J.V. McLaren

Synthesis and characterization of poly(acryloyl morpholine) xerogel networks suitable for the gel permeation chromatography of small molecules

Abstract The synthesis and characterization of a crosslinked poly(acryloyl morpholine) network, Enzacryl® Gel KO, calculated to be effective as a matrix for the g.p.c. of small molecules, is described. Chromatographic performance was evaluated in water, chloroform, dimethylformamide and pyridine. Characteristic g.p.c. behaviour was observed for all standards tested although ‘additional exclusion’ was observed in the case of aliphatic hydrocarbons in the three organic solvents and with aromatic hydrocarbons in chloroform and pyridine. Similar molecular weight fractionation ranges were obtained for poly(ethylene glycols) in the four solvents investigated the molecular weight exclusion limit being less than 1500.

Water-insolubilisation of glycoside hydrolases with cross-linked poly(acryloylaminoacetaldehyde dimethyl acetal) (Enzacryl Polyacetal).

Abstract The preparation of water-insoluble derivatives of alpha-amylase and dextranase by chemical binding with a polyaldehyde derived from cross-linked poly(acryloyl-aminoacetaldehyde dimethyl acetal) (Enzacryl Polyacetal) is described. The water-insoluble derivatives could be used repeatedly against their respective substrates. Bound alpha-amylase and at least a proportion of the bound dextranase molecules were more stable to heat denaturation than are the corresponding free enzymes.

Enzacryl® gel packings: Inorganic salt compatibility, pH stability and ion-exchange derivatives

Abstract The elution behaviour of simple inorganic salts on Enzacryl® gel K1 was found to depend on both radius of the hydrated anion and electrostatic interaction. An approximately linear, inverse relationship was found to exist between the distribution coefficient, K ds , measured under conditions of static equilibrium, and the charge transfer to solvent absorption maximum, ν max. , for the anions studied. Transition metal cations were strongly adsorbed but could be eluted with sodium chloride solution. At 25°, Enzacryl gel was shown to be stable to 1 M HCl and the pendant acylmorpholino residues were only slowly hydrolysed by 1 M NaOH. Hydrolysis of Enzacryl gel K2 with 1 M NaOH at 100° gave a cation exchanger, Enzacryl-CO 2 H. An anion exchanger Enzacryl-DEAE was obtained by reaction of Enzacryl gel K2 with N,N-diethylethylene diamine in boiling ethylene glycol. The efficacy of Enzacryl-CO 2 H and Enzacryl-DEAE as packings for ion-exchange chromatography has been demonstrated.

Characterisation of cross-linked poly(acryloylmorpholines) as matrices for aqueous gel permeation chromatography

The characterisation of two cross-linked poly(acryloylmorpholines), Enzacry Gel K1 and Enzacryl Gel K2, as matrices for aqueous gel permeation chromatography is described. Near ideal plots of logarithm molecular weight versus distribution coefficient, Kd, are obtained for polyethylene glycols and linear, oligomeric α,ω-diols approximate molecular weight (Mn) fractionation ranges being 0–4000 and 0–20,000 for Enzacryl Gel K1 and Enzacryl Gel K2, respectively. Anomalous retardation of the Schardinger dextrins, cyclomaltohexose and cyclomaltoheptose, is observed although linear maltosaccharides behave normally. The internal gel volumes, calculated from column elution data in water, are significantly larger than the volumes of solvent imbibed by the dry column packings on constituting the gel. Internal gel volumes and solvent imbibition volumes in water are compared with the corresponding values obtained in chloroform and tetrahydrofuran. The two parameters are discussed, in the case of Enzacryl Gel K2 in water and chloroform, in the light of plots of logarithm viscometric hydrodynamic volume versus Kd for polyethylene glycols.

Crosslinked poly(acryloylaminoacetaldehyde dimethylacetal) and derived urease conjugates

Abstract The synthesis of crosslinked poly(acryloylaminoacetaldehyde dimethylacetal) is described. Controlled hydrolysis of this material with aqueous acid, led to polyaldehydrol gels, two of which were used to immobilize urease. With the more expanded urease derivative a modified substrate concentration-activity profile was observed in which saturation kinetics were evident between 50mM and 500mM urea concentration. This conjugate exhibited greater stability during continuous use in perfused columns, on storage in aqueous buffer suspension, to removal of substrate and to temperature change. The strategy of synthesizing reactive polyaldehydrol gels, their mode of binding enzymes and the mechanism of substrate inhibition in polyaldehydrol-urease conjugates are discussed.

Water-insoluble polyacrylamide-thermolysin conjugates.

Abstract 1. 1.|Water-insoluble conjugates of thermolysin have been prepared by chemical binding to two cross-linked copolymers of acrylamide. For one copolymer, binding was effected by diazo coupling at both pH 7.5 and 10.0. For the other, acid azide binding was employed. 2. 2.|Practical activity retention following insolubilisation has been measured using furacryloylglycyl- l -leucinamide (FAGLA) and casein as substrates. 3. 3.|With FAGLA as substrate, extremely large decreases in both Km and V were observed for the water-insoluble conjugates. 4. 4.|The water-insoluble conjugates were more resistant to heat denaturation than the native enzyme.

Gel permeation chromatography of metallocenes

Abstract The elution behaviour of ferrocene, ruthenocene and a series of substituted ferrocenes on a gel permeation chromatography support, cross-linked poly- (acryloyl morpholine) (Enzacryl® Gel), has been investigated. For most of the solutes an approximately linear relationship was obtained between logarithm molecular weight and the Wheaton Baumann absolute distribution coefficient (Kd), indicating that the separations were effected via a molecular sieving mechanism.

Poly(N-acryloyl-l-prolyl morpholine): a g.p.c. packing exhibiting novel temperature dependence in solute distribution coefficients

Abstract The synthesis of poly (N- acryloyl- l -prolyl morpholine ) , a new bead packing for the g.p.c. of small molecules, is described. Characteristic g.p.c. behaviour and high column efficiencies were observed in water and benzyl alcohol. Novel, reversible temperature variations in solute Wheaton and Bauman distribution coefficients were observed. This effect, which is most marked in water, is caused by thermo-reversible changes in network pore size distribution in the bead packing.