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Description  |
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BACKGROUND OF THE INVENTION
The present invention relates to polymer chemistry, and, more particularly,
to a method of minimizing defects due to shrinkage during polymerization.
While not so limited in scope, the present invention arose in the context
of polyacrylamide gel electrophoresis, in which columns of gels are
prepared in tubes. During electrophoresis, an ionic sample is located at
one end of the column. The ionized components migrate differentially
according to charge and bulk under the influence of an axially applied
electric field. After a predetermined time, the electric field is removed
and the components analyzed according to axial position along the tube.
The columns of gel can be prepared by filling a tube with an aqueous
mixture of acrylamide monomer, and then polymerizing the monomer. In the
case of acrylamide, as is generally true in polymer chemistry, the polymer
is substantially denser than the original pre-polymer, e.g., the monomer,
dimer, or oligomer, from which the polymer is formed. Accordingly,
significant shrinkage occurs during polymerization.
As a consequence of this shrinkage, the forming gel has a tendency to pull
away from the interior walls of the tube. The voids thus formed between
the tube and the gel can disturb the uniformity of an applied electrio
field, and seriously diminish the resolution of the electrophoresis
process Furthermore, the separation of the gel from the tube aggravates a
tendency of the gel to migrate out of the tube during electrophoresis.
These problem can be addressed by coating the interior of the tube with a
bonding agent which forms covalent bonds between the surface of the tube
and the polymer chains While separation and resulting migration are
mitigated, the tension introduced by tendency to shrink during
polymerization can cause bubble-like voids within the gel itself. These
internal voids also distort an applied electrio field and diminish the
resolution of the electrophoresis process.
The voids due to shrinkage are not amenable to procedures used to minimize
bubbles formed from dissolved gasses. The latter can be minimized by
degassing the prepolymer or by conducting polymerization under moderate
pressure of one hundred and some odd pounds per square inch (psi). While
effective at preventing bubbles formed from dissolved gasses, these
methods have negligible impact on voids induced by shrinkage.
The problem of shrinkage-induced voids is not limited to electrophoresis
gels. Polymerization is generally accompanied by increases in density,
viscosity, and, particularly in the cases of cross-linked polymers,
rigidity. Where the polymerizing substance is constrained in some way,
e.g., by vessel walls and/or a bonding agent, it can occur that the
polymerizing substance cannot respond to continuing increases in density
in a fluid or elastic manner. In such cases, the forming polymer structure
can rupture haphazardly. This is problematic where it is desired to
precisely control the dimensions and uniformity of the resulting polymer.
Thus, what is needed generally is a method of preventing defects induced by
shrinkage during polymerization. Specifically, it is desired to reliably
produce columns of polyacrylamide gel without voids internal to the gel or
between the gel and the interior wall of the confining vessel, or
shrinkage of the gel along the axis of the column.
SUMMARY OF THE INVENTION
Defects due to shrinkage during polymerization can be minimized by
decreasing or eliminating the shrinkage itself by compressing the
prepolymer and maintaining increased density during polymerization In one
realization of the present invention, the density of the polymer product
is achieved in the prepolymer form and during polymerization; thus
shrinkage, along wih any defects induced thereby, is eliminated. However,
this is a special case within an empirically determinable range of options
that result in substantially shrinkage-defect-free polymers.
In a reduction to practice of the present invention, an aqueous solution of
10% by weight acrylamide monomer was introduced into a meter long
capillary tube, previously treated with a bonding agent. The tube was
placed in a pressurizing chamber, which then pressurized to 10,000 psi
until polymerization was completed. The result was a substantially
defect-free electrophoresis gel.
This example illustrates four boundary conditions on the range of
time-functions of compression covered by the present invention. (1) the
pressures applied in the prior art to minimize bubbles from dissolved
gasses are negligible compared to the pressures required by the present
invention to minimize shrinkage defects; (2) the compression can exceed
that needed to achieve the density of the polymer product; (3) the
compression need not be constant; and (4) it is not necessary to eliminate
all tendency to shrink to substantially eliminate the defects. This fourth
point leads to a fifth: (5) the compression can be less than that needed
to achieve the density of the polymer product.
With respect to the first point above, the 10,000 psi pressure is two
orders of magnitude greater than that used to eliminate bubbles caused by
dissolved gasses. While 10,000 psi is by no means the minimum pressure
prescribed by the present invention in all situations, this magnitude
helps explain the inability of pressures selected to minimize gaseous
discharge to impact shrinkage defects. The issue of pressure magnitudes is
addressed more precisely once the remaining of the previous paragraph's
five points are elaborated.
Regarding the second point, it was calculated that the density increase
during polymerization in the absence of compression would be about 2.2%
and, accordingly, a pressure of about 8200 psi applied to the monomer
mixture would suffice to achieve the desired pre-compression. It would be
difficult to conjecture how, if a certain high pressure were effective at
preventing shrinkage, an even higher pressure would cause such defects to
reappear. The experiment suggests that, in fact, shrinkage defects do not
reappear at higher pressures. Thus, upper limits on applicable pressures
are to be imposed by costs and equipment limitations rather than by the
principle of the present invention.
Turning to the third point, to the extent that an applied pressure is
effectively constant, the density of the substance increases with
polymerization. To illustrate, assume that the constant pressure applied
is that required to achieve a density in the monomer mixture equal to the
density of the polymer product in the absence of compression. However,
upon completion of polymerization, the polymer product is under the
constant pressure, and is thus compressed relative to its uncompressed
state and thus is compressed relative to the compressed monomer mixture.
This illustrates that both constant pressure and constant compression, and
a wide range of alternative time-functions of compression are provided for
by the present invention.
This leads into the fourth point that it is not necessary to eliminate all
shrinkage. In other words, some shrinkage can be accommodated without
causing defects. However, as is well known, the ratio of compression to
pressure generally decreases with density so that compression of the
monomer mixture is greater than compression of the polymer product, and
thus the shrinkage is less with greater pressure.
The fifth point is that since some shrinkage is tolerable, it is not
necessary that the initial compression completely achieve the density of
the uncompressed polymer product. In some cases, the fluidity or
elasticity of the polymerizing substance or the polymer product can
accommodate the increase in density without inducing defects.
Alternatively, the structural strength of the polymer product can be great
enough to withstand the negative pressure induced by the tendency to
shrink; accordingly such a polymer can maintain itself in a meta-stable
state.
In practice, an applied constant pressure is not necessarily constant in
effect. As applied to the reduction to practice described above, the
increase in viscosity at the ends of the tube during polymerization serves
to partially isolate the substance in the more central portions of the
tube from the effects of the applied pressure. Thus, even where the
externally applied pressure is constant, the effective internal pressure
may not be. This phenomenon and its implications are elaborated in the
detail description below. Briefly, while qualifying the understanding of
the reduction to practice described above, the qualifications are readily
aooommodated by the present invention.
Returning to the issue of the appropriate range of compressions or
pressures to be applied to the monomer or other prepolymer substance, the
minimum must normally be established empirically. Furthermore, the
creation of defects is a stochastic event, so that one must establish
acceptable "yields" to define a precise minimum. Notwithstanding the
above, there are certain general limitations to how small a compression,
and hence pressure, can be applied in accordance with the present
invention.
The present invention only addresses reactions in which shrinkage defects
can occur when polymerization is conducted at constant ambient pressure.
This would exclude, by way of example, very dilute aqueous mixtures for
two reasons. In the first place, the shrinkage of the mixture during
polymerization would be fluid, so that shrinkage would be accommodated by
flow rather than defect formation. In the second place, even if the
polymer product were not free-flowing, the shrinkage would be small enough
to be accommodated by limited fluidity, elasticity and structural strength
in the polymer product Thus, the present invention is constrained to
"defect prone" processes during which sufficient shrinkage could occur and
which result in a sufficiently rigid polymer product for defects to be
induced.
Given a "defect-prone" process, sufficient compression is required to make
a significant difference statistically. While this is in part a function
of the particular application, it is safe to say that the compression
should at least reduce shrinkage by half to substantially prevent
shrinkage defects that would otherwise occur.
Using acrylamides for example, a 1% aqueous mixture would not result in a
polymer product rigid enough for shrinkage defects to occur. Furthermore,
the about 0.2% shrinkage that would occur during polymerization under
ambient pressure could easily be accomodated without unduly stressing even
a more rigid polymer product. Yet, to reduce compression by one half would
require about 330 psi. What this indicates is that even tripling the
pressures applied to prevent gas discharge bubbles would not amount to
practicing the present invention.
In accordance with the foregoing, the present invention can be applied to
provide polymers substantially free of defects induced by shrinkage during
polymerization. Specifically, the substance does not pull away from vessel
walls or form voids during polymerization. Moreover, shrinkage along any
axis can be prevented or minimized for reasons other than for eliminating
voids.
The invention is applicable to any composition of pre-polymer,
cross-linker, catalyst, initiator, and/or accelerator. A solvent or buffer
system can be used, and the product can be a gel or other at least
minimally rigid polymer. The polymer can be cast in any shape vessel,
including a tube of any length and diameter. Since compression can be
effected by immersion, the vessel can be quite fragile. The polymer can be
formed unbonded or covalently bonded to an interior wall of the vessel so
that the polymer does not migrate or otherwise move. Other features and
advantage are apparent in the context of the detailed description below in
conneotion with the following figures.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 is a perspective cutaway view of a pressure vessel containing a
capillary tube confining a polymerizing substance in accordance with the
present invention.
FIG. 2 is a schematic representation of a pressurizing system incorporating
the pressure vessel of FIG. 1.
DESCRIPTION OF THE PREFERRED EMBODIMENTS
In accordance with the present invention, a polymerizable substance is
compressed prior to polymerization to substantially avoid defects due to
shrinkage during polymerization. The compression can be effected by
applying pressure to the polymerizable substance.
In one embodiment of the present invention, a one meter length of fused
silica capillary tube 11 is treated with a wall-bonding agent 12, such as
3-methacryloxypropyltrimethoxysilane. An aqueous mixture 17 including 10%
by weight acrylamide monomer and bisacrylamide cross-linker is then
introduced into the treated capillary tube 11. This filled tube 11 is
inserted into a pressure chamber 13, previously filled with water 15.
Sufficient ammonium persulfate initiator and tetramethylethyenediamine
promoter are included in the mixture 17 to facilitate polymerization.
The pressure chamber 13 is attached to a pumping system 19 including a
compressed air, or other gas, cylinder 21, a regulator valve 23, and a
hydraulic amplification pump 25. As is well known, generally, the
polymerization process is accompanied by shrinkage so that there is a
positive density differential between the resultant polymer and the
prepolymer.
A "T" connector 29 splits the pump 25 output between two delivery arms 31
so that pressure can be applied to both ends of the stainless steel
cylindrical pressure chamber 13. The delivery arms 31 are sealed to the
ends of the chamber 13 by means of swage-lock fittings 33.
The pumping system 19 is arranged to provide pressures sufficient to
compress the prepolymer so that this differential is at least halved.
Preferably, the pumping system 19 should be able to compress the
prepolymer to densities beyond that of the resultant polymer.
While the monomer mixture remains in a free-flowing form, the pumping
system 19 is activated to apply pressure to the polymerizable substance to
increase its density beyond the expected density of the gel to be formed.
In the present case, a typical cross-linked acrylamide gel (10%) by weight
in water increases in density about 2.2% during the process of
polymerization. Thus, pressure to be applied is selected to increase the
density of the prepolymer 2.2% or more.
In order to effect this density change, a pressure of at least about 8200
psi can be applied. In the present method, a static pressure of 10,000 psi
was established. Satisfactory results can usually be achieved with at
least 3700 psi applied, but the probability of defects diminishes with
increasing pressures. In an embodiment using a 5% monomer mixture, full
compression is achieved at about 3700 psi, and 1700 psi suffices to
diminish shrinkage by one half.
In the illustrated embodiment, the applied pressure is essentially
maintained throughout the polymerization process. After about an hour, the
polymerization process is oomplete. After depressurizing the chamber, the
residual pressure in the gel is allowed to equilibrate with the atmosphere
for about 12 hours.
The capillary tube is then removed from the pressure chamber. Except at its
ends, the tube 11 now contains a uniform, polyacrylamide gel, which is
substantially free of voids. The few centimeters at each end of the tube
where the pressurizing water mixes with the aqueous solution are filled
with unpolymerized material. If desired, the ends can be removed to
provide a uniform gel. The gel is bonded to the internal wall of the tube
11.
While the illustrated embodiment used 10% by weight of monomer, 5% to 20%
monomer is a suitable range. Other applications of the present invention
can involve concentrations in excess of pb 20%. In iso-electric focusing
applications, concentrations as low as 1% monomer can be used. However,
below about 1% the change in density due to polymerization would not tend
to induce srinkage defects, and so such dilute substances are not
addressed by the present invention.
Pressure can be applied in various ways to the polymerizable substance, and
this can effect the time function of pressure applied during
polymerization. In the illustrated embodiment, pressure is applied by
immersion in a bath which maintains constant pressure throughout
polymerization. Thus, when polymerization is completed the pressure is
reflected in internal pressure in the polymer. This internal pressure is
gradually relieved after the pressur is removed.
However, pressure system 19 is equipped with a programmable pressure
function controller 27 which controls the valve 23. Thus, the pressure can
be adjusted during polymerization to effect constant compression or other
time functions of pressure. Alternatively, constant compression can be
applied using pistons at both ends of a tube containing a polymerizable
substance. The pistons can be forced a fixed distance inward to achieve
the desired compression. As polymerization progresses, the fixed distance
remains unchanged so that pressure is gradually relieved during
polymerization.
In one approach, the fixed distance is selected to establish in the
prepolymer the expected density of the polymer product in the absence of
compression. Thus, upon completion of polymerization, the polymer product
is at atmospheric pressure and substantially free of defects due to
shrinkage.
The pressure function controller 27 can be programmed to execute a wide
variety of time functions of pressure provided for by the present
invention to yield shrinkage-defect-free polymers. The range of pressure
functions is, itself, a function of the polymers and prepolymers involved
and the concentrations of these components in the substances carrying
them. Also, the nature and presence of cross-linkers, initiators, and
promoters, can affect the relationship between pressure and compression
during polymerization. The resulting polymer can be free of negative
pressure or can be meta-stable within limits of the polymer's structural
strength.
As indicated above, in practice, constant external pressure is not always
equated with constant internal pressure. By way of explanation, and not of
limitation, the following, more detailed view of the polymerization
process is presented.
During polymerization, monomers join together, increasing the viscosity of
solution. If the monomer solution contains a cross-linking agent, a
cross-linked network is formed. At some point during the polymerization,
i.e., the gelation point, this network becomes extended and rigid enough
to resist flow, and a gel results.
This gel includes water essentially trapped in the network of polymer
strands. The possibility of bulk flow of water through the matrix is
greatly reduced, while the tendency for small molecules to diffuse is
relatively unchanged. Shrinkage results from the fact that the volume
oooupied by a polymer strand is less than the sum of the volumes occupied
by its constituent monomers prior to polymerization.
Among the approaches to polymerization under pressure provided for by the
present invention are: constant volume or compression, constant pressure,
and constant external pressure yielding constant volume. In the constant
volume, the monomer solution can be compressed to the anticipated
uncompressed volume of the resulting gel. The vessel bearing the monomer
solution can then be sealed. This is equivalent to compressin the liquid
in a cylinder with a piston and then locking the piston.
As polymerization progresses, the pressure inside decreases, while the
density remains constant. At the end of polymerization, the gel is at
atmospheric pressure, both internally and externally. In the piston and
cylinder implementation, the piston would not moved up or down if unlocked
at this point.
In the constant pressure approach, the monomer solution is compressed using
a constant pressure. Sufficient pressure can be applied so that the
density of the monomer solution is substantially that of the anticipated
unpressurized polymer product. This can be implemented by placing a large
weight on a piston capping a cylinder which holds the monomer solution.
After polymerization is completed, and after extended equilibration, the
gel is under an internal pressure equal to the applied constant external
pressure. When the external pressure is removed, the water that has been
compressed slowly expands and flows out of the gel. The polymer network
substantially maintains its shape and size. After extended
re-equilibration with the atmosphere, the gel is at atmospheric pressure
internally.
The constant external pressure yielding constant volume approach most
closely characterizes the preferred method applied to aqueous monomer
mixture 17. Although a constant pressure is applied, the polymerization is
fast enough so that, at some point before completion of the polymerization
process, the forming gel resists the flow of water. The ends of the gel
column act as caps, or locked pistons and keep the rest of the gel
substantially at constant volume.
As the polymerization proceeds, the pressure in the middle of the gel
diminishes. At the completion of polymerization, the ends of the gel are
under the applied pressure, while the middle is about at atmospheric
pressure. Thus, the ends are effectively at constant pressure, while the
center is effectively at constant volume, and intermediate reqions are
effectively at intermediate pressures. If the external pressure is
maintained after polymerization, the gel slowly approaches a constant
pressure condition.
Applying a constant pressure greater than that required to achieve a
monomer solution density equal to the density of the normally formed
polymer product can yield a polymer network of greater than normal density
because the polymer itself is compressed. This compressed polymer can have
a tendency to expand when the pressure is released, even after water in
the gel has escaped and equilibrated with the ambient atmospheric
pressure. However, a cross-linked polymer can retain its compressed form
along with some internal expansion tension.
If, on the other hand, the applied pressure is less than that needed to
achieve a monomer solution density equal to the normal density of the
polymer product, and if no shrinkage or voids are formed, the polymer
network remains with some internal contraction tension.
While most of the foregoing has addressed polymer solutions and mixtures,
the invention can be applied to a solvent-free polymer system. In this
oase, a gel does not form and water cannot flow in and out of the network.
However, the present invention can still be applied to reduce shrinkage
and minimize void formation.
In addition to the foregoing variations, it is recognized that the nature
of the constraints imposed on the polymerizing substance is a factor in
determining appropriate pressure functions. For example, the shape of the
vessel, and the nature of any bonding agents must be considered. Those
skilled in the art can recognize other variations and modifications of the
foregoing embodiments that are within the scope of the present invention,
which is, accordingly, limited only by the following claims.
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