Development of a pHrodo-based assay for the assessment of in vitro and in vivo erythrophagocytosis during experimental trypanosomosis.

Extracellular trypanosomes can cause a wide range of diseases and pathological complications in a broad range of mammalian hosts. One common feature of trypanosomosis is the occurrence of anemia, caused by an imbalance between erythropoiesis and red blood cell clearance of aging erythrocytes. In murine models for T.

Nanostructured porous polymer monolithic columns for capillary liquid chromatography of peptides.

The macroporous structure of poly(styrene-co-divinylbenzene) monolithic capillary columns has been optimized for the gradient separation of peptides. To exploit monolithic supports with porosity exceeding 70%, the thermodynamic properties of the polymerization mixture were carefully tailored to yield homogeneous monolithic materials featuring macropore and polymer microglobule sizes in the range of 50–200 nm. The effects of (i) initiator content, (ii) composition of porogenic mixture, comprising tetrahydrofuran and 1-decanol, (iii) percentage of divinylbenzene crosslinker, and (iv) monomers to porogen ratio on the morphology was investigated.

Titanium-scaffolded organic-monolithic stationary phases for ultra-high-pressure liquid chromatography.

Organic-polymer monoliths with overall dimensions larger than one millimetre are prone to rupture - either within the monolith itself or between the monoliths and the containing wall - due to the inevitable shrinkage accompanying the formation of a cross-linked polymeric network. This problem has been addressed by creating titanium-scaffolded poly(styrene-co-divinylbenzene) (S-co-DVB) monoliths. Titanium-scaffolded monoliths were successfully used in liquid chromatography at very high pressures (up to 80MPa) and using gradients spanning the full range of water-acetonitrile compositions (0 to 100%).

Gradient-elution parameters in capillary liquid chromatography for high-speed separations of peptides and intact proteins.

This contribution relates to the assessment of gradient-elution parameters in capillary liquid chromatography affecting the peak widths in the reversed-phase separation of peptides and intact proteins. Gradient separations were performed using both a poly(sytrene-co-divinylbenzene) monolithic column and a microparticulate fused-core column (silica C18, 2.7μm).

Monitoring the morphology development of polymer-monolithic stationary phases by thermal analysis.

Thermal analysis and SEM were employed to gain insights in the different stages of morphology development and the thermal properties of polymer-monolithic stationary phases. The studied system was a thermally initiated free-radical copolymerization reaction at 70°C of styrene and divinylbenzene in the presence of tetrahydrofuran and 1-decanol. The key events in the early stages of morphology development are initiation, chain growth, branching, and cyclization, leading to microgel particles.

High-speed gradient separations of peptides and proteins using polymer-monolithic poly(styrene-co-divinylbenzene) capillary columns at ultra-high pressure.

For the first time, polymer monolithic capillary columns have been employed at ultra-high-pressure liquid chromatographic conditions (UHPLC) to investigate their potential for high-speed separations of peptides and intact proteins. In comparison to conventional flow rates and gradient conditions, a substantial decrease in analysis time (>factor 4) can be achieved when operating monolithic columns such as ultra-high-pressure conditions while scaling the gradient volume. The effects of flow rate and column length on the peak capacity and the gradient performance limits were assessed for the separation of peptide and protein mixtures applying the maximum system pressure (80MPa) and a fixed gradient steepness.

High-resolution peptide separations using nano-LC at ultra-high pressure.

We report on the optimization of nano-LC gradient separations of proteomic samples that vary in complexity. The gradient performance limits were visualized by kinetic plots depicting the gradient time needed to achieve a certain peak capacity, while using the maximum system pressure of 80 MPa. The selection of the optimal particle size/column length combination and corresponding gradient steepness was based on scouting the performance of 75 μm id capillary columns packed with 2, 3, and 5 μm fully porous silica C18 particles.

Investigation of carryover of peptides in nano-liquid chromatography/mass spectrometry using packed and monolithic capillary columns.

This article relates on reversed-phase column technology as the main cause of carryover in the LC-MS/MS analysis of proteomics samples. The separation performance and column carryover was investigated using four capillary columns with different morphologies by monitoring the remaining traces of tryptic peptides of bovine serum albumin in subsequent blank LC-MS runs. The following trend in column carryover was observed: capillary column packed with 3μm porous C18 particles≫2.

High-resolution separations of tryptic digest mixtures using core-shell particulate columns operated at 1,200 bar.

Combining two recent advances in instrumentation and column technology (ultra-high-pressure LC instruments and core-shell particles), the current peak-capacity generation limits in one-dimensional LC have been explored for the case of tryptic digest separations. To operate as close as possible to the Knox and Saleem limit of the particles, and hence to operate the 2.6 μm core-shell particles at their kinetic optimum, the separations were conducted in a coupled column systems at 1200 bar.

Maximizing the peak capacity using coupled columns packed with 2.6 μm core-shell particles operated at 1200 bar.

We report on the peak capacity that can be produced by operating a state-of-the-art core-shell particle type (d(p)=2.6 μm) at its kinetic optimum at ultra-high pressures of 600 and 1200 bar. The column-length optimization needed to arrive at this kinetic optimum was realized using column coupling.

Comparison of the gradient kinetic performance of silica monolithic capillary columns with columns packed with 3 μm porous and 2.7 μm fused-core silica particles.

The kinetic-performance limits of a capillary silica C18 monolithic column and packed capillary columns with fully-porous 3 μm and fused-core 2.7 μm silica C18 particles (all 5 cm long) were determined in gradient-elution mode for the separation of peptides. To establish a kinetic plot in gradient-elution mode, the gradient time to column dead time ratio (t(G)/t₀) was maintained constant when applying different flow rates.