Column-Only Band Broadening in a Porous Shell Radially Elongated Pillar Array Column.

In the quest for better performing separation media for liquid chromatography, micropillar array columns have received great interest over the past years. While previous research was mainly focused around micropillar array columns (µPACs) filled with cylindrical pillars, this contribution discusses µPACs with rectangular pillars, which, for the first time, have been anodized and hence carry a mesoporous shell. We report on a series of on-chip measurements of the band broadening and flow permeability in a µPAC with very wide radially elongated pillars (3·75 µm) and with an interpillar distance (2 µm) between that of the first (2.5 µm) and second generation (1.25 µm) of cylindrical µPACs. Because of the extreme flow path tortuosity, this type of µPAC can produce very large plate numbers over a short distance. Despite the relatively large interpillar distance, we obtain Hmin = 0.26 µm for a nearly unretained component (phase retention factor, k' ≈ 0.24) and Hmin = 0.79 µm for a retained component with k' ≈ 3. The kinetic performance in terms of separation impedance (Ei = 19) is considerably improved compared to cylindrical pillar µPACs (Ei in range 40-50) and is in excellent agreement with the theoretical value for an open tubular channel with a rectangular cross-section (Ei = 18). This shows that rectangular µPACs can be represented as a parallel bundle of interconnected open-tubular channels.

An In-Depth Investigation of Micropillar Array Columns for the Separation of Finite-Sized Particles by Hydrodynamic Chromatography.

The exact moment approach (EMA) is adopted to predict, without any fitting parameters, the plate height curves for polystyrene microparticles of different sizes in micropillar array columns performed by hydrodynamic chromatography. The EMA allows us to decouple the contribution of horizontal and vertical dispersion terms and thus investigate the influence of pillar height and interpillar distance on separation performance. In the convection-controlled regime, we found that axial dispersion is mainly controlled by the vertical dispersion term, the latter being due to the flow-arresting top and bottom walls. This vertical contribution can be estimated from the axial dispersion in rectangular, open tubular channels formed between the pillars. Henceforth, plate height curves can be accurately predicted by simply adding the estimated vertical term to the horizontal dispersion term evaluated from 2D simulations. This finding allowed us to understand that, to improve separation performance, it is advisible to decrease the interpillar distance (expected result) and decrease the pillar height (counterintuitive result).

Selectivity and Resolving Power of Hydrophobic Interaction Chromatography Targeting the Separation of Monoclonal Antibody Variants.

This study presents a comprehensive investigation of the mechanistic understanding of retention and selectivity in hydrophobic interaction chromatography. It provides valuable insights into crucial method-development parameters involved in achieving chromatographic resolution for profiling molecular variants of trastuzumab. Retention characteristics have been assessed for three column chemistries, i.e., butyl, alkylamide, and long-stranded multialkylamide ligands, while distinguishing column hydrophobicity and surface area. Salt type and specifically chloride ions proved to be the key driver for improving chromatographic selectivity, and this was attributed to the spatial distribution of ions at the protein surface, which is ion-specific. The effect was notably more pronounced on the multialkylamide column, as proteins intercalated between the multiamide polymer strands, enabling steric effects. Column coupling proved to be an effective approach for maximizing resolution between molecular variants present in the trastuzumab reference sample and trastuzumab variants induced by forced oxidation. Liquid chromatography-mass spectrometry (LC-MS)/MS peptide mapping experiments after fraction collection indicate that the presence of chloride in the mobile phase enables the selectivity of site-specific deamidation (N30) situated at the heavy chain. Moreover, site-specific oxidation of peptides (M255, W420, and M431) was observed for peptides situated at the Fc region close to the CH2-CH3 interface, previously reported to activate unfolding of trastuzumab, increasing the accessible surface area and hence resulting in an increase in chromatographic retention.

Microfluidic Validation of the Diffusional Bridging Effect Suppressing Dispersion in Multicapillary Flow Systems.

The efficiency of liquid chromatography separations could be strongly improved by changing the current packed bed columns by a bundle of parallel capillary tubes. In practice, however, the polydispersity effect, which emanates from the inevitable small differences in capillary diameter, completely ruins this potential. The concept of diffusional bridging, introducing a diffusive cross talk between adjacent capillaries, has recently been proposed to resolve this. The present contribution provides the first experimental proof for this concept and quantitatively validates its underlying theory. This has been accomplished by measuring the dispersion of a fluorescent tracer in 8 different microfluidic channels with different degrees of polydispersity and diffusional bridging. The observed degree of dispersion reduction agrees very well with the theoretical predictions, hence opening the road to the use of this theory to design a new family of chromatographic beds, potentially offering unprecedented performance.

Computational Fluid Dynamics Study of the Dispersion Caused by Capillary Misconnection in Nano-Flow Liquid Chromatography.

It is well known that high-speed/high-efficiency separations in nano-flow liquid chromatography (LC) are very sensitive to the quality of the connections between the column and the rest of the instrument. In the present study, two types of connection errors (capillary misalignment and the occurrence of an inter-capillary gap) have been investigated using computational fluid dynamics. Interestingly, it has been found that large degrees of capillary misalignment (assuming an otherwise perfect contact between the capillary end-faces) can be afforded without introducing any significant dispersion over the entire range of investigated relative misalignment errors (0 ≤ ε/dcap ≤ 75%), even at the largest flow rates considered in nano-LC. On the other hand, when an inter-capillary gap is present, the dispersion very rapidly increases with the radial width Dc of this gap (extra variance ∼Dcn with n even reaching values above 4). The dependency on the gap length Lc is however much smaller. Results show that, when Dc ≤ 30 µm and Lc ≤ 200 µm, dispersion losses can be limited to the order of 1 nL2 at a flow of 1.5 µL/min, which is generally very small compared to the dispersion in the capillaries (20 µm i.d.) themselves. This result also reconfirms that zero-dead volume connectors with a sufficiently narrow bore can in theory be used without compromising peak dispersion in nano-LC, at least when the capillaries can be matched perfectly to the connector in- and outlet faces. The results are also indicative of the extra dispersion occurring inside microfluidic chips or in the connections between a microfluidic chip and the outer world.

On-Chip Comparison of the Performance of First- and Second-Generation Micropillar Array Columns.

Because of its dimensions, the recently introduced micropillar array columns are most suited for high-efficiency liquid chromatography separations in proteomics. Unlike the packed bed columns and capillary-based column formats, the micropillar array concept still has significant room to progress in terms of the reduction of its characteristic size (i.e., pillar diameter and interpillar distance) to open the road to even higher-efficiency separations and their applications. We report here on the on-chip comparison between first-generation (Gen 1) and second-generation (Gen 2) micropillar array columns wherein the pillar and interpillar size have been halved. Because of the on-chip measurements, the observed plate heights H represent the fundamental band broadening, devoid of any extra-column band-broadening effects. The observed reduction of H with a factor of 2 around the uopt-velocity and with a factor of 4 in the C-term dominated regime of the van Deemter-curve is in full agreement with the theoretically expected gain. This shows the pillar and interpillar size reduction could be effectuated without affecting the theoretical separation potential of the micropillar arrays. Compared to Gen 1, Gen 2 offers a 4-fold reduction of the required analysis time around the optimal velocity and about a 16-fold reduction in the C-term-dominated range.

Nonadditivity and Nonlinearity of Mobile and Stationary Zone Mass Transfer Resistances in Chromatography.

Using a two-zone moment analysis (TZMA) method based on Brenner's generalized dispersion theory for two-dimensional (2D) and three-dimensional (3D) periodic media, we investigated the mechanisms for dispersion in particulate media for liquid chromatography. This was done using a set of plate height data covering an unprecedented wide range of retention factors, diffusion coefficients, and velocities, all computed with unequaled accuracy. Applying Giddings' additivity test, based on alternatingly making the diffusion coefficient in the mobile and stationary zones infinitely large, the dispersion data clearly indicate a lack of additivity. Although this lack could be directly understood by identifying the existence of multiple parallel mass transfer paths, the additivity assumption interestingly overestimates the true C term band broadening (typically by more than 10%, depending on conditions and dimensionality of the system). However, Giddings originally asserted the occurrence of parallel paths would always lead to an underestimation of the dispersion. The origin of the lack of additivity is analyzed in detail and qualitatively explained. Finally, we also established a generic framework for the modeling of the effect of the reduced velocity and the retention coefficient on the C term in ordered chromatographic media. This led to the introduction of a new expression for the mobile zone mass transfer term, which, unlike the currently used literature expression, contains the complete k″ dependency.

Minimize Precolumn Band Broadening with Immiscible Solvent Sandwich Injection.

We investigated the possibility of reducing the effect of precolumn band broadening (PreCBB) by sandwiching the sample between two small plugs of an immiscible liquid. It has been found that in cases of severe PreCBB, improvements in peak efficiency can amount up to 20 times for the early-eluting compounds. For smaller degrees of PreCBB, the gain on the efficiency of early-eluting compounds is smaller (order of 50%), yet it is still significant. It has been verified that the presence of the immiscible fluid sandwich does not affect the repeatability of the analysis nor the linearity of the calibration curves used for analyte quantitation. It is also shown that the main effect of the two sandwich plugs is the minimization of the dispersion in the precolumn transfer tubing itself, which makes the method fundamentally different from pure on-column focusing methods such as the performance optimizing injection sequence (POISe) method. It is further demonstrated that both halves of the sandwich are needed, since the beneficial effect is clearly much smaller when only one plug is present. A drawback of the method is that some of the late-eluting peaks are slightly adversely affected by the presence of the sandwich liquid in the case where 127 µm i.d. tubing was used. The mechanism for this peak deterioration effect is at present still unclear but only occurs under gradient conditions and is clearly linked to the size of the sandwich plugs (the smaller the plugs, the smaller the adverse effect) and the internal diameter of the tubing used between the injection valve and the column.

Development of a 3D-Printable, Porous, and Chemically Active Material Filled with Silica Particles and its Application to the Fabrication of a Microextraction Device.

We report on the first successful attempt to produce a silica/polymer composite with retained C18 silica sorptive properties that can be reliably printed using three-dimensional (3D) FDM printing. A 3D printer provides an exceptional tool for producing complex objects in an easy and inexpensive manner and satisfying the current custom demand of research. Fused deposition modeling (FDM) is the most popular 3D-printing technique based on the extrusion of a thermoplastic material. The lack of appropriate materials limits the development of advanced applications involving directly 3D-printed devices with intrinsic chemical activity. Progress in sample preparation, especially for complex sample matrices and when mass spectrometry is favorable, remains a vital research field. Silica particles, for example, which are commonly used for extraction, cannot be directly extruded and are not readily workable in a powder form. The availability of composite materials containing a thermoplastic polymer matrix and dispersed silica particles would accelerate research in this area. This paper describes how to prepare a polypropylene (PP)/acrylonitrile-butadiene-styrene (ABS)/C18-functionalized silica composite that can be processed by FDM 3D printing. We present a method for producing the filament as well as a procedure to remove ABS by acetone rinsing (to activate the material). The result is an activated 3D-printed object with a porous structure that allows access to silica particles while maintaining macroscopic size and shape. The 3D-printed device is intended for use in a solid-phase microextraction (SPME) procedure. The proposed composite's effectiveness is demonstrated for the microextraction of glimepiride, imipramine, and carbamazepine. The complex honeycomb geometry of the sorbent has shown to be superior to the simple tubular sorbent, which proves the benefits of 3D printing. The 3D-printed sorbent's shape and microextraction parameters were fine-tuned to provide satisfactory recoveries (33-47%) and high precision (2-6%), especially for carbamazepine microextraction.

In-Depth Performance Analysis and Comparison of Monolithic and Particulate Zwitterionic Hydrophilic Interaction Liquid Chromatography Polymer Columns.

The kinetic performance of different zwitterionic hydrophilic interaction liquid chromatography polymer columns is evaluated and compared in-depth. For this purpose, two lab-made monolithic columns, synthesized with different crosslinkers, and a commercial particle packed column are considered. It is found that performance evaluation techniques, such as comparing plate height curves or fitted A-, B- and C-terms, obtained by fitting experimental plate height data to a plate height model, are complicated by the determination of a reliable characteristic length. This is due to the very different morphology of these column types, and the heterogeneity of the monolithic columns. The occurrence of a convective flow through the packed particle column further complicates the interpretation of the obtained fitting parameters, as part of the C-term is wrongfully attributed to the A-term. Therefore, the use of the kinetic plot method is suggested for the comparative evaluation of these columns, as kinetic plots do not require the determination of a characteristic length, nor rely on any fitting parameters. With the kinetic plot method, it is demonstrated that the lab-made monolithic columns outperform the packed particle column for plate counts between 10,000 and 800,000. This is attributed to the higher column efficiency of these columns, due to their small domain and skeleton sizes, and their high permeability, resulting from their high external porosity and the occasional occurrence of preferential flow paths.

Shape-Enhanced Open-Channel Hydrodynamic Chromatography.

Hydrodynamic chromatography (HDC) is a well-established analytical separation method for the size separation of nano- and microparticles and large molecular weight solutes such as synthetic polymers and proteins. We report on a theoretical study showing that the separation resolution of open-tubular HDC can be significantly enhanced by changing the cross-sectional shape of the separation channel. By enforcing Brenner's macro-transport approach, we provide theoretical/numerical evidence showing how the shape of the cross section influences quantitatively both the selectivity and the axial dispersion of the suspended particles in HDC. The separation performance of square-, triangle-, and star-shaped channel cross sections is compared to that of a cylindrical capillary over three decades of the particle Péclet number in terms of the minimal separation length and time to obtain the unit resolution of a two-particle mixture. Enhancement factors up to 400% are found in the case of triangular shapes, with the best performing case being the 70.6° angle, which can be obtained by KOH etching of bulk silicon.

Evaluation of Gas Chromatography Columns with Radially Elongated Pillars as Second-Dimension Columns in Comprehensive Two-Dimensional Gas Chromatography.

The present study investigated the use of a dedicated gas chromatography (GC) column (L = 70 cm, 75 µm deep, and 6.195 mm wide) with radially elongated pillars (REPs) as the second column in a comprehensive two-dimensional gas chromatography (GC × µGC) system. Three stationary phases [apolar polydimethylsiloxane (PDMS), medium polar room-temperature ionic liquid (RTIL) based on monocationic phosphonium, and polar polyethylene glycol (PEG-1000)] have been coated using the static method at constant pressure or using an original vacuum pressure program (VPP) from 400 to 4 mbar. The best efficiency reached up to N = 62,000 theoretical plates for a film thickness of 47 nm at 100 °C for an iso-octane peak (k = 0.16) at an optimal flow rate of 4.8 mL/min. The use of the VPP improved the efficiency by approximately 15%. Efficiencies up to 28,000 and 47,000 were obtained for PEG-1000 and RTIL, respectively. A temperature-programmed separation of a mixture of 11 volatile compounds on a PDMS-coated chip was obtained in less than 36 s. The PDMS-, PEG-1000-, and RTIL-coated chips were tested as the second column using a microfluidic reverse fill/flush flow modulator in a GC × µGC system. The REP columns were highly compatible with the operating conditions in terms of flow rate and with more than 30,000 plates for the iso-octane peak. Moreover, a commercial solvent called white spirit containing alkanes and aromatic compounds was injected in three sets of columns in normal and reverse modes, demonstrating the great potential of the chip as a second-dimension separation column.

Enhanced Sensitivity in Comprehensive Liquid Chromatography: Overcoming the Dilution Problem in LC × LC via Temperature-Responsive Liquid Chromatography.

In comprehensive two-dimensional liquid chromatography (LC × LC), solvents of high eluotropic strength are frequently used in the first dimension (1D), which lead to peak broadening in the second dimension (2D). In the majority of the current LC × LC column combinations, analytes are less than optimally refocused upon transfer to the second column, which negatively affects sensitivity. Furthermore, the typical combination of 1 or 2.1 mm columns in the 1D paired with a 3 mm (or broader) column in the 2D leads to at least a 9- or 4-fold dilution and a corresponding loss of sensitivity when using concentration-sensitive detectors. This occurs due to the enhanced radial dilution of the analytes in a broader column, while the sensitivity problem is further exacerbated in LC × LC due to the high flow operated 2D. In this paper, we introduce a solution to neutralize and inverse this dilution problem through a reconcentrating solution using temperature-responsive liquid chromatography (TRLC) in the 1D, which is a purely aqueous separation mode. Full solute refocusing at the 2D column head is thereby obtained when TRLC is combined with reversed-phase liquid chromatography (RPLC). This is shown for the combination of a 2.1 mm I.D. TRLC column with decreasing RPLC column diameters (3-2.1-1 mm) operated at the same linear velocities, hence a resulting decrease in dilution, respectively. Ultraviolet (UV) and electrospray ionization time-of-flight mass spectrometry (ESI-TOF-MS) detection were used to determine the experimental detection limits. Sensitivity improvements with UV detection were somewhat lower than expected, but represent ∼1.5- and 3-fold sensitivity enhancement when using a 1 mm I.D. column compared to 2.1 or 3 mm I.D. columns in the 2D, respectively. This is attributed to extra-column dispersion and the poorer performance of 1 mm I.D. columns. A major benefit of the use of 1 mm I.D. columns in the 2D is that it allows split-free coupling of 2D effluent with ESI-MS (at 450 µL/min), making the coupling robust and simple. When using ESI-MS even better, albeit more variable, sensitivity enhancements were obtained on the narrower columns. The benefits of the methodology are demonstrated for paraben test solutes and for phenolic compounds in a blueberry extract by TRLC × RPLC-UV-ESI-TOF-MS.

Wafer-Scale Particle Assembly in Connected and Isolated Micromachined Pockets via PDMS Rubbing.

The present contribution reports on a study aiming to find the most suitable rubbing method for filling arrays of separated and interconnected micromachined pockets with individual microspheres on rigid, uncoated silicon substrates without breaking the particles or damaging the substrate. The explored dry rubbing methods generally yielded unsatisfactory results, marked by very large percentages of empty pockets and misplaced particles. On the other hand, the combination of wet rubbing with a patterned rubbing tool provided excellent results (typically <1% of empty pockets and <5% of misplaced particles). The wet method also did not leave any damage marks on the silicon substrate or the particles. When the pockets were aligned in linear grooves, markedly the best results were obtained when the ridge pattern of the rubbing tool was moved under a 45° angle with respect to the direction of the grooves. The method was tested for both silica and polystyrene particles. The proposed assembly method can be used in the production of medical devices, antireflective coatings, and microfluidic devices with applications in chemical analysis and/or catalysis.

Self-organization of agitated microspheres on various substrates.

The vibration dynamics of relatively large granular grains is extensively treated in the literature, but comparable studies on the self-assembly of smaller agitated beads are lacking. In this work, we investigate how the particle properties and the properties of the underlying substrate surface affect the dynamics and self-organization of horizontally agitated monodisperse microspheres with diameters between 3 and 10 µm. Upon agitation, the agglomerated hydrophilic silica particles locally leave traces of particle monolayers as they move across the flat uncoated and fluorocarbon-coated silicon substrates. However, on the micromachined silicon tray with relatively large surface roughness, the agitated silica agglomerates form segregated bands reminiscent of earlier studies on granular suspensions or Faraday heaps. On the other hand, the less agglomerated hydrophobic polystyrene particles form densely occupied monolayer arrangements regardless of the underlying substrate. We explain the observations by considering the relevant adhesion and friction forces between particles and underlying substrates as well as those among the particles themselves. Interestingly, for both types of microspheres, large areas of the fluorocarbon-coated substrates are covered with densely occupied particle monolayers. By qualitatively examining the morphology of the self-organized particle monolayers using the Voronoi approach, it is understood that these monolayers are highly disordered, i.e., multiple symmetries coexist in the self-organized monolayers. However, more structured symmetries are identified in the monolayers of the agitated polystyrene microspheres on all the substrates, albeit not all precisely positioned on a hexagonal lattice. On the other hand, both the silica and polystyrene monolayers on the bare silicon substrates transition into less disordered structures as time progresses. Using Kelvin probe force microscopy measurements, we show that due to the tribocharging phenomenon, the formation of particle monolayers is promoted on the fluorocarbon surface, i.e., a local electrostatic attraction exists between the particle and the substrate.

Graph Convolutional Networks for Improved Prediction and Interpretability of Chromatographic Retention Data.

Machine learning is a popular technique to predict the retention times of molecules based on descriptors. Descriptors and associated labels (e.g., retention times) of a set of molecules can be used to train a machine learning algorithm. However, descriptors are fixed molecular features which are not necessarily optimized for the given machine learning problem (e.g., to predict retention times). Recent advances in molecular machine learning make use of so-called graph convolutional networks (GCNs) to learn molecular representations from atoms and their bonds to adjacent atoms to optimize the molecular representation for the given problem. In this study, two GCNs were implemented to predict the retention times of molecules for three different chromatographic data sets and compared to seven benchmarks (including two state-of-the art machine learning models). Additionally, saliency maps were computed from trained GCNs to better interpret the importance of certain molecular sub-structures in the data sets. Based on the overall observations of this study, the GCNs performed better than all benchmarks, either significantly outperforming them (5-25% lower mean absolute error) or performing similar to them (<5% difference). Saliency maps revealed a significant difference in molecular sub-structures that are important for predictions of different chromatographic data sets (reversed-phase liquid chromatography vs hydrophilic interaction liquid chromatography).

Methods to determine the kinetic performance limit of contemporary chromatographic techniques.

By combining separation efficiency data as a function of flow rate with the column permeability, the kinetic plot method allows to determine the limits of separation power (time vs. efficiency) of different chromatographic techniques and methods. The technique can be applied for all different types of chromatography (liquid, gas, or supercritical fluid), for different types of column morphologies (packed beds, monoliths, open tubular, micromachined columns), for pressure and electro-driven separations and in both isocratic and gradient elution mode. The present contribution gives an overview of the methods and calculations required to correctly determine these kinetic performance limits and their underlying limitations.

Advances and Challenges in Extremely High-Pressure Liquid Chromatography in Current and Future Analytical Scale Column Formats.

The present study speculates on the potential gain we can expect of a further leap in pressure by moving from the current 1500 bar to a futuristic 3000 bar as well as reviews the main impediments to be expected when trying to realize such systems. The study focuses on so-called "analytical scale" separations, i.e., separations that are currently carried out in 2.1 mm columns, as this is the current state-of-the-art in ultrahigh-pressure liquid chromatography (UHPLC) instrumentation.

Prototyping of a Microfluidic Modulator Chip and Its Application in Heart-Cut Strong-Cation-Exchange-Reversed-Phase Liquid Chromatography Coupled to Nanoelectrospray Mass Spectrometry for Targeted Proteomics.

A novel multilayer modulator chip offering a robust miniaturized interface for multidimensional liquid chromatography has been developed. The thermoplastic microfluidic device comprises five tailor-made functional layers, and the chip is compatible with commercially available switching-valve technology. The modulator chip allows for robust ultrahigh-pressure operation up to 65 MPa. Peak-dispersion characteristics of system peaks were assessed directly at the valve outlet by monitoring fluorescein injection profiles with laser-induced fluorescence detection. Integration of a microporous monolithic mixing entity in the microchannels significantly narrows the resulting peak profile. Proof-of-concept of the applicability of the microfluidic modulator chip is demonstrated in a heart-cut multidimensional strong-cation-exchange-reversed-phase liquid chromatography proteomics analysis workflow coupled to nanoelectrospray mass spectrometry for the target analysis of Glu-1-Fibrinopeptide B spiked in a protein digest mixture of bovine serum albumin.

Spatial Segregation of Microspheres by Rubbing-Induced Triboelectrification on Patterned Surfaces.

Particle (monolayer) assembly is essential to various scientific and industrial applications, such as the fabrication of photonic crystals, optical sensors, and surface coatings. Several methods, including rubbing, have been developed for this purpose. Here, we report on the serendipitous observation that microparticles preferentially partition onto the fluorocarbon-coated parts of patterned silicon and borosilicate glass wafers when rubbed with poly(dimethylsiloxane) slabs. To explore the extent of this effect, we varied the geometry of the pattern, the substrate material, the ambient humidity, and the material and size of the particles. Partitioning coefficients amounted up to a factor of 12 on silicon wafers and even ran in the 100s on borosilicate glass wafers at zero humidity. Using Kelvin probe force microscopy, the observations can be explained by triboelectrification, inducing a strong electrostatic attraction between the particles and the fluorocarbon zones, while the interaction with the noncoated zones is insignificant or even weakly repulsive.