Chemistry: electrical and wave energy – Processes and products – Electrophoresis or electro-osmosis processes and electrolyte...
Reexamination Certificate
2000-03-22
2003-07-01
Warden, Jill (Department: 1743)
Chemistry: electrical and wave energy
Processes and products
Electrophoresis or electro-osmosis processes and electrolyte...
C204S470000, C526S261000, C526S258000, C526S286000, C526S288000, C526S301000, C526S302000, C526S303100, C526S304000, C526S305000, C526S306000, C526S307300, C526S307400, C526S307500, C526S307700
Reexamination Certificate
active
06585873
ABSTRACT:
TECHNICAL FIELD
This invention relates to polymer gels and membranes. In particular it relates to polymer gels and membranes that are suitable for separation of molecules. The invention is also concerned with the preparation of novel polymer gels and membranes, the separation of molecules by techniques such as electrophoresis using these gels and membranes, and crosslinking agents useful in their preparation. The invention also relates to polymer gels of interest in areas that include bio-compatible applications such as prosthetic devices and optical and eye lenses.
The invention is especially suitable for electrophoretic applications and accordingly, for convenience, the invention will be further described with reference to electrophoresis. It is to be understood, however, that the gels, membranes, processes and crosslinking agents of the present invention are not so limited.
BACKGROUND OF THE INVENTION
Polyacrylamide gels used for electrophoresis are conventionally prepared by the copolymerization of the acrylamide (AAm) with methylene-bis-acrylamide (BIS) as the crosslinker (Scheme 1).
Such polyacrylamide gels have a number of limitations in electrophoretic applications, such as a limited porosity range
1
, high background silver staining
2
, low resistance to hydrolysis when stored in alkaline media, and restriction to concentration of the gel solution due to the lack of clarity.
Since both double bonds of BIS are of the same type, their reactivities are essentially the same. We have previously found an improvement in the separation of polyacrylamide gels when they are prepared using particular asymmetrical crosslinking agents (see PCT/AU97/00437, the disclosure of which is incorporated herein by reference).
DISCLOSURE OF THE INVENTION
We have now discovered that using multifunctional crosslinkers, that is crosslinkers having at least three crosslinkable functional groups, gives some unexpected improved properties. These improvements include:
1) Greater control when designing gels with a different pore size range;
2) Control of pore size distribution;
3) Greater resistance to hydrolysis in alkaline media;
4) Greater clarity of gels prepared with high concentration of the crosslinkers;
5) Reduced background after silver staining.
Accordingly, in one aspect, the present invention provides a crosslinked polymer gel formed from a least one monomer and at least one crosslinker having at least three crosslinkable functional groups, wherein at least one of the crosslinkable functional groups is an acryloyl or methacryloyl group as hereinafter defined.
The said at least three crosslinkable functional groups of the crosslinker may be the same or different.
The crosslinker may be a linear, branched or cyclic compound. Preferably all functional groups of the crosslinker have an ethylenic double bond. More preferably, the crosslinker has at least three acryloyl functional or methacryloyl groups or a combination thereof. Preferably, each acryloyl or methacryloyl group is attached to a nitrogen or oxygen atom.
The crosslinked polymer gel may be formed in the presence of one or more conventional crosslinker(s).
The monomer or monomers used to prepare the gel may be any suitable monomer. The gel may be formed from two or more different monomers.
The crosslinked polymer gel may be prepared from one or more monomers having the formula H
2
C=CR
5
—CO—NR
3
R
4
where R
3
, R
4
are each independently H, alkyl, alcohol (—(CH
2
)
a
—OH), or ester (—(CH
2
)
a
—OCH
3
), where n is 1-6, and R
5
is H or optionally substituted alkyl. Examples of monomers include acrylamide, acrylamide derivatives or acrylamide substitutes known to the art such as N,N-dimethylacrylamide, methacrylamide, N-methyloylacrylamide, propylacrylamide, dipropyl acrylamide, isopropyl acrylamide, diisopropyl acrylamide, lactyl acrylamide, methoxyacrylamide and mixtures thereof. Preferably the, or at least one of, the monomer(s) is acrylamide.
In a preferred form of the invention, the crosslinker used in the crosslinked polymer gel of the invention is a compound selected from Formula I and/or Formula II
wherein, in Formula I:
C represents a ring structure of the crosslinker molecule which is connected with at least 3 functional groups —Y—CZC(R)═CH
2
which functional groups may be the same or different;
Y in each functional group may be the same or different and selected from a single bond, N, O or S;
Z in each functional group may be the same or different and selected from O or S; or Z may be two hydrogens, a hydrogen and an optionally substituted alkyl, or two optionally substituted alkyl groups; and
R in each functional group may be the same or different and selected from hydrogen or substituted or unsubstituted alkyl, preferably H or CH
3
.
Ring C may be a 3 to 12-membered carboxyclic or heterocyclic ring. Preferably C is a six-membered heterocyclic ring. The heteroatom)s) in the heterocyclic ring may be independently selected from N, O or S. Examples of suitable ring structures include heterocyclic amines and oxides. Y in each functional group may be the same or different and selected from N, O or S when it is connected to a carbon atom that is part of the ring system. Y may be a single bond if the functional group is connected to a ring nitrogen.
Preferably, each functional group is connected to the ring C through a heteroatom. Preferably the heteroatom is N. The heteroatom may be a ring atom or a heteroatom of a ring C substituent.
Ring C may be a heterocyclic nitrogen-containing ring, for example, a ring having the structure:
In Formula II:
D represents a backbone chain of the crosslinker which is connected with at lest three functional groups —Y—CzC(R)═CH
2
which functional groups may be the same or different;
Y in each functional group may be the same or different and selected from a single bond, N, O or S;
Z in each functional group may be the same or different and selected from O or S; and
R in each functional group may be the same or different and selected from hydrogen or substituted or unsubstituted alkyl, preferably H or CH
3
.
The backbone chain of the compound of Formula II may be linear, branched or cyclic. The backbone may optionally be substituted and/or optionally interrupted by one or more heteroatoms O, S, N and/or one or more aromatic, saturated or unsaturated carboxyclic or heterocyclic radicals. The backbone may be a small molecule (monomer), oligomer or polymer. Y in each functional group may be the same or different and selected from N, O or S if the backbone chain contains only carbons. Y may also be a single bond if the backbone chain contains N within the chain or contains N or O or S at the ends of the main backbone chain or a branched chain to connect with the functional groups.
Preferably, each functional group is connected to the backbone via a heteroatom. Preferably, the heteroatom is N. The heteroatom may be a heteroatom interrupting the backbone, a heteroatom at the end(s) of the backbone chain, or it may be a heteroatom of a branching group of the backbone chain.
The backbone chain may be a relatively small molecule of sufficient length to allow substitution of 3 to about 6 crosslinkable functional groups.
The backbone chain may be a linear, branched or cyclic oligomer having approximately 3-20 repeat units, which may be the same or different. Examples of suitable oligomer backbones are polyalkylene imine oligomers (eg polyethylene imine oligomers) and polyalkylene oxides oligomers.
The backbone chain of the compound of formula II may be a linear, branched or cyclic polymer having a length up to about one million atoms. The polymer may be a polyalkylene imine (eg polyethylene imine) or polyalkylene oxide. The polymer may have a degree of substitution of up to 100% of the functional group.
The term “acryloyl” as used herein denotes the group CH
2
═CH—CO— and;
The term “methacryloyl” as used herein denotes the group CH
2
═C(R)—CO—, where R is optionally substituted alkyl, preferably C1-C4 alkyl, more preferably CH
3
.
In this specification the term “optional
Patras Georgia
Qiao Greg GuangHua
Solomon David Henry
Baker & McKenzie
Noguerola Alex
University of Melbourne
Warden Jill
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