No abstract

A new method is described for producing highly porous polyacrylamide matrices: polymerization in presence of a preformed hydrophilic polymer. If a standard mixture of monomers (e.g., 5%T, 4%C) is polymerized in presence of, e.g., polyethylene glycol (PEG) 10 kDa, lateral chain aggregation occurs, with formation of large pore sizes. In PEG 10 kDa, the transition from a small- to a large-pore gel is clearly apparent at 0.5% PEG addition and reaches a plateau already at 2.5% PEG. Even with shorter PEG fragments (6.2 and 1 kDa) this transition occurs, but with progressively larger amounts of PEG in solution (up to 25% for the 1 kDa species). Other polymers such as hydroxymethyl cellulose (1000 kDa) and polyvinyl-pyrrolidone (360 kDa and 25 kDa) are also able to elicit this phenomenon. It appears that lateral chain aggregation (before the cross-linking event) is induced via intra-chain hydrogen bonding, since urea and temperature strongly inhibit it, whereas tetramethylurea (an agent quenching hydrophobic interactions) does not hamper it. By scanning electron microscope, it is found that the maximum pore size obtained in a 5%T, 4%C gel in presence of 2.5% PEG 10 kDA is of the order of 0.5 micron, whereas the same 5%T, 4%C control gel would have an average pore diameter of 5 nm. Thus, an increment of pore size of about 2 orders of magnitude is obtained: in these new matrices, a 21000 bp DNA fragment exhibits a much greater migration than in a control gel in which the sample is entrapped at the application site.

Photopolymerization of polyacrylamide gels in the presence of methylene blue (100 microM) and a redox couple (1 mM sodium toluenesulfinate, a reducer, and 50 microM diphenyliodonium chloride, an oxidizer) has been investigated. The gel point, i.e. the time needed for onset of gelation upon illumination, has been found to lengthen progressively at lower temperatures and at lower light intensities. If the three catalysts are progressively diluted, the gel point does not vary for a threefold dilution, but gelation is greatly hampered below a 1:5 dilution of the three effectors. Photobleaching has been assessed as a function of liquid layer thickness (from 0.5 to 2 mm), of a progressive dye dilution (down to a fourfold dilution) and as a function of temperature. A maximum of elastic modulus is located in correspondence to a minimum of permeability (both situated at 5% cross-linker). It is found that methylene blue-activated polymerization produces polyacrylamide gels with elastic properties which are higher than in persulfate-activated gels, so far the most popular matrices for electrokinetic separations. Due to the ease of preparation, the full control of all experimental parameters, and the lack of oxidizing power of this catalyst system (as opposed to the strong oxidation power of persulfate catalysis), methylene blue catalysis is advocated as a valid alternative to other redox systems.

The conversion efficiency of 5 different catalyst systems for polyacrylamide gel polymerization has been assessed as a function of pH of the gelling solution, in the pH 4.0-10.0 range. The system of persulfate-N,N,N',N'-tetramethylethylene-diamine (TEMED) gives optimal incorporation of monomers only in the pH 7.0-10.0 range; at progressively acidic pH values, the conversion drops markedly until, at pH 4.0, no gelation occurs. In the system of riboflavin-TEMED, the opposite behavior is observed: good conversions in the pH 4.0-7.0 interval (with a peak at pH 6.2), followed by progressive worsening at alkaline pH values until, at pH 10.0, no gelation occurs. An excellent catalyst system appears to be the complex comprising methylene blue, toluene-4-sulfinic acid and diphenyliodonium chloride, which is able to guarantee > 95% conversion in the pH 4.0-8.0 range. Lower efficiencies are obtained at pH 9.0 and 10.0 (down to 81% conversion) but gelation still occurs in the entire pH range. Two other systems (ascorbic acid, ferrous sulfate and hydrogen peroxide in one case, and persulfate, TEMED and hydrosulfite, in another case) have been investigated. The former has some efficiency only at pH 4 (81% conversion), with a rapid drop as pH increases (only 48% incorporation at pH 6). The latter exhibits the opposite behavior: 70% efficiency at pH 4.0, increasing to 92% conversion at pH 6.0.

The kinetics of monomer incorporation into a polyacrylamide gel have been studied in a photopolymerization system comprising 100 microM methylene blue in presence of a red-ox system, 1 mM sodium toluenesulfinate (reducer) and 50 microM diphenyliodonium chloride (oxidizer). A precise assessment of gel point (pc) was obtained in a droplet chamber, in which argon was gently bubbled with a fused silica capillary into the reaction mixture. At pc, 50% (+/- 3) acrylamide was incorporated into the matrix, vs. 80% (+/- 4) N,N'-methylenebisacrylamide. This incorporation level remained the same when polymerized in the 2-36 degrees C temperature range. Incorporation continued almost linearly for acrylamide up to 80% conversion. The reaction was continued up to 55 min (at 2 degrees C), at which point bisacrylamide had been essentially consumed (> 99.5% incorporation) and acrylamide had reacted (95%). At 2 degrees C, after gelation, the gel became progressively turbid (the Tyndall effect plateauing at 50 min), but it remained fully transparent if, at the gel point, reaction was continued at 50 degrees C. The consumption of the pendant double bonds of Bis followed the progression of turbidity. It is concluded that, by gelation at 2 degrees C, the nascent chains form clusters held together by hydrogen bonds (melting point at 28 degrees C); such clusters are subsequently "frozen" in the three-dimensional space as the pendant double bonds in the chains react progressively. Such turbid matrices are more porous and less elastic than when the gel is polymerized at 50 degrees C. This process is similar to the "lateral aggregation" occurring when gels are formed in presence of a polymer in solution (e.g. 10 KDa polyethylene glycol; Righetti et al., Electrophoresis 1992, 13, 587-594).