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. Interparticle reactions through pendant vinyl groups lead to the formation of microgel clusters. The rapid increase in molecular weight and cross-link density of the microgel clusters causes a reaction-induced phase separation, and the formation of a macroscopic network of interconnected globules was observed (macrogelation) at around 45 min. After 3 h or 65% conversion, a space-filling macroporous monolithic network was observed. Afterwards, mainly growth of existing globules takes place, reducing the macropore size. The porogen ratio affects the timing of the reaction-induced phase separation, strongly influencing the morphology of the polymer material. The use of a mixture of divinylbenzene isomers yielded a monolithic material that is less cross-linked at the surface compared to the central part of the polymer backbone due to copolymerization-composition drift. The less cross-linked outer layer starts devitrifying at 100°C.

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. At optimal gradient conditions, peak capacities up to 500 can be obtained within a 120 min gradient using 2 µm particle-packed capillary columns. Separations of proteomic samples including a cytochrome c tryptic digest, a bovine serum albumin tryptic digest, a six protein mix digest, and an Escherichia coli digest are demonstrated while operating at the kinetic-performance limit, i.e. using 2-µm packed columns, adjusting the column length and scaling the gradient steepness according to sample complexity. Finally, good run-to-run retention time stability (RSD values below 0.18%) was demonstrated applying ultra-high pressure conditions.

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. brucei trypanosomosis, anemia is marked by a very sudden non-hemolytic loss of RBCs during the first-peak parasitemia control, followed by a short recovery phase and the subsequent gradual occurrence of an ever-increasing level of anemia. Using a newly developed quantitative pHrodo based in vitro erythrophagocytosis assay, combined with FACS-based ex vivo and in vivo results, we show that activated liver monocytic cells and neutrophils as well as activated splenic macrophages are the main cells involved in the occurrence of the early-stage acute anemia. In addition, we show that trypanosomosis itself leads to a rapid alteration of RBC membrane stability, priming the cells for accelerated phagocytosis.

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 50­200 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. The resulting column structures were investigated using scanning electron microscopy and the prepared monolithic columns were tested for the separation of a tryptic digest of cytochrome c while applying a fixed flow rate and gradient time. To obtain a better understanding of the effects of macropore and microglobule size, and structure homogeneity on the separation performance in gradient elution, both in terms of peak capacity and gradient plate height, separations were also carried out at different flow rates while maintaining a constant gradient steepness. Furthermore, performance limits were determined applying ultra-high pressure conditions up to the maximum system pressure of 80 MPa. The potential of monolithic nanostructured columns is demonstrated for the separation of tryptic digests of cytochrome c and bovine serum albumin.

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). The applicability of the conventional linear (LSS) and non-linear solvent-strength model (Neue-Kuss) were investigated to describe the retention behaviour of the compounds as a function of the mobile-phase composition. This was performed by using a wide range of gradient conditions, including different gradient slopes (ß, ranging from 0.05 to 0.65min(-1)) and mobile-phase compositions (Δϕ, i.e. gradient span). Although the LSS-model provided accurate retention time predictions (<1.3% deviation) of scouting runs with more conventional gradient slopes, the prediction of high-speed separations with a high degree of accuracy (<2%) could only be obtained with the non-linear model. The solvent-strength parameters resulting from the use of both models, as well as the retention factors at the moment of elution (ke), further served as input parameters to assess the influence of the gradient slope on the expected peak-compression effects in gradient mode, with a focus on high-speed separations. The importance of the correct model choice was emphasized in terms of compression; while the LSS-model lead to the conclusion of peak broadening rather than peak sharpening, the use of a more accurate non-linear model showed the existence of peak compression effect. The results presented in this manuscript show the occurrence of gradient-related focusing effects, which appear to be more prevalent for extremely fast separations.

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%). The kinetic-performance of (50-mm long) titanium-scaffolded monoliths was compared to that of similar monolith created in 1-mm i.d. glass-lined tubing at pressures up to 50MPa. The peak capacities obtained with the titanium-scaffolded column was about 30% lower. An increased Eddy-diffusion, due to the pillar-structure, and a decreased permeability are thought to be the main reasons for this reduced kinetic-performance. No decrease in performance was observed when the titanium-scaffolded columns were operated at pressures of 80MPa for up to 12h. The column-to-column repeatability (n=5) was acceptable in terms of observed peak widths at half heights (RSD ca. 10%) The run-to-run repeatability (n=135) in terms of retention times and peak widths at half height were found to be good. Titanium-scaffolded columns coupled in series up to a combined length of (200mm) were used for the analyses of a complex Escherichia coli protein sample. Our experiments demonstrate that columns based on titanium-scaffolded organic-polymer monolith can be operated under strenuous conditions without loss in performance. The titanium-scaffolded approach makes it feasible to create organic-polymer monoliths in wide-bore columns with accurate temperature control.

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≫2.7µm fused-core C18 packed column>silica C18 monolith≫poly(styrene-co-divinylbenzene) monolith. This is mainly related to the intrinsic properties of the different chromatographic materials, related to surface area and the presence and size of mesopores (stagnant zones where mass transfer is controlled by diffusion). Both isocratic and gradient wash steps with 2-propanol/acetonitrile mixtures were not effective to reduce column carryover. An isocratic wash step using a high acetonitrile percentage or blank gradient reduced carryover with approximately 50%. Nevertheless, it is important to note that effects of column carryover were still observed in a fifth subsequent gradient blank. Although the polymer monolith clearly outperformed the silica materials in terms of carryover, this material exhibited also the lowest loadability, which may be a disadvantage when profiling proteomics mixtures with a broad dynamic range.

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. The potential for ultra-fast gradient separations of large biomolecules was further demonstrated for very steep gradients (gradient times≪1min). A tryptic digest of cytochrome c was separated using a gradient time of only 1min. Finally, the run-to-run repeatability and column robustness were assessed at ultra-high pressure conditions (after>800 runs) with consecutive steep 1min separations of peptides, yielding RSD values below 0.12% in retention time.

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. The normalized gradient approach was validated by dimensionless chromatograms, obtained at different flow rates and gradient times by plotting them as a function of the retention factor. The separation performance of the different column types was visualized via kinetic plots depicting the gradient time required to achieve a certain peak capacity when operating at a maximum system pressure of 350 bar. The gradient steepness (applying t(G)/t0=10, 20, and 40) did not significantly affect the gradient performance limits for low (< 250) peak-capacity separations. For high peak-capacity separations the peak capacity per unit time increases when increasing the t(G)/t0 ratio. The C-term contribution of the porous 3 µm and fused-core 2.7 µm was comparable yielding the same gradient kinetic-performance limits for fast separations at a column temperature of 60 °C. The capillary silica monolithic column showed the lowest contribution in mass transfer and permeability was higher than the packed columns. Hence, the silica monolith showed the best kinetic performance for both fast and high peak-capacity gradient separations.

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. Whereas the traditional operating mode (using a single 15 cm column operated at its optimum flow rate of 0.4 mL/min) offered a peak capacity of 162 in 10.8 min, a fully optimized train of 60 cm (4×15 cm) columns offered a peak capacity of 325 in 61 min when operated at 1200 bar. Even though the particles have a reputed low flow resistance and a relatively large size (>2 µm), it was found that the increase in performance that can be generated when switching from a fully optimized 600 bar operation to a fully optimized 1200 bar operation is significant (roughly 50% reduction of the analysis time for the same peak capacity and approximately a 20% increase in peak capacity if compared for the same analysis time). This has been quantified in a generic way using the kinetic plot method and is illustrated by showing the chromatograms corresponding to some of the data points of the kinetic plot curve.

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. Using coupled columns with a total length of 450 mm at 1,200 bar and applying 40 and 120 min gradients (t(G)/t(0)=17 and 52, respectively), peak capacities of n(c)=480 and 760 were measured. The kinetic performance was further improved by coupling six 150 mm long columns and applying 1,200 bar, yielding a flow rate close to the optimum of the van Deemter curve while scaling the gradient volume. At t(G)/t(0)=52 a peptide separation yielding a peak capacity of 1360 was achieved, applying a 480 min gradient. The observed increase of peak capacity with column length agrees well with the theoretical expectations based on the linear solvent strength (LSS) model.