Preparation of polymeric monoliths by copolymerization of acrylate monomers with amine functionalities for anion-exchange capillary liquid chromatography of proteins

Two novel polymeric monoliths for anion-exchange capillary liquid chromatography of proteins were prepared in a single step by a simple photoinitiated copolymerization of 2-(diethylamino)ethyl methacrylate and polyethylene glycol diacrylate (PEGDA), or copolymerization of 2-(acryloyloxy)ethyl trimethylammonium chloride and PEGDA, in the presence of selected porogens. The resulting monoliths contained functionalities of diethylaminoethyl (DEAE) as a weak anion-exchanger and quaternary amine as a strong anion-exchanger, respectively. An alternative weak anion-exchange monolith with DEAE functionalities was also synthesized by chemical modification after photoinitiated copolymerization of glycidyl methacrylate (GMA) and PEGDA. Important physical and chromatographic properties of the synthesized monoliths were characterized. The dynamic binding capacities of the three monoliths (24 mg/mL, 56 mg/mL and 32 mg/mL of column volume, respectively) were comparable or superior to values that have been reported for various other monoliths. Chromatographic performance was also similar to that provided by a modified poly(GMA-ethylene glycol dimethacrylate) monolith. Separation of standard proteins was achieved under gradient elution conditions using these monolithic columns. Peak capacities of 34, 58 and 36 proteins were obtained with analysis times of 20–30 min. This work represents a successful attempt to prepare functionalized monoliths via direct copolymerization of monomers with desired functionalities. Compared to earlier publications, additional surface modifications were avoided and the PEGDA crosslinker helped to improve the biocompatibility of the monolithic backbone.     

Bilinear electric field gradient focusing

Electric field gradient focusing (EFGF) uses an electric field gradient and a hydrodynamic counter flow to simultaneously separate and focus charged analytes in a channel. Previously, most EFGF devices were designed to form a linear field gradient in the channel. However, the peak capacity obtained using a linear gradient is not much better than what can be obtained using conventional CE. Dynamic improvement of peak capacity in EFGF can be achieved by using a nonlinear gradient. Numerical simulation results indicate that the peak capacity in a 4-cm long channel can be increased from 20 to 150 when changing from a linear to convex bilinear gradient. To demonstrate the increased capacity experimentally, an EFGF device with convex bilinear gradient was fabricated from poly(ethylene glycol) (PEG)-functionalized acrylic copolymers. The desired gradient profile was confirmed by measuring the focusing positions of a standard protein for different counter flow rates at constant voltage. Dynamically controlled elution of analytes was demonstrated using a monolith-filled bilinear EFGF channel. By increasing the flow rate, stacked proteins that were ordered but not resolved after focusing in the steep gradient segment were moved into the shallow gradient segment, where the analyte peak resolution increased significantly. In this way, the nonlinear field gradient was used to realize a dynamic increase in the peak capacity of the EFGF method.     

Monolithic bed structure for capillary liquid chromatography

Monolithic stationary phases show promise for LC as a result of their good permeability, ease of preparation and broad selectivity. Inorganic silica monoliths have been extensively studied and applied for separation of small molecules. The presence of a large number of through pores and small skeletal structure allows the chromatographic efficiencies of silica monoliths to be comparable to columns packed with 5 μm silica particles, at much lower back pressure. In comparison, organic polymeric monoliths have been mostly used for separation of bio-molecules; however, recently, applications are expanding to small molecules as well. Organic monoliths with high surface areas and fused morphology rather than conventional globular morphology have shown good performance for small molecule separations. Factors such as domain size, through-pore size and mesopore size of the monolithic structures have been found to govern the efficiency of monolithic columns. The structure and performance of monolithic columns are reviewed in comparison to particle packed columns. Studying and characterizing the bed structures of organic monolithic columns can provide great insights into their performance, and aid in structure-directed synthesis of new and improved monoliths.     

A new controlled release system of chlorhexidine and chlorhexidine:βcd inclusion compounds based on porous silica

The purpose of this study was to prepare and characterize a controlled release system based on porous silica loaded with chlorhexidine (Cx) and its inclusion compounds in β-cyclodextrin (βcd), and to evaluate its antimicrobial activity. Acetate chlorhexidine (CxA), gluconate chlorhexidine (CxG), βcd:chlorhexidine acetate 2:1 (βcd:CxA) and βcd:chlorhexidine gluconate 2:1 (βcd:CxG) were incorporated into porous silica. Drug loading was characterized by FTIR, powder X-ray diffraction, thermal analysis and BET, and was shown to be in an amorphous state and porous matrix. The kinetics release parameter of the drug was established, which showed that the Cx systems release profile followed zero order release until 400 h and Higuchi model release until 750 h, after the burst effect at the first 8 h. Chlorhexidine therapeutic range was reached near first hour for all systems. The chlorhexidine porous silica system was biologically active against Enterococcus faecalis and Candida albicans in vitro. The systems showed an efficient Cx controlled release modulated by the presence of the β-cyclodextrin and by the porous silica matrices, providing effective antimicrobial activity.     

Phenylurethans of pyrazolidinols and piperidazinols as anticonvulsants

The synthesis of 1,2-diethyl-4-hydroxymethylpyrazolidine ( II ) was accomplished in two steps from 1,2-diethyl-4-ethoxycarbonylpyrazolidine ( I ). Also, 1,2-dimethyl-4-hydroxypiperidazine ( VI ) was prepared in three steps from 1,2-diethoxycarbonyl-1,2,3,6-tetrahydropyridazine ( IV ). The two alcohols were added to several aryl isocyanates and afforded fourteen new phenylurethan derivatives which showed moderate anticonvulsant activity in mice.     

Preparation and evaluation of hydrophilic C18 monolithic sorbents for enhanced polar compound retention in liquid chromatography and solid phase extraction

Hydrophilic C18 monolithic polymer sorbents were synthesized for use in solid phase extraction (SPE) and in capillary liquid chromatography (LC). The approach involved incorporating both hydrophobic and hydrophilic monomers into a monolithic material, by copolymerization of stearyl methacrylate (SMA), poly(ethylene glycol) methyl ether methacrylate (PEGMEMA) and ethylene dimethacrylate (EDMA) in the presence of selected porogens, to produce translucent mesoporous monolithic materials in bulk (SPE) or white macroporous monoliths inside fused silica capillary columns (capillary LC). A capillary column containing one of the hydrophilic C18 monoliths (i.e. poly(SMA-co-PEGMEMA-co-EDMA) with 15% (w/w) PEGMEMA) demonstrated nearly 35% reduction in retention of polycyclic aromatic compounds and greater than 40% increase in retention of phenols compared to a hydrophobic C18 monolithic column. In addition, the hydrophilic monolith demonstrated significantly improved resolution of phenols. Similar monolithic materials prepared in bulk were ground and sieved to obtain 45-65 μm particles with desired rigidity for SPE. To achieve optimum extraction performance for phenols, several parameters, including sample pH and volume, and eluent type and volume, were investigated. Under optimized experimental conditions, the method demonstrated good sensitivity (1.6 ng/mL LOD) and linearity (R(2)>0.97 for 10-200 ng/mL). Again, incorporation of 15% (w/w) PEGMEMA in the monolith increased the extraction efficiency of phenols in water from approximately 20% to 67-92% compared to a hydrophobic C18 monolithic material. Increased wettability of the sorbent by the aqueous sample matrix and the presence of hydrogen-bonding interactions are responsible for the improved retention of polar compounds.     

Surfactant addition and alternating current electrophoretic oscillation during size fractionation of nanoparticles in channels with two or three different height segments

An array of parallel planar nanochannels containing two or three segments with varying inner heights was fabricated and used for size fractionation of inorganic and biological nanoparticles. A liquid suspension of the particles was simply drawn through the nanochannels via capillary action. Using fluorescently labeled 30 nm polyacrylonitrile beads, different trapping behaviors were compared using nanochannels with 200-45 nm and 208-54-30 nm height segments. Addition of sodium dodecyl sulfate (SDS) surfactant to the liquid suspension and application of an AC electric field were shown to aid in the prevention of channel clogging. After initial particle trapping at the segment interfaces, significant particle redistribution occurred when applying a sinusoidal 8V peak-to-peak oscillating voltage with a frequency of 150 Hz and DC offset of 4V. Using the 208-54-30 nm channels, 30 nm hepatitis B virus (HBV) capsids were divided into three fractions. When the AC electric field was applied to this trapped sample, all of the virus particles passed through the interfaces and accumulated at the channel ends.     

Synthesis of simple heparanase substrates

Heparanase degrades heparan sulfate (HS) chains on proteoglycans; elevated levels of heparanase expression correlate with tumour cell metastatic potential and vascularity, and reduced post-operative survival of cancer patients. Consequently, heparanase expression is considered a biomarker for cancer detection. Although several heparanase assays have been developed, most require the preparation of heterogeneous, (radio)labelled HS substrates and rely on the separation of enzymatically-degraded products on the basis of molecular size. In studies directed towards the development of a more direct heparanase assay, a series of glucuronides and glycosyl glucuronides were synthesised as putative heparanase substrates. These compounds were designed with various aryl aglycones that could be measured spectrophotometrically upon hydrolysis of the glycosidic linkage by heparanase. It was found that the N-sulfated 4-nitrophenyl glycosyl glucuronide 24 and the N-sulfated methylumbelliferyl glycosyl glucuronide 26 were hydrolysed by recombinant human heparanase. These compounds represent the simplest substrates of heparanase reported to date.