container between uses. Working solutions are prepared daily
by dilution of concentrated silicate stock with water, preferably in
plastic tubes.
The container is generally made of any CO.sub.2 -impermeable material which
is chemically inert with respect to the silicate reagent. Glass containers
should be avoided since the high alkalinity of the silicate reagent can
cause leaching of variable amounts of silicates from the glass surface and
into the stock solution. Suitable container materials include
polypropylene, polyethylene, "TEFLON" and the like The container may
additionally include a septum to allow removal of aliquots of the reagent
by syringe. For long-term storage or transport, the vial is preferably
placed in a sealed bag (e.g., a polyethylene bag) flushed with argon gas.
In studies conducted in support of the invention, the applicants have
found that silicate reagent solutions in accordance with the invention
remain stable (i.e., retain full efficacy) for at least two months when
packaged in a polypropylene container sealed in a polyethylene bag under
argon atmosphere.
II. Applications in Electrophoresis
In one 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 surfaces. As applied to electrophoretic applications, the
method encompasses several related embodiments. First, the method can be
used to increase endoosmotic flow of an underivatized silica surface, by
increasing the number of negatively charged silanol groups along the
surface. Second, the method can be used to expand the pH range over which
high EOF can be obtained, by reducing the average bulk pKa of the silica
surface. Third, by increasing the number of silanol groups on the silica
surface, the method can be used to enhance the reactivity of the surface
towards coating agents used to reduce or reverse EOF, to reduce
interactions with analytes and medium components, or to anchor a
cross-linked separation matrix to the silica surface.
As will be seen further below, the method provides a number of significant
improvements over prior art methods. When used to increase EOF, the method
provides rate enhancements and overall run-to-run consistency better than
previously available using non-silicate alkaline solutions such as 0.1 to
1N NaOH. When the silicate reagent of the invention is used periodically
between successive runs, the method is effective to maintain EOF at
consistent levels over many electrophoretic runs, simplifying the analyses
of comparative samples. Furthermore, the silicate reagent can be used to
regenerate the condition of the silica surface after exposure to harsh
conditions (e.g., acidic pH), extending the useful lifetime of the surface
for electrophoresis. When used as a preliminary step for derivatization
with a coating, the method can be used to control the density and
uniformity of coating, improving batch-to-batch reproducibility of such
coatings and the coating's durability.
A. Electrophoresis System
General considerations regarding electrophoresis protocols are illustrated
in this section using capillary tube electrophoresis as an example.
However, it will be recognized that the discussion is also applicable to
other narrow channel formats, such as capillary electrophoresis performed
in a micro-channel formed in the glass or silica substrate of a microchip.
FIG. 13 shows a simplified schematic view of a capillary electrophoresis
system 20 (Applied Biosystems, Foster City Calif.) suitable for practicing
the method of the invention. The system includes a capillary tube 22
having a length preferably between about 10-200 cm, typically less than
about 100 cm, and an inner diameter (i.d.) of preferably between about
10-200 .mu.m (microns), typically about 50 .mu.m. Capillary tubes with
rectangular or square cross-sections are also contemplated. One preferred
capillary tube is a fused silica tube having an inner diameter of 50 .mu.m
(available from Polymicro Technologies, Phoenix, Ariz.).
More generally, the capillary tube may be any channel capable of supporting
a column of electrolyte solution, preferably having an inner diameter of
200 .mu.m or less.
A cathodic reservoir 26 in system 20 contains an electrolyte solution 28.
The cathodic end of the tube, indicated at 22a, is sealed within reservoir
26 and is immersed in electrolyte solution, as shown, during
electrophoresis. Second tube 30 in reservoir 26 is connected to a finely
controlled air pressure system (not shown) which can be used to control
the pressure in the head space above the solution, e.g., for loading
electrolyte solution into the tube by positive pressure. The pressure
system is able to generate a pressure differential across the ends of the
capillary tube of about 100-300 psi or less. Alternatively or in addition,
the air pressure system can include a vacuum system for drawing solution
through the capillary tube.
A sample reservoir 31 in system 20 contains the sample mixture to be loaded
into the inlet end of the tube (assumed here to be the cathodic end). The
sample and cathodic reservoirs may be carried on a carousel or the like,
for placement at a position in which the cathodic end of the tube can be
immersed in the reservoir fluid. Although not shown here, the carousel may
carry additional reservoirs containing, for example, solutions for
cleaning and flushing the tube between electrophoretic runs, or different
electrophoretic media.
The opposite end of the tube (assumed here to be the anodic end), indicated
at 22b, is immersed in an anodic electrolyte solution 32 contained in an
anodic reservoir 34. A second tube 36 in reservoir 34, analogous to tube
30 in reservoir 26, can be included to control the pressure above solution
32, e.g., for loading solution into the tube, just as with tube 30 in
reservoir 26. Typically, the compositions of electrolyte solutions 28 and
32 are identical. However, in certain applications, particularly
isotachophoresis, the electrolyte solutions at each end may be different.
For sample loading and subsequent sample separation by electrophoresis, the
filled capillary tube and electrode reservoirs are preferably configured
so that there is little or no net hydrodynamic flow through the tube. This
can be effected by keeping the surfaces of the electrode reservoir
solutions at the same height, or by controlling the atmospheric pressures
above the two solutions.
A high voltage supply 40 in the system is connected to the cathodic and
anodic reservoirs as shown, for applying a selected electric potential
between the two reservoirs. The power supply leads are connected to
platinum electrodes 41, 42 in the cathodic and anodic reservoirs,
respectively. The power supply may be designed for applying a constant
voltage (DC) across the electrodes, preferably at a voltage setting of
between 6 kV and 30 kV.
Detector 44 in the system is positioned adjacent the anodic end of the
tube, for monitoring sample peaks migrating through an optical detection
zone 45 in the tube. Typically, the capillary tubing has been treated to
remove a small region of exterior polyimide coating (in the case of a
polyimide-coated capillary tube) to create a small window. The detector
may be designed for any mode of detection known in the art, including
UV/visible absorption detection, conductivity detection, fluorescence
emission detection, radioisotope detection, and mass-spectrometric
detection, for example.
In operation, the capillary tube is washed by flushing suitable rinsing
solutions through the tube by applying positive or negative pressure to
the head space above the appropriate solution reservoir. Alternatively,
the capillary can be washed manually by syringe. If a cleaning solution
different from the electrolyte solution (running buffer) is used, the tube
is finally flushed with several volumes of running buffer before use.
The sample is then loaded into the inlet end of the tube, typically by
electrokinetic injection for charged species, and by hydrostatic injection
for neutral species. After sample loading, the tube end is returned to the
solution in cathodic reservoir 26, and a separation voltage (e.g., 30 kV)
is applied until the desired number of fragment peaks have passed through
the detection zone.
For automated electrophoresis of multiple samples, the apparatus may be
adapted to include an array of capillary tubes and suitable detection
means for simultaneous monitoring of sample migration in the tubes. By
such an arrangement, the same sample or a number of different samples can
be analyzed in parallel using such an array.
B. Improved Surface Properties
The composition of the silicate reagent for a particular application may be
optimized for the particular analyte separation to be carried out, on the
basis of test studies over a range of silicate and monovalent metal
concentrations as illustrated below.
FIG. 1 shows a general trend of EOF mobilities achieved following
conditioning of a fused silica capillary tube with silicate reagent as a
function of concentrations of SiO.sub.2. As can be seen, the measured
mobilities generally increases as a function of silicate concentration
with maximal EOF being reached at about 0.4 wt % SiO.sub.2. Details of
this study are given in Example 1.
As discussed above, the silicate reagent of the invention may be prepared
from a number of silicate sources. Because of the relatively high
alkalinity of the silicate reagent, the reagent contains a number of
silicate structures in rapid equilibrium, independent of the initial
structures in the silicate source. The steady-state distribution of
species is dependent on several factors, including total concentration of
SiO.sub.2, concentration of hydroxide ion, and the identity of the alkali
metal.
The flexibility in terms of silicate source material is illustrated in FIG.
2, which shows EOF mobilities measured in 6 successive electrophoresis
runs following pre-conditioning of capillary tubes with either of two
silicate reagent solutions containing 0.25% SiO.sub.2 /100 mM NaOH
(Example 2). One of the reagent solutions was prepared from a commercial
stock solution of sodium silicate (27% SiO.sub.2 /14% NaOH). The other was
prepared from an aqueous stock solution made from anhydrous sodium
metasilicate powder (Na.sub.2 SiO.sub.3). As can be seen from FIG. 2, the
two reagent solutions provide substantially the same EOF profiles. These
results indicate that performance of the silicate reagent is independent
of whether the source is a commercial alkaline stock solution or a
polymeric silicate solid.
FIG. 3 illustrates how reagent performance can depend on the ratio of
SiO.sub.2 /Na.sub.2 O. In this study, detailed in Example 3, EOF
mobilities were measured following capillary treatment with silicate
reagent solutions containing 0.2, 0.4, and 0.6 wt % SiO.sub.2
concentrations over a range of NaOH concentrations (0.5 to 200 mM).
Electrophoresis of neutral marker, DMSO (dimethyl sulfoxide), was
conducted in 20 mM sodium acetate, pH 4.5, as with FIGS. 1 and 2. As can
be seen from the left-hand curve of FIG. 3, the series of solutions
containing 0.2% SiO.sub.2 show a maximum EOF (5.5.times.10.sup.4 cm.sup.2
/V.sec) in the presence of .about.37 mM NaOH. The 0.4% and 0.6% SiO.sub.2
series show similar EOF maxima at NaOH concentration of about 80 and 120
mM NaOH, respectively. These data indicate that for the SiO.sub.2
concentrations tested, maximum EOF mobility is obtained at a SiO.sub.2
/Na.sub.2 O ratio of about 1.5.
EOF mobilities obtained with a second running buffer (Tris-phosphate, pH
7.4, containing 0, 50 or 100 mM SDS) are shown in FIGS. 4A (0 mM SDS) and
4B (50 mM SDS). As can be seen, EOF mobility is virtually constant over a
broad range of SiO.sub.2 concentrations (0.03 to 0.8%), regardless of
whether SDS is present or absent. It should also be noted that the lower
mobilities obtained with the 50 mM SDS buffer (FIG. 4B) are attributable
to conductivity-related suppression of EOF due to the presence of the SDS
in the buffer.
FIGS. 5A and 5B show the mobilities obtained in the Tris-phosphate buffer
(containing 50 mM SDS) following capillary conditioning with 0.2 or 0.4%
SiO.sub.2 over a range of NaOH concentrations. For the 0.2% SiO.sub.2
solutions, a maximum mobility of about (5.7.times.10.sup.4 cm.sup.2
/V.sec) is obtained over a broad range of NaOH concentrations (80 mM
NaOH). By way of contrast, maximum EOF with 0.4% SiO.sub.2 is not reached
until the NaOH concentration had reached about 160 mM. These results
indicate that the optimal SiO.sub.2 /Na.sub.2 ratio, e.g., to obtain
maximum EOF, may depend on the separation conditions used.
The effects of varying the exposure time of the silica surface to silicate
reagent is illustrated by Example 5. In this study, EOF was measured for
five consecutive runs in 20 mM sodium acetate, pH 4.5, after conditioning
of the capillary tubes with silicate reagent (0.25% SiO.sub.2 /100 mM
NaOH) for different lengths of time (10, 20, and 30 minutes). As can be
seen from FIG. 6, under the conditions tested, the observed mobilities
correlate with exposure time, with longer exposure time giving greater
mobility. In addition, for the 10 and 20 minute exposures, EOF increases
somewhat over the five successive runs, whereas runs following the 30
minute exposure time remain substantially constant.
It will be appreciated how similar studies can be done for other separation
conditions to arrive at exposure conditions that afford high and
consistent EOF values.
As noted above, the silicate reagent of the invention can be used to
achieve greater maximum EOF rates than can be achieved using
hydroxide-containing solutions which are silicate-free. This is
illustrated in FIG. 7. In this study, detailed in Example 6, a neutral
marker (DMSO) and a set of three charged compounds were
electrophoretically separated in capillary tubes which had been
conditioned with 100 mM NaOH in the presence or absence of soluble
silicate (0.2% SiO.sub.2). The separation was repeated 20 times in each
case, with each run preceded by a pre-cycle wash with 100 mM NaOH or 0.2%
SiO.sub.2 /100 mM NaOH. In the runs performed following conditioning with
NaOH solution alone (without silicate), none of the charged compounds
eluted within the first 30 minutes for the first run (see FIG. 7). In
subsequent runs, the elution times of the charged compounds gradually
decreased, reaching relatively steady values after about the ninth run. In
contrast, pre-conditioning with silicate reagent afforded rapid migration
times for all of the charged compounds even in the first run, with
constant levels being reached within the first 5 to 7 runs (FIG. 7).
Furthermore, the migration times obtained with silicate reagent were in
all cases substantially faster than those obtained where conditioning had
been performed with NaOH solution alone. These results clearly demonstrate
the superiority of conditioning with the silicate reagent of the
invention, compared with conditioning using sodium hydroxide alone.
According to another important feature of the invention, contacting a
silica surface, e.g., the inner wall of a capillary tube, with the
silicate reagent of the invention is effective to lower the average bulk
pKa of the surface, extending the pH range in which EOF can be exploited
in electrophoretic separations. As discussed above, EOF is a phenomenon
that results from the bulk flow of electrophoretic medium generated by
movement of the shell of cations lining the capillary wall towards the
cathode. When the pH of the running buffer is substantially greater than
the bulk pKa of the capillary wall (e.g., greater than about 1.5 pH
units), most of the surface silanol groups are negatively charged by
virtue of being deprotonated. However, when the pH is lowered towards the
bulk pKa of these groups, a greater proportion of the surface silanol
groups become protonated, reducing the number of negatively charged groups
on the surface and hence, the size of the cationic shell responsible for
EOF. In MECC, for example, the bulk pKa of fused silica has limited the
use of non-coated capillary tubes to running conditions having a pH
greater than about 6.5. Ideally, for applications where a large EOF is
desired, the bulk pKa of the silica surface should be as low as possible
to enable good separation over a broader pH range.
Example 7 describes a study in which EOF was measured over a broad pH range
following exposure to alkaline solutions containing various concentrations
of silicate (0 to 0.3% SiO.sub.2). First, a new capillary tube was
conditioned with NaOH solution alone, and EOF mobilities were then
measured using DMSO marker and buffers having pH values ranging from 11 to
2.5. This procedure was then repeated after preconditioning with
increasing concentrations of silicate (0.05, 0.1, and 0.3% SiO.sub.2). The
0.3% SiO.sub.2 solution was tested twice, with exposure times of 3 and 20
minutes, respectively. The results are shown in FIG. 8.
With reference to the lowest curve in FIG. 8, preconditioning with NaOH
solution alone yields a pH profile in which EOF steadily decreases towards
a value of zero at lower pH. The bulk pKa under these conditions is about
6.7, based on the pH at which the EOF is half of maximum. However, upon
preconditioning with increasing concentrations of silicate, the bulk pKa
of the surface becomes gradually lower. As can be seen, the best results
are obtained with the highest silicate concentration tested, for which the
bulk pKa is estimated to be about 3.7 (highest curve). The data also show
that for the highest silicate concentration, EOF remain at a maximum
constant level over a pH range of about 6 to 11, and is still greater than
half-maximal at a pH of 4. It is also noted that upon each reconditioning
cycle prior to data collection for the next pH profile, the EOF is
returned each time to the same EOF maximum (or slightly higher) at pH 11,
indicating that use of the silicate reagent delays onset of capillary
degradation.
In using the silicate reagent of the invention to increase surface acidity
or maximum possible EOF, treatment of the selected silica surface may be
preceded by treatment with a concentrated hydroxide solution, preferably
with an aqueous solution of MOH having a pH greater than 11, where M is
selected from the group consisting of Li.sup.+, Na.sup.+, and K.sup.+.
Such pretreatment may be helpful to help activate the surface towards
reaction with the silicate reagent, as well as removing impurities and
residue from the manufacturing process or from previous electrophoretic
runs.
C. Illustrative Applications
The silicate reagent of the invention can be used in association with a
variety of capillary electrophoresis techniques, including moving boundary
electrophoresis, free zone electrophoresis, electrokinetic capillary
chromatography (ECC), isotachophoresis (displacement electrophoresis), and
isoelectric focusing.
In one general embodiment, the silicate reagent is used to increase maximum
electroendosmotic flow, particularly in free zone electrophoresis and ECC
techniques, where a large EOF is used to effectively lengthen the
separation window of the analyte mixture.
For example, in zone electrophoresis with un-derivatized capillary tubes
(i.e., having a negatively charged capillary walls), EOF toward the
cathode can be established in opposition to the direction of migration of
negatively charged analytes. Where the electrophoretic mobilities of such
analytes are faster than the rate of EOF, the analytes are detected at the
cathodic end in order of increasing electrophoretic mobility, as if the
separation had been carried out in a static (non-moving) medium in a
longer channel than actually used. More usually, the analytes migrate more
slowly than the rate of EOF, so that the analytes are detected at the
anodic end in order of decreasing mobility. In either case, the degree of
analyte separation can therefore be enhanced.
With ECC techniques, a variety of separation formats can be used in which
large EOF rates facilitate analyte separation, particularly in the context
of electrically neutral or uncharged analytes. In ECC, the separation
medium includes an additive, sometimes called "pseudo-phase", which is
capable of forming transient complexes with the analytes during
electrophoresis. In one approach, the additive has a net zero charge and
migrates at the rate of EOF. In this case, charged analytes are separated
based on a combination of electrophoretic mobility and differential
interactions with the additive, usually with electrophoretic migration in
the direction opposing the EOF. In a second approach, a charged additive
is used having a mobility opposite the direction of EOF, usually to
separate neutral analytes. Many suitable additives are known, such as
cyclodextrin compounds and micelle-forming surfactants (Grossman et al.,
1992, pp. 179-181 and 313-320).
Because of their dependencies on the magnitude of EOF, the methods above
have been limited by the magnitude of EOF rate obtainable, pH limitations
resulting from the bulk pKa of the silica surface, difficulties in
establishing consistent EOF rates between successive runs, and
inconsistencies among different capillary tubes. These problems are
substantially reduced as a result of the present invention.
Example 8 illustrates use of the silicate reagent of the invention to
achieve excellent and reproducible separations for a chiral mixture by
means of a counter-current electrophoresis format. The chiral mixture,
consisting of racemic atropine, "TROLOX" and
4-methyl-5-phenyl-2-oxazolidinone (MPO), was separated in capillary tubes
from six different suppliers using a sulfonated .beta.-cyclodextrin
derivative as the pseudophase. As a control, DMSO marker was initially run
alone in running buffer lacking cyclodextrin. As can be seen for the first
five runs with DMSO marker alone (FIG. 9), use of silicate reagent affords
high and consistent EOF rates of between about 4.7 and 5.0 .times.10.sup.4
cm.sup.2 /V.sec in all instances. Furthermore, this consistency extends to
the separation of the chiral mixture (runs 6-10), wherein the six sample
stereoisomers are all base-line resolved with very little change in
mobility (FIG. 9). FIG. 10 illustrates continued run-to-run
reproducibility, in terms of migration time, achieved with ten consecutive
runs. | | |
