Sebastiaan Eeltink

Monolithic porous polymer stationary phases in polyimide chips for the fast high-performance liquid chromatography separation of proteins and peptides

Poly(lauryl methacrylate-co-ethylene dimethacrylate) and poly(styrene-co-divinylbenzene) stationary phases in monolithic format have been prepared by thermally initiated free radical polymerization within polyimide chips featuring channels having a cross-section of 200micromx200microm and a length of 6.8cm. These chips were then used for the separation of a mixture of proteins including ribonuclease A, myoglobin, cytochrome c, and ovalbumin, as well as peptides. The separations were monitored by UV adsorption. Both the monolithic phases based on methacrylate and on styrene chemistries enabled the rapid baseline separation of most of the test mixtures. Best performance was achieved with the styrenic monolith leading to fast baseline separation of all four proteins in less than 2.5min. The in situ monolith preparation process affords microfluidic devices exhibiting good batch-to-batch and injection-to-injection repeatability.

Characterization of polymer-based monolithic capillary columns by inverse size-exclusion chromatography and mercury-intrusion porosimetry.

Organic-polymer monolithic capillary columns were prepared in fused-silica capillaries by a radical copolymerization reaction of butyl methacrylate and ethylene dimethacrylate monomers in the presence of 1,4-butanediol and 1-propanol as porogen solvents and azobisisobutyronitrile as the initiator. The porous properties could be influenced by changing the ratio of porogen solvents, while keeping the monomer content constant, or by changing the ratio of monomers to porogen in the polymerization mixture. The resulting chromatographic properties, such as porosity, permeability were determined under high-pressure liquid chromatography (HPLC) conditions. The mesopore size distributions of the monolithic materials determined with inverse size-exclusion chromatography (ISEC) were compared with those measured by mercury-intrusion porosimetry. Both techniques are complementary. While mercury-porosimetry measures the entire range of pore sizes and provides more physical information on the monoliths, ISEC is very suitable for determining the size of mesopores in the swollen monoliths.

High-efficiency liquid chromatography-mass spectrometry separations with 50 mm, 250 mm, and 1 m long polymer-based monolithic capillary columns for the characterization of complex proteolytic digests.

In this study, high-efficiency LC-MS/MS separations of complex proteolytic digests are demonstrated using 50 mm, 250 mm, and 1m long poly(styrene-co-divinylbenzene) monolithic capillary columns. The chromatographic performance of the 50 and 250 mm monoliths was compared at the same gradient steepness for gradient durations between 5 and 150 min. The maximum peak capacity of 400 obtained with a 50mm column, increased to 485 when using the 250 mm long column and scaling the gradient duration according column length. With a 5-fold increase in column length only a 20% increase in peak capacity was observed, which could be explained by the larger macropore size of the 250 mm long monolith. When taking into account the total analysis time, including the dwell time, gradient time and column equilibration time, the 50mm long monolith yielded better peptide separations than the 250 mm long monolithic column for gradient times below 80 min (n(c)=370). For more demanding separation the 250 mm long monolith provided the highest peak production rate and consequently higher sequence coverage. For the analysis of a proteolytic digest of Escherichia coli proteins a monolithic capillary column of 1m in length was used, yielding a peak capacity of 1038 when applying a 600 min gradient.

In-line system containing porous polymer monoliths for protein digestion with immobilized pepsin, peptide preconcentration and nano-liquid chromatography separation coupled to electrospray ionization mass spectroscopy

The use of two different monoliths located in capillaries for on-line protein digestion, preconcentration of peptides and their separation has been demonstrated. The first monolith was used as support for covalent immobilization of pepsin. This monolith with well-defined porous properties was prepared by in situ copolymerization of 2-vinyl-4,4-dimethylazlactone and ethylene dimethacrylate. The second, poly(lauryl methacrylate-co-ethylene dimethacrylate) monolith with a different porous structure served for the preconcentration of peptides from the digest and their separation in reversed-phase liquid chromatography mode. The top of the separation capillary was used as a preconcentrator, thus enabling the digestion of very dilute solutions of proteins in the bioreactor and increasing the sensitivity of the mass spectrometric detection of the peptides using a time-of-flight mass spectrometer with electrospray ionization. Myoglobin, albumin, and hemoglobin were digested to demonstrate feasibility of the concept of using the two monoliths in-line. Successive protein injections confirmed both the repeatability of the results and the ability to reuse the bioreactor for at least 20 digestions.

Kinetic plot based comparison of the efficiency and peak capacity of high-performance liquid chromatography columns: theoretical background and selected examples.

The present contribution reviews the foundations of the kinetic-plot method for the direct comparison of the kinetic performance of different chromatographic support and operating modes. The method directly uses experimental data collected for a specific sample and operating condition of ones interest, and is applicable both under isocratic- and gradient-elution conditions. Experimental proof is provided for the strong relation between the kinetic performance of a given support under isocratic and gradient conditions: a material offering superior kinetic performances under isocratic conditions will remain superior under gradient conditions and vice versa provided the comparison occurs under unbiased conditions. In addition, a review is made of the recent literature using the kinetic-plot method to compare and assess the kinetic performance of high performance HPLC columns and their operation mode.

Optimizing the peak capacity per unit time in one-dimensional and off-line two-dimensional liquid chromatography for the separation of complex peptide samples.

To obtain the best compromise between peak capacity and analysis time in one-dimensional and two-dimensional (2D) liquid chromatography (LC), column technology and operating conditions were optimized. The effects of gradient time, flow rate, column temperature, and column length were investigated in one-dimensional reversed-phase (RP) gradient nano-LC, with the aim of maximizing the peak per unit time for peptide separations. An off-line two-dimensional LC approach was developed using a micro-fractionation option of the autosampler, which allowed automatic fractionation of peptides after a first-dimension ion-exchange separation and re-injection of the fractions onto a second-dimension RP nano-LC column. Under the applied conditions, which included a preconcentration/desalting time of 5 min, and a column equilibration time of 12.5 min, the highest peak capacity per unit time in the 2D-LC mode was obtained when applying a short (10 min) first-dimension gradient and second-dimension RP gradients of 20 min duration. For separations requiring a maximum peak capacity of 375, one-dimensional LC was found to be superior to the off-line strong cation-exchange/x/RPLC approach in terms of analysis time. Although a peak capacity of 450 could be obtained in one-dimensional LC when applying 120-min gradients on 500-mm long columns packed with 3-mum particles, for separations requiring a peak capacity higher than 375 2D-LC experiments provide a higher peak capacity per unit time. Finally, the potential of off-line 2D-LC coupled to tandem mass spectrometry detection is demonstrated with the analysis of a tryptic digest of a mixture of nine proteins and an Escherichia coli digest.

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.

High-resolution separations of protein isoforms with liquid chromatography time-of-flight mass spectrometry using polymer monolithic capillary columns

The separation of intact proteins, including protein isoforms arising from various amino-acid modifications, employing a poly(styrene-co-divinylbenzene) monolithic capillary column in high-performance liquid chromatography coupled on-line to a time-of-flight mass spectrometer (MS) is described. Using a 250 mm × 0.2 mm monolithic capillary column high-sensitivity separations yielding peak capacities of >600 were achieved with a 2h linear gradient and formic acid added in the mobile phase as ion-pairing agent. The combination of high-resolution chromatography with high-accuracy MS allowed to distinguish protein isoforms that differ only in their oxidation and biotinylation state allowing the separation between structural isoforms. Finally, the potential to separate proteins isoforms due to glycosylation is discussed.

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.

1 mm ID poly(styrene‐co‐divinylbenzene) monolithic columns for high‐peak capacity one‐ and two‐dimensional liquid chromatographic separations of intact proteins

The LC performance of a 1x50 mm polymer monolithic column format was demonstrated with high-peak capacity one- (1D) and offline two dimensional (2D) LC separations of intact proteins. After optimizing the RP 1D-LC conditions, including column temperature, flow rate and gradient time, a peak capacity of 475 was achieved within a 2-h analysis. The suitability of the monolithic column was also demonstrated for fast 1 min protein separations yielding 1 s peak widths determined at half peak height. In addition, an offline 2D-LC method was developed using the micro-fraction collection capabilities of the autosampler allowing automatic fractionation of intact proteins after the weak-ion-exchange (WAX) separation, and re-injection of the fractions onto the second-dimension RP monolithic column. The best peak capacity-to-analysis time ratio was obtained when applying 10 min second-dimension RP gradients. At optimized conditions, the WAX/x/RPLC separation of intact Escherichia coli proteins was performed within 6 h yielding a maximum theoretical peak capacity of 4880.