Temperature control in large-internal-diameter scaffolded monolithic columns operated at ultra-high pressures.

Scaffolding makes it feasible to create organic-polymer monoliths in large confinements, such as wide-bore columns. By creating the scaffold from a metal good heat conductivity inside the column is obtained, which renders the relatively large columns (comparable with 4.6 mm i.d.) suitable for application under ultra-high-pressure LC conditions. It was anticipated that the metal scaffold would allow accurate control of the temperature within the columns, but the temperature profiles within the columns could not be characterized using the previously available small-internal-diameter scaffolded columns. In the current study the internal diameter of the scaffolded columns was increased up to square conduits of 4×4 mm. Prior to the formation of the stationary phase the heating efficiency in the empty scaffolded conduits was addressed. The performance of stationary phases created in the large scaffolds was investigated using the kinetic performance approach and the results were compared to those of the previous studies. Finally, scaffolded columns were tested under ultra-high-pressure LC conditions, where good temperature control is essential.

Comprehensive Two-Dimensional Liquid Chromatography with Stationary-Phase-Assisted Modulation Coupled to High-Resolution Mass Spectrometry Applied to Proteome Analysis of Saccharomyces cerevisiae.

Stationary-phase-assisted modulation is used to overcome one of the limitations of contemporary comprehensive two-dimensional liquid chromatography, which arises from the combination of a first-dimension column that is typically narrow and long and a second-dimension column that is wide and short. Shallow gradients at low flow rates are applied in the first dimension, whereas fast analyses (at high flow rates) are required in the second dimension. Limitations of this approach include a low sample capacity of the first-dimension column and a high dilution of the sample in the complete system. Moreover, the relatively high flow rates used for the second dimension make direct (splitless) hyphenation to mass spectrometry difficult. In the present study we demonstrate that stationary-phase-assisted modulation can be implemented in an online comprehensive two-dimensional LC (LC × LC) setup to shift this paradigm. The proposed active modulation makes it possible to choose virtually any combination of first- and second-dimension column diameters without loss in system performance. In the current setup, a 0.30 mm internal diameter first-dimension column with a relatively high loadability is coupled to a 0.075 mm internal diameter second-dimension column. This actively modulated system is coupled to a nanoelectrospray high-resolution mass spectrometer and applied for the separation of the tryptic peptides of a six-protein mixture and for the proteome-wide analyses of yeast from Saccharomyces cerevisiae. In the latter application, about 20000 MS/MS spectra are generated within 24 h analysis time, resulting in the identification of 701 proteins.

Post-polymerization photografting on methacrylate-based monoliths for separation of intact proteins and protein digests with comprehensive two-dimensional liquid chromatography hyphenated with high-resolution mass spectrometry.

Post-polymerization photografting is a versatile tool to alter the surface chemistry of organic-based monoliths so as to obtain desired stationary phase properties. In this study, 2-acrylamido-2-methyl-1-propanesulfonic acid was grafted to a hydrophobic poly(butyl methacrylate-co-ethylene glycol dimethacrylate) monolith to create a strong cation exchange stationary phase. Both single-step and two-step photografting were addressed, and the effects of grafting conditions were assessed. An experimental design has been applied in an attempt to optimize three of the key parameters of the two-step photografting chemistry, i.e. the grafting time of the initiator, the monomer concentration and the monomer irradiation time. The photografted columns were implemented in a comprehensive two-dimensional column liquid chromatography ( (t) LC × (t) LC) workflow and applied for the separation of intact proteins and peptides. A baseline separation of 11 intact proteins was obtained within 20 min by implementing a gradient across a limited RP composition window in the second dimension. (t) LC × (t) LC with UV detection was used for the separation of cytochrome c digest, bovine serum insulin digest and a digest of a complex protein mixture. A semi-quantitative estimation of the occupation of separation space, the orthogonality, of the (t) LC × (t) LC system yielded 75%. The (t) LC × (t) LC setup was hyphenated to a high-resolution Fourier transform ion cyclotron resonance mass spectrometer instrument to identify the bovine serum insulin tryptic peptides and to demonstrate the compatibility with MS analysis.

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.

Macroporous polymer monoliths as second dimension columns in comprehensive two-dimensional gas chromatography: a feasibility study.

When the typical column combinations are used, comprehensive two-dimensional gas chromatography (GC×GC) suffers from the impossibility to operate both dimensions at their optimum carrier gas velocities at the same time. This as a result of the flow mismatch caused by the different dimensions of the columns used. The objective of the present study was the development of monolithic second dimension columns which would allow simultaneous optimum-velocity operation. With monolithic GC columns the optimum performance can be obtained at any given flow rate by varying the bed structure and column diameter. Different divinylbenzene-based monolithic columns were prepared and evaluated in terms of permeability and performance. Plate heights of less than 0.18 mm and plate generation rates up to 600 plates/s were achieved. 1D-GC experiments performed on short monolithic columns showed a good resolving power thanks to the elevated retention and the good selectivity. A peak capacity up to 12 peaks per 4-5s was obtained for low-boiling alkanes, confirming the potential for fast separations. Excellent repeatability in terms of retention times (RSD<0.5%) and peak widths (RSD<1.5%) was observed. The columns prepared were successfully used in the second dimension of a GC×GC setup with a standard non-polar first dimension. Model experiments proved the possibility to operate both dimensions at their optimum linear velocity simultaneously. The suitability of the novel second dimension column format to perform multidimensional separations was shown for selected applications.