Advancing HIC method development: Retention-time modeling and tuning selectivity with ternary mobile-phase systems.

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 (k0) 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 k0. 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 %.

Highly sensitive two-dimensional ion chromatography mass spectrometry method for nitrite determination in hydroxypropyl methylcellulose.

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 (1D) 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 (2D) 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.

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

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

Digital light processing 3D printing of microfluidic devices targeting high-pressure liquid-phase separations.

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.

Toward Unrivaled Chromatographic Resolving Power in Proteomics: Design and Development of Comprehensive Spatial Three-Dimensional Liquid-Phase Separation Technology.

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.

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.

Comprehensive quantitative analysis of erythrocytes and leukocytes using trace volume of human blood using microfluidic-image cytometry and machine learning.

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.

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.

Exploiting ion-mobility mass spectrometry for unraveling proteome complexity.

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.

A protocol for fabrication of polymer monolithic capillary columns and tuning the morphology targeting high-resolution bioanalysis in gradient-elution liquid chromatography.

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.

Recent developments in digital light processing 3D-printing techniques for microfluidic analytical devices.

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.

Removal of Low Trace ppb-Level Perfluorooctanesulfonic Acid (PFOS) with ZIF-8 Coatings Involving Adsorbent Degradation.

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.

Insights in column packing processes of narrow bore and capillary-scale columns: Methodologies, driving forces, and separation performance - A tutorial review.

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.

Development of a generic ultra-high-pressure gradient liquid-chromatography method development protocol: The analysis of residual multi-class antibiotics in food products as a case study.

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.

Design Guidelines and Kinetic Performance Limits for Spatial Comprehensive Three-Dimensional Chromatography for the Analysis of Intact Proteins.

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 (xLC) in the first and second dimension and time domain (tLC) 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 xIEF × xSEC × tRPLC device. Novel equations are proposed to determine the peak capacity in xSEC 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 2D and 4096 3D 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 xIEF × xSEC × tRPLC separation stages.

A protocol for setting-up robust hydrophobic interaction chromatography targeting the analysis of intact proteins and monoclonal antibodies.

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 (C4) 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.

Assessment of the resolving power of hydrophobic interaction chromatography for intact protein analysis on non-porous butyl polymethacrylate phases.

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).