Packings and stationary phases for biopolymer separations by HPLC

Summary Packings and stationary phases applied to high resolution separations of proteins, enzymes, and nucleic acids must satisfy a series of distinct criteria that are different from those usually required by HPLC of low molecular weight non-biologically active analytes. These requirements have been met through substantial improvements in classical gel media together with novel developments in silica supports, and have led to a family of products with tailor-made and reproducible properties. Supports consisting of cross-linked organic gels, and inorganic materials (mostly silicas) are now available with graduated particle sizes, pore sizes, porosities and surface areas as well as non-porous beads. A whole range of stationary phases, such as reversed phase, hydrophobic interaction, ion exchanger and affinity packings, were designed for application as chemical sensors for biopolymer recognition in adsorptive chromatography. The phase systems are operated in the gradient mode, giving high resolution and high peak capacities. In addition, aqueous liquid-liquid partitioning systems have been developed for the fractionation of proteins and nucleic acids. Size exclusion media complete the set of HPLC variants enabling a discrimination of proteins according to their size and shape in an isocratic elution mode. Basically, protein purification and isolation is a multistage process where-by the HPLC variants are combined in a logistic sequence, utilizing the different selectivities of the phase systems and thus maximising resolution, speed and throughput.     

Biocompatible polymers for antibody support on gold surfaces

The elimination or minimization of non-specific protein adsorption from serum is critical for the use of surface plasmon resonance (SPR) sensors for in vitro and in vivo analysis of complex biological solutions. The ultimate goals in this application are to minimize non-specific adsorption of protein and to maximize analyte signal. A reduction of the non-specific protein adsorption from serum of up to 73% compared to carboxymethylated-dextran 500 kDa (CM-dextran) was achieved following a survey of eight biocompatible polymers and 10 molecular weights of CM-dextran. These coatings minimize non-specific adsorption on the sensor while also serving as immobilization matrices for antibody fixation to the probes. Polymers including polysaccharides: CM-dextrans, CM-hyaluronic acid, hyaluronic acid, and alginic acid were investigated. Humic acid, polylactic acid, polyacrylic acid, orthopyridyldisuldfide–polyethyleneglycol–N-hydroxysuccinimide (OPSS–PEG–NHS), and a synthesized polymer; polymethacrylic-acid-co-vinyl-acetate (PMAVA) were also used. The non-specific protein adsorption reduction was measured over a 14 day period at 0 °C for each polymer. Calibration curves using some of these polymers were constructed to show the performance and low detection limit possibilities of these new antibody supports. For many of the polymers, this is the first demonstration of employment as an antibody support for an optical or surface active sensor. CM-dextran is the polymer offering the largest signal for the antigen detection. However, the biocompatible polymers demonstrate a greater stability to non-specific binding in serum. These biocompatible polymers offer different alternatives for CM-dextran.     

Photopolymerization of enzymatically synthesized methacrylated poly(caprolactone) with poly(ethylene glycol) macromonomer

Polycaprolactone (PCL) based α,ω-methacrylated macromonomer (DMPCL) was synthesized via enzymatic ring-opening polymerization (eROP) by using Novozyme 435 as the enzyme immobilized catalyst. DMPCL was further photopolymerized with monofunctional poly(ethylene glycol) methyl ether methacrylate (PEGMA-950) macromonomer and trimethylolpropane triacrylate (TMPTA) as tri-functionalized crosslinking agent in glass vials when CHCl₃ was the solvent and Irgacure 819 was the photoinitiator. Ultraviolet (UV) Light Emitting Diode (LED) bulbs enabled photoinduced reactions at room temperature with low heat generation and high reaction efficiency. The obtained gels were characterized with Fourier Transform Infrared Spectroscopy (FT-IR) and Differential Scanning Calorimetry (DSC). DMPCL participated as an effective crosslinking agent in the photopolymerization of PEGMA-950. Combined usage of DMPCL and PEGMA-950 resulted in significantly more effective polymerization than the separate photopolymerizations of these macromonomers.     

Separation of poly-3-hydroxyvalerate-co-3-hydroxybutyrate through gradient polymer elution chromatography

Polyhydroxyalkanoates are biodegradable polyesters produced by bacteria that can have a wide distribution in molecular weight, composition of monomers, and functionalities. This large distribution often leads to unpredictable physical properties making commercial applications challenging. To improve polymer homogeneity and obtain samples with a clear set of physical characteristics, poly-3-hydroxyvalerate-co-3-hydroxybutyrate copolymers were fractionated using gradient polymer elution chromatography (GPEC) as opposed to extensively used bulk fractionation. Separation was achieved using a reversed-phase column with chloroform and ethanol as the solvent and non-solvent, respectively. A separation was also conducted on a normal-phase column to compare elution patterns between columns of varied polarity. The fractions were analyzed using Size Exclusion Chromatography (SEC) and NMR to determine the percentage of 3-hydroxyvalerate in the copolymer as well as its molecular weight. It was found that as the percentage of "good" solvent was increased in the mobile phase, the polymers eluted with decreasing percentage of 3-hydroxyvalerate and increasing molecular weight which indicates the importance of precipitation/redissolution in the separation. The elution pattern of the polymer remained unchanged when using both a normal- and reversed-phase column which also illustrates the dominance of precipitation/redissolution in GPEC of polyhydroxyalkanoates. As such, GPEC is shown to be an excellent choice to provide polyhydroxyalkanoate samples with a narrower distribution in composition than the original bulk copolymer sample.     

Visualisation of biopolymer mixtures using confocal scanning laser microscopy (CSLM) and covalent labelling techniques

Confocal scanning laser microscopy (CSLM) has been used to study the behaviour of mixtures of proteins, gelatine, whey proteins and β-lactoglobulin, and polysaccharides, dextran, gellan gum, carrageenan, gum Arabic, and starch. CSLM proved to be a suitable technique to visualise the microstructure of these (phase separated) mixtures in two and three-dimensional images. Contrast through fluorescence is obtained either by covalent labelling (polysaccharides and proteins) or non-covalent labelling (proteins and starch). Double and triple labelling allows the visualisation of individual components in a complex mixture of biopolymers.     

分离生物大分子的液相色谱固定相

本文介绍了各类用于生物大分子分离的液相色谱填料,并对它们在生物大分子分离及制备中的性能、基本特征作了评述。     

Biopolymer-Polysiloxane Double Network Aerogels

A new series of transparent aerogels of biopolymer-polysiloxane double networks is reported. Biopolymer aerogels have attracted much attention from green and sustainable aspects but suffered from strong hydrophilicity and difficulty to make homogeneous structures in nanoscale; these drawbacks are overcome by compositing with a polysiloxane network. Alginate-polymethylsilsesquioxane aerogel has high optical transparency, water repellency, comparable superinsulation property and improved bending flexibility compared to pure polymethylsilsesquioxane aerogel. The nanoscale homogeneity is realized by separating the crosslinking steps for two networks in a sequential protocol: condensation of siloxane bonds and metal-crosslinking of biopolymer. The crosslinking order, biopolymer-first or siloxane-first, and universality/limitation of biopolymer-crosslinker pairs are discussed to construct fundamental chemistry of double network systems for their further application potentials.     

Processing Parameters Matching Effects upon Rhizobium tropici Biopolymers’ Rheological Properties

The combined effects of the processing parameters upon rheological properties of biopolymers produced by Rhizobium tropici were studied as a function of the Ca(+2) ions' concentration variation, yeast extract concentration added to the medium, aeration, and agitation, maintaining the mannitol concentration in 10 g/L. The experiments were carried out using a fermenter with 20-L capacity as a reactor. All processing parameters were monitored online. The temperature [(30 +/- 1) degrees C] and pH values (7.0) were kept constant throughout the experimental time. As a statistical tool, a complete 2(3) factorial design with central point and response surface was used to investigate the interactions between relevant variables of the fermentation process: calcium carbonate concentration, yeast extract concentration, aeration, and agitation. The processing parameter setup for reaching the maximum response for rheological propriety production was obtained when applying mannitol concentration of 10.0 g/L, calcium carbonate concentration 1.0 g/L, yeast extract concentration 1.0 g/L, aeration 1.30 vvm, and agitation 800 rpm. The viscosimetric investigation of polysaccharide solutions exposed their shear-thinning behavior and polyelectrolytic feature.     

Seawater ageing of flax/poly(lactic acid) biocomposites

Natural fibre reinforced biopolymer composites, or biocomposites, are an alternative to the glass fibre reinforced thermoset composites widely used today in marine applications. Biocomposites offer good mechanical properties and total biodegradability, but if they are to be adopted for marine structures their durability in a seawater environment must be demonstrated. In the present study unreinforced PLLA (Poly(l-Lactic acid)), injected and film stacked flax composites with the same PLLA matrix have been examined. All the samples were aged in natural seawater at different temperatures in order to accelerate hygrothermal ageing. Changes to physico-chemical and mechanical behaviour have been followed by weight measurements, thermal and gel permeation chromatography (GPC) analyses, and tensile testing, completed by acoustic emission recording and scanning electron microscopy (SEM) examination. The matrix tensile stiffness is hardly affected by seawater at temperatures to 40 °C but the composite loses stiffness and strength. Fibre/matrix interface weakening is the main damage mechanism induced by wet ageing, but both matrix and fibre cracks also appear at longer periods.