U.S. patent application number 09/197896 was filed with the patent office on 2001-07-12 for coated surface comprising a polyvinyl alcohol (pva) based covalently bonded stable hydrophilic coating.
Invention is credited to GOETZINGER, WOLFGANG, KARGER, BARRY L..
Application Number | 20010007701 09/197896 |
Document ID | / |
Family ID | 23498901 |
Filed Date | 2001-07-12 |
United States Patent
Application |
20010007701 |
Kind Code |
A1 |
KARGER, BARRY L. ; et
al. |
July 12, 2001 |
COATED SURFACE COMPRISING A POLYVINYL ALCOHOL (PVA) BASED
COVALENTLY BONDED STABLE HYDROPHILIC COATING
Abstract
Coatings suitable for surfaces such as are found in capillary
electrophoresis columns and methods for their preparation are
disclosed. A coated surface of the invention, preferably an
interior surface of a microcapillary, generally includes a surface
having an interconnected polymeric coating that includes a
functional group attached to the surface and capable of
copolymerizing with an organic compound in an organic solvent, and
a polymer of the organic compound copolymerized with the functional
group. The coating can be covalently or non-covalently attached to
the surface and can further include an additional layer of coating
material. Preferably, the organic compound is a vinyl ester, and
most preferably, vinyl acetate, and the attached polymer forming
the exposed surface of the coating is a polyvinyl alcohol, the
hydroxyl groups of which can be further derivatized in any desired
manner. The coating of the invention creates a new, stable surface,
appropriate for CE columns or general surface modification. The
coating is stable over a wide pH range and allows highly efficient
grafting and/or adsorption of a variety of additional layers, if
desired.
Inventors: |
KARGER, BARRY L.; (NEWTON,
MA) ; GOETZINGER, WOLFGANG; (BOSTON, MA) |
Correspondence
Address: |
WEINGARTEN SCHUGIN GAGNEBIN &HAYES
TEN POST OFFICE SQUARE
BOSTON
MA
02109
|
Family ID: |
23498901 |
Appl. No.: |
09/197896 |
Filed: |
November 23, 1998 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09197896 |
Nov 23, 1998 |
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08861906 |
May 22, 1997 |
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5840388 |
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Current U.S.
Class: |
428/36.91 ;
428/522 |
Current CPC
Class: |
Y10T 428/1321 20150115;
B01J 20/262 20130101; B01J 20/285 20130101; Y10T 428/1352 20150115;
Y10T 428/31663 20150401; Y10T 428/31935 20150401; Y10T 428/31931
20150401; G01N 27/44752 20130101; Y10T 428/1383 20150115; Y10T
428/131 20150115; Y10T 428/1393 20150115 |
Class at
Publication: |
428/36.91 ;
428/522 |
International
Class: |
B32B 001/08 |
Claims
What is claimed is:
1. A coated surface comprising: a surface having an interconnected,
polymeric, surface-modifying coating comprising a polyvinyl alcohol
based polymer covalently attached to said surface, said coating
remaining stable and covalently attached to said surface at pH
values comprising pH 4.4 to pH 10.0.
2. The coated surface of claim 1 wherein, in said coating, said
polyvinyl alcohol based polymer is covalently attached to said
surface by Si--O--Si bonds.
3. The coated surface of claim 1 wherein said surface is made of a
material selected from the group consisting of fused silica, glass,
polytetrafluorethylene and polyether ether ketone.
4. The coated surface of claim 1 wherein said coating is further
modified.
5. The coated surface of claim 1 wherein said coating further
comprises an additional layer of coating material.
6. The coated surface of claim 5 wherein said additional layer of
coating material is covalently bonded to hydroxyl groups of said
polyvinyl alcohol based polymer.
7. The coated surface of claim 1 wherein, in said coating, said
polyvinyl alcohol based polymer comprises free hydroxyl groups.
8. The coated surface of claim 1 wherein, in said coating, said
polyvinyl alcohol based polymer comprises derivatized hydroxyl
groups.
9. The coated microcapillary column of claim 8 wherein said
derivatized hydroxyl groups of said polyvinyl alcohol based polymer
comprise hydroxyl groups on adjacent carbon atoms derivatized to
form an epoxide or an acetal.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a divisional application of U.S.
application Ser. No. 08/861,906, filed May 22, 1997, which is a
file wrapper continuation application of U.S. application Ser. No.
08/379,834, filed Jan. 27, 1995, the whole of which are hereby
incorporated by reference herein.
BACKGROUND OF THE INVENTION
[0002] Capillary electrophoretic separation techniques find wide
application in the biologically related sciences. Molecular species
such as peptides, proteins, oligonucleotides, and oligosaccharides
are separated by causing them to migrate in a buffer solution under
the influence of an electric field. The separation is normally
carried out in thin-walled, narrow-bore capillary tubes to minimize
the evolution of heat during electrophoretic separation, which
would cause zone deformation.
[0003] Among the other mechanisms that can cause zone deformation
are non-uniform electroendosmosis, excess electroosmotic flow, and
solute adsorption to the inner surface of the capillary. However,
these problems can be minimized or overcome by coating the inner
wall of the electrophoresis tube with various polymeric
substances.
[0004] In U.S. Pat. No. 4,680,201, Hjerten discloses a method for
coating the inner wall of a narrow bore capillary with a
monomolecular polymeric coating of polyacrylamide bonded to the
capillary wall by means of a bifunctional reagent, e.g.,
.gamma.-methacryloxypropyltrimethoxysilane. These capillaries can
be used for free-zone electrophoresis in open tubes.
[0005] Novotny et al., U.S. Pat. No. 5,074,982, discloses that the
inner wall of silica capillaries used in electrophoretic
separations can be coated with bifunctional reagent using a
Grignard reagent, for hydrolytic stability.
[0006] Thermal immobilization of adsorbed polyvinyl alcohol (PVA)
as a coating on fused silica capillary surfaces is described in
Gilges et al., Anal. Chem. 66:2038-2046 (1994). These coatings are
stable for separations over a wide range of pH; however, at high
buffer pH, the adsorption of PVA molecules and the suppression of
analyte/wall interaction is weakened.
SUMMARY OF THE INVENTION
[0007] The present invention generally features coatings suitable
for surfaces such as are found in capillary electrophoresis columns
and methods for their preparation. A microcapillary column of the
invention generally includes a microcapillary having an interior
cavity and a wall with an inner surface, the inner surface of the
wall having an interconnected polymeric coating that includes a
functional group attached to the inner surface and capable of
copolymerizing with an organic compound in an organic solvent and a
polymer of the organic compound copolymerized with the functional
group. The coating can be covalently or non-covalently attached to
the column wall and can further include an additional layer of
coating material. Preferably, the organic compound is a vinyl
ester, and most preferably, vinyl acetate, and the attached polymer
forming the exposed surface of the coating is a polyvinyl alcohol,
the hydroxyl groups of which can be further derivatized in any
desired manner. In a most preferred capillary column, the coating
material includes a polyvinyl alcohol based polymer covalently
attached to the column wall by Si--O--Si bonds.
[0008] The method of the invention generally includes providing a
microcapillary, modifying the inner surface of the capillary wall
to provide attached functional groups capable of copolymerizing
with an organic compound in an organic solvent, introducing a
solution of the organic compound in an organic solvent into the
interior cavity of the microcapillary; and causing molecules of the
organic compound to copolymerize with the attached functional
groups to form an interconnected polymeric coating material
attached to the inner surface of the microcapillary column.
Preferably, the attached functional groups are covalently bonded
vinyl groups, the organic compound is a vinyl ester, and the
resulting interconnected polymeric coating material of polyvinyl
ester is modified in a polymer homologous reaction to produce the
desired polyvinyl alcohol coating.
[0009] In another aspect, the method of the invention features, in
general, forming a column with a hydrophilic polymeric coating by
directly converting an attached hydrophobic polymeric coating
material to a hydrophilic coating material. The resulting
hydrophobic polymeric coating can contain acidic, basic or neutral
functionalities depending on the intended use of the column.
[0010] The terms "CE column" or "microcapillary column" are meant
to include a vessel of any shape in which capillary electrophoresis
can be carried out. For example, it is also known to use chips with
open grooves microfabricated into the surface of the chip for
capillary electrophoresis.
[0011] The coating of the invention creates a new, stable surface,
appropriate for CE columns or general surface modification. The
coating is stable over a wide pH range and allows highly efficient
grafting and/or adsorption of a variety of additional layers, if
desired. As used in capillary electrophoresis, the coating
suppresses or controls electroosmotic flow and prevents adsorption
of analytes to the surface of the column.
BRIEF DESCRIPTION OF THE DRAWINGS
[0012] Other features and advantages of the invention will be
apparent from the following description of the preferred
embodiments thereof, taken in conjunction with the accompanying
drawings, in which:
[0013] FIG. 1 shows a cross-sectional view of a coated
microcapillary column of one embodiment of the invention in which
an interconnected polyvinyl alcohol based polymeric coating is
covalently attached to the inner wall of the column by Si--O--Si
bonds;
[0014] FIG. 2 shows the hydrolysis and condensation of
vinyltrimethoxysilane to form a mixture of oligomeric vinylsilanol
compounds as carried out in the surface modification step of one
embodiment of the method of the invention;
[0015] FIG. 3 shows attachment and curing of the oligomeric
vinylsilanol compounds of FIG. 2 as carried out in the surface
modification step of one embodiment of the method of the
invention;
[0016] FIG. 4 shows copolymerization of the monomer vinylacetate
with the vinyl groups of the attached oligomeric vinylsilanol
compounds of FIG. 3 to form a covalently bonded hydrophobic polymer
(polyvinyl acetate), according to one embodiment of the method of
the invention;
[0017] FIG. 5 shows conversion of the covalently bonded hydrophobic
polymer (polyvinyl acetate) into its hydrophilic counterpart
(polyvinyl alcohol or PVA);
[0018] FIGS. 6-9 show open tube capillary zone electrophoresis of
proteins at pH 4.4, 8.8, 6.2, and 10.0, respectively, using a
microcapillary column of the invention;
[0019] FIG. 10 shows isoelectric focussing of proteins using a
microcapillary column of the invention;
[0020] FIG. 11 shows isoelectric focussing of hemoglobin variants
using a microcapillary column of the invention;
[0021] FIG. 12 shows open tube capillary zone electrophoresis of
.PHI.X174 digested with Hae III, using a microcapillary column of
the invention;
[0022] FIG. 13 shows polymer network separation of DNA sequencing
reaction products using a microcapillary column of the invention;
and
[0023] FIG. 14 shows polymer network separation of poly-dA
oligonucleotides of various lengths using a microcapillary column
of the invention.
DETAILED DESCRIPTION OF THE INVENTION
[0024] The invention, as applied in capillary electrophoresis,
provides a new method for obtaining highly stable hydrophilic
coated microcapillary columns with superior performance for the
separation of biopolymers. As shown in FIG. 1, a preferred
microcapillary column of the invention includes a fused silica
microcapillary 10 having an inner wall 12 and an interconnected
polyvinyl alcohol based polymeric coating 14 covalently attached to
inner wall 12 by Si--O--Si bonds 16.
[0025] A preferred method of the invention generally includes, as a
first step, a silanization procedure which modifies the silica
surface of the capillary with highly reactive vinyl silanol
oligomers, leaving free vinyl groups as a reactive functionality.
Next, a hydrophobic monomer (vinyl acetate) is copolymerized with
the anchored vinyl groups in an organic solvent in the interior
cavity of the surface modified, fused silica capillary. In the last
step, the covalently bonded hydrophobic polymer (polyvinyl acetate)
is converted into its hydrophilic counterpart (polyvinyl alcohol or
PVA). (It is possible that some small amount of acetate groups may
remain.) The preparation procedure for the polyvinyl alcohol-coated
capillaries of the invention is simple, reliable and reproducible.
No toxic chemicals are involved. The resulting hydrophilic coating
is chemically and hydrolytically very stable and allows the
efficient separation of biopolymers over a wide pH range.
[0026] The following procedure (which is described in more detail
in the EXAMPLES) will result in a stable coating of polyvinyl
alcohol, covalently attached to the surface of fused silica
capillaries:
[0027] Surface Modification of the Fused Silica Capillary to
Provide Stable, Covalently Bonded Groups for Copolvmerization with
Monomers of an Organic Compound.
[0028] To allow reproducible surface modification, the capillaries
are first treated with acid to compensate for the different surface
quality of the individual fused-silica capillaries (different
manufacturers, different aging history from batch-to-batch). This
procedure results in a more active capillary surface as well as a
more uniform surface by increasing the number of exposed, reactive
silanol groups. Actual modification of the capillary wall is
achieved by reacting the surface silanols with a mixture of
oligomeric vinylsilanol compounds. Referring to FIG. 2, these
compounds are obtained in solution by reacting
vinyltrimethoxysilane 22 with a limited amount of 0.1 M HCl. The
acid cleaves off the protective methoxy group and converts the
silane into a highly surface-reactive polysilanol species
(vinylsilanetriol) 24. In addition, the vinylsilanetriol condenses
to oligomers 26. The limited amount of water present prevents the
compounds in the mixture from polymerizing and cross-linking, which
would result in precipitation. It is to be noted that these
silanization mixtures are stable for days.
[0029] Referring to FIG. 3, the subsequent attachment of oligomers
26 to the capillary surface 12 (via the condensation of surface
silanol groups 28 with oligomer silanol groups 30) results in a
very stable Si--O--Si bond 16 and provides the surface with
covalently bonded vinyl groups 32 which can copolymerize with vinyl
acetate. The use of an olefinic, nonfunctional silane avoids any
hydrolytically unstable functional groups in the attached molecule.
In addition, due to the thermal stability of the vinylsilane group,
a curing of the modified surface can be performed at high
temperature (110.degree. C. for 1 h), which increases stability of
the coupling chemistry.
[0030] Polymerization of Vinyl Acetate Monomers within the
Capillary Tube in an Organic Solvent.
[0031] As shown in FIG. 4, copolymerization of vinyl acetate
monomers 34 with the vinyl groups 32 of the covalently bonded
vinylsilanes on the capillary surface 12 results in covalent
bonding of the resulting polyvinyl acetate 36 to the silica
surface. (The dotted lines in the representation of polyvinyl
acetate 36 in FIG. 4 indicate the earlier location of the double
bonds in the vinyl acetate monomers. Those skilled in the art use
the term polyvinyl acetate for the polymer of vinyl acetate
monomers even though the resulting polymer no longer contains vinyl
groups.)
[0032] Polymerization is initiated in an organic solvent, in the
capillary tube, by the thermal decomposition of radical initiators
such as .alpha., .alpha.'-azodiisobutyronitrile or benzoyl
peroxide, which start to decompose below the boiling point of the
monomer. In appropriate circumstances, the polymerization solutions
may be deposited as a thin film on the modified capillary wall.
This polymerization procedure is not sensitive to trace amounts of
oxygen, as is, e.g., the polymerization of acrylamide. Organic
solvents such as ethyl acetate produce good polymerization yields.
Since the viscosity of the resulting polymer solutions in organic
solvents is very low, up to 40% monomer can be used for the
polymerization, guaranteeing a high grafting density
(copolymerization with the surface vinyls and thus covalent
attachment of the polymer). Furthermore, excess polymer solution
can easily be pushed out of the capillaries. (With acrylamide in
aqueous solution, the density limits are about 7% monomer.)
[0033] Conversion of the Covalently Bonded Hydrophobic Polymer
(Polyvinyl Acetate) into its Hydrophilic Counterpart (Polyvinyl
Alcohol or PVA)
[0034] After the hydrophobic polyvinyl acetate polymer is bound to
the capillary surface, the coating has to be converted into its
desired hydrophilic form, polyvinyl alcohol (PVA). Referring to
FIG. 5, in a polymer homologous reaction, the covalently bonded
polyvinyl acetate 36 can easily be deacetylated by flushing a
solution of sodium methylate through the capillaries at
room-temperature. Base-catalyzed deacetylation is very efficient
even at ambient temperature; this step is conveniently carried out
by flushing the sodium methylate solution through the capillary for
a short period of time--about 15 minutes. As the hydrolysis of the
ester group is performed under non-aqueous conditions, the silane
chemistry is not affected by this strong base. Having anhydrous
methanol as a solvent for hydrolysis precipitates the resulting
polyvinyl alcohol 14, thus shielding the silane coupling chemistry
underneath the polymer. The result of this procedure is a
microcapillary with a hydrophilic, covalently attached coating of
polyvinyl alcohol, which can be stored until use.
[0035] In comparison with, e.g., the polymerization of aqueous
solutions of acrylamide within a capillary, the procedure described
above offers several advantages. As polymerization is initiated by
thermal decomposition of a radical initiator after the polymerizing
solution has been injected into the capillary, the whole procedure
is very convenient and controllable. In contrast, conventional
polymerization of acrylamide in aqueous solution is initiated as
soon as the ingredients (e.g., TEMED, APS, acrylamide monomers) are
combined, and the solution must be handled quickly to prevent
premature polymer formation. Polymerization in organic medium
results in much lower viscosities of the polymer solution so that
higher monomer concentrations can be used. This results in higher
grafting density and thus better performance and stability. With
40% vinyl acetate, excess organic solvent can still easily be
pushed out of the capillary after polymer formation, while in
water, 7% acrylamide is about the limit. Liquid organic compounds
that are polymerizable under the described conditions can, in fact,
be used without any additional solvent. In this case, excess
compound would be pushed out of the capillary after sufficient
polymerization had occurred.
[0036] Polymerization in an organic solvent is less sensitive to
oxygen than is polymerization in water, which allows the
elimination of degassing steps and permits the use of chemicals as
delivered from the manufacturer. This makes the overall procedure
much more convenient and gives more reliable results. Finally, the
chemicals (vinyl acetate, ethyl acetate and benzoyl peroxide) are
much less toxic than those used in conventional, e.g., aqueous
acrylamide, polymerization.
[0037] Use
[0038] The above method results in capillaries with excellent
performance for the separation of proteins over a wide range of
pH-values (see FIGS. 6-9). Even at almost neutral pH (pH=6.20, FIG.
8), excellent separation of proteins can be achieved under normal
buffer conditions. A capillary run constantly at pH 10.0 at a
voltage of 540 V/cm showed no loss in efficiency or shift in
migration times after 7 days. In contrast, it is known that all
acrylamide- or acrylate-based polymers become charged upon
hydrolysis of functional groups at high pH. This condition results
in peak deterioration and a shift in migration times for samples
separated at high pH in polyacrylamide coated CE columns.
Additionally, capillaries prepared according to the method of the
invention have been used successfully for isoelectric focusing. Due
to their chemical stability, excellent migration time
reproducibility could be achieved.
[0039] Any of a variety of other vinyl esters could be used
according to the method of the invention to make a polyvinyl
alcohol coating, covalently bonded to the inner surface of a
capillary column. In addition to the preferred ester, vinyl
acetate, other appropriate vinyl esters include vinyl propionate,
butyrate, benzoate, or laurate. Furthermore, the method of the
invention is effective in forming a coating layer from any organic
compound (monomer or oligomer) that is capable of copolymerizing in
an organic solvent with functional groups attached to the capillary
wall. Moderately hydrophobic monomers (e.g., vinylpyrrolidone,
hydroxyalkyl(meth)acrylates, etc.) may be used to produce attached
polymers that are sufficiently hydrophilic for use for CE
separation of biopolymers in aqueous buffers without subsequent
polymer homologous conversion.
[0040] The anchored functional groups can be attached covalently or
non-covalently to the column wall. Functional groups that can
copolymerize with vinyl esters preferrably include vinyl groups,
but can also include any other functional group that can
copolymerize in an organic solvent (such as allyl, acryl, methacryl
or any other double-bond containing group). Other functional groups
would be used for polymerizing with other polymerizable organic
compounds.
[0041] The capillary is preferrably made of fused silica and the
anchored functional groups are preferably covalently attached to
the inner surface of the column by the silane coupling chemistry
described; however, other coupling methods will be obvious to those
skilled in the art. The capillary may also be made of any organic
polymer that already contains an appropriate functional group or
that allows copolymerizable groups to be bonded to the surface.
[0042] Coating of other surfaces that are of importance in
separation technologies (such as silica gel, polystyrene, etc.)
could easily be carried out using the method of the invention. The
preferred method described herein of applying a PVA coating is
applicable to all silica surfaces (or polymer surfaces) modified
with groups that can copolymerize with the monomer. This procedure
would be of particular importance for HPLC, where surfaces with low
protein adsorption are highly desirable, especially for size
exclusion chromatography of proteins.
[0043] Coatings prepared by the method of the invention can easily
be derivatized or modified to change surface chemistry or to attach
an additional coating layer. For example, modifications to a
covalently bonded PVA-coating (prepared as described) in a
capillary or on the surface of silica gel can include crosslinking
by bi-(or poly)functional reagents (such as diepoxides,
diisocyanates, acid anydrides); chemical conversion of the
polyhydroxy-coating into polyethers, polyesters, etc., by chemical
reaction of the hydroxy-functionality with monomeric or polymeric
reagents; linking of adjacent hydroxy-functionalities as in an
epoxide or acetal; or reacting the functional surface with groups
that would introduce charges and may result in an ion-exchange
capacity of the surface. Thermal treatments may result in physical
(orientation of the polymeric layer, hydrogen bonding, partial
crystallization) or chemical (partial or complete condensation and
thus crosslinking) modifications of the coating. Modification of
the polymerization conditions or chemical conversion after
polymerization to affect the distribution of 1,2- and 1,3-diols is
also possible.
[0044] Use of borate containing buffers with a PVA coated column
could result in complex formation with PVA hydroxyl groups. The
resulting surface charge, at high pH, gives an EOF comparable to a
bare fused silica capillary. Thus, PVA coated capillaries may be
considered as capillaries with switchable (buffer dependent) EOF;
with PVA coated HPLC-supports, borate buffers could be used to
generate a dynamic cation exchanger.
[0045] The surface chemistry possible with PVA-coated silica
surfaces allows for a broad range of chemical reactions as the
surface can be considered in general as a polyhydroxy-compound
(polyalcohol). This property is useful for forming affinity
matrices for affinity CE or HPLC as antibodies, or other
biospecific reagents, can easily be bonded to the coated surface
via hydroxy-reactive groups. The PVA-coating beneath the attached
antibodies would guarantee low adsorption and thus eliminate
non-specific interaction.
[0046] The following examples are presented to illustrate the
advantages of the present invention and to assist one of ordinary
skill in making and using the same. These examples are not intended
in any way otherwise to limit the scope of the disclosure.
EXAMPLE I
[0047] Preparation of a covalently bonded polyvinyl alcohol (PVA)
coated capillary:
[0048] A) A length of about 60 cm of polyimide-coated fused silica
capillary with an internal diameter of 75 .mu.m is rinsed for 2 h
with conc. HCl/H.sub.2O (1:1) at a temperature of 110.degree. C. to
provide a surface with high silanol concentration for successful
silane coupling chemistry. The capillary is then rinsed to neutral
pH and dried in a gentle He stream.
[0049] B) The silanization solution is prepared by mixing 2 ml of
vinyltrimethoxysilane and 0.4 ml of 0.1 M HC1. Upon stirring, the
initial two phase mixture homogenizes due to hydrolysis of
alkoxysilane groups and liberation of methanol, a very exothermic
reaction. One hour is allowed for hydrolysis and condensation of
the vinylsilanols. The solution of oligomeric vinylsilanols is
pushed through the capillaries for 1 h and allowed to stand
overnight. The next day the capillaries are rinsed with methanol.
Curing is performed at 110.degree. C. in a gentle He stream.
[0050] C) The polymerization solution is prepared by mixing 3 ml of
ethyl acetate with 2 ml of vinyl acetate, resulting a concentration
of vinyl acetate of 40% (V/V). The addition of 100 .mu.l of a 5%
solution of benzoyl peroxide in ethyl acetate yields an initiator
concentration of 0.25%. The vinyl modified capillaries from B) are
filled with the polymerization solution and the polymerization is
performed at 75.degree. C. for 20 h. The polymer solution is then
pushed out, and the capillaries are rinsed with ethyl acetate and
methanol to remove any non-bonded polyvinyl acetate.
[0051] D) To convert the covalently bonded polyvinyl acetate into
polyvinyl alcohol, the coated capillaries from C) are rinsed for 15
min with a solution of 0.5 M sodium methylate in methanol. The
capillaries are rinsed afterwards with methanol and then dried in a
He stream.
EXAMPLE II
(FIG. 6)
[0052] Sample: (1) lysozyme, (2) cytochrome C, (3) myoglobin, (4)
trypsinogen, (5) .alpha.-chymotrypsinogen A (0.1 mg/ml each).
Coating: PVA as described in Example I. Conditions: i.d.=75 .mu.m;
L=30/37 cm; buffer: 20 mM E-aminocaproic acid, pH=4.40; injection:
4 sec at 5 kV; separation voltage: 20 kV.
EXAMPLE III
(FIG. 7)
[0053] Sample: (1) glucose-6-phosphatedehydrogenase, (2) trypsin
inhibitor, (3) L-asparaginase, (4) .alpha.-lactalbumin (0.1 mg/ml
each). Coating: Example I. Conditions: i.d.=75 .mu.m; L=30/37 cm;
buffer: 20 mM TAPS/AMPD, pH=8.80; injection: 4 sec at 5 kV;
separation voltage: 20 kV.
EXAMPLE IV
(FIG. 8)
[0054] Sample: (1) lysozyme, (2) cytochrome C, (3) myoglobin, (4)
trypsinogen, (5) .alpha.-chymotrypsinogen A (0.1 mg/ml each).
Coating: Example I. Conditions: i.d.=75 .mu.m; L=30/37 cm; buffer:
20 mM TRIS/cacodylic acid, pH=6.20; injection: 5 sec with pressure
injection (PAC/E); separation voltage: 20 kV.
EXAMPLE V
(FIG. 9)
[0055] Sample: (1) glucose-6-phosphatedehydrogenase, (2) trypsin
inhibitor, (3) L-asparaginase, (4) .alpha.-lactalbumin (0.1 mg/ml
each). Coating: Example I. Conditions: i.d.=75 .mu.m; L=30/37 cm;
buffer: 20 mM CAPS/NaOH, pH=10.0; injection: 4 sec at 5 kV;
separation voltage: 20 kV.
EXAMPLE VI
(FIG. 10)
[0056] Sample: (A) myoglobin (pl=7.2), (B) carbonic anhydrase 1
(pl=6.6), (C) carbonic anhydrase II (pl=5.9), (D)
.beta.-lactoglobulin A(pl=5.1) (0.1 mg/ml of each protein mixed 1:1
with 2% Pharmalyte (3-10)). Coating: Example I. Conditions: i.d.=50
.mu.m; L=30/37 cm; focussing: at 25 kV; mobilization: low pressure
(PAC/E) starting after 10 min (25 kV); anolyte: 20 mM H.sub.3PO4;
catholyte: 20 mM NaOH.
EXAMPLE VII
(FIG. 11)
[0057] Sample: hemoglobin variants C (pl=7.45), S (pl=7.20), F
(pl=7.00) and A (pl=6.95) (0.1 mg/ml each mixed with 1:1 with a 2%
ampholine mixture (Pharmalyte, Servalyte and Ampholyte)). Coating:
Example I. Conditions: i.d.=50 .mu.m; L=30/40 cm; focussing: at 30
kV; mobilization: hydrodynamic (d H=5 cm) starting after 15 min (30
kV); anolyte: 0.5% acetic acid; catholyte: 0.25% ammonium
hydroxide.
EXAMPLE VIII
(FIG. 12)
[0058] Sample: .PHI.X174 digested with Hae III. Coating: Example I.
Conditions: i.d.=100 .mu.m; L=26.75/27.50 cm; sieving matrix: 1%
methylcellulose (2% gives 4000 cps) in 40 mM TAPS/TRIS; injection:
5 sec at 5 kV; separation voltage: 5 kV.
EXAMPLE IX
(FIG. 13)
[0059] Sample: FAM labeled primer sequencing reaction terminated
with dideoxythymidinetriphosphate on M13mp18. Coating: Example I.
Conditions: i.d.=100 .mu.m; L=30/40 cm; sieving matrix: 4%T LPA in
40 mM TRIS/TAPS with 30% formamide and 3.5 M urea; separation
voltage: 8 kV; injection: 5 sec at 8 kV.
EXAMPLE X
(FIG. 14)
[0060] Sample: poly-da oligonucleotides 12-18, 25-30 and 40-60.
Coating: Example I. Conditions: i.d.=100 .mu.m; L=20/27 cm; sieving
matrix: 10% polyamide+45% DMSO+10% urea+35% TAPS/TRIS (50 mM);
injection: 3 sec at 10 kV; separation voltage: 20 kV; temperature:
40.degree. C.
EXAMPLE XI
[0061] Coating a stationary phase for use in HPLC with polyvinyl
alcohol. A silica gel of 300 .ANG. pore size and 5 .mu.m particle
diameter is modified with vinyl groups as described in Example I
(B) for a fused silica surface or according to other procedures
well known to those skilled in the art (E. P. Plueddemann, Silane
Coupling Agents. Plenum, N.Y. (1982)) 5 g of the silica gel is
suspended in a solution of 10 ml vinyl acetate and 30 ml ethyl
acetate. After 25 mg of benzoyl peroxide is added, the suspension
is heated up to 75.degree. C. for 20 hours under stirring and
reflux. The polymer solution is then removed with a G4 filter
funnel, and the polyvinyl acetate coated silica gel is washed with
ethyl acetate and methanol to remove any non-covalently adsorbed
polymer. Homologous conversion of the bonded polyvinyl acetate into
PVA is carried out by stirring the silica gel for 15 min in a 0.1 M
solution of sodium methylate in methanol. Then the silica gel is
washed with methanol and dried at a temperature of 60.degree.
C.
EXAMPLE XII
[0062] The PVA-coated silica gel of Example XI is packed into a
HPLC column, and high performance (or high pressure) size exclusion
chromatography (SEC) of proteins, nucleotides and synthetic
polymers can be performed in organic or aqueous solvents.
EXAMPLE XIII
[0063] Preparation of affinity matrices from PVA-coated surfaces
can be prepared according to procedures well known to those skilled
in the art, as disclosed in, e.g., G. T. Hermanson, A. K. Mallia,
P. K. Smith, Immobilized Affinity Ligand Techniques. Academic
Press, San Diego (1992).
[0064] Other embodiments of the invention will be apparent to those
skilled in the art from a consideration of this specification or
practice of the invention as disclosed herein. It is intended that
the specification and examples be considered as exemplary only,
with the true scope and spirit of the invention being indicated by
the following claims.
* * * * *