Optimization of elution conditions and comparison of emerging biocompatible columns on the resolving power and detection sensitivity of oligonucleotides by ion-pairing reversed-phase liquid chromatography mass spectrometry.

A generic performance comparison strategy has been developed to evaluate the impact of mobile-phase additives (ion-pairing agent / counter ion systems), distinct stationary phases on resulting resolving power, and MS detectability of oligonucleotides and their critical impurities in gradient IP-RPLC. Stationary-phase considerations included particle type (core-shell vs. fully porous particles), particle diameter, and pore size. Separations were carried out at 60°C to optimize mass transfer (C-term). The incorporation of an active column preheater mitigated thermal mismatches, leading to narrower peaks and overcoming peak splitting. Acetonitrile as organic modifier outweighed methanol in terms of peak-capacity generation and yielded a 30% lower back pressure. Performance screening experiments were conducted varying ion-pairing agents and counter ions, while adjusting gradient span achieved an equivalent effective retention window. Hexafluoromethylisopropanol yielded superior chromatographic resolution, whereas hexafluoroisopropanol yielded significantly higher MS detection sensitivity. The 1.7 µm core-shell particle columns with 100 Å pores provided maximum resolving power for small (15-35 mers) oligonucleotides. Sub-min analysis for 15-35 polyT ladders was achieved operating a 50 mm long column at the kinetic performance limits. High-resolution separations between a 21-mer modified RNA sequence oligonucleotides and its related (shortmer and phosphodiester) impurities and complementary strand were obtained using a coupled column set-up with a total length of 450 mm.

Recent advances in analytical techniques for high throughput experimentation.

High throughput experimentation is a growing and evolving field that allows to execute dozens to several thousands of experiments per day with relatively limited resources. Through miniaturization, typically a high degree of automation and the use of digital data tools, many parallel reactions or experiments at a time can be run in such workflows. High throughput experimentation also requires fast analytical techniques capable of generating critically important analytical data in line with the increased rate of experimentation. As traditional techniques usually do not deliver the speed required, some unique approaches are required to enable workflows to function as designed. This review covers the recent developments (2019-2020) in this field and was intended to give a comprehensive overview of the current "state-of-the-art."

Practical constraints in the kinetic plot representation of chromatographic performance data: theory and application to experimental data.

It is demonstrated that the kinetic plot representation of experimental plate height data can also account for practical constraints on the column length, the peak width, the viscous heating, and the mobile-phase velocity without needing any iterative solution routine. This implies that the best possible kinetic performance to be expected from a given tested support under any possible set of practical optimization constraints can always be found using a directly responding calculation spreadsheet template. To show how the resulting constrained kinetic plots can be used as a powerful design and selection tool, the method has been applied to a series of plate height measurements performed on a number of different commercial columns for the same component (butyl-parabene) and mobile-phase composition. The method, for example, allows one to account for the fact that the advantageous solutions displayed by the silica monolith and 5 microm particle columns in the large plate number range of the free kinetic plot are no longer accessible if applying a maximal column length constraint of Lmax = 30 cm. In the plate number range that remains accessible, the investigated sub-2 mum particle columns in any case perform (at least for the presently considered parabene separation) better than the 3.5 mum particle columns or silica monolith, especially if considering the use of system pressures exceeding 400 bar. The constrained kinetic plot method can also be used to select the best-suited column length from an available product gamma to perform a separation with a preset number of plates. One of the optimization results that is obtained in this case is that sometimes a significant gain in analysis time can be obtained by selecting a longer column, yielding the desired plate number at a larger velocity than that for a shorter column.

Experimental validation of the tetrahedral skeleton model pressure drop correlation for silica monoliths and the influence of column heterogeneity.

This paper describes the use of computational fluid dynamics for the calculation of the flow resistance through computer-generated models resembling silica monoliths. This study was undertaken to determine the effect of skeleton heterogeneity on the flow resistance and, more precisely, to test the hypothesis that increased skeleton heterogeneity decreases the flow resistance. To evaluate the proposed model, 24 real silica monoliths have been prepared using the same method, covering a wide range of skeleton sizes (2.2 microm < d(s) < 8 microm) and porosities (0.47 < epsilon < 0.66). The permeability of these monoliths was determined by pressure drop measurements, and structural information was obtained by image analysis of laser scanning confocal microscopy-generated 3D images of the skeleton structure. The results indicate that the presence of preferential flow paths due to an increased heterogeneity of the flow through pore space reduces the flow resistance of monolithic media. It is also shown that the pore size is hence a much better suited scaling dimension than the skeleton size to reduce the permeability of monolithic columns.

General rules for the optimal external porosity of LC supports.

We present a series of numerically calculated plate height and flow resistance data obtained for an idealized chromatographic support mimic with variable bed porosity (0.3

A computational study of the porosity effects in silica monolithic columns.

We report on a theoretical study of the influence of the through-pore porosity on the main chromatographic performance parameters (reduced theoretical plate height, flow resistance, and separation impedance) of silica monoliths. To investigate this problem devoid of any structural uncertainties, computer-generated structural mimics of the pore geometry of silica monolithic columns have been studied. The band broadening in these synthetic monoliths was determined using a commercial Computational Fluid Dynamics (CFD) software package. Three widely differing external porosities (epsilon = 0.38, epsilon = 0.60, and epsilon = 0.86) are considered and are compared on the basis of an identical intra-skeleton diffusivity (Ds = 5 x 10(-10)m2/s), internal porosity (epsilon(int) = 0.5), and for the same phase retention factor (k' = 1.25). Since the data are obtained for perfectly ordered structures, the calculated plate heights and separation impedances constitute the ultimate performance ever to be expected from a monolithic column. It is found that, if silica monoliths could be made perfectly homogeneous, domain size-based reduced plate heights as small as h(min) approximately 0.8 (roughly independent of the porosity) and separation impedances as small as Emin approximately 130 (epsilon = 0.60) and Emin approximately 40 (epsilon = 0.86) should be achievable with pure water as the working fluid. The data also show that, although the domain size is a much better reduction basis than the skeleton size, the former is still not capable of bringing the van Deemter curves of different porosity columns into perfect agreement in the C term dominated velocity range. It is found that, in this range, large porosity monoliths can be expected to yield smaller domain size-based reduced plate heights than small porosity monoliths.

Importance and reduction of the sidewall-induced band-broadening effect in pressure-driven microfabricated columns.

The influence of the detailed design of the sidewall region upon the over-all band-broadening in microfabricated packed-bed or collocated monolithic support structure (COMOSS) columns has been investigated using computational fluid dynamics (CFD) simulation techniques. It is shown that, under unretained solute conditions, very small structural variations of the order of only 5% of the particle diameter can give rise to a 4-fold increase of the band-broadening. A comprehensive study has been made to quantify this effect as a function of the fluid velocity, the particle diameter, the channel widths, and of course, the sidewall region design. Because the sidewall effect can be fully attributed to a mismatch between the flow rates in the column center and in the sidewall region, it is fortunately also quite straightforward to avoid it. A very simple design, yielding band-broadening values identical to that of a hypothetical sidewall-less column for all possible values of the flow velocity, the particle diameter, or the channel width is proposed.

Theoretical comparison of the band broadening in nonretained electrically and pressure-driven flows through an ordered chromatographic pillar packing.

Using a well-validated computational fluid dynamics simulation method, based on a multi-ion transport model, a detailed analysis of the differences in band broadening between pressure-driven (PD) and electrically driven (ED) flows through perfectly ordered, identical chromatographic pillar packings has been made. It was found that, although the eddy-diffusion band-broadening contributions were nearly completely absent in the considered structure, the ED flow still yields much smaller plate heights than the PD flow. This difference could be fully attributed to the different ways in which the ED and PD velocity profiles reshape when passing through a tortuous pore structure with undulating cross section. Whereas in the PD case the parabolic tip of the band front is continually squeezed and extended each time it passes a pore constriction, the ED flow displays some kind of band front restoring mechanism, with which the fluid elements of the band front are (at least partly) laterally re-aligned after each pore constriction passage. This could be clearly visualized from a series of step-by-step images of the progression of a sharply "injected" species band moving through the packing under ED and PD conditions.

Model column structure for the analysis of the flow and band-broadening characteristics of silica monoliths.

We report on the use of commercial computational fluid dynamics software to study the band broadening in a perfectly ordered three-dimensional model structure, the so-called tetrahedral skeleton column (TSC), selected for its close geometrical resemblance to the specific pore network topology of silica monoliths. Van Deemter plots are presented for the case of a species flow through a non-porous skeleton and for the case of a retained component (k' = 1) in a porous skeleton (mesopore porosity epsilon = 0.6 in both cases). Using the flow domain as the characteristic scaling dimension, the TSC model yields reduced plate heights as small as h(min) = 0.8 and separation impedances as small as Emin = 120 for a retained component with k' = 1. The very small reduced plate heights for the TSC model can without any doubt largely be attributed to the perfect homogeneity of the considered model structure: the B and C terms are similar to those obtained in real silica monoliths with similar external porosity, whereas the A term is significantly (about a factor of 10) smaller. The present study hence suggests that further experimental work to obtain more homogeneous silica networks could yield large gains in reduced plate height and separation impedance. Comparing the three-dimensional TSC model with a 2D array of cylinders, it was found that the use of the domain size as the characteristic dimension in the reduced plate height expression is much more appropriate than the use of the skeleton size, hence validating earlier approaches adopted in the literature.

Experimental Van Deemter plots of shear-driven liquid chromatographic separations in disposable microchannels.

We present a new stationary phase coating method, yielding a monolayer of densely arrayed porous HPLC beads (d(p)=4 microm) for use in a disposable shear-driven flow LC system. The system is inherently suited for whole-column detection through the small voids between the individual particles of the layer. The chromatographic performance of the system has been characterized by performing a series of coumarin dye separation experiments (reversed-phase mode) and by measuring the theoretical plate height as a function of the mobile phase velocity. The resulting Van Deemter curve, yielding a value of about 90,000 plates/m near the u=u(opt) velocity, shows good agreement with the theoretical expectations, and hence constitutes the first full validation of the theory of shear-driven chromatography.

A correlation for the pressure drop in monolithic silica columns.

To gain insight into how the pressure drop in monolithic silica columns is determined by the microscopic details of the pore structure, a series of well-validated computational fluid dynamics simulations has been performed on a simplified model structure, the so-called tetrahedral skeleton column. From these simulations, a direct correlation between the pressure drop and two main structural properties (skeleton thickness and column porosity) of the monolithic skeleton could be established. The correlation shows good agreement with the experimental pressure-drop data available from the literature on silica monoliths, especially when a correction for the flow-through pore size heterogeneity is made. The established correlation also yields a much more accurate representation of the relation between the flow resistance and the bed porosity than does the Kozeny-Carman model, making it much better suited for porosity optimization calculations.