WO2003087170A1

WO2003087170A1 - Polyacrylamide hydrogels

Info

Publication number: WO2003087170A1
Application number: PCT/AU2003/000431
Authority: WO
Inventors: David Henry Solomon; Greg Guanghua Qiao; Marcus Julian Caulfield; Xiaojuang Hao
Original assignee: Gradipore Limited
Priority date: 2002-04-10
Filing date: 2003-04-10
Publication date: 2003-10-23
Also published as: AUPS166702A0

Abstract

A polyacrylamide gel network formed from an acrylamide-based cross-linker, the gel network being treated with a chemical scavenger such that the gel is substantially free of labile precursor structures and the treated gel network is not substantially capable of releasing acrylamide monomers under normal handling conditions.

Description

POLYACRYLAMIDE HYDROGELS

Technical Field

The present invention relates to chemically stable polyacrylamide gels and membranes formed from such gels.

Background Art

Polyacrylamides (PAAm) find wide use as water purification flocculants, as soil conditioning agents, as hydrogels including contact lenses, and in many biomedical applications. In particular, gels or membranes made from polyacrylamides have been extensively used in recent years for protein separations.

Polyacrylamide is generally, but not universally accepted as being non-toxic. However, acrylamide monomer is carcinogenic. Thus the level of acrylamide monomer in commercial polymers has been an important issue particularly for applications where human contact is involved. For example, polyacrylamide used as a water purification agent has strict specifications on the amount of monomer that is allowed. Similarly, polyacrylamide membranes used in blood purification require no detectable monomer or have undergone techniques for removing residual monomer.

Polyacrylamide (PAAm) gel networks are formed in an aqueous medium and are widely used as electrophoresis gels (Hames, B.D., Rickwood, D., (Eds), Gel Electrophoresis of Proteins; A practical Approach, second edition, Oxford University

Press, Oxford 1994) for protein separations (Horvath, Z.S.; Orthals, G.L.; Wrigley, C.W.; Margolis, J. Electrophoresis, 1994, 15, 968) or as membranes for protein isolations or blood purifications. Because these hydrogels are used in some applications where human contact is involved, the products are required to be either non-toxic and/or biocompatible. Although acrylamide monomer is toxic, it is generally, but not universally accepted that polyacrylamide is non-toxic. Consequently, purification of polyacrylamide gels by removing residual monomers and then keeping gels in a stable form becomes an important issue.

Recently, it has been suggested in reports that an additional but vitally important concern is the possibility of degradation of polyacrylamide to acrylamide. Such reports have been challenged but the question remains as to whether or not polyacrylamide can release acrylamide monomer, and if so, the extent of such degradation processes. In polyacrylamide network formation, the polymer is covalently cross-linked by using acrylamide (AAm) monomer and at least one vinyl type cross-linker. The present inventors have studied the stability of a number of PAAm's prepared with different initiation systems, when heated, when exposed to irradiation of fluorescent light (indoor laboratory condition), when exposed to ultraviolet irradiation (outdoor condition) and when kept at room temperature. There have not been any previous studies on the degradation behaviour of these cross-linked gels. The purification of polyacrylamide gels prepared with various cross-linkers was investigated and gel behaviour under different handling conditions is described. These results have been used to rationalize the conflicting reports in the literature and produce gels being substantially free of acrylamide monomer, more stable to degradation, and having improved separation performance. Furthermore and importantly, a treatment system has been devised which removes labile precursor structures in gels which can lead to AAm monomer being released during use of untreated gels.

Disclosure of Invention

The present invention provides a polyacrylamide gel network formed utilizing an acrylamide-based cross-linker, wherein the gel is substantially free of labile precursor structures capable of releasing acrylamide monomers in use. The gels according to the present invention have been modified such that the gels substantially lack the ability to release acrylamide during normal handling conditions. Furthermore, it has been found that the gels according to the present invention are more stable to handling conditions, have better separation performance in membrane-based electrophoresis, and are more chemically inert with regard to unwanted interaction with many proteins or other compounds.

In a first aspect, the present invention provides a polyacrylamide gel network ' formed utilizing an acrylamide-based cross-linker, the gel network being treated with a chemical scavenger such that the gel is substantially free of labile precursor structures and the treated gel network is substantially not capable of releasing acrylamide monomers under normal handling conditions.

The term "normal handling conditions" is used herein to include hot aqueous environments, usually less than about 95°C, preferably less than about 75°C, or storage and handling under a laboratory or factory environment. The term "labile precursor structures" is used herein to include structures having unsaturated double bonds of residual acrylamide or any pendant double bonds from cross-linkers in the gel network.

Preferably, the gel network is prepared by cross-linking one or more monomers selected from suitable monomers which are, without this being an exhaustive list, hydroxyl-substituted lower alkyl acrylates and methacrylates, methacrylamide, (loweralkyl)acrylamides and -methacrylamides, ethoxylated acrylates and methacrylates, hydroxyl-substituted (lower alkyl)acrylamides and -methacrylamides, hydroxyl-substituted lower alkylvinyl ethers, sodium vinyisulfonate, sodium styrenesulfonate, 2-acrylamido-2- methylpropanesulfonic acid, N-vinylpyrrole, N-vinyl-2-pyrrolidone, 2-vinyloxazoline, 2- vinyl-4,4'-dialkyloxazolin-5-one, 2- and 4-vinylpyridine, vinylically unsaturated carboxylic acids having a total of 3 to 5 carbon atoms, amino(lower alkyl)- (where the term "amino" also includes quaternary ammonium), mono(lower alkylamino)(lower alkyl) and di(loweralkylamino)(lower alkyl) acrylates and methacrylates, allyl alcohol and the like. Preference is given, for example, to N-vinyl-2-pyrrolidone, acrylamide, methacrylamide, hydroxyl-substituted lower alkyl acrylates and methacrylates, hydroxy-substituted (loweralkyl)acrylamides and -methacrylamides, or mixtures thereof. More preferably, the one or more monomers are selected from acrylamide or methacrylamide or substituted derivatives of acrylamide or methacrylamide. The gel network is prepared using cross-linking agents containing or comprising acrylamide residues. Examples of suitable cross-linkers include N,N'- methylenebisacrylamide (Bis), triacryloyl-tris(2-aminoethyl)amine, trimethacryloyl-tris(2- aminoethyl)amine, tetraalcryloyl triethylene tetramine, tetramethacryloyl triethylene tetramine, tetra acryloyl triethylene tetramine, and tetra methacryloyl triethylene tetramine, mixtures of two or more thereof. These compounds are described in

Examples 2, 3, 4, and 5 from WO 00/56792 by the present inventors, incorporated herein by reference. More preferably, the cross-linker is selected from Bis, triacryloyl-tris(2- aminoethyl)amine, or trimethacryloyl-tris(2-aminoethyl)amine.

The polyacrylamide gels according to the present invention are typically cross- linked acrylamide formed by treating acrylamide with a cross-linking agent, usually N,N'- methylenebisacrylamide (Bis) under suitable initiating conditions. Although Bis is the cross-linker of choice for most standard gel-forming processes, a number of other cross- linking agents have been developed or are being developed. Examples of cross-linking agents are described above and can be found in Examples 2, 3, 4, and 5 from WO 00/56792, incorporated herein by reference. The polymerization can be carried out by any free-radical initiating system including Redox, thermal, photoactivation including UV initiated systems. Particularly preferred systems are UV initiated and Redox initiated using e.g. ammonium persulphate (APS) and TEMED (/v,/v,Λ/',Λ/' tetramethyethylene diamine). More preferably, the polyacrylamide gel network is formed by cross-linking a monomer system of acrylamide with N,N'-methylenebisacrylamide (Bis).

Preferably, the gel has undergone treatment with a chemical scavenger to react with any unsaturated double bonds of residual acrylamide or any pendant double bonds from cross-linkers in the gel network. The chemical scavengers are used to react with pendent double bond to either saturate the double bond or react with it to form small removable molecules. Examples of these scavengers include but not limited to ammonia, ammonia salts, alkylamine or hydroxylamine or their salts (that react with double bond and contains nitrogen); hydrogen halides, acetoacetate, malonate (that contains an active nitrogen); bromine, bromosuccinimide, pyridum bromide or dioxane perbromide (that contain bromine); permanganates, bichromates, chromates, selenium dioxide, ozone, or hydrogen peroxide (that oxidize the double bonds); alkali sulfites, disulfites, alkali or ammonium hydrogen sulphur or thio compounds (that contain sulphur).

In another preferred form, the chemical scavenger is an oxidation agent, which can oxidize residual acrylamide or pending double bonds to water-soluble small molecules which can then be removed by washing, preferably washing with water.

One of preferred oxidation agents is potassium permanganate which can oxidize any residual acrylamide or pending double bonds to water-soluble formic acids which can then be removed by washing with water. Possible mechanism of this oxidation reaction is shown below:

=o + Mn0₄ + H₂0 )=o + NH₃ + Mn0₂ + C0₂ + KOH (1 )

H,N HO

Mn0₂ + C0₂ + KOH (2)

Another preferred oxidation agent is hydrogen peroxide. Pendent double bond on polyacrylamide gels can be saturated by addition of hydroxyl groups as shown below:

The use of chemical scavengers can not only remove or saturate the pendent double bonds that can be the precursor of acrylamide; it will also provide a process to control the hydrophilicity of the membrane gels. For example, brominated membrane will be more hydrophobic, due to the addition of bromine atoms on the gel and this is reflected by the move of proteins to be separated by the membranes. The membrane after oxidation will cause the gel to be more hydrophilic. Depending on the degree of oxidation, hydrophilicity is controlled. For example, a mild oxidation such as with hydrogen peroxide will convert the pendent double bonds to glycol moity that is hydrophilic. Further oxidation will break the double bond and form amine groups and in some other case carboxylic acid groups. The process will produce a much more hydrophilic surface of the gel.

In another preferred form, the chemical scavenger is a halogen, preferably bromine.

In another preferred form, the chemical scavenger is selected from halogens or other compounds capable of adding across the double bond. Examples include, but not limited to, sodium sulphite, ammonia or amines.

Preferably, the gel network does not release acrylamide monomer under storage conditions of less than about 95°C for at least about 120 days.

Substantially free from acrylamide monomers is defined herein as having less than about 1 ppb (parts per billion). The gels according to the present invention can be prepared having various T and C ratios. Importantly, the T and C ratio does not seem to have any undue influence on the treatment process to remove unwanted labile precursor structures. The term T is used to indicate the total amount of monomer in the solution and C represents the % cross-linker based on T. The polyacrylamide gels according to the present invention are suitable for many uses including electrophoresis media, separation of bio-molecules for human or animal use, and in particular where long term contact with body fluids is encountered.

In a second aspect, the present invention provides a method for forming a polyacrylamide gel network substantially free of acrylamide monomers or potential sources of acrylamide monomers, the method comprising treating a polyacrylamide gel with a chemical scavenger to react with any unsaturated double bonds of residual acrylamide or any pendant double bonds from cross-linkers in the gel network which could be precursors for the release of acrylamide. In one preferred form, the method comprises: reacting an acrylamide monomer and a cross-linking agent containing acrylamide residues under suitable conditions to form a polyacrylamide gel network; and treating the gel with a chemical scavenger to react with any unsaturated double bonds of residual acrylamide or any pendant double bonds from cross-linkers in the gel network.

In a preferred form, the method includes the following step after the reacting step: removing unreacted monomer and/or cross-linking agent.

This step can be achieved by washing or rinsing in a suitable liquid such as water. This intermediate step is useful to assist in subsequent efficient reaction of any unsaturated double bonds of residual acrylamide or any pendant double bonds from cross-linkers in the gel network.

Substantially free from acrylamide monomers is defined herein as having less than about 1 ppb.

The gels according to the present invention can be prepared with having various T and C ratios. Importantly, the T and C ratio does not seem to have any undue influence on the treatment process to remove unwanted labile precursor structures. Examples include sodium sulphite, ammonia or amines. In one preferred method form, treating step is carried out by treating the gel with bromine for sufficient time to ensure that substantially all unsaturated double bonds of residual acrylamide or any pendant double bonds from cross-linkers in the gel network have been reacted. Treating the gel with a saturated solution of bromine until no further bromine reacts with the gel has been found to be particularly suitable. Excess bromine can then be removed by adding a few drops of Na₂S₂O₃ solution and then washing the gel if required. Time of treatment may vary from minutes to hours, depending on the gel components, conditions of polymerization, and the like. The method according to the present invention is applicable to any polyacrylamide gel system where free acrylamide may be present after the polymerization process or potential sources of acrylamide monomer are present. Examples include but are not limited to membranes-based electrophoresis, blood purification or renal dialysis units. In a third aspect, the present invention provides a polyacrylamide gel network produced by the method according to the second aspect of the present invention.

In a fourth aspect, the present invention provides an electrophoretic membrane including a polyacrylamide gel network in accordance with the present invention formed on a porous substrate. The substrate supplies the support frame for the electrophoretic medium. The substrate may be a porous paper or fabric. The substrate may be woven or non-woven sheet, for example, a non-woven PET.

Preferably, the membrane has a defined pore size and pore size distribution.

The polyacrylamide gel network according to the present invention is suitable for use in applications where there maybe concerns regarding the presence of potentially toxic amounts of free acrylamide. Uses include but not limited to blood purification, protein electrophoresis and renal dialysis.

Throughout this specification, unless the context requires otherwise, the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps.

Any discussion of documents, acts, materials, devices, articles or the like which has been included in the present specification is solely for the purpose of providing a context for the present invention. It is not to be taken as an admission that any or all of these matters form part of the prior art base or were common general knowledge in the field relevant to the present invention as it existed in Australia before the priority date of each claim of this application. In order that the present invention may be more clearly understood, preferred forms will be described with reference to the following drawings and examples.

Brief Description of the Drawings Figure 1. AAm released from an AAm/BIS gel (Redox initiation) under thermal degradation at 95°C.

Figure 2. AAm released from an AAm/BIS gel (APS initiation at 60°C) under thermal degradation at 95°C.

Figure 3. AAm released from mAAm/BIS gel (APS initiation) under thermal degradation at 95°C.

Figure 4. AAm released from an AAm/BIS gel (initiated by a thermal method with APS at 60°C) under UV irradiation at 254 nm.

Figure 5. AAm released from an AAm/mBIS gel (initiated by a thermal method with APS at 60°C) under UV irradiation at 254 nm. Figure 6. AAm released from a mAAm/BIS gel (initiated by a thermal method with APS at 60°C) under UV irradiation at 254 nm.

Figure 7. AAm released from a non-brominated AAm/PIP gel (initiated by a redox method with TEMET/APS at room temperature) under UV irradiation at 254 nm.

Figure 8. AAm released from AAm/BIS gels, initiated by a thermal APS and redox APS, under UV irradiation at 254 nm

Mode(s) for Carrying Out the Invention EXPERIMENTAL AND RESULTS Materials Electrophoresis-grade (>98%) acrylamide (AAm) and methacrylamide (mAAm) were purchased from ICN Biomedicals Inc and Aldrich Chemical Co. Crosslinkers, N,N'- methylenebisacrylamide (BIS) and Λ/,Λ/-methylenebismethacrylamide (mBIS), were purchased from BDH Laboratory Supplies and Polyscience Inc, respectively. Bisacryloyl-piperazine (PIP) was obtained from Lancaster Synthesis. Ammonium persulfate (APS, >98.8) was obtained from Sigma Chemical Co, H₂O₂ (AR grade, 30% w/v) from AJAX Chemicals, and Λ/,Λ/,Λ/',Λ/'-tetramethylethylene-diamine (TEMED, >99.5%) was from Aldrich. Bromine (AR grade) was obtained from FSE Pty Ltd, and sodium thiosulphate (>99.5%) was purchased from AJAX Chemicals. Phenol red solution was from Merck Pty. Ltd.

Saturated bromine water was made by shaking Milli Q water with bromine and then standing the mixture overnight at 4°C. The aqueous phase was used.

Sodium thiosulphate was used as 1 M solution.

Glassware

All glassware was washed with tap water, rinsed with distilled water, and then heated in an oven at 450°C overnight to remove any organic residues.

Polymerization methods

APS/TEMED redox initiated polymerization

AAm/BIS and AAm/PIP gels were obtained by redox initiated polymerization. Monomer solutions (20%T/5%C) were prepared in distilled water, where %T (w/w) refers to the concentration of total monomer in the solution and %C (w/w) refers to the concentration of cross-linking monomer in total monomers. The monomer solutions were purged with high purity argon until the content of oxygen was below 1 %. The initiators, APS and TEMED in their 10% (w/w) solutions (2% and 1% (w/w) in total monomers, respectively), were then added into the monomer solution. Polymerization was allowed to proceed overnight at room temperature.

APS thermally-initiated polymerization

The compositions of monomers, initiators and cross-linkers for all gels of AAm/BIS, mAAm/BIS, and AAm/mBIS (see Scheme 1) with thermally initiated polymerization using APS are shown in Table 1. Table t Compositions of monomers, cross-linkers and initiators

* 2000%, T of mAAm/BIS was not soluble.

Monomers, crosslinkers and initiators were initially dissolved in distilled water in a 100 ml round bottom flask. The freeze-thaw degassing technique was used to remove oxygen in the solution. Then the flask containing the monomer mixture was immersed in an oil bath at 60°C overnight to allow the polymerization to take place. The flask remained sealed throughout the entire process of polymerization.

AAm BIS

mAAm mBIS

PIP Scheme 1 Molecular structure of monomers and cross-linkers Purification methods for polyacrylamide gels

In carrying out tests, it was important to ensure that all gels had no detectable acrylamide in the gel suspension before degradation studies. Therefore we initially investigated methods to purify the polyacrylamide gels.

Washing

Once the polymerization was finished, the gel was collected and ground. The ground gel was then washed by rinsing with distilled water through a glass-sintered filter (size no 3). As acrylamide is highly soluble in water and gel is insoluble, rinsing with water allowed selective removal of acrylamide from the gel. After physical washing with water (at least 10 times) the gel was then washed using methanol (twice) to replace the water in the gel network, and then dried in vacuo below 50°C overnight.

Bromination A further purification procedure was performed in order to remove trace amount of residual AAm in gels. In this method, bromine (Br₂) was used to react with the double bond of residual acrylamide. It also reacted with the pendant double bonds from the cross-linkers in the network.

The previously washed and dried gel (2 g) was soaked in Milli-Q water (100 ml) and stirred. The saturated bromine water was then added to the solution until it became brown indicating the presence of excess bromine. The solution was allowed to stand for a few minutes. The solution turned from brownish to colorless, indicating that all bromine had been reacted. More bromine water was then added. This step was repeated until there was no color change in the solution. Normally, a total reaction time of at least overnight was needed for this bromination. Excess bromine was then removed by a few drops of Na₂S₂O₃ solution (1 M). The gel was then washed again with distilled water for 15 times and finally rinsed with methanol twice.

With an AAm/BIS gel, repeated cold water washing could remove monomer residues to a level where acrylamide was undetectable. However, it was found that if the gel was washed by hot water (85°C), acrylamide kept leaching out due to unreacted double bonds in the gel matrix. Acrylamide detection (HPLC method)

Acrylamide was detected by HPLC with UV detector at λ=196 nm. Sample solution (50 μl) was injected and delivered through a 3 x 4 mm reverse-phase 5 μm C18 guard column and a reverse-phase 5 μm Aqua column (C18125A, Phenomenex) with size of 250 x 4.6 mm. Filtered Milli-Q water was used as mobile phase and all HPLC samples were made up in water solution (1% (w/w)) using the same filtered Milli-Q water. The minimum sensitivity of detectable AAm level on an Aqua column was determined to be 1 part per billion (ppb).

Degradation methods

Thermal degradation at 95 °C

Gel/water mixtures (10 ml, 1% (w/w)) were prepared using filtered Milli-Q water. Duplicates were made for each gel. No AAm was present in the solutions as measured by HPLC. Thermal degradation of gel mixtures was carried out in an oven at 95°C. Each

10 ml volumetric flask was covered with a glass vial to minimize water vapour escaping during heating at 95°C. When necessary, water was added at regular intervals to prevent the gels from drying out.

Before taking samples; water was added to each volumetric flask to ensure a total solution volume of 10 ml, and then the mixture was mixed and allowed to stand for at least 4 hours. This was intended to allow all the gel to settle to the bottom of the flask so that samples taken from the top did not contain any fine gel particles. The amount of sample taken was approximately 0.5 ml.

AAm/BIS gel

Thermal degradation was performed on AAm/BIS gels. These gels were either washed without treatment with bromine (non-brominated), or washed and treated with bromine for 1 hour (1 hour-brominated) or overnight (overnight-brominated). The AAm/BIS gels were prepared using APS and APS/TEMED as initiators and the polymerization was carried out at 60°C and room temperature, respectively. The number of AAm molecules released per 10⁶ polymer repeat units in the gel from these samples was plotted against degradation time (Figure 1 and 2). AAm/mBIS gel and AAm/PIP gel

Polyacrylamide gels crosslinked with either mBIS or PIP were synthesized and were subject to similar thermal degradation.

For the AAm/mBIS gel, no AAm or mAAm was detected in the degradation solution after 7 days. A separate experiment on the degradation of a 10 ppm mBIS solution under the same conditions showed the release of mAAm. Similarly the brominated AAm/mBis gels did not liberate AAm or mAAm.

For the AAm/PIP gel, no AAm or acrylic acid (an expected hydrolysis product) was observed from the degradation solution. A separate experiment on the degradation of 10 ppm PIP solution under the same conditions showed PIP was stable and did not release any acrylic acid. This may be because the cyclic diamide ring is more stable and does not hydrolyze easily under the degradation conditions of this study. In this respect, the PIP amide can be regarded as a tertiary amide and these are known to be more stable hydrolytically than secondary or primary amides.

mAAm/BIS gel

The formation of AAm (in ppm of polymer repeat units) with degradation time for mAAm/BIS gel is shown in Figure 3. Similar to AAm/BIS gel, the thermal degradation on mAAm/BIS gel resulted in the formation of AAm. Additionally, brominated mAAm/BIS gel gave less AAm compared to the non-brominated gel.

The amount of AAm formed during thermal degradation of a mAAm/BIS gel was approxmately 10 times greater (Figure 5) than the amount of AAm formed from an AAm/BIS gel. This indicates there are many more pendant double bonds in the mAAm/BIS gel.

Determination of hydrolysis content of gels after hot aqueous treatment at 95° C

The hydrolysis content of polyacrylamide gels from thermal degradation was determined by titration. Firstly, the gel suspension was acidified by adding a small amount of HCI solution (2.5 M) and the pH value of the solution was adjusted to around 2. The gel was then filtered, rinsed with distilled water (10 x) and methanol (2 x), and then dried in vacuo overnight before titration. Gel suspension (1 % (w/w), 8-10 ml) was then made up in water and NaOH solution (0.0100 ± 0.0006M) used to titrate the acid content of the gel. Phenol red indicator was used to show the end point (pH = 7.9). From the volume of NaOH solution at the end point the amount of acid groups, formed by hydrolysis of amide groups in the polymer chain was calculated. These results are shown in Table 2.

UV degradation when irradiated at 254 nm

Gel/water mixtures (10 ml, 1% (w/w)) were prepared in 28 ml sample vials (75 x 25 mm), covered with plastic film, which allows UV light to go through the gel solutions and minimize water loss. Filtered Milli-Q water was used to make up the solution. Similar to the thermal degradation experiments, samples were taken from each gel before degradation to ensure no AAm was present in solutions.

Sample vials containing gel mixtures were placed in a black box, where the UV irradiation (wavelength of 254 nm) was from the top of the box. Samples (about 0.5 ml) were taken, and 50 μl was injected directly into the HPLC column to determine the AAm level of the solution. The same amount of Milli-Q water was added back to the solution after sampling in order to keep the volume constant.

Polyacrylamide gels were made with different monomer and cross-linker compositions using different initiation methods. These gels were non-brominated, brominated for one hour or brominated overnight. Using gels thermally initiated with APS at 60°C a comparison of the gel degradation behaviour with different monomer and cross-linker compositions was studied. The effect of initiation methods for polymerization was also investigated with the AAm/BIS gels.

Effect of monomer/crosslinker compositions

After an AAm/BIS gel was purified by washing, a 1 % (w/w) gel suspension was subject to UV irradiation at λ=254 nm. AAm monomer was released to the solution. The amount of AAm increased with the time of irradiation before levelling out at about 4 ppm polymer repeat units after 15 days (Figure 4). When partially brominated AAm/BIS gel was irradiated under the same conditions, an increased amount of the AAm monomer was observed. A completely brominated gel gave much more AAm in the solution. The maximum amount of AAm in the solution after 15 day was approxmately 50ppm polymer repeat units.

When an AAm/mBIS gel, thermally-initiated by APS, was used under the same irradiation condition, similar AAm release was observed. As shown in Figure 5, we also observed the increase of AAm when the gel was brominated.

In contrast to the previous two cases, although the 1 hr brominated mAAm/BIS gel showed an increase in AAm release, an overnight brominated gel caused reduction of the AAm release from the polymer (Figure 6). In Figure 7, it is shown that AAm/PIP gel initiated by TEMED/APS redox system also gives AAm under UV degradation. The non-brominated AAm/PIP gel gave much higher levels of AAm than the AAm/mBIS or AAm/BIS gels.

Effect of initiation methods

An AAm/BIS gel was also polymerized by using an APS/TEMED redox initiation system at room temperature. The resultant gels, both non-brominated and overnight- brominated, were irradiated at 254 nm. The comparisons of these gels' degradation behaviour to the one initiated by APS only are shown in Figure 8. The overnight-brominated gel released more AAm than the non-brominated gel, which is similar to the results for APS initiated gels observed previously. Gel made by APS only gave much more AAm than the gel made by a redox method. This difference . is particularly enhanced when these gels were brominated.

Stability under fluorescent light ^■

Gel/water mixtures (10 ml, 1 % (w/w)) in 28 ml sample vials (75 x 25 mm), covered with plastic film, were subject to continuous irradiation of fluorescent light at 30 cm. Samples (about 0.5 ml solution) were taken and 50 μl injected directly into the HPLC to determine the AAm level of the solution. No AAm was detected in a period of 15 days. Similar results were observed for a range of monomer/crosslinker compositions such as AAm/BIS, AAm/mBIS, AAm/PIP or mAAm/BIS gels.

Oxidation treatment of membrane with potassium permanganate

A 20T/5C AAm/Bis membrane was merged in milli-Q water and potassium permanganate (1 mg/l gel solution) was added. The solution was allowed to react for 4 hours before the membrane was washed with milli-Q water three times. Upon analysis, the gels did not release AAm.

Oxidation treatment of membrane with hydrogen peroxide A 20T/5C AAm/Bis membrane was merged in milli-Q water and hydrogen peroxide (about 1 ppm in the gel solution) was added. The solution was allowed to react for 4 hours before the membrane was washed with milli-Q water three times. Upon analysis, the gels did not release AAm.

Separation Properties of Brominated Gels The following experiments clearly demonstrate that the separation properties of the brominated gels are superior to the non-treated gels.

Experiment 1: Synthesis of isolation membrane (AAm/BIS 20T/5C)

AAm/BIS stock solutions were made up at 40T/10C and 40T/0C concentrations. The reaction solution was made by mixing 62.5 ml of each stock solution and then making up to 250 ml with distilled water. The monomer solution was degassed with argon until oxygen content was below 1 %. An aliquot (0.5 ml) TEMED solution (10% w/v) was added into the solution at this stage, followed by adding .0 ml APS solution (10% w/v). The mixture was poured into a glass tank and membranes were made in the tank by putting PET sheets between glass plates and leaving reaction for a few hours. Once the reaction was completed the membranes were washed with distilled water a few times and then cut into small pieces. Small pieces of membranes were further washed in distilled water until no AAm was detectable in the water by HPLC measurement. The membranes, which we describe as non-brominated membranes, were now ready for protein isolation. Some membranes were put in another box and treated with saturated bromine water overnight. These treated membranes were washed in distilled water many times to get rid of impurities, to give the brominated membranes, which, were ready for protein isolation.

Experiment 2: Protein isolation by membrane-based electrophoresis

Non-brominated and brominated membranes were used for protein isolation to compare properties of these two kinds of membranes. A membrane-based electrophoresis apparatus (Gradiflow™ system produced by Gradipore Limited) was used to test the efficiency of the polyacrylamide hydrogel membranes. Bovine serum albumin (BSA) was used for the test. The operation conditions were: 2 mg/ml BSA, 40 mM TG buffer, 200V, 500 mA, 10 ml. Samples were taken at 0, 10, 20, 30 min from both stream 1 (sample side of membrane) and stream 2 (product side of membrane). Experiment 3: electrophoresis analysis of membrane separation iGels™ having 4-20% polyacrylamide gradients (produced by Gradipore Limited) were used for running PAGE analysis of samples. Each gel has provision for running up to 10 samples in 10 separate sample wells. An aliquot (10 μL) of each sample from electrophoresis runs outlined above was injected in a well. Each PAGE gel can run 10 samples at a time. The operation conditions were set at 200V, 500 mA, 1.5 hour. After 10, 20, and 30 minutes, the BSA proteins transferred through brominated membrane significantly more than non-brominated membrane. That means brominated membrane gave a faster transfer rate of BSA protein.

DISCUSSION

Purification of gels

The need for careful purification of linear PAAm has been studied previously and a similar situation applies with the gel structures. However, there are distinct and vitally important differences between purifying the soluble linear polymers and the hydrogel networks.

Thus whilst it was difficult to remove AAm from linear PAAm by precipitation, the insoluble nature of the gels made for easy removal of AAm by simple cold water washing procedures. The use of a hot water (85°C) wash, designed to speed-up the washing process, gave an unexpected but nevertheless, very important result; AAm monomer was released from the hydrogel. This AAm comes from BIS residues in which only one double bond has been incorporated into the network. In other words there are pendant structures which are the precursors for the release of AAm. As with the linear PAAm, purification with a chemical scavenger, bromine, is also possible. But with the gels there is the added complication that pendant unsaturation can also be brominated. And unlike the brominated AAm which can be removed by washing, the brominated Bis units remain in the gel structure.

Because chemical scavenging is recommended for the commercial purification of PAAm, we have compared systems in which removal of AAm has been by simple washing or by washing followed by various levels of bromine or other chemical scavenging. Stability under laboratory conditions

Gels produced according to the present invention showed no sign of hydrolysis or of the release of AAm after 60 days exposure in an aqueous environment to continuous irradiation by laboratory fluorescent lights at room temperature. Similar results were obtained for the range of gels, AAm/mBIS, AAm/PIP, mAAm/BIS and for AAm/BIS gels at a range of T and C values.

Thus the gels are stable under these conditions.

Degradation in hot aqueous suspension of AAm/BIS gels (thermal degradation) It was observed from these experiments that non-brominated gels released AAm into the solution during the first 10 days and then the AAm level reached a plateau. The amount of AAm released was within 10 ppm of repeat units. When 1 hour-brominated gel was used, the amount of AAm released was substantially reduced (Figure 1 and 2). With an ovemight-brominated gel (APS as initiator), no AAm monomer was observed from the polymer solution during the whole degradation period of 13 days. These results suggest that residual double bond of BIS is the precursor to the released AAm.

Evidence for pendant AAm units in BIS gels

Firstly we noted that the amount of AAm released decreases eventually to zero as the extent of bromination increased.

Secondly, by heating BIS under our thermal conditions a low yield of AAm results. When a 10 ppm BIS solution was heated at 95°C, AAm formation was observed. This indicates that BIS itself can hydrolyze and form AAm. Therefore, we would expect that a pendant double bond of BIS in the gel network would undergo a similar degradation. BIS can be synthesized by the condensation reaction of N-methylol acrylamide and AAm with the elimination of water. Hydrolysis of BIS is the reverse of this reaction.

In AAm/BIS gels, AAm may be released either by the scission of the main polymer chain or detachment of pendant double bonds in BIS. During bromination, the pendant double bonds in the cross-linkers would react with bromine. If the bromination (or other chemical scavenger treatment) is complete, all pendant double bonds will be reacted. The above results suggest that the AAm monomer released from the polymer network comes from the pendant double bonds being hydrolyzed to AAm monomer. Therefore, partially brominated (1 hour bromination) gels caused the reduction of the AAm released and completely brominated (overnight bromination) gels (APS as initiator) did not give any AAm under thermal degradation at 95°C. Supporting evidence for this theory is the absence of AAm from linear PAAm, confirming that it is not the result of backbone scission. In the brominated BIS gel we could not identify hydrolysis products which might be expected, the dibromo propionamide and α-bromo acrylamide (amide). It is possible that the brominated product is more resistant to hydrolysis. Alternatively bromination may alter the pathway of hydrolysis and lead to the unstable N-methylol bromoacrylamide. A possible reason is that the electron withdrawing bromine atoms alter the hydrolytic fission as shown in Scheme 2.

Scheme 2

Thirdly, in AAm/BIS gels there is a correlation between the amount of BIS and the level of AAm released.

Fourthly, changing the crosslinker from BIS to mBIS or PIP results in no AAm being released, confirming that AAm was released from the crosslinker and not the backbone. These gels are discussed below in further detail. Finally, changing to a mAAm/BIS gel still gave AAm, confirming that AAm is not from the backbone. AAm/mBIS and AAm/PIP gels (thermal degradation)

If AAm were released from pendant double bonds of an AAm/BIS gel, changing the type of the crosslinker should prevent the formation of AAm during the thermal degradation. Replacement of the BIS crosslinker with either mBIS or PIP gave gels that did not release AAm. This is further evidence that the polyacrylamide backbone in the gel is stable under the thermal degradation condition and does not release any AAm.

PIP was stable when heated at 95°C for 3 days and hence we conclude that hydrolysis of pendant PIP units is unlikely.

mAAm/BIS gel (thermal degradation)

Again, since BIS crosslinker was used in this gel, AAm formation would be expected. When the amount of pendant double bonds in BIS was reduced by bromination, less AAm formation was observed under thermal degradation, and no AAm was observed with completely brominated gel (ovemight-brominated). This is further evidence that the release of AAm from gels under thermal degradation comes from the pendant double bond of the crosslinker.

The mAAm/BIS gel release more AAm than the AAm/BIS gel and this observation can be explained by the reactivity of mAAm. The mAAm is more reactive than AAm, while AAm has similar reactivity to BIS. Hence, this influences the reaction path and the final structure of the gels. During polymerization of AAm/BIS gels, since AAm and BIS have similar reactivity, BIS is statistically incorporated to the network. However, in the formation of mAAm/BIS gel, mAAm is more reactive than BIS and BIS will incorporate into the network later in comparison to the AAm/BIS case. Therefore, the chance for the second double bond of the BIS in mAAm/BIS gel being reacted is reduced. This will result in more pendant double bonds in the mAAm/BIS gels.

From the thermal degradation results of the gels with different monomer compositions we can conclude that the carbon-carbon backbone of polyacrylamide gels is quite stable under our thermal condition (95°C). However, the BIS crosslinker can result in unreacted pendant double bonds that can degrade to AAm. Once these pendant double bonds are eliminated by bromination, no AAm is released. Hydrolysis of amide side chain

Polyacryamide gels showed less hydrolysis content in comparison with linear polyacryamide, as shown in Table 2. The gel of mAAm/BIS hydrolysed to a very small extent upon heating, which reflects the known greater stability of methacrylic to acrylic amides/esters. The gels initiated by APS usually produced more acid groups upon heating than ones initiated by redox system. In addition, the AAm/BIS gel with higher C gave less hydrolysis than the one with lower C (Table 2).

It will be appreciated by persons skilled in the art that numerous variations and/or modifications may be made to the invention as shown in the specific embodiments without departing from the spirit or scope of the invention as broadly described. The present embodiments are, therefore, to be considered in all respects as illustrative and not restrictive.

Claims

Claims:

1. A polyacrylamide gel network formed utilizing an acrylamide-based cross-linker, the gel network being treated with a chemical scavenger such that the gel is substantially free of labile precursor structures and the treated gel network is not substantially capable of releasing acrylamide monomers under normal handling conditions.

2. The gel network according to claim 1 wherein the normal handling conditions include hot aqueous environments less than about 95°C, preferably less than about 75°C, or storage and handling under a laboratory or factory environment.

3. The gel network according to claim 1 or 2 wherein the labile precursor structures include structures having unsaturated double bonds of residual acrylamide or any pendant double bonds from cross-linkers in the gel network.

4. The gel network according to any one of claims 1 to 3 wherein the gel network is prepared by cross-linking one or more monomers selected from the group consisting of hydroxyl-substituted lower alkyl acrylates and methacrylates, methacrylamide, (loweralkyl)acrylamides and -methacrylamides, ethoxylated acrylates and methacrylates, hydroxyl-substituted (lower alkyl)acrylamides and -methacrylamides, hydroxyl-substituted lower alkylvinyl ethers, sodium vinyisulfonate, sodium styrenesulfonate, 2-acrylamido-2-methylpropanesulfonic acid, N-vinylpyrrole, N-vinyl- 2-pyrrolidone, 2-vinyloxazoline, 2-vinyl-4,4'-dialkyloxazolin-5-one, 2- and 4- vinylpyridine, vinylically unsaturated carboxylic acids having a total of 3 to 5 carbon atoms, amino(lower alkyl)- (where the term "amino" also includes quaternary ammonium), mono(lower alkylamino)(lower alkyl) and di(loweralkylamino)(lower alkyl) acrylates and methacrylates, allyl alcohol and mixtures thereof.

5. The gel network according to claim 4 wherein the monomer is selected the group consisting of N-vinyl-2-pyrrolidone, acrylamide, methacrylamide, hydroxyl-substituted lower alkyl acrylates and methacrylates, hydroxy-substituted (loweralkyl)acrylamides and -methacrylamides, and mixtures thereof.

6. The gel network according to claim 5 wherein the monomer is selected from the group consisting of acrylamide, methacrylamide, substituted derivatives of acrylamide and substituted derivatives of methacrylamide.

7. The gel network according to any one of claims 1 to 6 wherein the gel network is prepared using cross-linking agents containing or comprising acrylamide residues.

8. The gel network according to claim 7 wherein the cross-linking agents are selected from the group consisting of N,N'-methylenebisacrylamide, triacryloy l-tris (2- aminoethyl)amine, trimethacryloyl-tris(2-aminoethyl)amine, tetraalcryloyl triethylene tetramine, tetramethacryloyl triethylene tetramine, tetra acryloyl triethylene tetramine, tetra methacryloyl triethylene tetramine, and mixtures of two or more thereof.

9. The gel network according to claim 8 wherein the cross-linking agents are selected from the group consisting of N,N'-methylenebisacrylamide, triacryloyl-tris(2- aminoethyl)amine, and trimethacryloyl-tris(2-aminoethyl)amine.

10. The gel network according to any one of claims 1 to 9 wherein polymerization is carried out by any free-radical initiating system including Redox, thermal, photoactivation including UV initiated systems.

11. The gel network according to claim 10 wherein the Redox initiated system comprises ammonium persulphate (APS) and TEMED (Λ/,Λ/,Λ/',Λ/'tetramethyethylene diamine).

12. The gel network according to claim 11 formed by cross-linking a monomer system of acrylamide with N,N'-methylenebisacrylamide.

13. The gel network according to any one of claims 1 to 12 wherein the chemical scavenger reacts with any unsaturated double bonds of residual acrylamide or any pendant double bonds from cross-linkers in the gel network.

14. The gel network according to claim 13 wherein the chemical scavenger reacts with pendent double bond to saturate the double bond or reacts with the double bond to form small removable molecules.

15. The gel network according to claim 14 wherein the chemical scavenger is selected from the group consisting of ammonia, ammonia salts, alkylamine or hydroxylamine or their salts (that react with double bond and contains nitrogen); hydrogen halides, acetoacetate, malonate (that contains an active nitrogen); bromine, bromosuccinimide, pyridum bromide or dioxane perbromide (that contain bromine); permanganates, bichromates, chromates, selenium dioxide, ozone, or hydrogen peroxide (that oxidize the double bonds); alkali sulfites, disulfites, alkali or ammonium hydrogen sulphur or thio compounds (that contain sulphur), and combinations thereof.

16. The gel network according to claim 15 wherein the chemical scavenger is a halogen, preferably bromine.

17. The gel network according to claim 14 wherein the chemical scavenger is an oxidation agent which can oxidize residual acrylamide or pending double bonds to water-soluble small molecules which can then be removed by washing with water.

18. The gel network according to claim 17 wherein the chemical scavenger is potassium permanganate which can oxidize residual acrylamide or pending double bonds to water-soluble formic acids which can then be removed by washing with water.

19. The gel network according to claim 17 wherein the chemical scavenger is hydrogen peroxide.

20. The gel network according to any one of claims 1 to 19 wherein the gel network does not release acrylamide monomer under storage conditions of less than about 95°C for at least about 120 days.

21. A method for forming a polyacrylamide gel network substantially free of acrylamide monomers or potential sources of acrylamide monomers under normal handling conditions, the method comprising treating a polyacrylamide gel with a chemical scavenger to react with any unsaturated double bonds of residual acrylamide or any pendant double bonds from cross-linkers in the gel network which could be precursors for the release of acrylamide.

22. The method according to claim 21 comprising: reacting an acrylamide monomer and a cross-linking agent containing acrylamide residues under suitable conditions to form a polyacrylamide gel network; and treating the gel network with a chemical scavenger to react with any unsaturated double bonds of residual acrylamide or any pendant double bonds from cross-linkers in the gel network.

23. The method according to claim 22 further comprising prior to the treating step: removing any unreacted monomer or cross-linking agent.

24. The method according to claim 23 comprising washing or rinsing the gel network in a suitable liquid such as water.

25. The method according to any one of claims 21 to 24 wherein the gel network is prepared by cross-linking one or more monomers selected from the group consisting of hydroxyl-substituted lower alkyl acrylates and methacrylates, methacrylamide,

(loweralkyl)acrylamides and -methacrylamides, ethoxylated acrylates and methacrylates, hydroxyl-substituted (lower alkyl)acrylamides and -methacrylamides, hydroxyl-substituted lower alkylvinyl ethers, sodium vinyisulfonate, sodium styrenesulfonate, 2-acrylamido-2-methylpropanesulfonic acid, N-vinylpyrrole, N-vinyl- 2-pyrrolidone, 2-vinyloxazoline, 2-vinyl-4,4'-dialkyloxazoIin-5-one, 2- and 4- vinylpyridine, vinylically unsaturated carboxylic acids having a total of 3 to 5 carbon atoms, amino(lower alkyl)- (where the term "amino" also includes quaternary ammonium), mono(lower alkylamino)(lower alkyl) and di(loweralkylamino)(lower alkyl) acrylates and methacrylates, allyl alcohol and mixtures thereof together with a cross-linking agent selected from the group consisting of Bis, triacryloyl-tris(2- aminoethyl)amine, trimethacryloyl-tris(2-aminoethyl)amine, tetraalcryloyl triethylene tetramine, tetramethacryloyl triethylene tetramine, tetra acryloyl triethylene tetramine, tetra methacryloyl triethylene tetramine and polyethylene acrylates, and mixtures of two or more thereof.

26. The method according to any one of claims 21 to 25 wherein polymerization is carried out by any free-radical initiating system including Redox, thermal, photoactivation including UV initiated systems.

27. The method according to any one of claims 21 to 26 wherein the chemical scavenger is selected from the group consisting of ammonia, ammonia salts, alkylamine or hydroxylamine or their salts (that react with double bond and contains nitrogen); hydrogen halides, acetoacetate, malonate (that contains an active nitrogen); bromine, bromosuccinimide, pyridum bromide or dioxane perbromide (that contain bromine); permanganates, bichromates, chromates, selenium dioxide, ozone, or hydrogen peroxide (that oxidize the double bonds); alkali sulfites, disulfites, alkali or ammonium hydrogen sulphur or thio compounds (that contain sulphur), and combinations thereof.

28. The method according to claim 27 wherein the chemical scavenger is a halogen, preferably bromine.

29. The method according to claim 27 wherein the chemical scavenger is an oxidation agent selected from potassium permanganate or hydrogen peroxide.

30. The method according to any one of claims 21 to 29 wherein the gel network does not release acrylamide monomer under storage conditions of less than about 95°C for at least about 120 days.

31. The method according to any one of claims 21 to 30 wherein the gel network has less than about 1 part per billion (ppb) free acrylamide.

32. A polyacrylamide gel network produced by the method according to any one of claims 21 to 31.

33. An electrophoretic membrane including a polyacrylamide gel network according to any one of claims 1 to 20 or claim 32 formed on a porous substrate.

34. The membrane according to claim 33 wherein the substrate supplies support for the electrophoretic medium and is selected from a porous paper, porous fabric, porous plastics, woven or non-woven sheet.

35. The membrane according to any one of claims 33 to 35 having a defined pore size and pore size distribution.