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The vast majority of separations in liquid chromatography (LC) still use the typical packed particle bed format, most commonly with fully or superficially porous particles in particle sizes as low as 1.3 urn. As an alternative, monolithic columns have been the topic of many studies, but they are currently used only in some niche applications. Research into perfectly ordered microfabricated columns has shown tremendous possibility for these high performance columns for use in nano-LC, but their development is still ongoing. The possibilities that emerging three-dimensional (3D) printing technology offers make it theoretically possible to develop any imaginable structure with high precision, but the technology is currently limited. This article provides a critical review of all these technologies and demonstrates how further development of chromatographic columns will be of paramount importance in the future., The improvements in instrument and column performance in liquid chromatography (LC) over the past 15-20 years have resulted in an almost 10-fold reduction in analysis time and threefold increase in [...]

The band dispersion in channels with an orderly pillar structure with a pressure-driven liquid flow was determined. Several channels with different geometries were etched in a silicon wafer and enclosed by a glass wafer. The microchannels obtained had the same depth, pillar disposition, and overall porosity, but different pillar diameters and channel widths. The broadening of narrow bands of a fluorescent sample solution flowing through the channels was measured using a fluorescence microscope. It was shown that the peak dispersion occurring in the channels can be much lower than in conventional packed columns, as a consequence of the higher degree of order of the solid structure. Reduced plate heights of ~0.2 could be obtained for (nonretained) bands. No correlation was found between the aspect ratio (the ratio of the channel width and the pillar diameter) and band dispersion. The geometrical construction of the sidewall region was shown to play a critical role for channel performance. A good agreement was found with predictions for the optimal sidewall geometry obtained previously with simulation studies.

A key bottleneck in developing chromatographic material is the chemically entangled control of morphology, pore structure, and material chemistry, which holds back precision material manufacture in order to pursue advanced separation performance. In this work, a precision manufacture strategy based on droplet microfluidics was developed, for production of highly efficient chromatographic microspheres with independent control over particle morphology, pore structure and material chemistry. The droplet-synthesized microspheres display extremely narrow particle size distribution (CV<3 %), enabling a 100 % production yield due to complete elimination of sieving steps. More importantly, the size of the droplet-synthesized microspheres is freely adjustable without the need for re-optimizing chemical recipes or reaction conditions. The resulting materials exhibit excellent separation efficiencies, achieving a reduced plate height of h min =1.67. This precision manufacture strategy also allows for flexible pore design and continuous pore size adjustment across three orders of magnitudes, providing a novel vehicle for resolution fine-tuning targeting protein separation. Besides traditional silica, organic-inorganic hybrid silica, zirconia, and titania microspheres can also be precisely synthesized on the same platform, supporting various separation applications and operating conditions. Powered by precision manufacture, super-throughput production, and versatile chemistry, the high-performance droplet-synthesized separation material will pave the way towards green and precision chromatographic industry., (© 2024 Wiley-VCH GmbH.)

A proof-of-concept system is presented for the hyphenation of spatial comprehensive three-dimensional liquid chromatography (3D-LC) to mass spectrometry (MS) detection via a robotic-microfluidic interface. A three-dimensional fractal microflow distributor, incorporating 16 parallel RP monolithic capillary columns arranged in a 4 × 4 configuration, was connected to an X-Y-Z robotic system. This setup facilitated the deposition of successive arrays of microdroplets onto an MS target plate. To minimize carryover during droplet deposition, a strategy was implemented in which the distance between the target plate and the capillary was gradually increased during the deposition process. System-level variation in travel time and subsequent flow rates across parallel columns was assessed and translated in retention alignment based on injection of a protein standard. The successful separation of intact proteins was demonstrated through a parallel 4 × 4 column configuration, applying MALDI-MS detection after microdroplet spotting on an MS target plate. Furthermore, the discussion encompasses high-throughput MS imaging detection within the framework of spatial 3D-LC.

Due to their potential adverse health effects, some N-nitrosamines in drug products are strictly regulated with very low maximum daily intake limits. Nitrosamines can be formed from the reaction of nitrite and secondary or tertiary amines when both species co-exist in the drug synthesis or formulation process. One key strategy to mitigate nitrosamine risk in drugs is to select low-nitrite containing pharma excipients for formulation. It is necessary to develop a sensitive method for trace nitrite determination in pharma excipients as it enables drug producers to study nitrosamine formation kinetics and select excipient suppliers. This study details the development and validation of a two-dimensional ion chromatography mass spectrometry (2D-IC/MS) method for trace nitrite determination in hydroxypropyl methylcellulose (HPMC), one of the most important pharmaceutical excipients used in many drug formulations. The 2D-IC system was operated in heart-cutting mode with a concentrator column coupling the two dimensions. A standard bore anion-exchange column was used in the first dimension ( 1 D) to enable a large volume injection for increased sensitivity and provide improved resolution between nitrite and the interfering chloride peak. A high efficiency microbore anion-exchange column with different selectivity was used in the second dimension ( 2 D) to resolve nitrite from other interfering species. The use of 2D-IC resulted in significantly improved resolution, solving the sensitivity loss issue due to ion suppression from an otherwise 1D separation. MS detection with selective ion monitoring and isotope labeled nitrite internal standard further improve the method specificity, accuracy, and ruggedness, as compared with conductivity detection. For trace determination, it is also extremely important to have a clean blank. For this purpose, a novel cleaning procedure using a strong anion wash was developed to remove nitrite contamination from labware. The optimized method was validated with linearity of nitrite in the concentration range of 18.5-5005.8 ng/g having a regression coefficient of >0.9999, precision with RSD at 3.5-10.1 % and recovery of 90.5-102.4 %. The limit of detection and limit of quantitation were 8.9 and 29.6 ng/g relative to the HPMC sample, or equivalent to 89 and 296 pg/g in the sample solution, respectively., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

The use of a ternary mobile-phase system comprising ammonium sulphate, sodium chloride, and phosphate buffer was explored to tune retention and enhance selectivity in hydrophobic interaction chromatography. The accuracy of the linear solvent-strength model to predict protein retention with the ternary mobile-phase system based on isocratic scouting runs is limited, as the extrapolated retention factor at aqueous buffer conditions (k 0 ) cannot be reliably established. The Jandera retention model utilizing a salt concentration averaged retention factor (k¯ 0 ) in aqueous buffer for ternary systems overcomes this bottleneck. Gradient retention factors were derived based on isocratic scouting runs after numerical integration of the isocratic Jandera model, leading to retention-time prediction errors below 11 % for linear gradients. Furthermore, an analytical expression was formulated to predict HIC retention for both linear and segmented linear gradients, considering the linear solvent-strength (LSS) model within ternary salt systems, relying on a fixed k 0 . The approach involved conducting two gradient scouting runs for each of the two binary salt systems to determine model parameters. Retention-time prediction errors for linear gradients were below 12 % for lysozyme and 3 % for trypsinogen and α-chymotrypsinogen A. Finally, the analytical expression for a ternary mobile-phase system was used in combination with a genetic algorithm to tune the HIC selectivity. With an optimized segmented ternary gradient, a critical-pair separation for a mixture of 7 proteins was achieved within 15 min with retention-time prediction errors ranging between 0.7 and 15.7 %., Competing Interests: Declaration of competing interest The authors declare that they have no conflict in interest., (Copyright © 2024. Published by Elsevier B.V.)

Spatial comprehensive three-dimensional chromatography (3D-LC) offers an innovative approach to achieve unprecedented resolving power in terms of peak capacity and sample throughput. This advanced technique separates components within a 3D separation space, where orthogonal retention mechanisms are incorporated. The parallel development of the second- and third-dimension stages effectively overcomes the inherent limitation of conventional multidimensional approaches, where sampled fractions are analyzed sequentially. This review focuses on the design aspects of the microchip for spatial 3D-LC and the selection of orthogonal separation modes to enable the analysis of intact proteins. The design considerations for the flow distributor and channel layout are discussed, along with various approaches to confine the flow during the subsequent development stages. Additionally, the integration of stationary phases into the microchip is addressed, and interfacing to mass spectrometry detection is discussed. According to Pareto optimality, the integration of isoelectric focusing, size-exclusion chromatography, and reversed-phase chromatography in a spatial 3D-LC approach is predicted to achieve an exceptional peak capacity of over 30,000 within a 1-h analysis, setting a new benchmark in chromatographic performance.

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., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

This paper focuses on 3D printing using digital light processing (DLP) to create microchannel devices with inner diameters of 100, 200, and 500 µm and cater flow-through applications within the realm of analytical chemistry, in particular high-pressure liquid chromatographic separations. Effects of layer thickness and exposure time on channel dimensions and surface roughness were systematically investigated. Utilizing a commercially accessible 3D printer and acrylate resin formulation, we fabricated 100-500 µm i.d. squared and circular channel designs minimizing average surface roughness (< 20%) by applying a 20-µm layer thickness and exposure times ranging from 1.1 to 0.7 s. Pressure resistance was measured by encasing microdevices in an aluminum chip holder that integrated flat-bottom polyetheretherketon (PEEK) nanoports allowing to establish the micro-to-macro interface to the HPLC instrument. After thermal post-curing and finetuning the clamping force of the chip holder, a maximum pressure resistance of 650 bar (1.5% RSD) was reached (n = 3). A polymer monolithic support structure was successfully synthesized in situ with the confines of a 500 µm i.d. 3D printed microchannel. A proof-of-concept of a reversed-phase chromatographic gradient separation of intact proteins is demonstrated using an aqueous-organic mobile-phase with isopropanol as organic modifier., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Austria, part of Springer Nature.)

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 ( N 30 ) situated at the heavy chain. Moreover, site-specific oxidation of peptides ( M 255 , W 420 , and M 431 ) 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.

A diagnostic test based on microfluidic image cytometry and machine learning has been designed and applied for accurate classification of erythrocytes and leukocytes, including a unique fully-automated 5-part quantitative differentiation into neutrophils, lymphocytes, monocytes, eosinophils, and basophils, using minute amounts of whole blood in a single counting chamber. A low-cost disposable multilayer microdevice for microfluidic image cytometry was developed that comprises a 1 mm × 22 mm × 70 μm ( w × l × h ) rectangular microchannel, allowing the analysis of trace volume of blood (20 μL) for each assay. Automated analysis of digitized binary images applying a border following algorithm was performed allowing the qualitative analysis of erythrocytes. Bright-field imaging was used for the detection of erythrocytes and fluorescence imaging for 5-part differentiation of leukocytes after acridine orange staining, applying a convolutional neural network enabling unparalleled speed for identification and automated morphology classification yielding 98.57% accuracy. Blood samples were obtained from 30 volunteers and count values did not significantly differ from data obtained using a commercial automated hematology analyzer.

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.

Polymer monolithic stationary phases are designed as a continuous interconnected globular material perfused by macropores. Like packed column, where separation efficiency is related to particle diameter, the efficiency of monoliths can be enhanced by tuning the size of both the microglobules and macropores. This protocol described the synthesis of poly(styrene-co-divinylbenzene) monolithic stationary phases in capillary column formats. Moreover, guidelines are provided to tune the macropore structure targeting high-throughput and high-resolution monolith chromatography. The versatility of these columns is exemplified by their ability to separate tryptic digests, intact proteins, and oligonucleotides under a variety of chromatographic conditions. The repeatability of the presented column fabrication process is demonstrated by the successful creation of 12 columns in three different column batches, as evidenced by the consistency of retention times (coefficients of variance [c.v.] = 0.9%), peak widths (c.v. = 4.7%), and column pressures (c.v. = 3.1%) across the batches., (© 2023 Wiley-VCH GmbH.)

Ion mobility spectrometry-mass spectrometry (IMS-MS) is experiencing rapid growth in proteomic studies, driven by its enhancements in dynamic range and throughput, increasing the quantitation precision, and the depth of proteome coverage. The core principle of ion mobility spectrometry is to separate ions in an inert gas under the influence of an electric field based on differences in drift time. This minireview provides an introduction to IMS operation modes and a description of advantages and limitations is presented. Moreover, the principles of trapped IMS-MS (TIMS-MS), including parallel accumulation-serial fragmentation are discussed. Finally, emerging applications linked to TIMS focusing on sample throughput (in clinical proteomics) and sensitivity (single-cell proteomics) are reviewed, and the possibilities of intact protein analysis are discussed., (© 2023 Wiley-VCH GmbH.)

Digital light processing (DLP) 3D printing is rapidly advancing and has emerged as a powerful additive manufacturing approach to fabricate analytical microdevices. DLP 3D-printing utilizes a digital micromirror device to direct the projected light and photopolymerize a liquid resin, in a layer-by-layer approach. Advances in vat and lift design, projector technology, and resin composition, allow accurate fabrication of microchannel structures as small as 18 × 20 µm. This review describes the latest advances in DLP 3D-printing technology with respect to instrument set-up and resin formulation and highlights key efforts to fabricate microdevices targeting emerging (bio-)analytical chemistry applications, including colorimetric assays, extraction, and separation., Competing Interests: Declaration of Competing Interest The authors declare no conflict of interest., (Copyright © 2023 Elsevier B.V. All rights reserved.)

For the first time, low trace-level removal of perfluorooctanesulfonic acid (PFOS), i.e., 20-500 μg/L (ppb), from aqueous solutions using zeolitic imidazolate framework-8 (ZIF-8)-coated copper sheet (ZIF-8@Cu) composite is reported here. In comparison with different commercial activated carbon (AC) and all-silica zeolites, the composite showed the highest removal rate of 98%, which remained consistent over a wide range of concentrations. Additionally, no adsorbent leaching from the composite was noticed, which eradicated pre-analysis steps such as filtration and centrifugation, unless needed for other adsorbents studied here. The composite displayed fast uptake with saturation reaching within 4 h, irrespective of the initial concentration. However, the morphological and structural characterization revealed surface degradation of ZIF-8 crystals, along with a decline in the crystal size. The adsorption of PFOS on ZIF-8 crystals was linked to chemisorption, as the surface degradation surges with an increase in PFOS concentration or with cyclic exposure at low concentrations. Methanol seemingly removed surface debris (partially), thus providing access to ZIF-8 beneath the surface debris. Overall, the findings demonstrate that at low trace ppb-level PFOS concentrations ZIF-8 can be considered as a possible candidate for PFOS removal, even though it suffers slow surface degradation, it also removes efficiently PFOS molecules from aqueous solutions.

Hydrophobic interaction chromatography (HIC) is a chromatographic technique that mainly targets the separation of biomolecules (intact proteins, monoclonal antibodies, etc.) based on the difference in surface hydrophobicity while applying non-denaturing conditions. This protocol paper provides guidelines for setting-up robust HIC analysis and considers the instrument configuration, mobile-phase and sample preparation, as well as chromatographic conditions and settings. The separation of a mixture of intact proteins and monoclonal antibodies is demonstrated by applying conventional HIC conditions, that is, using a mildly hydrophobic (C 4 ) stationary phase in combination with an inverse ammonium sulphate gradient dissolved in aqueous phosphate buffer. The effect of sample-preparation conditions on sample breakthroughs is presented. Finally, good run-to-run repeatability (relative standard deviation < 2%) is demonstrated for five different columns obtained from three different column lots, considering chromatographic retention, peak width, peak area and column pressure., Competing Interests: VUB and BOKU authors declare that they have no conflict in interest. Sebastiaan Eeltink is Editor‐in‐Chief of Analytical Science Advances. Daniel Eβer is an employee of YMC Europe., (© 2022 The Authors. Analytical Science Advances published by Wiley‐VCH GmbH.)

Having fundamental insights in the properties of stationary phases and in the driving forces during the column packing process, is crucial to obtain highly efficient and robust packed-bed column technologies for use in separation science. Here we discuss the properties of the most commonly-used stationary-phases, i.e., silica particulate materials, including fully-porous and core-shell silica particles and provide an overview of the most commonly used column hardware and available frit technologies. The different packing approaches that are considered are dry packing, high-pressure slurry packing, electrokinetic packing, and packing using centrifugal forces. In particular, sedimentation of particles in slurries and particle interaction during the packing process affecting the resulting sphere packing are discussed., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier B.V. All rights reserved.)

The present study discusses UHPLC method development allowing to establish ultra-high-resolution separations in gradient mode while operating at the kinetic performance limits, targeting the analysis of complex residual multi-class antibiotic samples in food products. The peak capacity and gradient occupation have been systematically assessed at different flow rates and gradient duration. The small particle size (1.5 µm core-shell particles) used in this study limits the mass-transfer contribution to band broadening when operating at high flow rate. As a result, for high-throughput analysis, high-pressure (1500 bar) operation leads to high resolving power where the gradient steepness dominates the peak capacity generation vs mass-transfer resistance. To reach the highest possible resolving power within a practically acceptable analysis time, one should use coupled-column systems at 1500 bar and adjust the gradient steepness correspondingly. Coupling four columns and applying a shallow gradient at 1500 bar led to a sample peak capacity of 379 in 140 min, allowing to resolve 71% of the analytes in a mixture composed of 61 milk antibiotics., Competing Interests: Declaration of Competing Interest Authors affiliated with VUB and Flanders Research Institute for Agriculture, Fisheries and Food declare no conflict of interest. Dr. De Pra and Dr. Steiner are employees of Thermo Fisher Scientific., (Copyright © 2022. Published by Elsevier B.V.)

The design aspects of microfluidic chips for spatial three-dimensional chromatography featuring an interconnected channel network and targeting protein analysis are discussed, and the corresponding kinetic performance limits have been established using a Pareto-optimality approach. The pros and cons to integrate different separation mechanisms (IEF, CE, SEC, RPLC, HILIC, HIC, and IEX) are discussed considering development stages in the spatial domain ( x LC) in the first and second dimension and time domain ( t LC) for the third dimension. Based on Pareto-optimization, we discuss the considerations of the channel length, particle diameter, and the effect of number of second- and third-dimension channels on the resulting peak capacity of a spatial x IEF × x SEC × t RPLC device. Novel equations are proposed to determine the peak capacity in x SEC and to account for sample modulation affected by the number of second- and third-dimension channels. The corresponding Pareto fronts have been constructed demonstrating the resolving power, in terms of peak capacity and analysis time, considering current state-of-the-art prototyping methodologies. A microfluidic spatial prototype chip with an integrated channel layout (64 2 D and 4096 3 D channels) has been created, which has the potential to yield a peak capacity of 32,600 within only 44 min of the total analysis time, by implementing x IEF × x SEC × t RPLC separation stages.

This study reports on the assessment of the separation performance of hydrophobic interaction chromatography for intact protein analysis using non-porous butyl polymethacrylate phases. The maximum peak capacity in inverse gradient mode was reached at a volumetric flow rate which was significantly (10-20 times) higher than the flow rate yielding the minimum plate height in isocratic mode, as the gradient volume dominates the peak-capacity generation. The flow rate yielding the maximum peak capacity increased with decreasing gradient volume, i.e., steeper gradients, and also depends on the magnitude of the mass-transfer contribution to peak dispersion (affected by particle size and molecular diffusion coefficient of proteins) at these high flow rates. The maximum peak capacity using a 100 mm long column packed with 4 µm particles for steep 7.5 min gradients was determined to be 60. Increasing the column length by coupling columns leads to better gradient performance than increasing the gradient duration for gradients of 60 min and longer. Using a coupled column system (2 × 100 mm long columns packed with 4 µm particles), the maximum peak capacity was determined to be 105, which was 33% higher compared to that of a single column while applying a similar gradient volume. Decreasing the particle size to 2.3 µm leads to higher peak capacities even though the column was operated at lower volumetric flow rate. The maximum peak capacity obtained with the 2.3 µm column was 128% higher than was obtained with the coupled column. Even at suboptimal conditions, the 2.3 µm column yields a higher peak capacity (14%) than when using two coupled columns packed with 4 µm at optimal conditions (gradient time of 120 min and a flow rate of 0.5 mL/min)., Competing Interests: Declaration of Competing Interest Dr Eβer is an employee of YMC Europe. Other authors declare that they have no conflict in interest., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Trifluoroacetic acid (TFA) is commonly used as mobile phase additive to improve retention and peak shape characteristics in hydrophilic interaction liquid chromatography (HILIC) of intact proteins. However, when using electrospray ionization-mass spectrometry (ESI-MS) detection, TFA may cause ionization suppression and adduct formation, leading to reduced analyte sensitivity. To address this, we describe a membrane-based microfluidic chip with multiple parallel channels for the selective post-column removal of TFA anions from HILIC. An anion-exchange membrane was used to physically separate the column effluent from a stripper flow solution comprising acetonitrile, formic acid, and propionic acid. The exchange of ions allowed the post-column removal of TFA used during HILIC separation of model proteins. The multichannel design of the device allows the use of flow rates of 0.2 mL/min without the need for a flow splitter, using mobile phases containing 0.1% TFA (13 mM). Separation selectivity and efficiency were maintained (with minor band broadening effects) while increasing the signal intensity and peak areas by improving ionization and reducing TFA adduct formation.

The present contribution discusses recent advances in ultra-high-pressure liquid chromatography (UHPLC) and multi-dimensional liquid chromatography (MDLC) technology. First, new developments in UHPLC column technology and system design are highlighted. The latter includes a description of a novel injector concept enabling method speed-up, emerging detectors, and instrument diagnostics approaches. Next, online MDLC workflows are reviewed and advances in modulation technology are highlighted. Finally, key applications published in 2020 are reviewed., (© 2021 The Authors. Analytical Science Advances published by Wiley‐VCH GmbH.)

This review highlights the current trends and the most recent advances in the field of preparation and application of organic polymer-based monolithic materials and covers literature published in 2020. A short historical background is provided and protocols to anchor monoliths covalently to the wall of the column/separation device are discussed. Furthermore, advances in tuning the macroporous structure and establishing its link to separation performance are conferred. Finally, method development and key applications using novel monolithic columns are discussed., Competing Interests: The authors have declared no conflict of interest., (© 2021 The Authors. Analytical Science Advances published by Wiley‐VCH GmbH.)

In spatial comprehensive three-dimensional chromatography (3D-LC) components are separated within a three-dimensional separation space that can lead to unprecedented resolving power, in terms of peak capacity and peak-production rate. The maximum peak capacity is the product of the peak capacities achieved in the individual dimensions when orthogonal retention mechanisms are incorporated. The parallel development of the second- and third-dimension separation stages overcomes the fundamental limitation of conventional multi-dimensional approaches, in which sampled fractions are analyzed sequentially. General considerations for chip design are discussed and possibilities and prospects to establish spatial comprehensive 3D-LC analysis are presented., Competing Interests: Declaration of competing interest The authors declare that they have no competing financial interest or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)

We report on the design and performance of in-house built column cartridges that can be directly screwed into the ports of a commercial rotor-stator valve to minimize extra-column band broadening and pressure-drop losses when pursuing ultra-fast separations such as those needed in 2D and 3D-LC separations. Two basic designs were evaluated and were compared with the results obtained with a commercial screw-in column cartridge. The system produces an extra-column band broadening as low as 0.05 to 0.1 μL 2 for the employed UV-detector set-up. Despite these very low values, the obtained separation efficiency of the in-house fabricated cartridge columns was very low, corresponding to a reduced minimal plate height around h=7 at the very best, which, for the 1.7 μm particle and 26.4 mm long columns corresponds to a number of theoretical plates of N=2200 under isocratic conditions. A similar poor performance was obtained with a commercial column cartridge with similar dimensions using the same set-up. One possible explanation of the observed performance could be found in the inner diameter of the column cartridges (i.d. =0.75 mm and 1 mm) which, for the employed sub 2-μm particles, falls into a region of column diameters that, according to literature models, is most likely to suffer from inherent packing problems., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020. Published by Elsevier B.V.)

This study involves the systematic assessment of the effects of system configuration on dispersion, pressure, and retention characteristics while operating a 1500 bar UHPLC system with 2.1 mm i.d. × 100 mm long columns packed with 1.5 µm core-shell particles in isocratic and gradient mode. Altering the system configuration by changing the i.d. of connection tubing and flow cells affects the elution time, dispersion characteristics, and the kinetic performance limits of the system. The gain in separation efficiency when decreasing tubing i.d. from 100 to 75 µm was found to contribute more to the decrease in separation impedance and the position of the kinetic performance curve than the loss in available column pressure induced by the narrower tubing. When applying steep gradients, characterized by gradient-to-column dead-time ratio < 7, optimizing instrument configuration leads to either a significant time gain factor of 3.9 without compromising peak capacity, or a gain in peak capacity with a gain factor of 1.3 while maintaining the analysis time constant. Due to the reduced fluidic volume of connection tubing of smaller i.d., a decrease in residence time is obtained. At the same time, an increase in k was observed due to a pressure-induced retention effect, and this effect is significant for late-eluting analytes., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020. Published by Elsevier B.V.)

This study targets the synthesis of high external-porosity poly(styrene-co-divinylbenzene) monolithic support structures with macropore and globule sizes in the sub-micron range, aiming at the realization of high-speed and high-resolution gradient separations of intact proteins and peptides. The thermodynamic and kinetic aspects of the free-radical polymerization synthesis were adjusted by tuning the porogen to monomer ratio, the porogen ratio, the initiator content, and polymerization temperature. Next, column morphology was linked to eddy-dispersion and mobile-phase mass-transfer contributions and the chromatographic performance limits were benchmarked against conventional packed columns and silica monoliths. Polymer monolithic structures yielding a separation impedance as low as 976 were created allowing to generate N > 1,000,000 (for an unretained marker), albeit the expense of very long analysis times. Decreasing the macropore and globule sizes below a certain threshold led to significant increase in eddy dispersion, as globular entities agglomerate, and a small number of large flow-through pores permeate the overall fine interconnected polymer network with small diameter flow-through pores. The potential of monolith chromatography for proteomics application is demonstrated with a ballistic 6 s gradient separation of intact proteins and a high-resolution nanoLC-Orbitrap mass spectrometric analysis of a tryptic E. coli digest applying a coupled-column system., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)

The performance of columns packed with 1.7 µm particles for aqueous size-exclusion chromatography was assessed at high-pressure conditions and linked to particle- and column-bed integrity. Decreasing the particle size from 3.5 µm to 1.7 µm increases the resolution due to the improved mass-transfer characteristics, allowing to significantly speed-up analysis without compromising the selectivity. A sub-minute separation of intact proteins was realized on a 4.6 mm i.d × 75 mm long column packed with 1.7 µm SEC particles applying a flow rate of 1.8 mL/min, corresponding to a column pressure of 530 bar. Ultra-high pressure operation (exceeding manufacturer's recommendations) resulted in peak deformation, a shift towards earlier retention times, and an alteration in selectivity. To gain insights in the mechanisms of column deterioration, short 30 mm long columns were operated at UHPLC conditions, maximizing the pressure drop over individual particles. This resulted in the presence of fractured particles situated at the column outlet, as verified by scanning electron micrographs. Mercury-intrusion porosimetry and argon-adsorption measurements did not reveal significant differences in intraparticle volume between particle batches sampled before and after pressure stress testing. As particles at the column outlet fracture (but not collapse) at high pressure operation, a void was formed at the column inlet. The degradation of the separation performance appeared to be the result of a decrease in interparticle pore volume., Competing Interests: Declaration of Competing Interests The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)

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.