Jelle De Vos

High-speed isocratic and gradient liquid-chromatography separations at 1500bar.

The need to improve either sample throughput on separation efficiency has spurred the development of ultra-high-pressure LC instrumentation, allowing to operate up to column pressures of 1500bar. In the present study, the isocratic and gradient performance limits were assessed at UHPLC conditions applying columns packed with core-shell particles. First, the extra-column band broadening contributions were assessed and minimized. Using an optimized system configuration minimum reduced plate heights of 1.8 were recorded on 2.1×100 columns packed with 1.5μm core-shell particles. Increasing the pressure limit from 500 to 1500bar and at the same time reducing the particle size from 2.6 to 1.5μm has allowed the analysis time to be decreased by a factor of 1.5 in isocratic mode, while maintaining separation efficiency (N=54,000). The kinetic time-gain factor in isocratic mode was proportional to the ratio of the separation impedance of both columns multiplied with the pressure ratio applied. In addition, the effect of operating pressure on the time gain factor was assessed in gradient mode. Using optimized gradient steepness (tG/t0=12) and increasing the operating pressure from 500 to 1500bar a time gain factor of almost 13 was achieved for the separation of a mixture of waste-water pollutants without compromising peak capacity.

High-resolution separations of tryptic digest mixtures using core–shell particulate columns operated at 1200 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.

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.

Design of a microfluidic device for comprehensive spatial two‐dimensional liquid chromatography

This study discusses the design aspects for the construction of a microfluidic device for comprehensive spatial two-dimensional liquid chromatography. In spatial two-dimensional liquid chromatography each peak is characterized by its coordinates in the plane. After completing the first-dimension separation all fractions are analyzed in parallel second-dimension separations. Hence, spatial two-dimensional liquid chromatography potentially provides much higher peak-production rates than a coupled column multi-dimensional liquid chromatography approach in which the second-dimension analyses are performed sequentially. A chip for spatial two-dimensional liquid chromatography has been manufactured from cyclic olefin copolymer and features a first-dimension separation channel and 21 parallel second-dimension separation channels oriented perpendicularly to the former. Compartmentalization of first- and second-dimension developments by physical barriers allowed for a preferential flow path with a minimal dispersion into the second-dimension separation channels. To generate a homogenous flow across all the parallel second-dimension channels, a radially interconnected flow distributor containing two zones of diamond-shaped pillars was integrated on-chip. A methacrylate ester based monolithic stationary phase with optimized macroporous structure was created in situ in the confines of the microfluidic chip. In addition, the use of a photomask was explored to localize monolith formation in the parallel second-dimension channels. Finally, to connect the spatial chip to the liquid chromatography instrument, connector ports were integrated allowing the use of Viper fittings. As an alternative, a chip holder with adjustable clasp locks was designed that allows the clamping force to be adjusted.

A generic approach to post-column refocusing in liquid chromatography

To increase detection sensitivity in liquid chromatography, a generic post-column refocusing strategy has been developed to enrich (target) analytes prior to detection. In this strategy, after separation on the analytical column, the analytes are led to a trap column preferably containing a stationary phase with strong retentive properties (e.g. silica C30). They are then eluted using a strong solvent in a backward-elution mode. A first key element of the proposed strategy is that the trapping time should be at least equal to the time the front of the remobilization solvent needs to cover the entire length of the trap column, divided by the ratio of the flow rates used for trapping and remobilization. This condition is independent of the retention properties of the analytes in the trapping and remobilization solvent. Another essential element is the addition of a third solvent (isopropanol in the present case) to the remobilization solvent to overcome viscous-fingering effects caused by the viscosity difference between the trap and the remobilization solvents. The potential of the proposed post-column refocusing strategy is demonstrated for an isocratic separation of KI (t0 marker), an antibiotic (sulfamethazine), and acetophenone as a case study. Using optimized remobilization conditions a maximum signal-enhancement factor of 8 was achieved. Higher enhancement factors using a remobilization solvent with slightly higher elution strength were prohibited by disturbances of the UV background signal.

Peak refocusing using subsequent retentive trapping and strong eluent remobilization in liquid chromatography: a theoretical optimization study.

The present paper presents the mathematical expressions for the concentration enhancement which can be expected when applying a subsequent retentive trapping and strong eluent remobilization process in a packed trap column connected to the end of an analytical separation column. The established expressions for the optimal loading times, trap dimensions and expected concentration enhancement are illustrated and confirmed using numerical simulations of the trapping and release process. These simulations also provide a direct insight in how the bands are deformed and sharpened during the different steps of the process. The simulations, as well as the established expressions, for example show that in the backward elution mode the loading time should exceed a minimum value to allow the strong eluent front to fully overtake the remobilized band as this is the necessary condition to get a completely sharpened peak (in both the backward and the forward elution modes). The simulations also show it is very critical that this occurs as close as possible to the trap exit, as the bands are most sensitive to band broadening once they have been sharpened. The refocusing traps should hence be filled with particles producing the smallest possible plate heights, and connected to the detector using very short and small i.d. connection tubing. It was also found that, if there would be no band broadening in the trap, the achievable concentration enhancement would be the same for either a strong or weakly retaining trap. The true advantage of a high retention in the trap is that it leads to small trap volumes, which in turn minimizes the distance the band has to travel to reach the detector. This then minimizes the band broadening (inevitable in practice) and helps keeping a high concentration enhancement.

Aqueous size‐exclusion chromatographic separations of intact proteins under native conditions: Effect of pressure on selectivity and efficiency

The selectivity and separation efficiency of aqueous size-exclusion chromatographic separations of intact proteins were assessed for different flow rates, using columns packed with 3 and 5 μm silica particles containing 150 and 290 Å stagnant pores. A mixture of intact proteins with molecular weights ranging between 17 000 and 670 000 Da was used to construct the calibration curves. Both the model fit and the predictive properties, using a leave-one-out strategy, of different polynomial models (up to fifth order) were evaluated for different flow rates. The best compromise between model fit and predictive properties was obtained using a third-order polynomial model. The accuracy of the predictive properties decreased with 10% with an eightfold increase in the flow rate. No changes in retention factors (hence selectivity) were observed in the flow-rate range applied. A strong correlation between molecular weight and plate height was observed. Exclusion of large-molecular-weight proteins led to a significant reduction in the stationary-phase mass-transfer contribution to the total plate-height value, and this effect was also independent of the flow rate applied. The kinetic-performance limits, in terms of plate number and time, and optimal column-length particle-size combinations were determined at the maximum recommended operating pressure of the size-exclusion chromatography columns (20 MPa). Finally, the possibilities of method speed-up using ultra-high-pressure size-exclusion chromatography in combination with columns packed with sub-2 μm particles are discussed.

A comprehensive study to protein retention in hydrophobic interaction chromatography.

The effect of different kosmotropic/chaotropic salt systems on retention characteristics of intact proteins has been examined in hydrophobic interaction chromatography (HIC). The performance was assessed using different column chemistries, i.e., polyalkylamide, alkylamine incorporating hydrophobic moieties, and a butyl chemistry. Selectivity in HIC is mainly governed by the salt concentration and by the molal surface tension increment of the salt. Typically, a linear relationship between the natural logarithm of the retention factor and the salt concentration is obtained. Using a 250mm long column packed with 5μm polyalkylamide functionalized silica particles and applying a 30min linear salt gradient, a peak capacity of 78 was achieved, allowing the baseline separation of seven intact proteins. The hydrophobicity index appeared to be a good indicator to predict the elution order of intact proteins in HIC mode. Furthermore, the effect of adding additives in the mobile phase, such as calcium chloride (stabilizing the 3D conformation of α-lactalbumin) and isopropanol, on retention properties has been assessed. Results indicate that HIC retention is also governed by conformational in the proteins which affect the number of accessible hydrophobic moieties.

Applicability of linear and nonlinear retention-time models for reversed-phase liquid chromatography separations of small molecules, peptides, and intact proteins.

The applicability and predictive properties of the linear solvent strength model and two nonlinear retention-time models, i.e., the quadratic model and the Neue model, were assessed for the separation of small molecules (phenol derivatives), peptides, and intact proteins. Retention-time measurements were conducted in isocratic mode and gradient mode applying different gradient times and elution-strength combinations. The quadratic model provided the most accurate retention-factor predictions for small molecules (average absolute prediction error of 1.5%) and peptides separations (with a prediction error of 2.3%). An advantage of the Neue model is that it can provide accurate predictions based on only three gradient scouting runs, making tedious isocratic retention-time measurements obsolete. For peptides, the use of gradient scouting runs in combination with the Neue model resulted in better prediction errors (<2.2%) compared to the use of isocratic runs. The applicability of the quadratic model is limited due to a complex combination of error and exponential functions. For protein separations, only a small elution window could be applied, which is due to the strong effect of the content of organic modifier on retention. Hence, the linear retention-time behavior of intact proteins is well described by the linear solvent strength model. Prediction errors using gradient scouting runs were significantly lower (2.2%) than when using isocratic scouting runs (3.2%).

Effect of gradient steepness on the kinetic performance limits and peak compression for reversed-phase gradient separations of small molecules

The effect of gradient steepness on the kinetic performance limits and peak compression effects has been assessed in gradient mode for the separation of phenol derivatives using columns packed with 2.6μm core-shell particles. The effect of mobile-phase velocity on peak capacity was measured on a column with fixed length while maintaining the retention factor at the moment of elution and the peak-compression factor constant. Next, the performance limits were determined at the maximum system pressure of 100MPa while varying the gradient steepness. For the separation of small molecules applying a linear gradient with a broad span, the best performance limits in terms of peak capacity and analysis time were obtained applying a gradient-time-to-column-dead-time (tG/t0) ratio of 12. The magnitude of the peak-compression factor was assessed by comparing the isocratic performance with that in gradient mode applying different gradient times. Therefore, the retention factors for different analytes were determined in gradient mode and the mobile-phase composition in isocratic mode was tuned such that the difference in retention factor was smaller than 2%. Peak-compression factors were quantitatively determined between 0.95 and 0.65 depending on gradient steepness and the gradient retention factor.