Search

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.

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.

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 (supix/i/supLC) in the first and second dimension and time domain (supit/i/supLC) 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 spatialsupix/i/supIEF ×supix/i/supSEC ×supit/i/supRPLC device. Novel equations are proposed to determine the peak capacity insupix/i/supSEC 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 (64sup2/supD and 4096sup3/supD 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 implementingsupix/i/supIEF ×supix/i/supSEC ×supit/i/supRPLC separation stages.

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.

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.

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).

Antibody tests are essential tools to investigate humoral immunity following SARS-CoV-2 infection. While first-generation antibody tests have primarily provided qualitative results with low specificity, accurate seroprevalence studies and tracking of antibody levels over time require highly specific, sensitive and quantitative test setups. Here, we describe two quantitative ELISA antibody tests based on the SARS-CoV-2 spike receptor-binding domain and the nucleocapsid protein. Comparative expression in bacterial, insect, mammalian and plant-based platforms enabled the identification of new antigen designs with superior quality and high suitability as diagnostic reagents. Both tests scored excellently in clinical validations with multi-centric specificity and sensitivity cohorts and showed unprecedented correlation with SARS-CoV-2 neutralization titers. Orthogonal testing increased assay specificity to 99.8%, thereby enabling robust serodiagnosis in low-prevalence settings. The inclusion of a calibrator permits accurate quantitative monitoring of antibody concentrations in samples collected at different time points during the acute and convalescent phase of COVID-19.

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.

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.

The ability to control the external porosity and to tune the dimensions of the macropore size on multiple length scales provides the possibility of tailoring the monolithic support structure towards separation performance. This paper discusses the properties of conventional polymer-monolithic stationary phases and its limitations regarding the effects of morphology on kinetic performance. Furthermore, guidelines to improve the macropore structure are discussed. The optimal monolithic macropore structure is characterized by high external porosity (while maintaining ultra-high-pressure stability), high structure homogeneity, polymer globule clusters in the submicron range, and macropores with a diameter tuned toward speed (small diameter in the 100-500 nm range using short beds) or efficiency (larger macropores in the range of 500 nm-1 μm allowing the use of longer column formats). Finally, promising approaches to control the morphology are discussed.

The current study comprises a systematic investigation to assess retention properties and selectivity of a mixed-mode reversed-phase/weak-anion-exchange (RP/WAX) stationary phase to aid method development. Retention was investigated for different compound classes which vary in hydrophobicity, van der Waals surface area, and charge as function of organic content, pH, and ionic strength of the mobile phase. The linear-solvent-strength model was successfully applied for aromatic hydrocarbons to obtain retention-time predictions based on log P values and van der Waals surface area values. For phenols, predictions were based on log P values and data from a single scouting run performed in isocratic mode to estimate the S parameter; the deviations between experimental and predicted retention times were smaller than 6%. To describe the mixed-mode (RP/WAX) retention behavior of singly and doubly negatively-charged aromatic acids, a novel model combining the linear-solvent-strength and the empirical stoichiometric-displacement-net-charge models is proposed and validated. Using combinations of three scouting runs that are not linearly dependent, the maximum prediction error was 11% and changes in selectivity were correctly forecasted when altering the mobile-phase composition, i.e., either organic modifier content or ionic strength. When using nine scouting runs in combination with a least-squares regression approach to determine the model parameters, the maximum prediction error was 6%.

The nucleocapsid protein (NP) of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is critical for several steps of the viral life cycle, and is abundantly expressed during infection, making it an ideal diagnostic target protein. This protein has a strong tendency for dimerization and interaction with nucleic acids. For the first time, high titers of NP were expressed in E. coli with a CASPON tag, using a growth-decoupled protein expression system. Purification was accomplished by nuclease treatment of the cell homogenate and a sequence of downstream processing (DSP) steps. An analytical method consisting of native hydrophobic interaction chromatography hyphenated to multi-angle light scattering detection (HIC-MALS) was established for in-process control, in particular, to monitor product fragmentation and multimerization throughout the purification process. 730 mg purified NP per liter of fermentation could be produced by the optimized process, corresponding to a yield of 77% after cell lysis. The HIC-MALS method was used to demonstrate that the NP product can be produced with a purity of 95%. The molecular mass of the main NP fraction is consistent with dimerized protein as was verified by a complementary native size-exclusion separation (SEC)-MALS analysis. Peptide mapping mass spectrometry and host cell specific enzyme-linked immunosorbent assay confirmed the high product purity, and the presence of a minor endogenous chaperone explained the residual impurities. The optimized HIC-MALS method enables monitoring of the product purity, and simultaneously access its molecular mass, providing orthogonal information complementary to established SEC-MALS methods. Enhanced resolving power can be achieved over SEC, attributed to the extended variables to tune selectivity in HIC mode., Graphical abstract Image 1

We report on a numerical and experimental study of the limits of peak refocusing and concentration enhancement that can be obtained with solvent front-assisted peak remobilization in a trap column receiving peaks eluting from a preceding analytical column. It is shown that the upper limit of peak refocusing can best be pursued by injecting a sufficiently large volume in a sufficiently narrow capillary and elute it with a sufficiently steep (ballistic) gradient. Corresponding equations offering a quantitative description have been derived and verified experimentally. For the latter purpose, peak volumes of the order of 0.5 to 2.0 μL were refocused in a dedicated set-up capable of trapping μL-sized peaks in a 75 μm i.d. capillary and remobilized using a nano-LC pump propelling an acetonitrile/isopropyl alcohol mixture with a viscosity matching that of the trapping solvent. Injecting 2.0 μL peaks, a peak refocusing factor of 17.3 could be achieved.

This review describes chromatographic dispersion and different plate-height models frequently used to assess the chromatographic performance of ultra-high-pressure liquid chromatography column technology. Furthermore, different performance indices, including the resolution, the separation impedance, and kinetic plots are discussed allowing to quantify and visualize the resolving power in liquid chromatography. The construction of kinetic plots is explained, and different visualization approaches are highlighted. Finally, key instrument and column-technology developments to advance the kinetic performance limits are discussed and selected state-of-the-art applications are highlighted.

Chapter in Encyclopedia of Analytical Science Worsfold, P., Townshend, A., Poole, C. & Miró, M. (eds.). 3 ed. Elsevier, 261–269

Background Antibody tests are essential tools to investigate humoral immunity following SARS-CoV-2 infection or vaccination. While first-generation antibody tests have primarily provided qualitative results, accurate seroprevalence studies and tracking of antibody levels over time require highly specific, sensitive and quantitative test setups. Methods We have developed two quantitative, easy-to-implement SARS-CoV-2 antibody tests, based on the spike receptor binding domain and the nucleocapsid protein. Comprehensive evaluation of antigens from several biotechnological platforms enabled the identification of superior antigen designs for reliable serodiagnostic. Cut-off modelling based on unprecedented large and heterogeneous multicentric validation cohorts allowed us to define optimal thresholds for the tests’ broad applications in different aspects of clinical use, such as seroprevalence studies and convalescent plasma donor qualification. Findings Both developed serotests individually performed similarly-well as fully-automated CE-marked test systems. Our described sensitivity-improved orthogonal test approach assures highest specificity (99.8%); thereby enabling robust serodiagnosis in low-prevalence settings with simple test formats. The inclusion of a calibrator permits accurate quantitative monitoring of antibody concentrations in samples collected at different time points during the acute and convalescent phase of COVID-19 and disclosed antibody level thresholds that correlate well with robust neutralization of authentic SARS-CoV-2 virus. Interpretation We demonstrate that antigen source and purity strongly impact serotest performance. Comprehensive biotechnology-assisted selection of antigens and in-depth characterisation of the assays allowed us to overcome limitations of simple ELISA-based antibody test formats based on chromometric reporters, to yield comparable assay performance as fully-automated platforms. Funding WWTF, Project No. COV20–016; BOKU, LBI/LBG

The applicability and predictive properties of the linear solvent strength model and two nonlinear retention-time models, i.e., the quadratic model and the Neue model, were assessed for the separation of small molecules (phenol derivatives), peptides, and intact proteins. Retention-time measurements were conducted in isocratic mode and gradient mode applying different gradient times and elution-strength combinations. The quadratic model provided the most accurate retention-factor predictions for small molecules (average absolute prediction error of 1.5%) and peptides separations (with a prediction error of 2.3%). An advantage of the Neue model is that it can provide accurate predictions based on only three gradient scouting runs, making tedious isocratic retention-time measurements obsolete. For peptides, the use of gradient scouting runs in combination with the Neue model resulted in better prediction errors (

Spatial multi-dimensional liquid chromatography can provide unprecedent resolving power by utilizing subsequent orthogonal separation mechanisms, while the analysis time is minimized thanks to parallel development of the analyzed fractions in each dimension. In this study, different engineering solutions have been realized aiming at achieving flow confinement and control during subsequent 1D and 2D chromatographic developments in a microfluidic device for spatial two-dimensional separations. First, the flow distributor design was optimized and physical barriers, i.e., reducing the cross section of microchannels as well as locally integrated monolithic substrates in chip segments, were assessed as a means of flow confinement. Furthermore, an on-chip activevalving, high-pressure approach was successfully developed. The flow in first-dimension separation is confined within a channel situated in a rotating axis containing through-holes. These are either closed during the 1st development or opened when aligned with the D-2 flow distributor and the D-2 channels after rotation of the axis by 90 degrees, allowing for sample transfer and executing the subsequent D-2 analysis. The on-chip active-valving concept has been successfully demonstrated in combination with stationary-phase analyte focusing prior to the D-2 development using a microfluidic spatial 2D-LC device containing polymer monolithic stationary phases locally synthesized in the parallel D-2 separation channels. Finally, this workflow was successfully employed to separate a mixture of dyes applying gradient RPLC during the D-2 development.

This chapter describes the theoretical fundamentals of capillary electrochromatography and gives an insight on the formation and characteristics of the electroosmotic flow and the respective factors to optimize retention. In electro-driven separations, greater efficiencies can be achieved compared to pressure-driven separations in liquid chromatography. Instrument setup and examples of hardware configurations are presented, and separation efficiency and kinetic performance limits are discussed. Finally, different column technologies and stationary-phase chemistry developments used to optimize separation efficiency and selectivity are described, and an overview of selected recent state-of-the-art applications is highlighted, focussing on biochemical and biopharmaceutical analysis, small molecule pharmaceuticals and enantiomer separations.

The selectivity and separation efficiency of aqueous size-exclusion chromatographic separations of intact proteins were assessed for different flow rates, using columns packed with 3 and 5 μm silica particles containing 150 and 290 A stagnant pores. A mixture of intact proteins with molecular weights ranging between 17 000 and 670 000 Da was used to construct the calibration curves. Both the model fit and the predictive properties, using a leave-one-out strategy, of different polynomial models (up to fifth order) were evaluated for different flow rates. The best compromise between model fit and predictive properties was obtained using a third-order polynomial model. The accuracy of the predictive properties decreased with 10% with an eightfold increase in the flow rate. No changes in retention factors (hence selectivity) were observed in the flow-rate range applied. A strong correlation between molecular weight and plate height was observed. Exclusion of large-molecular-weight proteins led to a significant reduction in the stationary-phase mass-transfer contribution to the total plate-height value, and this effect was also independent of the flow rate applied. The kinetic-performance limits, in terms of plate number and time, and optimal column-length particle-size combinations were determined at the maximum recommended operating pressure of the size-exclusion chromatography columns (20 MPa). Finally, the possibilities of method speed-up using ultra-high-pressure size-exclusion chromatography in combination with columns packed with sub-2 μm particles are discussed.

One decade has passed since the introduction of commercial ultra-high-pressure liquid chromatography (UHPLC) technology and columns packed with sub-2-µm particles, which led to significant improvements in separation efficiency and sample throughput. This review discusses fundamental and practical aspects of ultra-high-pressure liquid chromatography. Advances in system design addressing tubing configuration, pump and injector technology, oven configuration, and detection including MS interfacing are reviewed. In this context, practical aspects of method development are discussed and key UHPLC applications are highlighted.

This study discusses the design aspects for the construction of a microfluidic device for comprehensive spatial two-dimensional liquid chromatography. In spatial two-dimensional liquid chromatography each peak is characterized by its coordinates in the plane. After completing the first-dimension separation all fractions are analyzed in parallel second-dimension separations. Hence, spatial two-dimensional liquid chromatography potentially provides much higher peak-production rates than a coupled column multi-dimensional liquid chromatography approach in which the second-dimension analyses are performed sequentially. A chip for spatial two-dimensional liquid chromatography has been manufactured from cyclic olefin copolymer and features a first-dimension separation channel and 21 parallel second-dimension separation channels oriented perpendicularly to the former. Compartmentalization of first- and second-dimension developments by physical barriers allowed for a preferential flow path with a minimal dispersion into the second-dimension separation channels. To generate a homogenous flow across all the parallel second-dimension channels, a radially interconnected flow distributor containing two zones of diamond-shaped pillars was integrated on-chip. A methacrylate ester based monolithic stationary phase with optimized macroporous structure was created in situ in the confines of the microfluidic chip. In addition, the use of a photomask was explored to localize monolith formation in the parallel second-dimension channels. Finally, to connect the spatial chip to the liquid chromatography instrument, connector ports were integrated allowing the use of Viper fittings. As an alternative, a chip holder with adjustable clasp locks was designed that allows the clamping force to be adjusted.

The characterization of biotherapeutics represents a major analytical challenge. This review discusses the current state-of-the-art in analytical technologies to profile biopharma products under native conditions, i.e., the protein three dimensional conformation is maintained during liquid chromatographic analysis. Native liquid-chromatographic modes that are discussed include aqueous size-exclusion chromatography, hydrophobic interaction chromatography, and ion-exchange chromatography. Infusion conditions and the possibilities and limitations to hyphenate native liquid chromatography to mass spectrometry are discussed. Furthermore, the applicability of native liquid-chromatography methods and intact mass spectrometry analysis for the characterization of monoclonal antibodies and antibody-drug conjugates is discussed.

The present paper discusses practical aspects of prototyping of microfluidic chips using cyclic olefin copolymer as substrate and the application in high-performance liquid chromatography. The developed chips feature a 60 mm long straight separation channel with circular cross section (500 mu m i.d.) that was created using a micromilling robot. To irreversibly seal the top and bottom chip substrates, a solvent vapor-assisted bonding approach was optimized, allowing to approximate the ideal circular channel geometry. Four different approaches to establish the micro-to-macro interface were pursued. The average burst pressure of the microfluidic chips in combination with an encasing holder was established at 38 MPa and the maximum burst pressure was 47 MPa, which is believed to be the highest ever report for these polymer-based microfluidic chips. Porous polymer monolithic frits were synthesized in-situ via UV-initiated polymerization and their locations were spatially controlled by the application of a photomask. Next, high-pressure slurry packing was performed to introduce 3 mu m silica reversed-phase particles as the stationary phase in the separation channel. Finally, the application of the chip technology is demonstrated for the separation of alkyl phenones in gradient mode yielding baseline peak widths of 6 s by applying a steep gradient of 1.8 min at a flow rate of 10 mu L/min. (C) 2017 Elsevier B.V. All rights reserved.

We developed earlier the post-column refocusing strategy for isocratic separations, which employs trapping target analytes after an analytical separation and additionally focusing them using a strong remobilization solvent prior to detection, and have now extended it to high-speed gradient LC. A gradient separation of antibiotics and its metabolites, applying a linear aqueous acetonitrile gradient from 2 to 65% (v/v) ACN containing 0.1% FA in 10 min, performed on an analytical column was selected as an application. Eluted heart-cut fractions were directed from the analytical silica C-18 column to a trap column packed with Hypercarb particles. The remobilization of the target analytes was performed in back-flush mode using solvent mixtures tuned to maximize the solvent strength by mixing isopropanol into the remobilization solvent containing acetonitrile. Additionally, a viscosity-calibration experiment showed that the viscosity difference between trapping and remobilization solvents should be smaller than 0.15 mPa.s to prevent viscous fingering. To keep the viscosity difference below this limit, during the gradient separation performed on the analytical column, the composition of the remobilization solvent was changed in time. An empirical equation is provided that allows for the selection of the optimal remobilization-solvent composition. To maximize the signal enhancement, the loading time of target analytes on the trap column should be optimized. Peak dispersion was further minimized by applying a flow rate that corresponded to the optimal van-Deemter flow rate of the trap column (20 mu L/min). Finally, decreasing the diameter of the trap column from 1 mm to 0.3 mm led to a significant enhancement of the detection sensitivity with more than one order of magnitude. Using an optimized trap configuration and elution/remobilization conditions, a signal enhancement of a factor of 14 was achieved for sulfaguanidine (early-eluting compound in the gradient,separation) and 7.3 for furazolidone (late-eluting compound). (C) 2016 Elsevier B.V. All rights reserved.

The effect of different kosmotropic/chaotropic salt systems on retention characteristics of intact proteins has been examined in hydrophobic interaction chromatography (HIC). The performance was assessed using different column chemistries, i.e. polyalkylamide, alkylamine incorporating hydrophobic moieties, and a butyl chemistry. Selectivity in HIC is mainly governed by the salt concentration and by the molal surface tension increment of the salt. Typically, a linear relationship between the natural logarithm of the retention factor and the salt concentration is obtained. Using a 250 mm long column packed with 5 μm polyalkylamide functionalized silica particles and applying a 30 min linear salt gradient, a peak capacity of 78 was achieved, allowing the baseline separation of seven intact proteins. The hydrophobicity index appeared to be a good indicator to predict the elution order of intact proteins in HIC mode. Furthermore, the effect of adding additives in the mobile phase, such as calcium chloride (stabilizing the 3D conformation of α-lactalbumin) and isopropanol, on retention properties has been assessed. Results indicate that HIC retention is also governed by conformational in the proteins which affect the number of accessible hydrophobic moieties., SCOPUS: ar.j, info:eu-repo/semantics/published

We report on the peak capacity that can be produced by operating a state-of-the-art core–shell particle type (dp = 2.6 μm) at its kinetic optimum at ultra-high pressures of 600 and 1200 bar. The column-length optimization needed to arrive at this kinetic optimum was realized using column coupling. Whereas the traditional operating mode (using a single 15 cm column operated at its optimum flow rate of 0.4 mL/min) offered a peak capacity of 162 in 10.8 min, a fully optimized train of 60 cm (4 × 15 cm) columns offered a peak capacity of 325 in 61 min when operated at 1200 bar. Even though the particles have a reputed low flow resistance and a relatively large size (>2 μm), it was found that the increase in performance that can be generated when switching from a fully optimized 600 bar operation to a fully optimized 1200 bar operation is significant (roughly 50% reduction of the analysis time for the same peak capacity and approximately a 20% increase in peak capacity if compared for the same analysis time). This has been quantified in a generic way using the kinetic plot method and is illustrated by showing the chromatograms corresponding to some of the data points of the kinetic plot curve.

The applicability and predictive properties of the linear solvent strength model and two nonlinear retention-time models, i.e., the quadratic model and the Neue model, were assessed for the separation of small molecules (phenol derivatives), peptides, and intact proteins. Retention-time measurements were conducted in isocratic mode and gradient mode applying different gradient times and elution-strength combinations. The quadratic model provided the most accurate retention-factor predictions for small molecules (average absolute prediction error of 1.5%) and peptides separations (with a prediction error of 2.3%). An advantage of the Neue model is that it can provide accurate predictions based on only three gradient scouting runs, making tedious isocratic retention-time measurements obsolete. For peptides, the use of gradient scouting runs in combination with the Neue model resulted in better prediction errors (2.2%) compared to the use of isocratic runs. The applicability of the quadratic model is limited due to a complex combination of error and exponential functions. For protein separations, only a small elution window could be applied, which is due to the strong effect of the content of organic modifier on retention. Hence, the linear retention-time behavior of intact proteins is well described by the linear solvent strength model. Prediction errors using gradient scouting runs were significantly lower (2.2%) than when using isocratic scouting runs (3.2%).

a b s t r a c t The effect of gradient steepness on the kinetic performance limits and peak compression effects has been assessed in gradient mode for the separation of phenol derivatives using columns packed with 2.6 m core-shell particles. The effect of mobile-phase velocity on peak capacity was measured on a column with fixed length while maintaining the retention factor at the moment of elution and the peak- compression factor constant. Next, the performance limits were determined at the maximum system pressure of 100 MPa while varying the gradient steepness. For the separation of small molecules apply- ing a linear gradient with a broad span, the best performance limits in terms of peak capacity and analysis time were obtained applying a gradient-time-to-column-dead-time (tG/t0) ratio of 12. The magnitude of the peak-compression factor was assessed by comparing the isocratic performance with that in gradi- ent mode applying different gradient times. Therefore, the retention factors for different analytes were determined in gradient mode and the mobile-phase composition in isocratic mode was tuned such that the difference in retention factor was smaller than 2%. Peak-compression factors were quantitatively determined between 0.95 and 0.65 depending on gradient steepness and the gradient retention factor. © 2015 Elsevier B.V. All rights reserved.

The need to improve either sample throughput on separation efficiency has spurred the development of ultra-high-pressure LC instrumentation, allowing to operate up to column pressures of 1500 bar. In the present study, the isocratic and gradient performance limits were assessed at UHPLC conditions applying columns packed with core shell particles. First, the extra-column band broadening contributions were assessed and minimized. Using an optimized system configuration minimum reduced plate heights of 1.8 were recorded on 2.1 x 100 columns packed with 1.5 mu m core shell particles. Increasing the pressure limit from 500 to 1500 bar and at the same time reducing the particle size from 2.6 to 1.5 mu m has allowed the analysis time to be decreased by a factor of 1.5 in isocratic mode, while maintaining separation efficiency (N=54,000). The kinetic time-gain factor in isocratic mode was proportional to the ratio of the separation impedance of both columns multiplied with the pressure ratio applied. In addition, the effect of operating pressure on the time gain factor was assessed in gradient mode. Using optimized gradient steepness (t(G)/t(0)=12) and increasing the operating pressure from 500 to 1500 bar a time gain factor of almost 13 was achieved for the separation of a mixture of waste-water pollutants without compromising peak capacity. (C) 2015 Elsevier B.V. All rights reserved.

The selectivity and separation efficiency of aqueous size-exclusion chromatographic separations of intact proteins were assessed for different flow rates, using columns packed with 3 and 5 μm silica particles containing 150 and 290 Å stagnant pores. A mixture of intact proteins with molecular weights ranging between 17 000 and 670 000 Da was used to construct the calibration curves. Both the model fit and the predictive properties, using a leave-one-out strategy, of different polynomial models (up to fifth order) were evaluated for different flow rates. The best compromise between model fit and predictive properties was obtained using a third-order polynomial model. The accuracy of the predictive properties decreased with 10% with an eightfold increase in the flow rate. No changes in retention factors (hence selectivity) were observed in the flow-rate range applied. A strong correlation between molecular weight and plate height was observed. Exclusion of large-molecular-weight proteins led to a significant reduction in the stationary-phase mass-transfer contribution to the total plate-height value, and this effect was also independent of the flow rate applied. The kinetic-performance limits, in terms of plate number and time, and optimal column-length particle-size combinations were determined at the maximum recommended operating pressure of the size-exclusion chromatography columns (20 MPa). Finally, the possibilities of method speed-up using ultra-high-pressure size-exclusion chromatography in combination with columns packed with sub-2 μm particles are discussed.

This study discusses the design aspects for the construction of a microfluidic device for comprehensive spatial two-dimensional liquid chromatography. In spatial two-dimensional liquid chromatography each peak is characterized by its coordinates in the plane. After completing the first-dimension separation all fractions are analyzed in parallel second-dimension separations. Hence, spatial two-dimensional liquid chromatography potentially provides much higher peak-production rates than a coupled column multi-dimensional liquid chromatography approach in which the second-dimension analyses are performed sequentially. A chip for spatial two-dimensional liquid chromatography has been manufactured from cyclic olefin copolymer and features a first-dimension separation channel and 21 parallel second-dimension separation channels oriented perpendicularly to the former. Compartmentalization of first- and second-dimension developments by physical barriers allowed for a preferential flow path with a minimal dispersion into the second-dimension separation channels. To generate a homogenous flow across all the parallel second-dimension channels, a radially interconnected flow distributor containing two zones of diamond-shaped pillars was integrated on-chip. A methacrylate ester based monolithic stationary phase with optimized macroporous structure was created in situ in the confines of the microfluidic chip. In addition, the use of a photomask was explored to localize monolith formation in the parallel second-dimension channels. Finally, to connect the spatial chip to the liquid chromatography instrument, connector ports were integrated allowing the use of Viper fittings. As an alternative, a chip holder with adjustable clasp locks was designed that allows the clamping force to be adjusted.

The present paper presents the mathematical expressions for the concentration enhancement which can be expected when applying a subsequent retentive trapping and strong eluent remobilization process in a packed trap column connected to the end of an analytical separation column. The established expressions for the optimal loading times, trap dimensions and expected concentration enhancement are illustrated and confirmed using numerical simulations of the trapping and release process. These simulations also provide a direct insight in how the bands are deformed and sharpened during the different steps of the process. The simulations, as well as the established expressions, for example show that in the backward elution mode the loading time should exceed a minimum value to allow the strong eluent front to fully overtake the remobilized band as this is the necessary condition to get a completely sharpened peak (in both the backward and the forward elution modes). The simulations also show it is very critical that this occurs as close as possible to the trap exit, as the bands are most sensitive to band broadening once they have been sharpened. The refocusing traps should hence be filled with particles producing the smallest possible plate heights, and connected to the detector using very short and small i.d. connection tubing. It was also found that, if there would be no band broadening in the trap, the achievable concentration enhancement would be the same for either a strong or weakly retaining trap. The true advantage of a high retention in the trap is that it leads to small trap volumes, which in turn minimizes the distance the band has to travel to reach the detector. This then minimizes the band broadening (inevitable in practice) and helps keeping a high concentration enhancement. Copyright © 2015 Elsevier B.V. All rights reserved.

The diversification of the cellular proteome is the result of protein synthesis by translation of mRNA and the formation of protein isoforms. Using current liquid-chromatographic mass-spectrometry (LC-MS) techniques, including two-dimensional LC-MS strategies, it is only possible to assess the top 1000 proteins due to limited resolving power. In order to successfully tackle these complex separations, the development of novel multi-dimensional separation technology is required, addressing both the bottlenecks of efficiency and analysis time. In spatial chromatography, components are separated in the space domain with each peak being characterized by its coordinates in a plane or three-dimensional separation body. Due to parallel developments in the second and third dimension, analysis time is greatly reduced compared to a coupled-column multi-dimensional LC approach. Spatial three-dimensional LC has the potential to offer unrivaled peak capacity and peak-production rate. The design of microfluidic devicesfor comprehensive spatial multi-dimensional chromatography will be discussed. The device for two-dimensional chromatography features a first-dimension (1D) separation channel and 21 parallel second-dimension (2D) separation channels, orientated perpendicular to the 1D channel. Compartmentalization of 1D and 2D flow was achieved integrating a physical barrier that allowed for a preferential mobile-phase flow path in the 1D channel, with minimal dispersion into the flow distributor and 2D separation channels. Once the 1D separation is completed, a radially-interconnected flow distributor of diamond-shaped pillars introduces the mobile phase for the 2D separation. Monolithic stationary phase were created in-situ at the desired location via a UV-initiated polymerization reaction and employing photomasks. Finally, a prototype 3D spatial chip composed of three modules will be demonstrated. The top module is composed of a ramified 3D flow distributor consisting of eight generations of T-bifurcations resulting in 256 outlets. The middle module consists of a modified two-dimensional separation microfluidic device. The bottom module consists of a 256 parallel 3D channels, allowing for the final 3D separation.

The last decade has witnessed how liquid chromatography columns and instruments changed from long,bulky columns with relatively large fully porous particles operated at modest pressures (100-200 bar), to short compact columns with small superficially porous particles operated at ultrahigh pressures (12001500 bar). This (r)evolution has resulted in a tremendous increase in achievable separation performance or decrease in analysis time, but requires a good knowledge of optimal chromatographic conditions for each separation problem and, concomitant, the right instrument configuration.

To increase detection sensitivity in liquid chromatography, a generic post-column refocusing strategy has been developed to enrich (target) analytes prior to detection. In this strategy, after separation on the analytical column, the analytes are led to a trap column preferably containing a stationary phase with strong retentive properties (e.g. silica C-30). They are then eluted using a strong solvent in a backward-elution mode. A first key element of the proposed strategy is that the trapping time should be at least equal to the time the front of the remobilization solvent needs to cover the entire length of the trap column, divided by the ratio of the flow rates used for trapping and remobilization. This condition is independent of the retention properties of the analytes in the trapping and remobilization solvent, Another essential element is the addition of a third solvent (isopropanol in the present case) to the remobilization solvent to overcome viscous-fingering effects caused by the viscosity difference between the trap and the remobilization solvents. The potential of the proposed post-column refocusing strategy is demonstrated for an isocratic separation of KI (t(0) marker), an antibiotic (sulfamethazine), and acetophenone as a case study. Using optimized remobilization conditions a maximum signal-enhancement factor of 8 was achieved. Higher enhancement factors using a remobilization solvent with slightly higher elution strength were prohibited by disturbances of the UV background signal. (C) 2014 Elsevier B.V. All rights reserved.

A polymer-based microfluidic device has been designed and constructed for comprehensive spatial two-dimensional chromatography, with each peak being characterized by its coordinates in the plane. The device manufactured from cyclic olefin copolymer (COC) features a first-dimension (1D) separation channel and 21 parallel second-dimension (2D) separation channels orientated perpendicular to the 1D channel. Compartmentalization of 1D and 2D flow by a physical barrier allowed for a preferential mobile phase flow path mainly restricted to the 1D separation channel, with a minimal dispersion into the 2D separation channels. After a 1D separation was halted, a radially-interconnected flow distributor of diamond-shaped pillars with diverging side-walls introduced the mobile phase for the 2D separation, allowing eluent inside the 1D channel to be fractionated and simultaneously injected into the 2D channels. A hydrophobic methacrylate-based monolithic stationary phase was polymerized in-situ in desired locations via aUV-initiated polymerization reaction employing a photo mask.

xx