Magnus Glad

Use of silane monomers for molecular imprinting and enzyme entrapment in polysiloxane-coated porous silica

Abstract The use of organic silane monomers in the preparation of substrate-selective polymers by molecular imprinting is described. Silanes are allowed to polymerize on the surface of porous silica particles in aqueous solution. The resulting polysiloxane copolymer becomes covalently anchored to silanol groups of the original silica. such preparations retain the rigidity of the silica matrix and can therefore be used in high-performance liquid chromatography. Polysiloxane copolymers imprinted with the dyes rhodanile blue or safranine O showed preferential binding of the respective compound. The observed recognition is believed to occur because cavities containing specific binding groups for the dyes at defined positions are developed during the polymerization procedure. In this context the synthesis of a new silane, boronatesilane, was carried out. This compound was included in the monomer mixture used for the preparation of a polysiloxane-coated silica showing affinity for the glycoprotein transferrin. Organic silanes were also used for entrapment of enzymes, resulting in block polymers, which after fragmentation yielded relatively high recoveries of enzyme activity. Alternatively, the entrapment/polymerization was allowed to proceed on the surface of porous silica, in analogy with the imprinting procedure, resulting in entrapped enzyme preparations with high mechanical stability.

High-performance liquid affinity chromatography of nucleosides, nucleotides and carbohydrates with boronic acid-substituted microparticulate silica

High-Performance Liquid Affinity Chromatography of Nucleosides, Nucleotides and Carbonhydrates with Boronic Acid-Substituted Microparticulate Silica

High-Performance Liquid Affinity Chromatography of Proteins on Cibacron Blue F3G-A Bonded Silica

Abstract The reactive triazine dye, Cibacron Blue F3G-A, has been covalently attached to microparticulate porous silica and used for the resolution of a number of complementary proteins by high-performance liquid affinity chromatography (HPLAC). Cibacron Blue F3G-A was converted to its 6-aminohexyl derivative by reaction with 1,6-diaminohexane and coupled to γ-glycidoxypropyltrimethoxysilane-activated porous silica (5 μm) to generate an adsorbent containing 5.5–6.7 μmol dye/g silica. Cibacron Blue F3G-A silica columns, in conjunction with on-line monitoring of protein concentration and enzyme activity, may be exploited for the high speed fully automated resolution of dehydrogenases, isoenzymes, kinases and other proteins such as pancreatic ribonuclease A from simple or complex mixtures. The examples demonstrate the versatility of HPLAC on silica-immobilised Cibacron Blue F3G-A.

Acrylic polymer preparations containing recognition sites obtained by imprinting with substrates

Abstract Results from the “impriting” of various molecules in highly cross-linked synthetic polymers are described. In the procedure followed, polymerization of different acrylic monomers around a “substrate” was carried out. The bulk polymer so formed was then crushed to particles of size 300–500 μm, and the substrate was eluted. A number of dyes, notably rhodanile blue and safranine O, were used in the impriting procedure, and the polymer particles were tested for their recognition properties in column chromatography. It was found that polymers imprinted with rhodanile blue and safranine O showed preferential binding for rhodanile blue and safranine O, respectively. It is believed that the observed selective recognition occurs because cavities are formed that resemble the original substrate in size and have groups at fixed sites within them that allow non-covalent binding with the respective complementary groups of the original substrate. An alternative procedure for the preparation of substrate-selective polymers was subsequently developed. In this procedure a thin shell of acrylic polymer was formed under similar impriting conditions onto microparticulate porous silica carrying acrylate groups. Analysis of these composite particles under high-performance liquid chromatographic conditions showed similar recognition patterns but could be accomplished more rapidly.

Molecularly imprinted composite polymers based on trimethylolpropane trimethacrylate (TRIM) particles for efficient enantiomeric separations

Abstract A convenient method to prepare spherical microparticulate composite adsorbents is described. Molecularly imprinted crosslinked methacrylate polymers were copolymerized with preformed microparticulate support particles of trimethylolpropane trimethacrylate (TRIM) containing residual double bonds. The resulting composite adsorbent could be packed in HPLC columns after just a simple washing procedure and was used for efficient chiral separation of amino acid derivatives. The pore system of the TRIM-particles could be filled with large amounts of functional polymer. The enantioselectivity for the print molecule Boc-L-Phe-OH was in the same range as that obtained by traditional bulk phase molecular imprinting, while the performance in HPLC was improved, both in terms of efficiency and pressure stability. The effect of various degrees of crosslinking and of monomer concentration on polymer characteristics, such as porosity and specific surface area, and its impact on the structure of the final polymer is discussed as well as the chromatographic performance of the resulting matrices.

Boronic acid—silica: a new tool for the purification of catecholic compounds on-line with reversed-phase high-performance liquid chromatography

Abstract The properties of silica-immobilized benzeneboronic acids are discussed both in terms of coating of the silica and efficiency of these particles for use in a high-performance liquid chromatography column. The silica derivative displays an affinity for catecholic compounds, whose content in biological samples may be estimated with a column-switching system using boronic acid silica on-line followed by a reversed-phase chromatographic system. The dual system is suitable for direct injection of crude biological samples, and at least 1 ml of sample can be injected without additional band broadening.

High performance liquid affinity chromatography: a new tool in biotechnology

In high performance liquid affinity chromatography (HPLAC), the biospecificity of affinity chromatography is combined with the robust support materials of HPLC. HPLAC can provide specificity, speed and high resolution in chromatography both on analytical and preparative scales. The number of commercially available HPLAC matrices is an indicator of the growing use of the technique.

New methods for separation and recovery of biomolecules

New methods and applications in the separation of biomolecules are reviewed, with an emphasis on the large-scale recovery of proteins. Highlights include the advent of flow-through particles in perfusion chromatography, which allows for very high flow rates, while retaining a high chromatographic efficiency.