muramidase and ethylene-dimethacrylate

A porous zwitterionic monolithic column was prepared to rapidly and efficiently separate lysozyme from egg white. The monolith was synthesized in a stainless steel HPLC column (5 cm × 4.6 mm i.d.) by in-situ thermal initiated co-polymerization of N,N-dimethyl-N-methacryloxyethyl-N-(3-sulfopropyl) ammonium betaine (MSA) and ethylene dimethacrylate (EDMA). Due to the combination of quaternary ammonium and sulfonic groups on the monolithic matrix in one-pot process, the hydrophobic carbon chain and hydrophilic radical were obtained, which provided multiple driving forces for neutral, basic and acidic analytes, thus mix-mode chromatography mechanism contributed to the retention of different charged proteins. Properties such as composition, morphology and stability of the MSA-co-EDMA monolithic column were characterized by various analytical methods and the results showed that the monolith has large through-pores, good hydrophilicity and permeability. The effects of mobile phase pH and ionic strength on proteins were investigated, drawing the conclusion that the main adsorption and elution mechanism of lysozyme on MSA-co-EDMA monolith was electrostatic interaction, while those of other proteins included hydrophobic, hydrophilic and electrostatic interactions. Therefore, efficient separation of lysozyme and other proteins could be successfully achieved by switching the pH of mobile phase. Lysozyme can be adsorbed using 20 mmol/L phosphate buffer (pH 7.0) and eluted with 20 mmol/L phosphate buffer (pH 2.0). To prove the practicality of the monolithic column, it was also applied in the separation of lysozyme in egg white, which means the work has the potential for further development in proteome analysis of real biological samples.

Topics: Betaine; Egg White; Hydrogen-Ion Concentration; Hydrophobic and Hydrophilic Interactions; Methacrylates; Muramidase; Nucleosides; Permeability; Phenols; Polymerization; Polymers; Porosity; Static Electricity

Since sequential injection chromatography (SIC) emerged in 2003, it has been used for separation of small molecules in diverse samples, but separations of high molar mass compounds such as proteins have not yet been described. In the present work a poly(glycidyl methacrylate-co-ethylene dimethacrylate) (GMA-co-EDMA) monolithic column was prepared by free radical polymerization inside a 2.1-mm-i.d. activated fused silica-lined stainless steel tubing and modified with iminodiacetic acid (IDA). The column was prepared from a mixture of 24% GMA, 16% EDMA, 20% cyclohexanol, and 40% 1-dodecanol (all% as w/w) containing 1% of azobisisobutyronitrile (AIBN) (in relation to monomers). Polymerization was done at 60 °C for 24 h. The polymer was modified with 1.0 M IDA (in 2 M Na2CO3, pH 10.5) at 80 °C for 16 h. Separation of myoglobin, ribonuclease A, cytochrome C, and lysozyme was achieved at pH 7.0 (20 mM KH2PO4/K2HPO4) using a salt gradient (NaCl). Myoglobin was not retained, and the other proteins were separated by a gradient of NaCl created inside the holding coil (4 m of 0.8-mm-i.d. PTFE tubing) by sequential aspiration of 750 and 700 μL of 0.2 and 0.1 M NaCl, respectively. As the flow was reversed toward the column (5 μL s(-1)) the interdispersion of these solutions created a reproducible gradient which separated the proteins in 10 min, with the following order of retention: ribonuclease A < cytochrome C < lysozyme. The elution order was consistent with a cation-exchange mechanism as the retention increased with the isoelectric points.

Topics: Animals; Cattle; Chickens; Chromatography, Ion Exchange; Cytochromes c; Epoxy Compounds; Horses; Methacrylates; Muramidase; Myoglobin; Polymers; Ribonuclease, Pancreatic

A novel reverse-phase monolithic stationary phase containing double long alkyl chains was prepared based on the thermally initiated co-polymerization of 3-methylacryloyl-3-oxapropyl-3-(N,N-dioctadecylcarbamoyl)-propionate (AOD) and ethylene glycol dimethacrylate (EDMA) in the presence of 2-methyl-1-propanol and 1,4-butanediol as the selected porogens. The polymerization was carefully optimized and good permeability, stability and column efficiency were observed for the final monolithic columns. The column also showed good long term stability and reproducibility. The methylene selectivity demonstrates typical reversed phase characteristics. The optimized poly (AOD-co-EDMA) monolith exhibited good selectivity for a range of non-polar test analytes such as PAHs, tocopherols and alkylphenones. A good separation of intact proteins was also observed.

Topics: Animals; Chickens; Chromatography, High Pressure Liquid; Chromatography, Reverse-Phase; Cytochromes c; Ethylene Glycols; Methacrylates; Microscopy, Electron, Scanning; Muramidase; Polycyclic Aromatic Hydrocarbons; Reproducibility of Results; Ribonucleases; Tocopherols

A butyl methacrylate-co-ethylene dimethacrylate (BuMA-co-EDMA) monolith was synthesized by UV initiated polymerization at the inlet end of a 75 microm I.D. fused silica capillary that had been previously coated with a protein compatible polymer, poly(vinyl)alcohol. The monolith was used for on-line preconcentration of proteins followed by capillary electrophoresis (CE) separation. For the analysis of standard proteins (cytochrome c, lysozyme and trypsinogen A) this system proved reproducible. The run-to-run %RSD values for migration time and corrected peak area were less than 5%, which is typical of CE. As measured by frontal analysis using lysozyme as solute, saturation of a 1cm monolith was reached after loading 48 ng of protein. Finally, the BuMA-co-EDMA monolithic preconcentrator was coupled to a protein G monolithic column via a zero dead volume union. The coupled system was used for on-line removal of IgG, preconcentration of standard proteins and CE separation. This system could be a valuable sample preparation tool for the analysis of low abundance proteins in complex samples such as human serum, in which high abundance proteins, e.g., human serum albumin (HSA) and immunoglobulin G (IgG), hinder identification and quantification of low abundance proteins.

Topics: Cytochromes c; Electrophoresis, Capillary; Humans; Hydrophobic and Hydrophilic Interactions; Immunoglobulin G; Methacrylates; Microscopy, Electron, Scanning; Muramidase; Polymers; Proteins; Reproducibility of Results; Serum Albumin; Solid Phase Extraction; Trypsinogen

An affinity monolith with a novel immobilization strategy was developed leading to a tailored pore structure. Hereby the ligand is conjugated to one of the monomers of the polymerization mixture prior to polymerization. After the polymerization, a monolithic structure was obtained either ready to use for affinity chromatography or ready for coupling of additional ligand to further increase the binding capacity. The model ligand, a peptide directed against lysozyme, was conjugated to glycidyl methacrylate prior to the polymerization. With this conjugate, glycidyl methacrylate, and ethylene dimethacrylate, a monolith was formed and tested with lysozyme. A better ligand presentation was achieved indicated by the higher affinity constant compared to a conventional sorbent.

Topics: Chromatography, Affinity; Cross-Linking Reagents; Epoxy Compounds; Ligands; Methacrylates; Muramidase; Peptides; Polymers

A continuous rod of porous poly(glycidyl methacrylate-co-ethylene dimethacrylate) has been prepared by a free radical polymerization within the confines of a 300 x 8 mm I.D. chromatographic column. The epoxide groups of the rod have been modified by a reaction with diethylamine that affords ionizable functionalities required for the ion-exchange chromatographic mode. The properties of this rod column have been characterized and the column has been used successfully for the chromatographic separation of proteins. The column exhibits a dynamic capacity that exceeds 300 mg at a flow velocity of 200 cm/min. An excellent selectivity allows the separation of up to 300 mg of a protein mixture in a single run.

Topics: Animals; Chickens; Chromatography, High Pressure Liquid; Chromatography, Ion Exchange; Conalbumin; Epoxy Compounds; Methacrylates; Muramidase; Ovalbumin; Proteins; Serum Albumin, Bovine; Trypsin Inhibitors

A nonionic, high-water-content, high-strength hydrogel based on N-vinyl pyrrolidinone (NVP) and a novel hydrophilic-bulky monomer, 4-t-butyl-2-hydroxycyclohexylmethacrylate (TBCM), was developed. The TBCM was prepared in three relatively simple, high-yield steps. The copolymerization of NVP with varying concentrations of TBCM resulted in transparent hydrogel films possessing a wide range in mechanical and physical properties. A copolymer composition of 91.7 parts NVP, 8.0 parts TBCM, and 0.3 parts ethylene glycol dimethacrylate (EGDMA) gave a transparent, flexible film possessing a water content of 86%, a modulus of 130 g/mm2, and a tear strength of 3.4 g/mm. In contrast, a copolymer composition of 49.7 parts NVP, 50 parts TBCM, and 0.3 parts EGDMA gave a transparent, hard hydrogel film possessing a water content of 26% and a modulus of 86,000 g/mm2. All of the high-water copolymer compositions developed resulted in lysozyme uptake typical of nonionic high-water-content hydrogels and oxygen permeability levels greater than 50 (cm3-O2(STP).cm)/(s.cm2.mm Hg) x 10(-11).

Topics: Adsorption; Hydrogel, Polyethylene Glycol Dimethacrylate; Methacrylates; Muramidase; Oxygen; Permeability; Polyethylene Glycols; Pyrrolidinones; Spectrophotometry, Ultraviolet