Prototype sphere-on-sphere silica particles for the separation of large biomolecules.

The goal of this study was to evaluate the possibilities offered by a prototype HPLC column packed with ∼2.5µm narrow size distribution sphere-on-sphere (SOS) silica particles bonded with C4 alkyl chains, for the analytical characterization of large biomolecules. The kinetic performance of this material was evaluated in both isocratic and gradient modes using various model analytes. The data were compared to those obtained on other widepore state-of-the-art fully core-shell and fully porous materials commonly employed to separate proteins moreover to a reference 5µm wide pore material that is still often used in QC labs. In isocratic mode, minimum reduced plate height values of hmin=2.6, 3.3 and 3.3 were observed on butylparaben, decapeptide and glucagon, respectively. In gradient elution mode, the SOS column performs very high efficiency when working with fast gradients. This prototype column was also comparable (and sometimes superior) to other widepore stationary phases, whatever the gradient time and flow rate, when analyzing the largest model protein, namely BSA. These benefits may be attributed to the SOS particle morphology, minimizing the intra-particle mass transfer resistance. Finally, the SOS column was also applied for the analytical characterization of commercial monoclonal antibody (mAb) and antibody-drug conjugate (ADC) samples. With these classes of proteins, the performance of SOS column was similar to the best widepore stationary phases available on the market.

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.

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.

Monodisperse sphere-on-sphere silica particles for fast HPLC separation of peptides and proteins.

Monodisperse sphere-on-sphere (SOS) silica particles are produced in a one-pot reaction, removing the need for time-consuming preparation and classification steps. Analysis of peptides and proteins using HPLC displays faster separation at lower operating pressure than commercially available fused core materials.

Assessing the performance of curtain flow first generation silica monoliths.

Analytical scale active flow technology first generation silica monolithic columns kitted out in curtain flow mode of operation were studied for the first time. A series of tests were undertaken assessing the column efficiency, peak asymmetry and detection sensitivity. Two curtain flow columns were tested, one with a fixed outlet ratio of 10% through the central exit port, the other with 30%. Tests were carried out using a wide range in inlet flow segmentation ratios. The performance of the curtain flow columns were compared to a conventional monolithic column. The gain in theoretical plates achieved in the curtain flow mode of operation was as much as 130%, with almost Gaussian bands being obtained. Detection sensitivity increased by as much as 250% under optimal detection conditions. The permeability advantage of the monolithic structure together with the active flow technology makes it a priceless tool for high throughput, sensitive, low detection volume analyses.

Core-shell particles: preparation, fundamentals and applications in high performance liquid chromatography.

The challenges in HPLC are fast and efficient separation for a wide range of samples. Fast separation often results in very high operating pressure, which places a huge burden on HPLC instrumentation. In recent years, core-shell silica microspheres (with a solid core and a porous shell, also known as fused-core or superficially porous microspheres) have been widely investigated and used for highly efficient and fast separation with reasonably low pressure for separation of small molecules, large molecules and complex samples. In this review, we firstly show the types of core-shell particles and how they are generally prepared, focusing on the methods used to produce core-shell silica particles for chromatographic applications. The fundamentals are discussed on why core-shell particles can perform better with low back pressure, in terms of van Deemter equation and kinetic plots. The core-shell particles are compared with totally porous silica particles and also monolithic columns. The use of columns packed with core-shell particles in different types of liquid chromatography is then discussed, followed by illustrating example applications of such columns for separation of various types of samples. The review is completed with conclusion and a brief perspective on future development of core-shell particles in chromatography.

Enhancing the separation performance of the first-generation silica monolith using active flow technology: parallel segmented flow mode of operation.

Active flow technology (AFT) columns are designed to minimise inefficient flow processes associated with the column wall and radial heterogeneity of the stationary phase bed. This study is the first to investigate AFT on an analytical scale 4.6mm internal diameter first-generation silica monolith. The performance was compared to a conventional first-generation silica monolith and it was observed that the AFT monolith had an increase in efficiency values that ranged from 15 to 111%; the trend demonstrating efficiency gains increasing as the volumetric flow to the detector was decreased, but with no loss in sensitivity.