Publications by authors named "Fabrice Gritti"

246 Publications

Theoretical study of the efficiency of liquid chromatography columns with particle size gradient.

J Chromatogr A 2021 Aug 9;1651:462331. Epub 2021 Jun 9.

Research Group of Analytical Chemistry, University of Pannonia, Egyetem utca 10, Veszprém H-8200, Hungary.

Modern analytical applications of liquid chromatography require more and more efficient columns. In this work, the possibility of utilizing particle size gradient in the chromatographic column was studied by a theoretical approach. In the course of our work three different scenarios of particle size gradients were considered with different shapes (linear, convex and concave). The evolution of bandwidth inside the column was plotted for each scenario. As a reference point, the bandwidth of the uniform column was used, which had the same pressure drop as the non-uniform column. According to our calculations, in isocratic elution mode, the non-uniform column does not offer any advantage compared to the uniform column, regardless the type of the particle size gradient. In gradient elution mode, however, extra band compression occurs was found. For negative particle size gradients, the final physical bandwidth was found to be approximately 1-4 % smaller than for uniform columns. This slight gain in efficiency in terms of bandwidth compression can be expanded to 5-8 % by the optimization of the limiting particle sizes. These optimized results are obtained when the final particle size is approximately 40% of the initial particle diameter.
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http://dx.doi.org/10.1016/j.chroma.2021.462331DOI Listing
August 2021

Mitigation of analyte loss on metal surfaces in liquid chromatography.

J Chromatogr A 2021 Aug 19;1650:462247. Epub 2021 May 19.

Waters Corporation, 34 Maple Street, Milford, MA 01757, USA.

The adsorptive loss of acidic analytes in liquid chromatography was investigated using metal frits. Repetitive injections of acidic small molecules or an oligonucleotide were made on individual 2.1 or 4.6 mm i.d. column frits. Losses were observed for adenosine 5'-(α,β-methylene) diphosphate, 2-pyridinol 1-oxide and the 25-mer phosphorothioate oligonucleotide Trecovirsen (GEM91) on stainless steel and titanium frits. Analyte adsorption was greatest at acidic pH due to the positive charge on the metal oxide surface. Analyte recovery increased when a series of injections was performed; this effect is known as sample conditioning. Nearly complete recovery was achieved when the metal adsorptive sites were saturated with the analyte. A similar effect was achieved by conditioning the frits with phosphoric, citric or etidronic acids, or their buffered solutions. These procedures can be utilized to mitigate analyte loss. However, the effect is temporary, as the conditioning agent is gradually removed by the running mobile phase. Metal frits modified with hybrid organic/inorganic surface technology were shown to mitigate analyte-to-metal surface interactions and improve recovery of acidic analytes. Quantitative recovery of a 15-35 mer oligodeoxythymidine mixture was achieved using column hardware modified with hybrid surface technology, without a need for column conditioning prior to analysis.
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http://dx.doi.org/10.1016/j.chroma.2021.462247DOI Listing
August 2021

Perspective on the Future Approaches to Predict Retention in Liquid Chromatography.

Authors:
Fabrice Gritti

Anal Chem 2021 04 2;93(14):5653-5664. Epub 2021 Apr 2.

Waters Corporation, 34 Maple Street, Milford, Massachusetts 01757, United States.

The demand for rapid column screening, computer-assisted method development and method transfer, and unambiguous compound identification by LC/MS analyses has pushed analysts to adopt experimental protocols and software for the accurate prediction of the retention time in liquid chromatography (LC). This Perspective discusses the classical approaches used to predict retention times in LC over the last three decades and proposes future requirements to increase their accuracy. First, inverse methods for retention prediction are essentially applied during screening and gradient method optimization: a minimum number of experiments or design of experiments (DoE) is run to train and calibrate a model (either purely statistical or based on the principles and fundamentals of liquid chromatography) by a mere fitting process. They do not require the accurate knowledge of the true column hold-up volume , system dwell volume (in gradient elution), and the retention behavior ( versus the content of strong solvent φ, temperature , pH, and ionic strength ) of the analytes. Their relative accuracy is often excellent below a few percent. Statistical methods are expected to be the most attractive to handle very complex retention behavior such as in mixed-mode chromatography (MMC). Fundamentally correct retention models accounting for the simultaneous impact of φ, , pH, and in MMC are needed for method development based on chromatography principles. Second, direct methods for retention prediction are ideally suited for accurate method transfer from one column/system configuration to another: these quality by design (QbD) methods are based on the fundamentals and principles of solid-liquid adsorption and gradient chromatography. No model calibration is necessary; however, they require universal conventions for the accurate determination of true retention factors (for 1 < < 30) as a function of the experimental variables (φ, , pH, and ) and of the true column/system parameters (, , dispersion volume, σ, and relaxation volume, τ, of the programmed gradient profile at the column inlet and gradient distortion at the column outlet). Finally, when the molecular structure of the analytes is either known or assumed, retention prediction has essentially been made on the basis of statistical approaches such as the linear solvation energy relationships (LSERs) and the quantitative structure retention relationships (QSRRs): their ability to accurately predict the retention remains limited within 10-30%. They have been combined with molecular similarity approaches (where the retention model is calibrated with compounds having structures similar to that of the targeted analytes) and artificial intelligence algorithms to further improve their accuracy below 10%. In this Perspective, it is proposed to adopt a more rigorous and fundamental approach by considering the very details of the solid-liquid adsorption process: Monte Carlo (MC) or molecular dynamics (MD) simulations are promising tools to explain and interpret retention data that are too complex to be described by either empirical or statistical retention models.
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http://dx.doi.org/10.1021/acs.analchem.0c05078DOI Listing
April 2021

Multiple-open-tubular column enabling transverse diffusion. Part 2: Channel size distribution and structure optimization.

J Chromatogr A 2021 Apr 4;1642:462033. Epub 2021 Mar 4.

Department of Chemistry, Philipps-Universität Marburg, Hans-Meerwein-Strasse 4, Marburg 35032, Germany.

Multiple-open-tubular columns enabling transverse diffusion (MOTTD) are made of straight, parallel, and cylindrical flow channels separated by a mesoporous stationary phase. In Part 1, a model of band broadening along MOTTD columns accounting for longitudinal diffusion, the trans-channel velocity bias, and mass transfer resistance in the stationary phase was proposed and validated. In this Part 2, the model is completed by considering the impact of short-range inter-channel velocity biases on the MOTTD plate number. These velocity biases are caused by the wide distribution of the channel diameters. Different ratios, ρ, of the average inner diameter, 2, of the flow channels to their closest center-to-center distance d (d= 5 μm, ρ= 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, and 0.9) with a relative standard deviation (RSD) increasing from 0 to 50% are considered. The zone retention factor k was increased from 1 to 25. The complete model of band broadening is validated after adjustment to dispersion data obtained by 1) the lattice-Boltzmann method for modeling fluid flow, 2) a random-walk particle-tracking (RWPT) technique to address advective-diffusive transport, and 3) by considering two distinct populations of flow channels (inner radii r=(1-RSD) and r=(1+RSD)) arranged at the nodes of a hexagonal compact array. The completed model of band broadening in MOTTD columns reveals that the RSD of the channel diameters has only a moderate impact on the optimum plate number of MOTTD columns: the relative increase of the minimum plate height do not exceed 30% even for the largest RSDs. However, when the mass transfer of the analyte is governed by its slow rate of transverse diffusion across the MOTTD column, the plate height can be increased by up to 100% at high average velocities. Regarding the best trade-off between analysis speed and column performance at a fixed pressure drop of 400 bar, irrespective of the zone retention factor and RSD of the distribution of the channel diameters, the fastest analyses are recommended for MOTTD columns having a small structural parameter ρ. In contrast, for the longest analysis times, the largest values of ρ are required to maximize the performance of MOTTD columns.
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http://dx.doi.org/10.1016/j.chroma.2021.462033DOI Listing
April 2021

Theoretical performance of multiple size-exclusion chromatography columns connected in series.

Authors:
Fabrice Gritti

J Chromatogr A 2020 Dec 2;1634:461673. Epub 2020 Nov 2.

Waters Corporation, Instrument/Core Research/Fundamental, 34 Maple Street, Milford, MA, 01757, USA. Electronic address:

The fundamental relationships are derived for the retention, peak width, and peak capacity of non-retained polymers eluting from multiple standard size-exclusion chromatography (SEC) columns connected in series. The standard SEC columns may have different dimensions and are packed with particles having distinct average particle diameters (APDs) and average mesopore sizes (AMSs). The performances (peak capacity, local resolution power, and sensitivity) of three standard SEC columns connected in series (called a tri-SEC column) packed with bridged-ethylene-hybrid (BEH) fully porous particles (FPPs) having three different APDs (1.7, 2.5, and 3.5 μm) and AMSs (200, 450, and 900 Å, respectively) are calculated as a function of the applied flow rate and size of polystyrene standards. Irrespective of the APD and AMS, the present investigation assumes isomorphological materials relative to the mesopore space of the three different BEH particles. The advantage of a 15 cm long tri-SEC column over a single reference SEC column (APD=3.5 μm, AMS=900 Å), which generates the same back pressure and separation window as those of the tri-SEC column, is expected at flow rates larger than the optimum flow rate generating the maximum peak capacity. The calculations predict a significant relative increase of the peak capacity (from +25% to +85%), resolution of small molecules (from +75% to +225%), and of the detection limit of intermediate size (from +15% to +70%) and largest polymers (from +25 to +110%). This is explained by 1) the exclusion of the largest polymers from the internal volume of the particles having the smallest mesopores (restricted access media) and 2) the minimum dispersion along the columns packed with the smallest particle sizes in the tri-SEC column. The main benefit of multi-SEC columns is to easily adjust the desired pore size distribution by properly selecting the lengths of each individual SEC column. The user can then control the pore size distribution for any specific separation problem. A potential application is theoretically demonstrated for the fast purification of monoclonal antibodies from metabolites, host cell proteins, aggregated forms, and from virus-like particles.
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http://dx.doi.org/10.1016/j.chroma.2020.461673DOI Listing
December 2020

Thermodynamic interpretation of the drift and noise of gradient baselines in reversed-phase liquid chromatography using mobile phase additives.

Authors:
Fabrice Gritti

J Chromatogr A 2020 Dec 10;1633:461605. Epub 2020 Oct 10.

Waters Corporation, 34 Maple Street, Milford, MA 01757, USA. Electronic address:

The drift and noise of acetonitrile-water gradient baselines (5-95%, v/v, 5 min linear gradient) in reversed phase liquid chromatography (RPLC) are recorded at a wavelength of 215 nm using 0.1% trifluoroacetic acid (TFA) as the mobile phase additive, a 4.6 mm  ×  150 mm 5 μm Symmetry-C RPLC column, and an Arc system (low-pressure gradient proportioning valve or GPV, pump with a stroke volume of either 66 or 132 μL, no mixer) as the LC instrument. These observations are predicted from solid-liquid adsorption thermodynamics which requires the measurement of the excess adsorption isotherm of acetonitrile from water onto the RPLC column and of the variation of the Henry's constant of TFA as a function of the volume fraction of acetonitrile in the bulk mobile phase. The incomplete mixing of the acetonitrile and water packets delivered by the low-pressure GPV is represented by a sinusoidal perturbation of the programmed volume fraction of acetonitrile during the entire gradient. The variation of the TFA absorbance at 215 nm with increasing acetonitrile concentration is measured in order to transform TFA concentration into the observable absorbance unit. The drift and noise of the gradient baseline are calculated by solving numerically (Rouchon method) the equilibrium-dispersive (ED) mass balance equations of acetonitrile and TFA. The agreement between the calculated and observed gradient baselines is very good as the proposed model of chromatography accurately accounts for the displacement of TFA between stationary and mobile phases (early excess and late deficit of TFA concentration relative to 0.1%) and for the frequency (equal to the ratio of the applied flow rate to the stroke volume) and the amplitude of the periodic noise recorded during the gradient. From a practical viewpoint, the drift of the gradient baseline can be minimized by maximizing the ratio of the gradient volume to the hold-up volume ( > 10) and/or by minimizing the retention factor of the mobile phase additive in the water-rich eluent (k < 0.2). The reduction of the noise amplitude below 0.1 mAU as requested by the pharmaceutical industry imposes the ratio of the flow rate to the stroke volume of the pump to be larger than 1 Hz. This opens avenues towards the development of new GPV, pump, and mixers in order to mix efficiently the solvent packets delivered by conventional LC instrument.
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http://dx.doi.org/10.1016/j.chroma.2020.461605DOI Listing
December 2020

Multiple-open-tubular column enabling transverse diffusion. Part 1: Band broadening model for accurate mass transfer predictions.

J Chromatogr A 2020 Aug 10;1625:461325. Epub 2020 Jun 10.

Department of Chemistry, Philipps-Universität Marburg, Hans-Meerwein-Strasse 4, Marburg 35032, Germany.

We derive a model of band broadening in multiple-open-tubular columns enabling transverse diffusion (MOTTD). In MOTTD columns, the flow channels are straight, parallel, cylindrical tubes arranged in a hexagonal compact array. A mesoporous material or stationary phase (130 Å bridged-ethyl hybrid (BEH) silica support) is filling the volume between the flow channels. The model is based on Giddings' random-walk theory of non-equilibrium chromatography. It is calibrated for the unknown configuration factor, q, related to the specific geometry of the stationary phase in MOTTD columns. q values are found based on the best fit of the model to simulated dispersion data obtained by the lattice-Boltzmann method for modelling fluid flow and a random-walk particle-tracking technique to address advective-diffusive transport of the analytes. For the model calibration, simulations are performed for different ratios, ρ, of the average inner diameter of the flow channels to their closest center-to-center distance under retained and non-retained conditions. The model is successfully validated (average relative errors below 10%) under both retained and non-retained conditions. For the same column format (4.6 mm i.d.  ×  150 mm), external porosity, zone retention factor, and relative standard deviation of the distribution of the inner diameters of the flow channels, the derived model reveals the intrinsic advantage of MOTTD columns (center-to-center distance between flow channels of 5 µm and ρ = 0.62) over a conventional column packed with 5 µm 130 Å BEH silica particles and the same multiple porous-layer open-tubular column (MPLOT) disabling transverse dispersion. MOTTD columns are weakly affected by the polydispersity of the inner diameter of the flow channels. Provided MOTTD columns could be prepared at a small feature size of 5 µm or less, they are expected to deliver a significant improvement in column technology relative to current particulate and silica monolithic columns.
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http://dx.doi.org/10.1016/j.chroma.2020.461325DOI Listing
August 2020

Turbulent Supercritical Fluid Chromatography in Open-Tubular Columns for High-Throughput Separations.

Anal Chem 2020 06 12;92(11):7409-7412. Epub 2020 May 12.

Waters Corporation, Instrument/Core Research/Fundamental, 34 Maple Street, Milford, Massachusetts 01757, United States.

A method utilizing turbulent flow to perform ultrafast separations and screen chiral compounds in supercritical fluid chromatography (SFC) is described. Carbon dioxide at high flow rates (up to 4.0 mL/min) is delivered into gas chromatography (GC) open-tubular columns (OTC, 0.18 mm i.d., 20 m long, ∼0.2 μm stationary film thickness) to establish turbulent flow at Reynolds numbers () as high as 9000. Postcolumn dispersion is eliminated by using a modified UV detector that takes measurements directly on column. Upon crossing the laminar-to-turbulent flow transition regime, a significant reduction in plate height is observed resulting in a nearly 3-fold increase in peak capacity from the laminar regime. This is explained by the massive reduction of the mass transfer resistance in the mobile phase due to a flatter flow profile and faster analyte dispersion across the open-tubular column (OTC) i.d.. Demonstrated in this work is a 9 s separation of four polycyclic aromatic hydrocarbons (PAHs) over a 2.2 s separation window using a poly(dimethylsiloxane--methylphenylsiloxane) coated OTC. Additionally, three chiral compounds and three chiral cyclodextrin-incorporated OTCs were evaluated at high temperatures (90-120 °C) and CO flow rates (3.3-3.7 mL/min) to demonstrate column stability and application of this method for rapid screening. Turbulent SFC provides a separation method for users desiring to achieve separation speeds above what is currently available with very high-pressure LC systems and do so without the resolution loss commonly observed at maximum allowable speed.
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http://dx.doi.org/10.1021/acs.analchem.0c01578DOI Listing
June 2020

Morphology-transport relationships in liquid chromatography: Application to method development in size exclusion chromatography.

J Chromatogr A 2020 Jun 21;1620:460991. Epub 2020 Feb 21.

Department of Chemistry, Philipps-Universität Marburg, Hans-Meerwein-Strasse 4, 35032 Marburg, Germany. Electronic address:

We present relationships between the multiscale structure and the separation properties of size exclusion chromatography (SEC) columns. Physical bed reconstructions of wall and bulk regions from a 2.1 mm i.d. column packed with fully porous 1.7 µm bridged-ethyl hybrid (BEH) particles, obtained by focused ion-beam scanning electron microscopy, serve as geometrical models for the packing microstructure in wall and central regions of a typical narrow-bore SEC column. In addition, the intraparticle mesopore space morphology of the BEH particles is reconstructed using electron tomography, to ultimately construct a realistic multiscale model of the bed morphology from mesopore level via interparticle macropore space to transcolumn scale. Complemented by the results of eddy dispersion simulations in computer-generated bulk packings, relationships between packing microstructure and transchannel, short-range interchannel, as well as transcolumn eddy dispersion are used to analyze the fluid dynamics in the interparticle macropore space of the model. Further, we simulate hindered diffusion and accessible porosity for passive, finite-size tracers in the intraparticle mesopore space, to finally determine the effective particle and bed diffusion coefficients of these tracers in the hierarchical (macro-mesoporous) bed. Retention and transport properties of polystyrene standards with hydrodynamic diameters from 5 to 95 Å in tetrahydrofuran are subsequently predicted without introducing bias from arbitrary models. These properties include the elution volumes of the polystyrene standards, the global peak capacity (over the entire separation window), and the rate of peak capacity at any fixed elution volume. Optimal flow rates yielding maximal global peak capacity and a nearly uniform rate of peak capacity over the entire separation window are close to 0.04 and 0.20 mL/min, respectively. SEC column performance obtained for fully porous and superficially porous particles is compared by varying the core-to-particle diameter ratio ρ from 0 to 0.95. Because the separation window is narrowing more rapidly than the rate of peak capacity is growing with increasing ρ, core-shell particles always provide smaller global peak capacity; they still can be advantageous but only for simple sample mixtures. The presented morphology-performance approach holds great promise for method development in SEC.
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http://dx.doi.org/10.1016/j.chroma.2020.460991DOI Listing
June 2020

Evaluating MISER chromatography as a tool for characterizing HILIC column equilibration.

J Chromatogr A 2020 May 27;1619:460931. Epub 2020 Jan 27.

Waters Corporation, Milford, MA 01757, United States.

Hydrophilic Interaction Liquid Chromatography (HILIC) is a technique for retaining polar analytes that uses polar stationary phases and acetonitrile-rich mobile phases. While this technique has several advantages over reversed-phase liquid chromatography (RPLC), one main drawback is the reported need for longer column equilibration. The reason for this is not fully understood and is a topic of current investigation. In order to better understand and reduce the equilibration needs, accurate characterization of column equilibration under varying conditions is required. The current method of characterizing HILIC column equilibration produces limited data points per test, or low time resolution, and is highly dependent on the column and probe compounds being used. There is a need for an improved method for characterizing HILIC column equilibration, especially if trends across stationary phases are to be observed. In this work, MISER, or Multiple Injections in a Single Experimental Run, is evaluated as a possible tool for characterizing HILIC column equilibration. MISER improves time resolution by allowing for replicate injections without interruption of data collection, enabling a more thorough evaluation of column equilibration compared to traditional techniques. Experimental results gathered using MISER show that equilibration of a BEH Amide column is notably shorter when equilibrating from acetonitrile to mobile phases containing higher percentages of water. Column equilibration to a 10% aqueous mobile phase was found to be approximately 5-fold faster than equilibration to a 3% aqueous mobile phase.
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http://dx.doi.org/10.1016/j.chroma.2020.460931DOI Listing
May 2020

Utility of linear and nonlinear models for retention prediction in liquid chromatography.

J Chromatogr A 2020 Feb 9;1613:460690. Epub 2019 Nov 9.

Waters Corporation, 34 Maple Street, Milford, MA 01757, USA.

Linear solvation strength model in reversed-phase liquid chromatography assumes linear relationship between ln k and Φ. In this work we show that this assumption is true only in narrow range of mobile phase strength. The ln k versus Φ relationship could be more accurately described by three-parametric non-linear model in a wide range of eluent strength. We investigated the consequences of non-linearity on retention prediction accuracy and analyte retention behavior in reversed-phase chromatography. When the ln k versus Φ is measured in narrow range of mobile phase strength (ΔΦ ~ 0.1-0.2) both linear and nonlinear models provide comparable retention prediction results. We propose that the linear trend of ln k versus Φ relationship is obtained in the range flanking the elution factor k (value of retention factor at the column end). We calculated and plotted changes of retention factor of analytes along the column. The visualization illustrates the ranges of retention factor values participating in separation during gradient. For typical gradient slopes employed in liquid chromatography practice and small molecules the elution factor k value is between 2 and 8. As a simplified generalization for typical gradient slopes we propose using linear ln k versus Φ trend in the k range between 1 and 30. The spreadsheet was utilized to compare the retention prediction accuracy of linear and non-linear retention models. When fitting ln k versus Φ trend in k range 1-30 the simple linear model is in good agreement with nonlinear model with retention time prediction error 0.3-4.7% (for gradient slope 0.013-0.260).
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http://dx.doi.org/10.1016/j.chroma.2019.460690DOI Listing
February 2020

Retention loss of reversed-phase chromatographic columns using 100% aqueous mobile phases from fundamental insights to best practice.

J Chromatogr A 2020 Feb 29;1612:460662. Epub 2019 Oct 29.

Waters Corporation, Instrument/Core Research/Fundamental 34 Maple Street, Milford, MA, 01757, USA.

This work deals with experimental investigations pertaining to the impact of chemical (electrolyte concentration from 0 to 100 mM, dissolved nitrogen gas from 0 to 6.7  ×  10 M in water; surface chemistry including hexylphenyl, polyphenyl, C, C, and C; surface coverage in C-bonded chains from 1.5 to 3.5 µmol/m; presence of surface dopant), physical (hydrostatic pressure of water from 50 to 500 bar; temperature from 27 C to 75 C), and structural parameters (average pore size from 50 Å to 400 Å; pore connectivity) on the dewetting kinetics of water from the hydrophobic mesopores of particles packed in RPLC columns. The results are explained from physico-chemical viewpoints involving intrusion and extrusion Laplace pressures, advancing and receding contact angles, surface tension of water, vapor pressure of water, 3D reconstruction of the actual mesoporous structure, pore connectivity, and the hysteresis in nitrogen adsorption and desorption isotherm onto reversed-phase chromatographic materials. A model of water dewetting consistent with the observations and the physical interpretations is then proposed. Finally, the most relevant practical solutions (pressurizing the column in absence of flow, pore size enlargement, using phenyl-bonded phase, polar embedded or surface doped C-bonded phases, reducing the C surface coverage, doping the silica surface, lengthening of the alkyl-bonded chains, applying low temperatures, purging and degassing the mobile phase with helium gas) are suggested in order to eliminate or at least minimize the retention loss of RPLC columns when using fully aqueous mobile phases.
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http://dx.doi.org/10.1016/j.chroma.2019.460662DOI Listing
February 2020

Mismatch between sample diluent and eluent: Maintaining integrity of gradient peaks using in silico approaches.

J Chromatogr A 2019 Dec 6;1608:460414. Epub 2019 Aug 6.

Waters Corporation, 34 Maple Street, Milford, MA 01757, USA.

The mismatch of elution strength between the sample diluent and the eluent causes undesirable peak deformations for large sample volumes in gradient liquid chromatography. The solution to that problem consists in diluting the sample solution in a weak solvent. But the minimum dilution factor has to be determined by the user given some performance objectives. In silico approaches are applied in this work to find such adequate dilution factors. Two calculations methods are proposed for the prediction of peak distortions. The first comprehensive method is based on solving numerically the mass balance equations for all the analytes and the strong solvent. An excellent agreement between the experimental and the calculated gradient chromatograms is observed (sample diluent: acetonitrile/water, 50/50, v/v; injection volume: 15 μL; linear gradient: 5%-95% acetonitrile during 3 min) for five compounds (acetanilide, coumarin, benzoin, bi-naphthol, and dibutylphthalate) injected into a 2.1 × 50 mm column packed with 1.7 μm XBridge-C particles. This first method happens to be highly time-consuming and impractical for common users. Experimental work and calculation times are then minimized by applying a second method based on the basics of retention and dispersion of injected pulses. Despite being less accurate than the first method, the agreement between the experimental and calculated peak width remains physically meaningful allowing the experimenter to rapidly guess the required sample dilution factor for any combination of injected volume and strong solvent concentration in the sample solution.
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http://dx.doi.org/10.1016/j.chroma.2019.460414DOI Listing
December 2019

Theoretical Analysis of Efficiency of Multi-Layer Core-Shell Stationary Phases in the High Performance Liquid Chromatography of Large Biomolecules.

Molecules 2019 Aug 6;24(15). Epub 2019 Aug 6.

Department of Analytical Chemistry, University of Pannonia, Egyetem utca 10, H-8200 Veszprém, Hungary.

Modern analytical applications of liquid chromatography require columns with higher and higher efficiencies. In this work, the general rate model (GRM) of chromatography is used for the analysis of the efficiency of core-shell phases having two porous layers with different structures and/or surface chemistries. The solution of the GRM in the Laplace domain allows for the calculation of moments of elution curves (retention time and peak width), which are used for the analysis of the efficiency of bi-layer particles with and without a non-porous core. The results demonstrate that bi-layer structures can offer higher separation power than that of the two layers alone if the inner layer has smaller surface coverage (retentivity) and the pore size and pore diffusion of the outer layer is either equal to or higher than that of the inner layer. Even in the case of core-shell phases, there is an increase in resolution by applying the bi-layer structure; however, we can always find a mono-layer core-shell particle structure with a larger core size that provides better resolution. At the optimal core size, the resolution cannot be further improved by applying a bi-layer structure. However, in case of the most widely produced general-purpose core-shell particles, where the core is ∼70% of the particle diameter, a 15-20% gain of resolution can be obtained by using well-designed and optimized bi-layer core-shell phases.
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http://dx.doi.org/10.3390/molecules24152849DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6695945PMC
August 2019

Faster dewetting of water from C- than from C-bonded silica particles used in reversed-phase liquid chromatography: Solving the paradox.

J Chromatogr A 2019 Sep 25;1602:253-265. Epub 2019 May 25.

Department of Chemistry, Philipps-Universität Marburg, Hans-Meerwein-Strae 4, 35032 Marburg, Germany.

For comparable surface coverage of alkyl-bonded chains (∼3 μmol/m), the dewetting of 100% aqueous mobile phases from the mesopores of octyl(C)-bonded silica particles is found 70 times faster than that from the same but octadecyl(C)-bonded silica particles. This observation was made in this work for both fully porous (5 μm Symmetry) and superficially porous (2.7 μm CORTECS) particles. This experimental result is paradoxical because (1) the average pore size of C-bonded materials is 10-15 Å larger than that of C-bonded materials for the same unbounded silica gel and (2) the contact angle of water measured on smooth and planar C-bonded surface is about 6° smaller than that on the same but C-bonded surface (104° versus 110°). The equilibrium Laplace pressure is then expected to be smaller and the kinetics of water dewetting to be slower for silica-C than for silica-C stationary phases used in RPLC. The solution to this riddle is investigated based on (1) the calculation of the dewetting time assuming that the pores are monosized and the process is driven by the Laplace pressure, (2) the measurement of the advancing and receding contact angles of three different C- and C-bonded silica gels (4 μm NovaPak, 5 μm Symmetry, and 2.7 μm CORTECS) from the water porograms measured in a range of water pressure from normal pressure to 500 bar, and (3) on the calculation of the pore connectivity for both C and C-bonded silica. First, the experimental results show that the observed dewetting times are of the order of minutes or even hours instead of millisecond as predicted by the dewetting model. Secondly, the advancing and receding contact angles of water onto the C-bonded silicas are found larger (by an average of +7° and +2°, respectively) than those measured for the same but C-bonded silica (average of 112° and 92°). Finally, the calculated pore connectivity is decreasing by about 30% for 90 Å unbounded silica materials from C to C-bonded RPLC phases. Overall, the observed and much faster dewetting of water from C column than that from C column is primarily explained by a higher internal pore connectivity due to the thinner thickness of the alkyl-bonded layer (7 Å versus 15 Å) and, to a lesser extent, by a higher extrusion Laplace pressure of water (≃+10 bar).
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http://dx.doi.org/10.1016/j.chroma.2019.05.041DOI Listing
September 2019

The effect of column packing procedure on column end efficiency and on bed heterogeneity - Experiments with flow-reversal.

J Chromatogr A 2019 Oct 28;1603:412-416. Epub 2019 May 28.

Department of Analytical and Environmental Chemistry and Szentágothai Research Center, Ifjúság útja 6, H-7624 Pécs, Hungary; MTA-PTE Molecular Interactions in Separation Science Research Group, Ifjúság útja 6, H-7624 Pécs, Hungary; Institute of Bioanalysis, Medical School, University of Pécs, Szigeti út 12, H-7624 Pécs, Hungary. Electronic address:

The effect of column end structure and bed heterogeneity of six commercially available reversed-phase chromatographic columns for fast liquid chromatography with different column packing materials - such as fully porous (Waters XBridge C with 1.7 μm particles) and superficially porous (Waters CORTECS C with 1.6 μm particles), with column dimension of 2.1 × 50, 100 or 150 mm were tested with flow-reversal method. The method includes arresting the flow when a non-retained marker (thiourea) has penetrated to a given distance into the column and then reversing the column. Hence, when the flow has been restarted, the sample is eluted at the same end of the column where it entered. The experiments showed that all columns are axially heterogeneous, and some differences could be observed between the two respective column ends. Furthermore, we can conclude that the shorter columns are axially more homogeneous than the longer ones, thus the column length is an influencing factor on the column packing procedure.
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http://dx.doi.org/10.1016/j.chroma.2019.05.040DOI Listing
October 2019

Slow injector-to-column sample transport to maximize resolution in liquid chromatography: Theory versus practice.

J Chromatogr A 2019 Aug 24;1600:219-237. Epub 2019 Apr 24.

GL Sciences Inc., Iruma, Saitama 358-0032, Japan.

A simple chromatographic model including extra-column sample bandspreading is built and validated experimentally. It is used to quantify the advantage of slowly transporting under isocratic elution of the injected sample band along the pre-column volume of the chromatographic system compared to the conventional injection method at constant flow rate. For fast analyses (<30 s), the model predicts a maximum relative gain of 20% for the isocratic peak capacity of weakly retained compounds (k < 1) at the price of a 60% increase in retention time (+20 s) for short (3.0 cm long) narrow-bore (2.1 mm i.d.) columns packed with sub-2 μm particles and for low-dispersion (∼2 μL extra-column volume variance) vHPLC systems. These predictions were confirmed experimentally using a 2.1 mm × 30 mm column packed with 1.9 μm XBridge-C for the rapid separation of small molecules in 25 s at 40 °C using a mixture of methanol and water (75/25, v/v) as the eluent. For longer analyses (>30 s), the model allows for the determination of the optimum and slow injection speed that maximizes the trade-off between the analysis time and the isocratic peak capacity. For 2.1 mm × 20 mm columns packed with 2 μm particles and a highest retention factor of 10, the optimum injection speed is about 10-15% of the operating column flow rate for a maximum relative gain of 5% in overall isocratic peak capacity at constant elution time. Overall, the proposed injection method is directly applicable for enhanced resolution of weakly retained polar compounds in RPLC without the need of changing separation conditions, which may strongly increase the retention times of the late apolar eluters.
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http://dx.doi.org/10.1016/j.chroma.2019.04.060DOI Listing
August 2019

Gradient method transfer after changing the average pore diameter of the chromatographic stationary phase I - One-dimensional sample mixture.

Authors:
Fabrice Gritti

J Chromatogr A 2019 Jul 15;1597:119-131. Epub 2019 Mar 15.

Waters Corporation, 34 Maple Street, Milford, MA 01757, USA. Electronic address:

Three different approaches designed to transfer gradient methods from a chromatographic column 1 packed with particles (2.7 μm 90 ÅCORTECS-C or CORTECS-Triphenyl) to a column 2 packed with the same particles having a larger average pore diameter (APD = 120 Åand 450 Å) are proposed for a one-dimensional sample mixture. Two approaches are based on the variation of the experimental retention plots (lnk vs. the volume fraction, φ, of the strong solvent) with increasing the APD. They lead to so-called "vertical" (vertical shift in column phase ratio, lnϕϕ) and "horizontal" (horizontal shift in eluent composition, Δφ) gradient method transfers. The third method is based on in silico predictions of the gradient retention times when considering the actual non-linearity of the retention plots. The adjusted gradient parameters (starting eluent composition, φ, and temporal gradient steepness, β) are unambiguously determined by minimizing the distance between the calculated and targeted gradient retention times of all the analytes. The performances of the three approaches for gradient method transfer are compared for a sample mixture containing a non-retained compound (thiourea) and a series of five homologous compounds (n-alkanophenones). The ultimate goal is to keep unchanged the gradient retention times of all analytes when changing the APD of the particles. The results show that the in silico transfer systematically outperforms the "horizontal" transfer, which itself outperforms the "vertical" transfer. The first two approaches are the least successful ones because, even for a series of homologous compounds, the linear solvent strength model (LSSM) is only an approximate model and the best shifts in eluent composition that keeps retention factors unchanged are compound-dependent. In the end, the average relative deviations between the observed and targeted gradient retention times are 15.0, 1.7, and 0.4% (90 Åto 120 Åtransfer, C chemistry), 5.1, 5.8 and 0.8% (90 Åto 450 Åtransfer, C chemistry), 4.8, 0.5, and 0.4% (90 Åto 120 Åtransfer, Triphenyl chemistry), and 10.4, 7.1, and 2.2% (90 Åto 450 Åtransfer, Triphenyl chemistry) for the "vertical", "horizontal", and "in silico" gradient method transfers, respectively.
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http://dx.doi.org/10.1016/j.chroma.2019.03.024DOI Listing
July 2019

Kinetic mechanism of water dewetting from hydrophobic stationary phases utilized in liquid chromatography.

J Chromatogr A 2019 Jul 23;1596:41-53. Epub 2019 Feb 23.

Waters Corporation, Instrument/Core Research/Fundamental, 34 Maple Street, Milford, MA 01757, USA.

An experimental protocol was designed to accurately measure the dewetting kinetics of aqueous mobile phases from reversed-phase liquid chromatography (RPLC) columns. The protocol enables the determination of the losses in the wetted surface area and internal pore volume (leading to undesirable retention losses) of RPLC columns as a function of the dewetting time. It is used to evaluate the impact of the buffer/salt concentration in water (0-100 mM), nitrogen concentration dissolved in water (0-6.7 × 10 M), column temperature (300-358 K), and of the internal structure (pore connectivity) of the stationary phase on the dewetting kinetics of various RPLC packing materials. From a fundamental viewpoint, the experimental facts demonstrate that dewetting kinetics are not solely driven by the pore size of the stationary phase and the contact angle with water. Temperature has a major influence on dewetting kinetics as it controls the nucleation rate of isolated water vapor bubbles over the entire mesoporous network. Additionally, the internal microstructure of the stationary phase (characterized by its internal porosity, pore size distribution, and pore connectivity) influences the rate at which the water vapor bubbles grow and coalesce in the entire particle volume. From a more practical viewpoint, the retention loss of RPLC columns due to water dewetting can be eliminated or at least minimized by (1) adjusting the surface and bonding chemistries to reduce the receding contact angle, (2) elevating the column outlet pressure, (3) operating at the lowest possible temperature, (4) minimizing the pore connectivity of the stationary phase (e.g., by increasing the degree of surface functionalization from C to C-bonded phases), and (5) by degassing the aqueous mobile phase from any dissolved gases.
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http://dx.doi.org/10.1016/j.chroma.2019.02.051DOI Listing
July 2019

On the performance of conically shaped columns: Theory and practice.

J Chromatogr A 2019 May 23;1593:34-46. Epub 2019 Jan 23.

Waters Corporation, 34 Maple Street, Milford, MA 01757, USA.

The chromatographic performance (speed, efficiency, and gradient peak capacity for the same analysis time) of conical columns are investigated from fundamental and experimental viewpoints. A stainless steel, conically shaped column (2.1 mm i.d/4.2 mm i.d. × 15 cm long, 0.4° opening angle) was prepared in-house and packed with 5 μm XBridge-C fully porous particles. Its performance was compared to that of a conventional 3.0 mm × 15 cm cylindrical column packed with the same batch of particles. Both Giddings' theory of non-uniform columns and experiments agree and show that, irrespective of flow direction, the conical column is 15% less efficient than the conventional column. Remarkably, Blumberg's theory of band broadening in gradient elution mode predicts that conical columns may outperform conventional cylindrical columns if the ratio of their outlet i.d. to their inlet i.d. is 0.95 and 0.80 for small molecule and peptide mixtures, respectively. The maximum relative gain is marginal as it does not exceed a few percents. The theory reveals that the flow direction should be from the wide to the narrow end of the conical column in order to deliver the highest peak capacity. In agreement with the theory, the observed losses in absolute peak capacity for the same analysis time are 14.5% (narrow to wide end) and only 11.0% (wide to the narrow end) for small molecules (n-alkanophenones). They are 14.2% (narrow to wide end) and only 8.5% (wide to the narrow end) for peptide samples (bombesin). Additionally, conical columns reduce peak tailing with respect to standard columns. They are suitable column technology for ultra-fast gradient separations as they also minimize sample dispersion through the narrow i.d. outlet frit.
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http://dx.doi.org/10.1016/j.chroma.2019.01.055DOI Listing
May 2019

Impact of frit dispersion on gradient performance in high-throughput liquid chromatography.

J Chromatogr A 2019 Apr 11;1591:110-119. Epub 2019 Jan 11.

Waters Corporation, 34 Maple Street, Milford, MA 01757, USA.

The impact of porous frits (2.1 mm i.d. × 1 mm thick, 0.2 μm porosity, 20% void, 0.69 μL) in short 0.5-5 cm long × 2.1 mm i.d. columns packed with sub-2 μm superficially porous particles on gradient performance was investigated using a low dispersive i-class ACQUITY UPLC system (25 cm × 75 μm outlet tube + 250 nL optical cell). In order to maximize data accuracy, the sample dispersion through a single frit was measured from four independent methods: (1) plate height subtraction, (2) peak capacity versus efficiency plot, (3) flow reversal, and (4) direct measurement. Frit dispersion increases non-linearly with increasing flow rate. The corresponding volume variances of small molecules were measured at 0.16 ± 0.04, 0.24 ± 0.05, and 0.31 ± 0.06 μL at flow rates of 0.1, 0.2, and 0.3 mL/min, respectively. These observed variances are lower than and consistent with the maximum volume variance of 0 . 69 ∼ 0.5 μL expected for a mixer-like behavior. The peak capacity of short columns were then calculated for mixtures of peptides using a general model of gradient elution by considering (or not) the actual analyte dispersion taking place in the outlet frit, in the post-column connecting tube, and in the detection cell. The results show that the sole presence of the outlet frit is responsible for about 50% loss in peak capacity (relative to the expected gradient performance in absence of frit and post-column tube) for a 1 cm long column operated under standard gradient steepness. The actual structure and volume of column frits needs adjustment if one wants to take full advantage of the true performance of short high-throughput columns packed with sub-2 μm particles.
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http://dx.doi.org/10.1016/j.chroma.2019.01.021DOI Listing
April 2019

Increasing chromatographic resolution of analytical signals using derivative enhancement approach.

Talanta 2019 Jan 15;192:492-499. Epub 2018 Sep 15.

Department of Chemistry & Biochemistry, University of Texas at Arlington, United States. Electronic address:

A few decades ago, Giddings made a bleak statistical prediction stating that when using a chromatographic column with a peak capacity of n, one "has no real hope" of separating n compounds because of peak overlap. This statement holds true for today's far more complex separations including chiral, achiral or isotopic separations. Co-eluting chiral and isotopically labeled positional isomers pose a mass spectrometric challenge as isobaric analytes. Several advanced mathematical approaches exist to resolve and extract areas from overlapping data, such as Fourier self-deconvolution, wavelets, multivariate curve resolution, and iterative curve fitting. In this work, we develop a very straightforward approach to mathematically enhance signal resolution using the properties of derivatives while conserving peak area and its position. This technique is based on the fact that the area under a derivative of a distribution is equal to zero. Consequently, by alternately subtracting and adding multiples of even-derivatives (second, fourth, sixth, and so on) from the original peak, the area under a peak is conserved, and the bandwidth is reduced. Unlike multivariate curve resolution and iterative curve fitting approaches, this protocol does not require prior knowledge of the number of peaks. The concept is theoretically discussed for Gaussian and Lorentzian peaks. Several challenging chromatographic applications using deuterated benzenes, chiral separations, and biological applications are shown using twin-column recycling and conventional chromatography. The proposed protocol for a pair of overlapping peaks is currently limited to a R of 0.7 or greater with error < 1% under ideal conditions. Furthermore, tuning of peak shape by the first derivative is also described which can remove the exponential convolution of tailing peaks.
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http://dx.doi.org/10.1016/j.talanta.2018.09.048DOI Listing
January 2019

High-resolution turbulent flow chromatography.

Authors:
Fabrice Gritti

J Chromatogr A 2018 Oct 26;1570:135-147. Epub 2018 Jul 26.

Waters Corporation, Instrument/Core Research/Fundamental, Milford, MA 01757, USA. Electronic address:

The resolution power of turbulent flow chromatography using carbon dioxide as the mobile phase and coated (crosslinked methyl phenyl polysiloxane) open tube columns (OTCs) as the stationary phase was investigated under retentive conditions (0
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http://dx.doi.org/10.1016/j.chroma.2018.07.059DOI Listing
October 2018

Characterization of radial and axial heterogeneities of chromatographic columns by flow reversal.

J Chromatogr A 2018 Sep 7;1567:164-176. Epub 2018 Jul 7.

Waters Corporation, Instrument/Core Research/Fundamental, Milford, MA 01757, USA.

The impact of the column length (5, 10, and 15 cm) and packing mode (constant pressure and constant flow rate up to 15,000 psi) on the radial and axial heterogeneities of 3.0 mm i.d. research prototype columns packed with the same batch of 2.4 μm BEH-C particles was investigated by the flow reversal technique. The data were gathered for a non-retained marker (uracil, acetonitrile/water eluent mixture, 80/20, v/v, flow rate 0.5 mL/min, T = 297 K) and revealed that the radial heterogeneity of the packed bed, characterized by the center-to-wall relative velocity bias (ω) and its length scale, is nearly independent on the packing mode: the velocity biases extend over a same length scale estimated at 154 μm while ω is in between 4% and 6% for all columns. Secondly, the data revealed that the column length has a slight impact on ω: assuming a two-region (wall and center regions) stochastic model of transcolumn eddy dispersion, ω increases from 4.6% to 5.1% and to 6.1% for 5, 10, and 15 cm long columns, respectively, when packed at constant flow rate. For columns packed at constant pressure, ω increases from 5.0% to 5.2% and to 5.6%, respectively. Finally, it is found that all columns are axially heterogeneous: the bottom half, which is packed first (column inlet), is slightly more uniform than the top half (column outlet) which is packed last. Overall, the results of the flow reversal experiments corroborate recent observations (130 μm thick wall region and ω = 5.0%) based on flow simulations in a focused-ion-beam scanning electron microscopy (FIB-SEM) based 3D reconstruction from a 2.1 mm × 50 mm column packed with 2 μm BEH-C particles.
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http://dx.doi.org/10.1016/j.chroma.2018.07.011DOI Listing
September 2018

Semi-preparative high-resolution recycling liquid chromatography.

J Chromatogr A 2018 Sep 23;1566:64-78. Epub 2018 Jun 23.

Pfizer, Analytical Research and Development, Groton, CT 06340, USA.

A semi-preparative high-resolution system based on twin column recycling liquid chromatography was built. The integrated system includes a binary pump mixer, a sample manager, a two-column oven compartment, two low-dispersion detection cells, and a fraction manager (analytical). It addresses challenges in drug/impurity purification, which involve several constraints simultaneously: (1) small selectivity factors (α < 1.2, poor resolution), (2) mismatch of elution strength between the sample diluent and the eluent causing severe band fronting or tailing, (3) diluent-to-eluent mismatch of viscosity causing viscous fingering and unpredictable band deformation, (4) low abundance of the impurity relative to the active pharmaceutical ingredient (API) (<1/100), and (5) yield and purity levels to be larger than 99% and 90%, respectively. The prototype system was tested for the preparation of a trace impurity present in a concentrated solution of an API, estradiol. The ultimate goal was to collect ∼1 mg of impurity (>90% purity) for unambiguous structure elucidation by liquid state nuclear magnetic resonance (NMR 600 MHz and above). First, the particle size (3.5 μm) used to pack the 4.6 mm × 150 mm long twin columns is selected so that the speed-resolution of the recycling process is maximized at 4000 psi pressure drop. Next, the production rate of the process is also maximized by determining the optimum number (7) of cycles and the corresponding largest sample volume (160 μL) to be injected. Finally, the process is fully automated by programming the time events related to (1) sample cleaning, (2) transfer of the targeted impurity from one to the second twin column, and (3) impurity collection. The process was tested without interruption during one week for the collection of a trace impurity (α = 1.166, strong acetonitrile-methanol sample diluent, concentration ∼2 mg/L) from a concentrated (10 g/L) stock solution (60 mL total) of estradiol. The process enriches the impurity content relative to the API by about a factor ∼5000. For the lack of a sufficient collected amount (∼120 μg only) of the pure impurity (purity 50% only), NMR experiments could not provide reliable results. Instead, the combination of LC-MS (single ion monitoring) and UV absorption spectra (λ shift) revealed that the targeted impurity was likely the low-abundant enol tautomeric form of the ketone estrone, a possible intermediate or by-product of the synthesis reaction of estradiol.
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http://dx.doi.org/10.1016/j.chroma.2018.06.055DOI Listing
September 2018

Molecular dispersion in pre-turbulent and sustained turbulent flow of carbon dioxide.

J Chromatogr A 2018 Aug 5;1564:176-187. Epub 2018 Jun 5.

Waters Corporation, Instrument/Core Research/Fundamental, Milford, MA 01757, USA.

The average dispersion coefficients, D¯, of two small molecules (acetonitrile and coronene) were measured under laminar, transient, and sustained turbulent flow regimes along fused silica open tubular capillary (OTC) columns (180 μm inner diameter by 20 m length). Carbon dioxide was used as the mobile phase at room temperature (296 K) and at average pressures in the range from 1500 to 2700 psi. The Reynolds number (Re) was increased from 600 to 5000. The measurement of D¯ is based on the observed plate height of the non-retained analytes as a function of the applied Reynolds number. D¯ values are directly estimated from the best fit of the general Golay HETP equation to the experimental plate height curves. The experimental data revealed that under a pre-turbulent flow regime (Re < 2000), D¯ is 2-6 times larger (3.5 × 10 cm/s) than the bulk diffusion coefficients D of the analyte (1.6 × 10 and 5.8 × 10 cm/s for acetonitrile and coronene, respectively). This result was explained by the random formation of decaying or vanishing turbulent puffs under pre-turbulent flow regime. Yet, the peak width remains controlled exclusively by the slow mass transfer in the mobile phase across the inner diameter (i.d.) of the OTC. Under sustained turbulent flow regime (Re > 2500), D¯ is about four to five orders of magnitude larger than D. The experimental data slightly overestimated the turbulent dispersion coefficients predicted by Flint-Eisenklam model (D¯=4 cm/s). The discrepancy is explained by the approximate nature of the general Golay equation, which assumes that D¯ is strictly uniform across the entire i.d. of the OTC. In fact, both the viscous and buffer wall layers, in which viscous effects dominate inertial effects, cannot be considered as fully developed turbulent regions. Remarkably, the mass transfer mechanism in OTC under sustained turbulent flow regime is not only controlled by longitudinal dispersion but also by a slow mass transfer in the mobile phase across the thick buffer layer and the thin viscous layer. Altogether, these layers occupy as much as 35% of the OTC volume at Re = 4000. From a theoretical viewpoint, the general Golay HETP equation is only an approximate model which should be refined based on the actual profile of the analyte dispersion coefficient across the OTC i.d. In practice, the measured plate height of non-retained analytes under sustained turbulent flow of carbon dioxide are two orders of magnitude smaller than those expected under hypothetical laminar flow regime.
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http://dx.doi.org/10.1016/j.chroma.2018.06.005DOI Listing
August 2018

A stochastic view on column efficiency.

Authors:
Fabrice Gritti

J Chromatogr A 2018 Mar 9;1540:55-67. Epub 2018 Feb 9.

Waters Corporation, Instrument/Core Research/Fundamental, Milford, MA, 01757, USA. Electronic address:

A stochastic model of transcolumn eddy dispersion along packed beds was derived. It was based on the calculation of the mean travel time of a single analyte molecule from one radial position to another. The exchange mechanism between two radial positions was governed by the transverse dispersion of the analyte across the column. The radial velocity distribution was obtained by flow simulations in a focused-ion-beam scanning electron microscopy (FIB-SEM) based 3D reconstruction from a 2.1 mm × 50 mm column packed with 2 μm BEH-C particles. Accordingly, the packed bed was divided into three coaxial and uniform zones: (1) a 1.4 particle diameter wide, ordered, and loose packing at the column wall (velocity u), (2) an intermediate 130 μm wide, random, and dense packing (velocity u), and (3) the bulk packing in the center of the column (velocity u). First, the validity of this proposed stochastic model was tested by adjusting the predicted to the observed reduced van Deemter plots of a 2.1 mm × 50 mm column packed with 2 μm BEH-C fully porous particles (FPPs). An excellent agreement was found for u = 0.93u, a result fully consistent with the FIB-SEM observation (u = 0.95u). Next, the model was used to measure u = 0.94u for 2.1 mm × 100 mm column packed with 1.6 μm Cortecs-C superficially porous particles (SPPs). The relative velocity bias across columns packed with SPPs is then barely smaller than that observed in columns packed with FPPs (+6% versus + 7%). u=1.8u is measured for a 75 μm × 1 m capillary column packed with 2 μm BEH-C particles. Despite this large wall-to-center velocity bias (+80%), the presence of the thin and ordered wall packing layer has no negative impact on the kinetic performance of capillary columns. Finally, the stochastic model of long-range eddy dispersion explains why analytical (2.1-4.6 mm i.d.) and capillary (<400 μm i.d.) columns can all be packed efficiently (1 3) with sub-2 μm particles and with 1 μm particles, respectively.
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http://dx.doi.org/10.1016/j.chroma.2018.02.005DOI Listing
March 2018

On the relationship between radial structure heterogeneities and efficiency of chromatographic columns.

Authors:
Fabrice Gritti

J Chromatogr A 2018 Jan 14;1533:112-126. Epub 2017 Dec 14.

Waters Corporation, Instrument/Core Research/Fundamental, Milford, MA 01757, USA. Electronic address:

The general dispersion theory of Aris is applied to predict the virtual asymptotic dispersion behavior of packed columns. The derived model is also used to estimate the actual pre-asymptotic dispersion behavior of modern 2.1 mm × 50 mm columns packed with sub-2 μm fully porous particles (FPPs) during the transient dispersion regime. The model accounts for the actual radial distribution of the flow velocity across the column diameter. From the wall to the center of the column, focused-ion-beam scanning electron microscopy (FIB-SEM) experiments were recently performed to reveal the existence of a thin (0.15d wide, d is the average particle diameter) hydrodynamic boundary layer (THBL), a thin (3d wide) and loose orderly packed layer (TLOPL), a 60d wide and dense randomly packed layer (WDRPL), and a large (≃460d) randomly packed bulk central region [1]. The theoretical calculations of the actual pre-asymptotic reduced van Deemter curves (2.1 mm × 50 mm column, sub-2 μm BEH-C FPPs, n-hexanophenone analyte, acetonitrile/water eluent, 80/20, v/v, flow rate from 0.05 to 0.35 mL/min) confirm that the impact of the sole THBL on column dispersion can be neglected. In contrast, the contribution of the TLOPL to the reduced plate height (RPH) is about 0.2 h unit at optimum reduced velocity. Most remarkably, the negative impact of the TLOPL on column performance may be fully compensated by the presence of the adjacent WDRPL if the depth of the velocity well were to be 5% of the bulk velocity. In actual 2.1 mm × 50 mm columns packed with sub-2 μm FPPs, this velocity depth is as large as 25% of the bulk velocity causing a significant RPH deviation of 0.7 h unit from the RPH of the bulk packing free from wall effects. Maximum column performance is expected for a reduction of WDRPL density. This suggests optimizing the packing process by finding the proper balance between the stress gradient across the WDRPL (responsible for the deep velocity well) and the friction forces between the packed particles (responsible for the rearrangement of the particles during bed consolidation). Past and recently reported RPH data support the theoretical insights: the stress gradient/particle friction balance in the WDRPL is better realized when packing superficially porous particles (SPPs) rather than FPPs in 2.1-4.6 mm i.d. columns (the RPH deviation is reduced to 0.4 h unit) or sub-2 μm particles in 100 cm × 75 μm i.d. capillaries combining high slurry concentrations and sonication (the RPH deviation is reduced to only 0.15 h unit).
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http://dx.doi.org/10.1016/j.chroma.2017.12.030DOI Listing
January 2018

Chromatographic performance of microfluidic liquid chromatography devices: Experimental evaluation of straight versus serpentine packed channels.

J Chromatogr A 2018 Jan 14;1533:127-135. Epub 2017 Dec 14.

Waters Corporation, 34 Maple Street, Milford, MA 01757, USA.

We prepared a series of planar titanium microfluidic (μLC) columns, each 100 mm long, with 0.15, 0.3 and 0.5 mm i.d.'s. The microfluidic columns were packed with 1.8 μm C18 sorbent and tested under isocratic and gradient conditions. The efficiency and peak capacity of these devices were monitored using a micro LC instrument with minimal extra column dispersion. Columns with serpentine channels were shown to perform worse than those with straight channels. The loss of efficiency and peak capacity was more prominent for wider i.d. columns, presumably due to on-column band broadening imparted by the so-called "race-track" effect. The loss of chromatographic performance was partially mitigated by tapering the turns (reduction in i.d. through the curved region). While good performance was obtained for 0.15 mm i.d. devices even without turn tapering, the performance of 0.3 mm i.d. columns could be brought on par with capillary LC devices by tapering down to 2/3 of the nominal channel width in the turn regions. The loss of performance was not fully compensated for in 0.5 mm devices even when tapering was employed; 30% loss in efficiency and 10% loss in peak capacity was observed. The experimental data for various devices were compared using the expected theoretical relationship between peak capacity P and efficiency N; (P-1) = N × const. While straight μLC columns showed the expected behavior, the devices with serpentine channels did not adhere to the plot. The results suggest that the loss of efficiency due to the turns is more pronounced than the corresponding loss of peak capacity.
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http://dx.doi.org/10.1016/j.chroma.2017.12.031DOI Listing
January 2018

Performance optimization of ultra high-resolution recycling liquid chromatography.

J Chromatogr A 2018 Jan 22;1532:74-88. Epub 2017 Nov 22.

Waters Corporation, 34 Maple Street, Milford, MA 01757, USA.

The optimization of a twin-column recycling separation process (TCRSP) for maximum resolution or maximum speed-resolution was investigated. The general optimization method was based on the construction of kinetic plots by assuming an ideal TCRSP (no efficiency loss upon recycling). For the optimization, we examined three chromatographic parameters: operation pressure (3000, 6000, 9000, and 12,000psi), column length (10, 15, and 25cm), and column inner diameter (i.d.) (2.1, 3.0, and 4.6mm). Accordingly, the highest TCRSP resolution level is expected for 25cm long columns packed with 2.5, 2.0, 1.7, and 1.6μm particles at pressures of 3000, 6000, 9000, and 12,000psi, respectively. The maximum speed-resolution performance is expected for 10cm columns packed with 3.7, 3.0, 2.6, and 2.4μm particles. 3.0mm i.d. columns are best to minimize the negative impacts of thermal and inter-column dispersion effects on the TCRSP performance. The method was illustrated for the challenging separation (selectivity factor α<1.02) of small molecules in RPLC at a maximum pressure of 6000psi using commercially available columns. Accordingly, 3.0×150mm columns packed with 2.5μm cellulose-1 Trefoil particles (chiral separation, γ-phenylbutyrolactone, α=1.01, efficiency N=4500) and 2.7μm Cortecs-C particles (isotope separation, α=1.02, N=14, 500) particles were found to be the most suitable columns to maximize speed-resolution performance. Further optimization of the TCRSP performance was required by reducing the inter-column sample dispersion that could cause undesirable peak tailing. A standard 2.4μL Rheodyne valve and 100μm i.d. tubes were replaced with a home-made 0.5μL low-dispersion prototype valve and 75μm i.d. perfect connection tubes. As a result, the experimental resolution factors were increased by +60% (γ-phenylbutyrolactone, 25 cycles, R=0.7→1.1) and +80% (deuterated benzenes, 22 cycles, R=1.1→2.0). Direct comparison between the experimental and the predicted TCRSP performance unambiguously demonstrated that the resolution gain was explained by the significant reduction of the peak tailing after a large number of cycles (n>20).
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http://dx.doi.org/10.1016/j.chroma.2017.11.047DOI Listing
January 2018