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

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

Reversed-Phase Liquid Chromatography of Peptides for Bottom-Up Proteomics: A Tutorial.

The performance of the current bottom-up liquid chromatography hyphenated with mass spectrometry (LC-MS) analyses has undoubtedly been fueled by spectacular progress in mass spectrometry. It is thus not surprising that the MS instrument attracts the most attention during LC-MS method development, whereas optimizing conditions for peptide separation using reversed-phase liquid chromatography (RPLC) remains somewhat in its shadow. Consequently, the wisdom of the fundaments of chromatography is slowly vanishing from some laboratories. However, the full potential of advanced MS instruments cannot be achieved without highly efficient RPLC. This is impossible to attain without understanding fundamental processes in the chromatographic system and the properties of peptides important for their chromatographic behavior. We wrote this tutorial intending to give practitioners an overview of critical aspects of peptide separation using RPLC to facilitate setting the LC parameters so that they can leverage the full capabilities of their MS instruments. After briefly introducing the gradient separation of peptides, we discuss their properties that affect the quality of LC-MS chromatograms the most. Next, we address the in-column and extra-column broadening. The last section is devoted to key parameters of LC-MS methods. We also extracted trends in practice from recent bottom-up proteomics studies and correlated them with the current knowledge on peptide RPLC separation.

Investigation of the potential of mixed solvent mobile phases in temperature-responsive liquid chromatography (TRLC).

Temperature-responsive liquid chromatography (TRLC) allows for extensive retention and selectivity tuning through temperature in HPLC. This is mainly achieved through the use of a stationary phases comprising of a temperature-responsive polymer which undergoes a reversible change from hydrophilic to hydrophobic behaviour upon increasing the temperature. The approach can allow for reversed phase type separations to be achieved with purely aqueous mobile phases, whereby the retention is controlled through temperature instead of mobile phase composition. Despite the promising nature of such form of retention control under isocratic mobile phase conditions, TRLC can suffer from excessive retention of highly apolar solutes even at lower column temperatures whereby the polymer is considered hydrophilic. This is related both to a residual apolarity of the polymer chain and due to the high log P's and low water solubility of higly apolar compounds. While it was known that elution in TRLC doesn't necessarily has to be performed under purely aqueous conditions and that the use of organic co-solvents to the water is possible, the impact thereof on the temperature responsive behaviour itself had not yet been investigated in a systematic way. Therefore in this work the advantages and drawbacks of the use of the organic co-solvents methanol and acetonitrile in TRLC is assessed on two types of temperature reponsive phases: poly-N-N-propylacrylamide (PNNPAAm) and poly-N-isopropylacrylamide (PNIPAAm). The influence of organic co-solvents is investigated with two representative test mixtures (comprising 4 parabens and 5 apolar steroids).

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

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

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

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

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

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

Graphical data representation methods to assess the quality of LC columns.

We discuss the most important plot types for the kinetic performance of liquid chromatography columns and elaborate on how these plots should best be constructed and can be made dimensionless. Distinction is made between plots that are most suited for practitioners (column users) versus those most suited for theoreticians and column manufacturers.

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 %.

Comprehensive analysis of the effective and intra-particle diffusion of weakly retained compounds in silica hydrophilic interaction liquid chromatography columns.

A detailed analysis of intra-particle volumes and layer thicknesses and their effect on the diffusion of solutes in hydrophilic interaction liquid chromatography (HILIC) was made. Pycnometric measurements and the retention volume of deuterated mobile phase constituents (water and acetonitrile) were used to estimate the void volume inside the column, including not only the volume of the mobile phase but also part of the enriched water solvent acting as the stationary phase in HILIC. The mobile phase (hold-up) volume accessible to non-retained components was estimated using a homologous series approach. The joint analysis of the different approaches indicated the formation of enriched water layers on the hydrophobic silica mesopore walls with a thickness varying significantly with mobile phase composition. The maximal thickness of the enriched water layers, which corresponded to the minimum void volume accessible to unretained solutes, marked a transition in the retention behavior of the studied analytes. Discrepancies between deuterated solvent measurements and pycnometry were explained by the existence of an irreplaceable water layer adsorbed on the silica surface. Regarding the diffusion behavior in HILIC, peak parking experiments were used to interpret the influence of the acetonitrile content on the effective diffusion coefficient Deff. A systematic decrease in Deff and molecular diffusion Dm was observed with decreasing acetonitrile concentration, primarily attributed to variations in mobile phase viscosity. Notably, Deff/Dm remained nearly unaffected by variations in mobile phase composition. Finally, the effective medium theory was used to make a comprehensive analysis of Dpart/Dm to study the contribution to band broadening when the solute resides in the mesopores. The obtained data unveiled a curvature with a minimum corresponding to conditions of maximum water-layer thickness and retention. For the weakly retained compounds (k' < 0.5) the Dpart/Dm-values were found to be relatively high (order of 0.35-0.5), which directly reflects the high γsDs/Dm-values that were observed (order 0.35-7).

Detailed analysis of the effective and intra-particle diffusion coefficient of proteins at elevated pressure in columns packed with wide-pore core-shell particles.

To determine the efficiency that can be obtained in a packed-bed liquid-chromatography column for a particular analyte, a correct determination of the molecular and effective diffusion coefficients (Dm and Deff) of the analyte is required. The latter is usually obtained via peak parking experiments wherein the flow is stopped. As a result, the column pressure rapidly dissipates and the measurement is essentially conducted at ambient pressure. This is problematic for analytes whose retention depends on pressure, such as proteins and potentially other large (dipolar) molecules. In that case, a conventional peak parking experiment is expected to lead to large errors in Deff. To obtain a better estimate ofDeff, the present study reports on the use of a set-up enabling peak parking measurements under pressurized conditions. This approach allowed us to report, for the first time, Deff for proteins at elevated pressure under retained conditions. First, Deff was determined at a (average) pressure of about 105 bar for a set of proteins with varying size, namely: bradykinin, insulin, lysozyme, ß-lactoglobulin, and carbonic anhydrase in a column packed with 400 Å core-shell particles. The obtained data were then compared to those of several small analytes: acetophenone, propiophenone, benzophenone, valerophenone, and hexanophenone. A clear trend between Deff and analyte size was observed. The set-up was then used to determine Deff of bradykinin and lysozyme at variable (average) pressures ranging from 28 bar to 430 bar. These experiments showed a decrease in intra-particle and surface diffusion with pressure, which was larger for lysozyme than bradykinin. The data show that pressurized peak parking experiments are vital to correctly determine Deff when the analyte retention varies significantly with pressure.

High-resolution peptide separations using nano-LC at ultra-high pressure.

We report on the optimization of nano-LC gradient separations of proteomic samples that vary in complexity. The gradient performance limits were visualized by kinetic plots depicting the gradient time needed to achieve a certain peak capacity, while using the maximum system pressure of 80 MPa. The selection of the optimal particle size/column length combination and corresponding gradient steepness was based on scouting the performance of 75 µm id capillary columns packed with 2, 3, and 5 µm fully porous silica C18 particles. At optimal gradient conditions, peak capacities up to 500 can be obtained within a 120 min gradient using 2 µm particle-packed capillary columns. Separations of proteomic samples including a cytochrome c tryptic digest, a bovine serum albumin tryptic digest, a six protein mix digest, and an Escherichia coli digest are demonstrated while operating at the kinetic-performance limit, i.e. using 2-µm packed columns, adjusting the column length and scaling the gradient steepness according to sample complexity. Finally, good run-to-run retention time stability (RSD values below 0.18%) was demonstrated applying ultra-high pressure conditions.

Fundamental investigation of the dispersion caused by a change in diameter in nano liquid chromatography capillary tubing.

We report on a Computational Fluid Dynamics (CFD) study of the extra dispersion caused by the change in diameter when coupling two pieces of capillary tubing with different diameter. In this first investigation into the problem, the focus is on the typical flow rates (0.25≤F≤2µL/min) and diameters (d≤40µm) used in nano-LC, considering both the case of either a doubling or halving of the diameter. The CFD simulations allow to study the problem from a fundamental point of view, i.e., under otherwise perfect conditions (perfect alignment, zero dead-volume). Flow rates, capillary diameters, diffusion coefficients and liquid viscosities have been varied over a range relevant for nano-LC (Reynolds-numbers Re ≤ 1), with also an excursion made towards high-temperature nano-LC conditions (Re ≥ 10 and more). The extra dispersion caused by the change in diameter has been quantified via a volumetric variance σ2conn, defined in such a way that the overall dispersion across the entire capillary system can be easily reconstructed from the known analytical solutions in the individual segments. When the two capillaries are longer than their diffusion entry length, covering most of the practical cases, σ2conn converges to a limiting value σ2conn,∞ which varies to a close approximation with the square of flow rate. Under the investigated nano-LC conditions, the σ2conn,∞-values are surprisingly small (e.g., on the order of 0.01 to 0.15 nL2 in a 20 to 40µm connection) compared to the dispersion occurring in the remainder of the capillaries.

On-column modification for the creation of temperature-responsive stationary phases.

Temperature-responsive liquid chromatography (TRLC) offers an alternative for retention and selectivity optimisation in HPLC. This approach thereby exploits temperature gradients on stimuli-responsive stationary phases and forfeits the necessity for solvent gradients, allowing analyses to be performed using aqueous mobile phases. Consequently, it can be employed as a green alternative to reversed-phase separations. However, current production to obtain temperature-responsive columns inherently require dedicated column packing processes with polymer-modified particles. To facilitate the development of temperature-responsive phases, a flow-through modification procedure was developed allowing on-column modification of aminopropyl silica columns. Three columns were manufactured using this novel flow-through approach, which achieved identical column efficiencies compared to existing TRLC column. Demonstrating the possibility of bypassing the dedicated packing processes without losing efficiency. Additionally, it was observed that flow-through produced columns yielded higher retention at elevated temperatures despite their reduced carbon load. Further investigation of the carbon load revealed the presence of stationary phase gradients, whose influence was studied via novel developed retention experiments, which revealed a negligible change reduction in retention upon a change of polymer modification from 19.8% to 14.5%. However, further decrease from 14.5% to 12.3% resulted in a larger change. Interestingly, a further enhancement in apparent plate numbers was observed when operating the column under a reversed flow, yielding an increasing stationary phase gradient. This on-column modification procedure demonstrates the potential for modification of existing (commercial) packed columns to achieve temperature-responsive phases without loss of efficiency or retention. Thus, not only facilitating accessibility to temperature-responsive phases, but also aiding with development of further generations of temperature-responsive phases by removing the need for packing optimisation. Additionally, a novel experiment was set up to easily investigate the effect of inhomogeneous stationary phases retention.

Theory of separation performance and peak width in gradient elution liquid chromatography: A tutorial.

Separation performance in chromatography has been extensively studied since the dawn of the technique. Although the basic principles of band broadening and the resulting separation performance in isocratic elution are in general well known and understood, this is much less the case for gradient separations. In this tutorial, first the basic principles, concepts and parameters that determine separation performance, peak width and variance and analysis time in isocratic separations are reviewed. This is subsequently used to discuss the parameters that affect peak width in gradient elution, together with the concepts of plate count and plate height in this elution mode. In addition, the effect of peak compression in gradient elution is elaborated. Finally, the effect of extra-column dispersion on separation performance in gradient elution is discussed, and an overview of how these contributions can be experimentally evaluated is given.

Detailed computational fluid dynamics study of the parameters contributing to the viscous heating band broadening in liquid chromatography at pressures up to 2500 bar in 2.1 mm columns.

Over the past years viscous heating band broadening occurring in high pressure liquid chromatography has been studied extensively. In the present numerical study, we investigate the fine details of this band broadening contribution under extreme high-pressure conditions (2500 bar). To analyze the results, we first show that viscous heating leads to two clearly distinguishable band broadening effects, one originating from the radial differences in the species migration velocity and the other from the axial variations. It was found that the radial contribution is independent of the intrinsic band broadening of the bed (i.e. band broadening in absence of viscous heating) while it strongly depends on the radial dispersion coefficient and the retention enthalpy of the analytes. On the other hand, the axial contribution is strongly dependent on the bed intrinsic band broadening and it is found to be 4 to 5 times lower than the radial contribution. We also show the strong effect of the endfittings on the temperature gradients inside the column thus on the resulting viscous heating band broadening.

Measurement of the molecular diffusion coefficient and the effective longitudinal diffusion under supercritical fluid chromatography conditions in packed bed columns.

The improvement of supercritical fluid chromatography (SFC) instrumentation enhanced its reliability and utility over the past decade. The further development of high speed and high resolution separations is however obstructed by the lack of accurate models for axial dispersion in SFC. This work is a first step to tackle this by developing more reliable methods to measure molecular (Dmol) and longitudinal diffusion (Deff) in SFC, as these affect all aspects of separation efficiency. In the present contribution, we report on an improved method, to enable more flexible, reliable and accurate measurements of Dmol in SFC using commercial instrumentation. A two-column variant of the stopped-flow experiment is proposed as an adapted set-up for measuring the effective longitudinal diffusion coefficient Deff in SFC-conditions. Using the set-ups for a number of test-compounds, it has been found that Deff, and the coefficients describing its constituent sub-processes (cf. particle diffusion Dpart and surface diffusion γsDs), all vary in a linearly proportional way with the bulk diffusion coefficient Dmol within a high degree of accuracy. It has also been found that Deff decreases much more sharply with increasing retention factor compared to LC. By applying the effective medium theory, it was found that the relative surface diffusion coefficient γsDs/Dmol decreases strongly with retention factor for the investigated solutes and column, in contrary to what is typically observed in reversed phase liquid chromatography. Results indicate that this might be related to a change in retention behavior of the analytes. Obviously, more analytes and conditions need to be explored to complete this picture and the extend range of applicability of these observations.

Diffusion coefficients of an extensive set of pharmaceutical compounds in supercritical fluid chromatography over a wide range of mobile phase compositions.

Diffusion data are essential for adequate analysis of the kinetic separation performance of any chromatographic system. Unfortunately, for Supercritical Fluid Chromatography (SFC), very little data is available of the diffusion coefficients in mobile phases typically used in contemporary methods, i.e. with a non-negligible amount of polar modifier such as methanol. In this work, a relative simple method which only requires minor modifications to a standard commercially available SFC instrument is used to determine the diffusion coefficient of an extensive set of pharmaceutical compounds in the range of 10-50 vol% of modifier (methanol) in CO2. By using a traditional SFC column, the solute is first separated from the sample solvent plug, before entering a long capillary, where the band broadening can be linked to its diffusion coefficient using the Taylor-Aris equation. By using two UV-detectors, before and after the capillary, the effect of the dispersion in the column can be eliminated and the true volumetric flow rate determined. It was found that in the investigated range of conditions, the change in mobile phase viscosity in a first approximation allows to predict the variation in diffusion coefficient. Chemical structure and more particularly functional groups can however have a significant effect on the diffusion coefficient.

Review of recent insights in the measurement and modelling of the B-term dispersion and related mass transfer properties in liquid chromatography.

In this contribution, we review the recent literature relating to the measurement and modelling of all diffusion-dominated processes contributing to the efficiency of a chromatographic column. In first instance, this involves the measurement and modelling of the overall effective diffusion coefficient Deff (determining the so-called B-term band broadening). The latter manifests itself most clearly during a so-called peak parking experiment. Using effective medium theory modelling, the measured Deff-value can subsequently be decomposed into its constituent contributions, of which the intra-particle or the mesoporous zone and the surface diffusion coefficient are the most important ones. As an accurate estimation of the diffusion processes also allows computing the C-term plate height contribution terms, the review ends with some recent insights obtained when using the established B- and C-term contributions to compute the degree of eddy-dispersion in contemporary packed bed columns.

Kinetic performance optimisation for liquid chromatography: principles and practice.

This HPLC tutorial focuses on the preparation and use of kinetic plots to characterise the performance in isocratic and gradient LC. This graphical approach allows the selection of columns (i.e. optimum particle size and column length) and LC conditions (operating pressure and temperature) to generate a specific number of plates or peak capacity in the shortest possible analysis time. Instrument aspects including the influence of extra-column effects (maximum allowable system volume) and thermal operating conditions (oven type) on performance are discussed. In addition, the performance characteristics of porous-shell particle-packed columns and monolithic stationary phases are presented and the potential of future column designs is discussed.