•75 μm i.d. columns are packed with 1.9 μm BEH particles slurried at 5–200 mg/mL.•An optimal slurry concentration is found regarding column performance.•Packing microstructure is reconstructed using confocal laser scanning microscopy.•Increased slurry concentrations suppress transcolumn bed heterogeneities.•Too-concentrated slurries favour the formation of larger voids in the packing.Column wall effects and the formation of larger voids in the bed during column packing are factors limiting the achievement of highly efficient columns. Systematic variation of packing conditions, combined with three-dimensional bed reconstruction and detailed morphological analysis of column beds, provide valuable insights into the packing process. Here, we study a set of sixteen 75 μm i.d. fused-silica capillary columns packed with 1.9 μm, C18-modified, bridged-ethyl hybrid silica particles slurried in acetone to concentrations ranging from 5 to 200 mg/mL. Bed reconstructions for three of these columns (representing low, optimal, and high slurry concentrations), based on confocal laser scanning microscopy, reveal morphological features associated with the implemented slurry concentration, that lead to differences in column efficiency. At a low slurry concentration, the bed microstructure includes systematic radial heterogeneities such as particle size-segregation and local deviations from bulk packing density near the wall. These effects are suppressed (or at least reduced) with higher slurry concentrations. Concomitantly, larger voids (relative to the mean particle diameter) begin to form in the packing and increase in size and number with the slurry concentration. The most efficient columns are packed at slurry concentrations that balance these counteracting effects. Videos are taken at low and high slurry concentration to elucidate the bed formation process. At low slurry concentrations, particles arrive and settle individually, allowing for rearrangements. At high slurry concentrations, they arrive and pack as large patches (reflecting particle aggregation in the slurry). These processes are discussed with respect to column packing, chromatographic performance, and bed microstructure to help reinforce general trends previously described. Conclusions based on this comprehensive analysis guide us towards further improvement of the packing process.

•Hypercarb shows increased retention of metabolites compared to a C18 bonded phase.•Retention on Hypercarb allows for focusing of large injections in gradient elution.•Potential impact of effective diffusion on gradient runs studied using peak parking.•Diffusion characteristics make Hypercarb suitable for long gradient runs.The study of metabolites in biological samples is of high interest for a wide range of biological and pharmaceutical applications. Reversed phase liquid chromatography is a common technique used for the separation of metabolites, but it provides little retention for polar metabolites. An alternative to C18 bonded phases, porous graphitic carbon has the ability to provide significant retention for both non-polar and polar analytes. The goal of this work is to study the retention and effective diffusion properties of porous graphitic carbon, to see if it is suitable for the wide injection bands and long run times associated with long, packed capillary-scale separations. The retention of a set of standard metabolites was studied for both stationary phases over a wide range of mobile phase conditions. This data showed that porous graphitic carbon benefits from significantly increased retention (often >100 fold) under initial gradient conditions for these metabolites, suggesting much improved ability to focus a wide injection band at the column inlet. The effective diffusion properties of these columns were studied using peak-parking experiments with the standard metabolites under a wide range of retention conditions. Under the high retention conditions, which can be associated with retention after injection loading for gradient separations, Deff/Dm ∼ 0.1 for both the C18-bonded and porous graphitic carbon columns. As C18 bonded particles are widely, and successfully utilized for long gradient separations without issue of increasing peak width from longitudinal diffusion, this suggests that porous graphitic carbon should be amenable for long runtime gradient separations as well.

•75 μm i.d. columns are slurry-packed with 1.3 μm frictional, cohesive particles.•The slurry concentration is increased gradually from 5 to 50 mg/mL.•Packing microstructure is reconstructed using confocal laser scanning microscopy.•Increased slurry concentrations suppress transcolumn bed heterogeneities.•Too concentrated slurries favour the formation of larger voids in the packing.Lateral transcolumn heterogeneities and the presence of larger voids in a packing (comparable to the particle size) can limit the preparation of efficient chromatographic columns. Optimizing and understanding the packing process provides keys to better packing structures and column performance. Here, we investigate the slurry-packing process for a set of capillary columns packed with C18-modified, 1.3 μm bridged-ethyl hybrid porous silica particles. The slurry concentration used for packing 75 μm i.d. fused-silica capillaries was increased gradually from 5 to 50 mg/mL. An intermediate concentration (20 mg/mL) resulted in the best separation efficiency. Three capillaries from the set representing low, intermediate, and high slurry concentrations were further used for three-dimensional bed reconstruction by confocal laser scanning microscopy and morphological analysis of the bed structure. Previous studies suggest increased slurry concentrations will result in higher column efficiency due to the suppression of transcolumn bed heterogeneities, but only up to a critical concentration. Too concentrated slurries favour the formation of larger packing voids (reaching the size of the average particle diameter). Especially large voids, which can accommodate particles from >90% of the particle size distribution, are responsible for a decrease in column efficiency at high slurry concentrations. Our work illuminates the increasing difficulty of achieving high bed densities with small, frictional, cohesive particles. As particle size decreases interparticle forces become increasingly important and hinder the ease of particle sliding during column packing. While an optimal slurry concentration is identified with respect to bed morphology and separation efficiency under conditions in this work, our results suggest adjustments of this concentration are required with regard to particle size, surface roughness, column dimensions, slurry liquid, and external effects utilized during the packing process (pressure protocol, ultrasound, electric fields).

•High efficiency columns packed with high slurry concentrations and sonication.•Minimum plate heights of 1 particle diameter achieved.•500,000 theoretical plates per meter realized in one meter-long columns.Slurry packing capillary columns for ultrahigh pressure liquid chromatography is complicated by many interdependent experimental variables. Previous results have suggested that combination of high slurry concentration and sonication during packing would create homogeneous bed microstructures and yield highly efficient capillary columns. Herein, the effect of sonication while packing very high slurry concentrations is presented. A series of six, 1 m × 75 μm internal diameter columns were packed with 200 mg/mL slurries of 2.02 μm bridged-ethyl hybrid silica particles. Three of the columns underwent sonication during packing and yielded highly efficient separations with reduced plate heights as low as 1.05.

•Highly efficient capillary columns were created with superficially porous particles.•Highest calculated efficiency seen is over 500,000 plates/m for 31.7 cm long column.•At least one highly efficient column created in 30, 50, 75 μm internal diameters.•Serial capillary column packing can yield a number of efficient columns.Highly efficient capillary columns packed with superficially porous particles were created for use in ultrahigh pressure liquid chromatography. Superficially porous particles around 1.5 μm in diameter were packed into fused silica capillary columns with 30, 50, and 75 μm internal diameters. To create the columns, several capillary columns were serially packed from the same slurry, with packing progress plots being generated to follow the packing of each column. Characterization of these columns using hydroquinone yielded calculated minimum reduced plate heights as low as 1.24 for the most efficient 30 μm internal diameter column, corresponding to over 500,000 plates/m. At least one highly efficient column (minimum reduced plate height less than 2) was created for all three of the investigated column inner diameters, with the smallest diameter columns having the highest efficiency. This study proves that highly efficient capillary columns can be created using superficially porous particles and shows the efficiency potential of these particles.

•Sub-2 μm fully porous and superficially porous particle columns are compared in an isothermal setting.•Isothermal environments can be used to maximize radial thermal broadening.•The higher thermal conductivity of the superficially porous particles limits heating effects.•Temperature and plate height increases are limited with superficially porous particles when isothermal.At high flow rates and pressures, columns packed with sub-2 μm particles suffer from efficiency losses due to frictional heating. The thermal environment of the column (insulated or isothermal) can decrease or magnify these losses. While a number of studies have been conducted demonstrating the improved performance (partially due to the benefits of enhanced thermal conductivity) of columns packed with superficially porous particles, none have made a comparison between sub-2 μm fully and superficially porous particles in an isothermal environment where radial thermal gradients are maximized and thermal broadening is amplified. Here we show that when such columns are characterized in a recirculating water jacket (providing an isothermal environment), efficiency loss and changes in retention and mobile phase temperature are reduced for sub-2 μm superficially porous particles compared to sub-2 μm fully porous particles.

The need for multidimensional separations for bottom-up proteomic analyses has been well demonstrated by many over the past decade. The vast majority of reported approaches has focused primarily on improving the separation once the sample has already been digested. The work presented in this study shows an improvement in multidimensional approaches by prefractionation of intact proteins prior to digestion and separation of the peptides. Two modes of intact protein separation were compared, anion-exchange and reversed-phase, to assess the utility of each mode for the purpose of fractionation. Each of the samples was then enzymatically digested and analyzed by RP-UPLC-MSE. To assess the validity of each approach, baker’s yeast (Saccharomyces cerevisiae) was grown on two different carbon sources, glycerol and dextrose. More proteins were identified by the reversed-phase prefractionation approach (546) than were found by the anion-exchange method (262). As a result, there was much greater coverage of the metabolic pathways of interest for the reversed-phase method than for the anion-exchange method.

•Slurry concentration effects in the slurry packing process are investigated.•30–75 μm i.d. capillaries are packed with 0.9, 1.7, and 1.9 μm particles.•Packing microstructure is reconstructed using confocal laser scanning microscopy.•Radial profiles of porosity, mean particle size, and mean particle distance are analyzed.•Higher slurry concentrations reduce transcolumn bed heterogeneities.Transcolumn dispersion limitations on the separation efficiency of chromatographic columns suggest the need for packing methods that increase bed homogeneity and minimize potential wall effects. Here we address the influence of the slurry concentration in the slurry packing process on the resulting morphology and separation efficiency of ultrahigh-pressure liquid chromatography capillary columns. 30–75 μm i.d. capillaries were packed with fully porous 0.9, 1.7, and 1.9 μm bridged-ethyl hybrid particles and 1.9 μm Kinetex core–shell particles. Capillaries prepared with higher slurry concentrations (20–100 mg/mL) showed higher separation efficiencies than those prepared using a low slurry concentration (2–3 mg/mL). The effect is explained by an analysis of transcolumn bed heterogeneities in three-dimensional reconstructions acquired from the packed capillaries using confocal laser scanning microscopy. The three-dimensional analysis of porosity distributions and local particle size illustrates that beds packed with higher slurry concentrations suppress particle size segregation, however, at the expense of a larger amount of packing voids. In core–shell packings, where only few packing voids were found, the higher slurry concentration allowed for an additional densification of the bed's wall region, as revealed by a radial analysis of the mean particle distances. Overall, wall effects are attenuated in packed columns prepared with both wide and narrow particle size distributions, which will allow for improved chromatographic performance.

We derive a quantitative relationship between the bed morphology and the chromatographic separation efficiency of capillary columns packed with sub-2 μm particles, covering capillary inner diameters from 10 to 75 μm. Our study focuses on wall effects and their impact on band broadening at increasing column-to-particle diameter (aspect) ratios. We approach these complex effects by a morphological analysis of reconstructed column segments composed of several thousand particles that were imaged by confocal laser scanning microscopy. Radial interparticle porosity profiles including wall effects are quantified through an integral porosity deviation, a scalar measure that proves to be a general descriptor of transcolumn porosity heterogeneity. It correlates with the associated transcolumn eddy dispersion, which dominates band broadening in the capillaries and is visualized in the plate height curves by a simple velocity-proportional term. Our comprehensive approach identifies the packing structure features that contribute to decreased efficiency as reflected, e.g., in subtle variations of the wall effect at different aspect ratios, or a particle size-segregation effect in larger-diameter columns as a result of an increased number of packing voids near the wall–bed interface.

Superficially porous particles are characterized by a non-porous particle core surrounded by a thin porous layer. Superficially porous particles have been shown to have chromatographic advantages over traditional totally porous particles by reducing the resistance to mass transfer and the eddy diffusion contributions to the theoretical plate height, particularly for biomolecule separations. Currently, 1.7 μm superficially porous particles are commercially available, but a further decrease in the particle diameter and reduction in the porous layer thickness has the potential to further improve the efficiency of the column packing material. In this study, the synthesis of smaller diameter superficially porous particles was investigated. As the particle diameter was decreased, however, synthesis parameters previously reported were rendered unsuitable due to particle agglomeration, non-uniform coating, and porous layer disintegration. Parameters such as colloidal silica size, drying process, and sintering temperature were investigated to improve the structural characteristics of smaller diameter superficially porous particles. Reported is a synthetic route for production of 1.1 μm superficially porous particles having a 0.1 μm porous layer. Based on the revised method, the particles produced have a surface area, pore diameter, and particle size distribution RSD of 52 m2/g, 71 Å, and 2.2%, respectively.Highlights► The synthesis method produced monodisperse 1.1 μm superficially porous particles. ► Drying by lyophilization led to uniform surface coverage with colloidal silica. ► The pore diameter can be varied by modifying the colloidal silica diameter.

The development of a photothermal absorbance detector for use with microfluidic devices is described. Unlike thermo-optical techniques that rely on measuring refractive index changes, the solution viscosity is probed by continuously monitoring solution conductivity. Platinum electrodes microfabricated on a quartz substrate and bonded to a substrate containing the microchannels enable contact conductivity measurements. The effects of excitation frequency and voltage, electrode spacing, laser power, and laser modulation (chopping) frequency were evaluated experimentally. In the current configuration, a limit of detection of 5 nM for DABSYL-tagged glucosamine was obtained using long injections (to give flat-topped peaks). This corresponds to an absorbance of 4.4 × 10−7 AU. Separation and detection of DABSYL-tagged glycine, proline, and tryptophan are also shown to demonstrate the feasibility of the method. In addition, simulations were used to investigate the applicability of the technique to small volume platforms.

An instrument based on the Poiseuille flow principle capable of measuring solution viscosities at high pressures has been modified to observe UV-absorbent analytes in order to allow for the simultaneous measurement of analyte diffusivity. A capillary time-of-flight (CTOF) instrument was used to measure the viscosity of acetonitrile−water mixtures in all decade volume percent increments and the corresponding diffusion coefficients of small aromatic molecules in these solvent mixtures from atmospheric pressure to 2000 bar (∼30 000 psi) at 25 °C. The instrument works by utilizing a relatively small pressure drop (<100 bar) across a fused-silica capillary which has both the inlet and outlet pressurized so that the average column pressure can be significantly elevated (up to 2000 bar). Measurements with this instrument agree with high-pressure viscosity data collected previously using a CTOF viscometer, as well as with literature values obtained with falling-body viscometers of the Bridgman design. It has been further determined that, for the small molecules included in this study, trends in solute diffusivity with respect to pressure follow the predictions of the Stokes−Einstein equation when the solvent viscosity is corrected as a function of pressure. Because the instrument described herein determines viscosity and diffusivity independently, the effect of pressure on analyte hydrodynamic radius can also be monitored. An analysis of ultrahigh pressure liquid chromatography (UHPLC) data was performed using the pressure-corrected diffusion coefficient of hydroquinone to demonstrate the effect of this phenomenon on the analysis of chromatographic performance.

A novel instrument for profiling the current density of nanoelectrospray ionization plumes in three dimensions has been developed. A hemispherically-shaped electrostatic lens at atmospheric pressure is found to be able to compress the space-charge in nano-ESI and increase the average current density in the plume to three times the nominal value. Ion transmission into a single-quadrupole mass spectrometer is found to roughly double using the electrostatic lens. Data also suggest that ion transmission into the first vacuum region for a skimmer-type mass spectrometer interface using nano-ESI may be typically 40% or better with no special focusing device used.

•Development of a constant pressure UHPLC gradient system.•Development of a fully-automated constant pressure UHPLC gradient system.•Peak capacities exceeding 800 for peptide separations.•Gradient separations at 45 kpsi.Commercial chromatographic instrumentation for bottom-up proteomics is often inadequate to resolve the number of peptides in many samples. This has inspired a number of complex approaches to increase peak capacity, including various multidimensional approaches, and reliance on advancements in mass spectrometry. One-dimensional reversed phase separations are limited by the pressure capabilities of commercial instruments and prevent the realization of greater separation power in terms of speed and resolution inherent to smaller sorbents and ultrahigh pressure liquid chromatography. Many applications with complex samples could benefit from the increased separation performance of long capillary columns packed with sub-2 μm sorbents. Here, we introduce a system that operates at a constant pressure and is capable of separations at pressures up to 45 kpsi. The system consists of a commercially available capillary liquid chromatography instrument, for sample management and gradient creation, and is modified with a storage loop and isolated pneumatic amplifier pump for elevated separation pressure. The system’s performance is assessed with a complex peptide mixture and a range of microcapillary columns packed with sub-2 μm C18 particles.