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Natural estrogens (estrone: E1, 17β-estradiol: E2, estriol: E3) and synthetic 17α-ethynylestradiol (EE2) are reported as strong endocrine disruptors even at extremely low concentrations. Therefore, the watch list from the European Commission regarding emerging aquatic pollutants recommended maximum detection limits of 0.035 ng/L for EE2 and 0.4 ng/L for E1 and E2. In this study, a UHPLC-ESI-MS/MS method allowing quantification of E1, E2, E3 and EE2 in aqueous matrices was developed. The analytes were derivatized using pyridine-3-sulfonyl chloride and a broad range of product ions were generated and their specificity was assessed by analyzing both surface and groundwater. At least two product ions for each estrogenic compound were proved to be specific and hence suitable for quantification and confirmation. In complex aqueous matrices, analyte responses were particularly affected by ion suppression. This phenomenon was reduced by optimizing the clean-up and selecting a suitable stationary phase for the chromatographic separation. The limits of quantification assessed in surface water with the optimized method ranged from 0.098 ng/L (EE2) to 2.73 ng/L (E3)., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Recently, gas chromatography tandem mass spectrometry (GC-MS/MS) has been added in European Union (EU) legislation as an alternative to magnetic sector high resolution mass spectrometry (HRMS) for the analysis of dioxins and dioxin like polychlorinated biphenyls (dl-PCB) in food and feed. In this study the performance of APGC-MS/MS compared to GC-HRMS is investigated and compared with EU legislation. The study includes the legislative parameters, relative intermediate precision standard deviation (S Rw ,rel), trueness, sensitivity, linear range and ion ratio tolerance. In addition, over 200 real samples of large variety and spanning several orders of magnitude in concentration were analyzed by both techniques and the selectivity was evaluated by comparing chromatograms. The S Rw ,rel and trueness were evaluated using (in-house) reference samples and fulfill to EU legislation, though the S Rw ,rel was better with GC-HRMS. The sensitivity was considerably better than of GC-HRMS while the linear range was similar. Ion ratios were mostly within the tolerable range of ±15%. A (temporary unresolved) systematic deviation in ion ratio was observed for several congeners, yet this did not lead to exceeding of the maximum ion ratio limits. The APGC-MS/MS results for the non-dioxin-like-PCBs (ndl-PCBs) were negatively biased, particularly for PCB138 and 153 in contaminated samples. The selectivity of APGC-MS/MS was lower for several matrices. Particularly for contaminated samples, interfering peaks were observed in the APGC chromatograms of the native compounds (dioxins) and labeled internal standards (PCBs). These can lead to biased results and ultimately to false positive samples. It was concluded that the determination of dioxins and PCBs using APGC-MS/MS meets the requirements set by the European Commission. However, due to generally better selectivity and S Rw ,rel, GC-HRMS is the preferred method for monitoring purposes., (Copyright © 2016 Elsevier B.V. All rights reserved.)

The kinetic performances of 3.0 × 100 mm columns packed with 1.9 μm Titan-C18 particles with average mesopore sizes of 80 Å and 120 Å were investigated quantitatively for the analysis of biomolecules. Large mesopores are expected to speed up the rate of diffusivity of high-molecular-weight compounds across the stationary phase and to generate higher plate counts at high velocities. The mass transfer mechanism of bradykinin acetate salt (1060 Da) and insulin (5733 Da) was determined over a range of flow rates from 0.025 to 1.0 mL/min. The pore diffusivities of these two biomolecules were accurately measured from the peak parking method. Even though the gain in column efficiency was not found significant for small molecules such as valerophenone (162 Da), enlarging the average pore size from 80 to 120 Å induces a measurable diminution of the reduced plate height, h, of bradykinin (from 17 to 11 or -35% at a reduced velocity of 50) and a significant reduction for insulin (from 43 to 12 or -72% at a reduced velocity of 90). Remarkably, while the increase of the column efficiency for bradykinin is consistent with a faster diffusivity of bradykinin across the 120 Å Titan-C18 particles, the higher column efficiencies measured for insulin are mostly due to a faster absorption kinetics into the 120 Å than that into the 80 Å Titan-C18 particles. This result is supported by the fact that the effective pore diffusivity of insulin is even slightly smaller across the 120 Å than that across the 80 Å 1.9μm Titan-C18 particles., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Previous data have shown that could deliver a minimum reduced plate height as small as 1.7. Additionally, the reduction of the mesopore size after C18 derivatization and the subsequent restriction for sample diffusivity across the Titan-C18 particles were found responsible for the unusually small value of the experimental optimum reduced velocity (5 versus 10 for conventional particles) and for the large values of the average reduced solid-liquid mass transfer resistance coefficients (0.032 versus 0.016) measured for a series of seven n-alkanophenones. The improvements in column efficiency made by increasing the average mesopore size of the Titan silica from 80 to 120Å are investigated from a quantitative viewpoint based on the accurate measurements of the reduced coefficients (longitudinal diffusion, trans-particle mass transfer resistance, and eddy diffusion) and of the intra-particle diffusivity, pore, and surface diffusion for the same series of n-alkanophenone compounds. The experimental results reveal an increase (from 0% to 30%) of the longitudinal diffusion coefficients for the same sample concentration distribution (from 0.25 to 4) between the particle volume and the external volume of the column, a 40% increase of the intra-particle diffusivity for the same sample distribution (from 1 to 7) between the particle skeleton volume and the bulk phase, and a 15-30% decrease of the solid-liquid mass transfer coefficient for the n-alkanophenone compounds. Pore and surface diffusion are increased by 60% and 20%, respectively. The eddy dispersion term and the maximum column efficiency (295000plates/m) remain virtually unchanged. The rate of increase of the total plate height with increasing the chromatographic speed is reduced by 20% and it is mostly controlled (75% and 70% for 80 and 120Å pore size) by the flow rate dependence of the eddy dispersion term., (Copyright © 2015 Elsevier B.V. All rights reserved.)

We propose an original model of effective diffusion along packed beds of mesoporous particles for HILIC developed by combining Torquatos model for heterogeneous beds (external eluent+particles), Landauers model for porous particles (solid skeleton+internal eluent), and the time-averaged model for the internal eluent (bulk phase+diffuse water (W) layer+rigid W layer). The new model allows to determine the analyte concentration in rigid and diffuse W layer from the experimentally determined retention factor and intra-particle diffusivity and thus to distinguish the retentive contributions from adsorption and partitioning. We apply the model to investigate the separation of toluene (TO, as a non-retained compound), nortriptyline (NT), cytosine (CYT), and niacin (NA) on an organic ethyl/inorganic silica hybrid adsorbent. Elution conditions are varied through the choice of a third solvent (W, ethanol, tetrahydrofuran (THF), acetonitrile (ACN), or n-hexane) in a mobile phase (MP) of ACN/aqueous acetate buffer (pH 5)/third solvent (90/5/5, v/v/v). Whereas NA and CYT retention factors increase monotonously from W to n-hexane as third solvent, NT retention reaches its maximum with polar aprotic third solvents. The involved equilibrium constants for adsorption and partitioning, however, do not follow the same trends as the overall retention factors. NT retention is dominated by partitioning and NA retention by adsorption, while CYT retention is controlled by adsorption rather than partitioning. Our results reveal that the relative importance of adsorption and partitioning mechanisms depends in a complex way from analyte properties and experimental parameters and cannot be predicted generally., (Copyright © 2014 Elsevier B.V. All rights reserved.)

A new brand of BEH-C18 hybrid particles chemically bonded to a leash carrying an amine group permits the implementation of electrostatic repulsive interactions chromatography. Using columns packed with this material, the influence of the concentration of positive charges bonded to the BEH-C18 surface on the overloaded band profiles of a few positively charged peptides and proteins was investigated in the gradient elution mode. Three columns packed with endcapped BEH-C18 particles bonded with three different surface-charge densities (LOW, MEDIUM and HIGH) were used and compared with those provided by a column packed with non-doped, endcapped BEH-C18 particles. The surface concentrations of fixed charges in the LOW, MEDIUM and HIGH columns were estimated at 0.029, 0.050, and 0.064μmol/m(2), for example, about two orders of magnitude smaller than the surface density of bonded C18 chains (2.1μmol/m(2)). Three different mobile phase additives (0.1% v/v of trifluoro-acetic, phosphoric, and formic acid) were used to optimize the purification levels of proteins under different loading conditions. The weak ion-pairing ions (formate and phosphate) generate smaller retention but broader, more fronting band profiles than those eluted with a stronger ion-pairing ion (trifluoroactate). This effect is worse in the presence of fixed charges at the surface of the BEH-C18 particles. This was explained by an enhanced anti-Langmuirian adsorption behavior of the charged proteins in the presence of fixed surface charges. As the protein concentration increases in the bulk, so does the internal ionic strength, the electrostatic repulsive interactions weaken, and retention increases. Band fronting is mostly eliminated by replacing weak ion-pairing acids with TFA with which the adsorption isotherm remains weakly langmuirian. Faster but still complete gradient separation of insulin and myoglobin were achieved with the HIGH column than with the reference neutral column, despite a measurable loss in selectivity., (Copyright © 2014 Elsevier B.V. All rights reserved.)

The effects of the surface concentration of positive charges attached to the surface of research BEH-C 18 hybrid particles on the overloaded band profiles and the adsorption isotherms of a neutral (caffeine) and a positively charged (nortryptilinium hydrochloride) compounds were measured and investigated. The inverse method (IM) of chromatography was used to determine the isotherm parameters. Three columns were packed with endcapped BEH-C 18 particles doped with three different charge densities on their surfaces (LOW, MEDIUM and HIGH). Two other columns packed with unbonded, non-endcapped, and endcapped BEH-C 18 particles served as standard reference materials. Minor disturbance method (MDM) experiments were conducted with acetonitrile/water mixtures in order to assess qualitatively the surface densities of the fixed positive charges. A more quantitative approach based on the solution of the linearized Poisson-Boltzmann equation and the decrease of the experimental Henry constant was also applied. The results show that the surface concentrations of the fixed charges in the LOW, MEDIUM and HIGH columns were 0.029, 0.050, and 0.064μmol/m 2 , e.g., close to two orders of magnitude smaller than the surface density of bonded C 18 chains (2.1μmol/m 2 ). The adsorption isotherm of the ionizable compound nortryptilinium onto the BEH-C 18 columns is consistent with a two-sites adsorption model. The density of the high energy sites correlates directly to the total amount of the fixed charges and isolated silanols amidst the C 18 -bonded chains. The amount of low energy sites reflects the specific surface area of the adsorbent. The binding constants on the high- and low-energy adsorption sites are respectively ten and two times lower on the HIGH column than on the reference endcapped column. The active sites are closer to the adsorbent surface than the weak adsorption sites. Finally, a higher production rate of ionizable compounds can be achieved in preparative chromatography with the charge doped than with the reference RPLC-C 18 columns. For the same analysis time, larger recovery yields are observed despite a slight loss in selectivity due to non-selective repulsive electrostatic interactions., (Copyright © 2014. Published by Elsevier B.V.)

We propose to use constant enthalpy or isenthalpic diagrams as a tool to estimate the extent of the temperature variations caused by the mobile phase pressure drop along a chromatographic column, e.g. of its cooling in supercritical fluid and its heating in ultra-performance liquid chromatography. Temperature strongly affects chromatographic phenomena. Any of its variations inside the column, whether intended or not, can lead to significant changes in separation performance. Although instruments use column ovens in order to keep constant the column temperature, operating conditions leading to a high pressure drop may cause significant variations of the column temperature, both in the axial and the radial directions, from the set value. Different ways of measuring these temperature variations are available but they are too inconvenient to be employed in many practical situations. In contrast, the thermodynamic plot-based method that we describe here can easily be used with only a ruler and a pencil. They should be helpful in developing methods or in analyzing results in analytical laboratories. Although the most effective application area for this approach should be SFC (supercritical fluid chromatography), it can be applied to any chromatographic conditions in which temperature variations take place along the column due to the pressure drop, e.g. in ultra-high pressure liquid chromatography (UHPLC). The method proposed here is applicable to isocractic conditions only., (Copyright © 2014 Elsevier B.V. All rights reserved.)

The mass transport properties of a non-retained (thiourea) and three retained low molecular weight compounds (acetophenone, valerophenone, and octanophenone) along a 4.6mm×45mm PROSWIFT™ RP-1S monolithic column made of rigid cross-linked poly(styrene-divinylbenzene) copolymer was investigated in depth. Accurate protocols (peak parking experiments, measurement of the first and second central moments of peak profiles by numerical integration) combined with the use of validated models of effective diffusion along monolithic structures were applied for the determination of the longitudinal diffusion, the eddy dispersion, and the skeleton-eluent mass transfer resistances due to the finite analyte diffusivity across the polymer skeleton and to the slow absorption kinetics into the polymer volume. Experimental results show by increasing order of importance evidence that the resolution performance of this short and wide polymer-based monolithic HPLC column is limited by the slow analyte diffusivity across the polymer skeleton (smaller than one tenth of the bulk diffusion coefficient for k'>1), its large eddy dispersion HETP (Heddy≃100μm), and the slow rate of absorption (≃10Hz only) in the polymer volume for retained analytes. The column performance could be improved by preparing a more homogeneous material with a rigid internal mesoporous structure. This would provide a column bed having a larger specific surface area, allowing faster analyte diffusion across the mesoporous skeleton, a smaller eddy dispersion HETP, and a faster absorption kinetics in the polymeric monolith than those observed for the currently available materials., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Computer calculations of gradient chromatograms were performed by taking into account the adsorption behavior of the strong eluent in RPLC and the true Henry constant of the analytes. This improves the accuracy of classical gradient calculations, which all assume no affinity of the eluent modifier for the stationary phase and that the linear solvation strength model (LSSM) applies. The excess adsorption isotherm of acetonitrile with respect to water was measured by the minor disturbance method onto a Symmetry-C₁₈ RPLC adsorbent. The variations of the Henry constants of a nine compound mixture with the volume fraction of acetonitrile in the aqueous mobile phase were measured. The equilibrium dispersive model of chromatography combined with orthogonal collocation on finite elements was used to calculate chromatograms of the sample mixture for four gradient times decreasing from 25 to 1 min. The results predict a loss of resolution for the less retained analytes when the gradient times becomes smaller than 4 min. They also predict that this behavior can be eliminated when applying a quadratic gradient profile rather than a classical linear gradient. The predictions were validated by the agreement between the calculated and experimental chromatograms., (Copyright © 2014 Elsevier B.V. All rights reserved.)

The mass transfer mechanism in four prototype columns (2.1 and 3.0×50mm, 2.1 and 3.0×100mm) packed with 1.9μm fully porous Titan-C18 particles was investigated by using two previously reported home-made protocols. The first one was used to measure the eddy dispersion HETP of these new columns, the second one to estimate their intrinsic (corrected for HPLC system contribution) HETPs. Titan particles are fully porous particles with a narrow particle size distribution (RSD of 9.2%). The mean Sauter diameter (dSauter=2.04μm) was determined from Coulter counter measurements on the raw silica material (before C18 derivatization) and in the absence of a dispersant agent (Triton X-100) in a 2% NaCl electrolyte solution. The results show that these RPLC Titan columns have intrinsic minimum reduced HETPs ranging from 1.7 to 1.9 and generate up to 290,000 plates per meter. The 3.0mm i.d. columns are more efficient than the 2.1mm i.d. ones and short columns are preferred to minimize efficiency losses due to frictional heating at high speeds. This work also revealed that (1) the lowest h values of the Titan columns are observed at low reduced velocities (νopt=5); (2) this is due to the unusually small diffusivity of analytes across the porous Titan-C18 particles; and (3) the Titan columns are not packed more uniformly than conventional columns packed with fully porous particles. Earlier and recent findings showing that the PSD has no direct physical impact on eddy dispersion and column efficiency are confirmed by these results., (Copyright © 2014 Elsevier B.V. All rights reserved.)

In a previous report, it was reported that columns packed with fully porous 1.9μm Titan-C18 particles provided a minimum reduced plate height as small as 1.7 for the most retained compound (n-octanophenone) under RPLC conditions. These particles are characterized by a relatively narrow size distribution with a relative standard deviation (RSD) of only 10%. A column packed with classical 5μm Symmetry-C18 particles, used as a reference RPLC column, generated a minimum reduced plate height of 2.1 for the same retained compound. This work demonstrates that this was due to an unusually low intra-particle diffusivity across these particles, which leads to a small longitudinal diffusion coefficient along the column. The demonstration is based on the combination of accurate measurements of the height equivalent to a theoretical plate (HETP), inverse size exclusion chromatography (ISEC), peak parking (PP), and minor disturbance method (MDM) experiments. The experimental results show that the reduced eddy dispersion HETP term (A=0.8 for a reduced velocity of 5), the internal particle porosity (ϵp=0.35), and the enrichment of acetonitrile in the pore volume (75% acetonitrile in the bulk, 85% inside the mesoporous volume) are identical on both the Titan-C18 and Symmetry-C18 columns. The difference between the internal structures of these two brands of RPLC-C18 fully porous particles lies in the values of the internal obstruction factor γp, which is 0.42 for the Symmetry-C18 but only 0.26 for the Titan-C18 particles. This is in part related to the diffusion hindrance due to the small average pore size of the Titan-C18 particles, around 59Å versus 77Å for Symmetry-C18 particles. A simple model of constriction along diffusion paths having the shape of a truncated cone suggests that the width of the pore size distribution (RSD of 30% and 20% for Titan-C18 and Symmetry-C18 particles) is mostly responsible for the difference in their obstruction factors., (Copyright © 2014 Elsevier B.V. All rights reserved.)

The effect of the pressure on the dispersion of a low molecular weight compound along 0.508 and 1.016 mm i.d. × 50 cm long open circular tubes was investigated theoretically and experimentally. The theoretical predictions were based on the early models of dispersion derived by Aris and Taylor (1953) and on the approximate model of Alizadeh for the time moments (1980). Experimentally, the system pressure was increased at constant flow rate (0.15 mL/min) from less than 20 to nearly 1,000 bar by using a series of capillary tubes (25 μm i.d. PEEKSIL tubes) of increasing flow resistances placed upstream the detection cell of a commercial very high pressure liquid chromatograph (vHPLC) but downstream the 50 cm long tube. Theoretical and experimental results agree that the peak variance increases linearly with increasing pressure in the tube volume. The relative increase of the peak variance is 7% above that measured at low pressure (<20 bar) for each 100 bar increment in the tube volume. This result confirms that accurate measurements of the column efficiency corrected for extra-column contribution cannot be made by replacing the column with a zero dead volume union connector, because the pressures applied in the pre-column volume are significantly different in these two cases. This work shows also that increasing the pressure in the pre-column volume by increasing the flow rate affects the apparent column efficiency that is measured, independently of the direct effect of the flow rate. For a 2.1 × 50mm column packed with 1.3 μm core-shell particles run with a classic Acquity system, the associated relative decreases of the column efficiency are expected to be -30%, -20%, and -5% for retention factors of 1, 3, and 10, respectively. The column HETP is no longer independent of its length., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Whether columns packed with core-shell particles may outperform those packed with fully porous particles for chiral separations is controversial. The potential advantages of such columns are investigated from a theoretical viewpoint. The height equivalent to a theoretical plate (HETP) associated to the slow adsorption-desorption process typical of chiral chromatography was derived from the Laplace transform of the general rate model of chromatography for core-shell particles. The relationship between the resolution factor and the core-to-particle diameter ratio is predicted at constant selectivity. The calculations are based on a complete set of actual kinetic parameters (longitudinal diffusion, eddy dispersion, intra-particle diffusivity, and adsorption-desorption constant) measured for a reference column packed with Lux Cellulose-1 fully porous particles. If we compare columns packed with the best procedure available in either case, the results demonstrate that those packed with core-shell particles may outperform to a degree those packed with fully porous particles. The minimum reduced HETP could decrease from 2.0 to 1.7. The maximum relative gain in resolution is about 10%, which is not negligible for critical enantioselective-separations. This gain is observed only if the packing uniformity of the core-shell particles is achieved., (Copyright © 2014 Elsevier B.V. All rights reserved.)

The adsorption isotherms of the Naproxen enantiomers eluted with a mixture of carbon dioxide and methanol on a (R,R)-Whelk-O1 column were measured using frontal analysis and the inverse method. Sharp breakthrough fronts provided using a modified design of the instrument allowed an accurate determination of these isotherms. The adsorption model that best accounts for the data was derived from the analysis of the adsorption isotherm data. The affinity energy distribution of the adsorption of the two enantiomers were calculated to determine the heterogeneity of the adsorbent surface for each compound. The adsorption model chosen based on the adsorbent heterogeneity was fitted to the adsorption isotherm data points and the best fitted isotherm parameters were derived from this fit. The inverse method was used as a second method to derive the parameters of the same adsorption models for the overloaded elution bands of (S)- and (R)-naproxen. The isotherm parameters derived from these independent numerical calculations are discussed and compared in the case of single component adsorption and of competitive adsorption as well., (Copyright © 2014 Elsevier B.V. All rights reserved.)

The formation of a concentration shock layer of either methanol or acetonitrile used as the modifier in steep RPLC aqueous/organic gradients was predictable from their excess adsorption isotherm and was directly observed from their elution profiles recorded in UV absorption. A discontinuity of the concentration profile of the organic modifier arises and grows along the column. Its effects on the peak shapes and the resolution levels achieved in steep gradient chromatography were measured in the recorded chromatograms of a sample mixture containing 14 different analytes uniformly distributed across the gradient retention window. The results showed severe peak shape deformation for some compounds and a significant degradation of the baseline resolution of weakly (when using acetonitrile) and moderately (when using methanol) retained analytes. Solutions to limit this loss of resolution may lie in changing the initial gradient conditions or in adjusting the curvature of the gradient profile at the column inlet., (Copyright © 2014 Elsevier B.V. All rights reserved.)

A critical aspect in fast gradient separations carried out under constant pressure, in the very high pressure liquid chromatography (VHPLC) mode is that time-based chromatograms may not yield highly reproducible separations. A proposed solution to improve the reproducibility of these separations involves plotting the chromatograms as functions of the volume eluted vs. UV absorbance instead of time vs. UV. To study the consequences of using the volume-based rather than the time-based chromatograms, separations were first performed under low pressures that do not generate significant amounts of heat and for which the variations of the eluent density along the columns are negligible. Secondly, they were performed under very high pressures that do generate heat and measurable variations of the local retention factor and eluent density along the column. Comparison of the results provides estimates of the improvements obtained when volume based chromatograms are used in gradient analyses. Using a column packed with fully porous particles, four different types of methods and several sets for each method were used to perform the gradient elution runs: two sets of constant flow rate operations, four sets of constant pressure operations, two sets of constant pressure operations with programmed flow rate, and one set using the constant heat loss approach. The differences between time-based and volume-based chromatograms are demonstrated by using eight replicates of early, middle, and last eluting peaks. The results show that volume-based chromatograms improve the retention time reproducibility of the four constant pressure methods by a factor of 3.7 on average. If the column is not thermally conditioned prior to performing a long series of separations, flow controlled methods (constant flow rate, programmed constant pressure, and constant wall heat approaches) are more precise. If one gradient run is used to bring the column to a relatively stable temperature, constant pressure separations have a factor of 3 times better reproducibility of retention times with respect to constant flow rate gradient separations., (Copyright © 2014 Elsevier B.V. All rights reserved.)

The advantages of using columns packed with stationary phases having a composition gradient so that retention factors increase toward the outlet and eluting them with an isocratic mobile phase may provide increased peak resolution in liquid chromatography. This approach is discussed from a theoretical viewpoint. The retention factor is assumed to increase linearly along the column. The peak width can be predicted under three different conditions: (1) the possible band compression is neglected (the Giddings model); (2) band compression is taken into account (the Poppe model); and (3) both band compression and extra-column effects are considered in the calculations. The impact of a stationary phase gradient on the resolution of a pair of compound difficult to separate (constant selectivity α=1.05) is illustrated for a 3mm×100mm silica monolithic column of the second generation (porosity ϵt=0.85, plate height H=6μm) operated with a new generation of liquid chromatograph (pre- and post-column volume variances of 10 and 5μL(2), respectively). The results show that the resolution factor RS becomes optimum for a specific positive value of the retention gradient along the column. This optimum depends on the retention factor at the column inlet., (Copyright © 2014 Elsevier B.V. All rights reserved.)

The adsorption isotherms of both naproxen enantiomers were measured on a (R,R)-Whelk-O1 chiral adsorbent from a mobile phase made of liquid carbon dioxide and methanol, using frontal analysis. These isotherms were used to model the competitive adsorption behavior of a racemic mixture of naproxen enantiomers. Their overloaded elution band profiles were calculated numerically using these isotherm data. The injected volume and the injected concentration of the samples of racemic mixture were carried out simultaneously, using two objective functions. The maximization of the production rate and of the product of the recovery yield and the production rate were performed. Based on the optimum points obtained, the two objective functions were compared for several recovery yield constraints in a range of injected volumes and concentrations. The consequences of choosing a separation design focused on the degree of band overlapping are discussed based on the results of these optimizations., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Severe distortions of the axial concentration profiles of modifiers in steep RPLC gradients were recently observed. These distortions are directly explained by the results of measurements of the excess adsorption isotherms of the strongest mobile phase component, the concentration of which is made to increase linearly with time at the column inlet. A front shock or a discontinuity of the organic modifier concentration may arise and grow along the column. The position where it forms is determined by the reciprocal of the second derivative of the excess adsorption isotherm with respect to the concentration of the strongest mobile phase component. It forms when two characteristic lines intersect for the first time. Gradient profiles are continuous and diffuse as long as characteristic lines do not intersect but diverge from each other. However, acetonitrile-water gradients are systematically distorted and deviate significantly from assumed ideal, linear, non-retained gradients. This challenges the validity of classical theories of gradient chromatography regarding the prediction of retention times, peak widths, and band compression factors when steep gradients are applied., (Copyright © 2014 Elsevier B.V. All rights reserved.)

This work reviews and discusses controversies and errors made in the determination of the average volumetric flow rate of a compressible mobile phase forced to flow through a chromatographic column. Proper estimates of the volumetric flow rate, which obviously changes along the column, are keys to understanding the retention mechanism that takes place inside the column and to achieve repeatable and reproducible separations. Each step of the calculation process will be discussed in detail, including how to estimate the variations of the pressure and the temperature along the column. The determination of the average volumetric flow rate requires the knowledge of the average density of the mobile phase and of its mass flow rate. The calculations were carried out under various experimental conditions, including different column temperatures and inlet pressures. The estimated values of the volumetric flow rate are validated by the conversion of the retention times to the retention volumes of nitrous oxide peaks, which is valid since this compound is assumed to be non retained, which makes it a hold-up time marker., (Copyright © 2014 Elsevier B.V. All rights reserved.)

The mass transfer mechanism in three prototype narrow-bore columns (2.1mm×100mm format) packed with 1.6μm superficially porous particles was investigated using different instruments. The heights equivalent to a theoretical plate of three small molecules were measured using a mixture of acetonitrile and water as the eluent. The values reported include the contributions of longitudinal diffusion, eddy dispersion, and the solid-liquid mass transfer resistance. The bulk diffusion coefficients of the analytes were measured using the capillary method, calibrated with thiourea in pure water. The reduced longitudinal diffusion coefficient was determined from the results of a series of peak parking experiments. The solid-liquid mass transfer resistance coefficient and the intra-particle diffusivities of the analytes in the porous region of the particles were estimated from Garnett-Torquato's model of effective diffusion in dense beds packed with core-shell particles. The eddy dispersion term, mostly due to trans-column and border effects, was obtained by subtracting the longitudinal diffusion and the solid-liquid mass transfer resistance terms from the total HETP obtained from the first and second central peak moments calculated by numerical integration (Simpson's approach) after baseline correction and systematic left and right cuts of the peak profiles. The results show that the eddy dispersion controls at least 66% of the overall column HETP for small molecules beyond the optimum velocity. This work illustrates how important it is to use ultra-low dispersive very high pressure liquid chromatography (vHPLC) systems to properly measure and to practically use the high efficiencies of narrow-bore columns packed with 1.6μm core-shell particles since these columns provide intrinsic efficiencies higher than 400,000 plates per meter., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Three methods were used to analyze elution bands of methanol on silica, using pure CO2 as the eluent. The results of these analyses were applied to calculate overloaded elution band profiles in supercritical fluid chromatography. The results obtained are compared. To ensure that the mobile phase density varies widely along the column bed, high volumetric flow rates of the mobile phase (CO2) were applied to two columns packed with neat, porous silica. Then, even a slight error made in the determination of the isotherm parameters or during the numerical calculations should be magnified compared to those obtained with a low pressure drop along the column. During the determination of the isotherms of adsorption of methanol from liquid carbon dioxide onto silica, the inlet and outlet pressure of the column, the mass flow rate and the temperature were monitored continuously. Based on these parameters, overloaded elution bands were calculated numerically using three calculation methods. The results are compared with experimental ones., (Copyright © 2014 Elsevier B.V. All rights reserved.)

The intrinsic heights equivalent to a theoretical plate (HETPs) of 31 narrow-bore and wide-bore columns packed with four different brands of core-shell particles were accurately measured on an optimized vHPLC instrument (1290 Infinity system) that has an extra-column volume variance of 13.6±0.3 μL(2). These results were derived from the slopes of the linear plots of the apparent plate heights of each column versus the reciprocal of (1+k')(2) for seven homologous compounds with a linear regression coefficient larger than 0.999. The results show that the kinetic performance of narrow-bore columns packed with core-shell particles increases almost linearly with decreasing particle diameter. The optimum reduced plate heights increase slightly from 1.6 to 1.9 with decreasing particle sizes from 4.6 to 1.3 μm. This confirms that wide-bore columns provide better efficiencies than narrow-bore columns., (Copyright © 2014 Elsevier B.V. All rights reserved.)

The effects of column length on performance in segmented flow chromatography were tested. Column efficiencies were measured for 4.6mm I.D. 3, 5, 7.5 and 10 cm long columns packed with 3.0 μm Hypurity-C18 fully porous particles and of 4.6mm I.D. 5, 10, 15 and 25 cm long columns packed with 5 μm Hypersil GOLD C18 particles. For each column length and particle type, two different configurations were tested: (1) both the inlet and outlet column endfittings were standard and (2) the inlet endfitting was standard but the outlet endfitting allowed parallel segmentation of the exiting flow into a central and a peripheral coaxial region. The segmentation flow ratio was set at 45% (for 3 μm) and at 43% or 21% (for 5 μm). Four samples were used, naphthalene, toluene, butylbenzene, and insulin, which has a ten times smaller diffusion coefficient than the small molecules. The column performance for the low molecular weight compound is significantly improved at velocities above the optimum value when the outlet flow rate is segmented because longitudinal diffusion and mass transfer resistance of this compound in the stationary phase are negligible sources of band broadening at reduced linear velocities between 5 and 25. At high flow rate (4 mL/min), the long-range eddy dispersion terms are about 3.9, 3.2, 2.6, and 1.8h unit lower for the 3, 5, 7.5 and 10 cm long columns, respectively. The longer the column, the lower the efficiency improvement because the border effects are smaller. This result was not systematically observed for the columns packed with 5 μm particles because the transverse dispersion is larger. In contrast, the gain in column efficiency is marginal for insulin because the mass transfer mechanism of this compound is mostly controlled by the slow diffusivity of insulin across Hypurity-C18 particles., (Copyright © 2014 Elsevier B.V. All rights reserved.)

The mechanism of mass transfer in chiral chromatography was investigated using an experimental protocol already applied in RPLC and HILIC chromatography. The different contributions to the reduced height equivalent to a theoretical plate (HETP) include the longitudinal diffusion HETP term, the solid-liquid mass transfer resistance HETP term, the short-range eddy dispersion HETP term, and the long-range eddy dispersion HETP term. Their accurate measurement permits the determination of the adsorption rate constant kads of trans-stilbene enantiomers on a column packed with Lux 5 μm Cellulose-1 particles. The experimental results demonstrate that the number of adsorption-desorption steps per unit time of chiral compounds on polysaccharide-based chiral stationary phases is four orders of magnitude smaller than that of achiral compounds., (Copyright © 2014 Elsevier B.V. All rights reserved.)

The implementation of the traditional FA method is difficult with classical supercritical fluid chromatography (SFC) instruments. The instrument mixer and other sources of extra-column volumes are large and significantly broaden the fronts of injected plugs, which diminishes the precision and accuracy of the FA method. An SFC instrument was modified to permit more accurate determinations of adsorption isotherm data. The sample, the modifier, and CO2 are separately pumped via small volume connection tubes into a small volume mixer (250μL), where they are mixed into a homogeneous fluid fed to the column. The extra-column volumes and the column hold-up volume were accurately measured at each back pressure from the retention times of tracers. This modified instrument was used to measure the adsorption isotherm of S-naproxen by frontal analysis (FA) on a (R, R)-Whelk-O1 column, using a mixture of methanol (20%, v/v) and CO2 as the mobile phase. Its performance is studied at several different back pressures from 100 to 210bar. In all the experiments, the total flow rate was kept to a low value (1mL/min) in order to minimize the variation of the equilibrium constant along the column. Although a suitable breakthrough curve could not be obtained at low back pressures (<150bar) due to the closeness to the critical point pressure of the methanol/CO2 mixture, excellent results were obtained at higher back pressures (>150bar), conditions remote from the critical point and breakthrough curves with very sharp front shocks are obtained. The RSDs of the profiles recorded at each back pressures are excellent, better than 1%., (Copyright © 2013 Elsevier B.V. All rights reserved.)

The mechanism of mass transfer was studied on a cellulose-based chiral stationary phase (CSP, Lux Cellulose-1) using aqueous mixtures of acetonitrile (50/50-90/10, v/v) or methanol (90/10 and 100/0, v/v) as the mobile phase. An experimental protocol validated in RPLC and HILIC chromatography and recently extended to chiral RPLC was applied. The five mass-transfer contributions (longitudinal diffusion, short-range and long-range eddy dispersion, solid-liquid mass transfer resistances due to finite intra-particle diffusivity and slow adsorption-desorption) to the reduced height equivalent to a theoretical plate (HETP) were measured. The experimental results show that the adsorption rate constants kads of trans-stilbene enantiomers onto the CSP are three times larger with acetonitrile than with methanol as the organic modifier. This is correlated to the decrease of enantioselectivity from 1.4 (in methanol) to only 1.1 (in acetonitrile). The amount of solvent needed to achieve a separation factor of exactly 2.0 was determined. This showed that analysis cost could be reduced seven times by selecting pure methanol as the eluent for a 5cm long column rather than an acetonitrile-water mixture for a longer (20-45cm) column., (Copyright © 2013 Elsevier B.V. All rights reserved.)

A rapid and simple validated experimental protocol is proposed for the accurate determination of the true intrinsic column efficiency and for that of the variance of the extra-column volume of the instrument used, the latter being obtained without requiring the removal of the chromatographic column from the HPLC system. This protocol was applied to 2.1mm×100mm columns packed with sub-3 (2.7μm Halo Peptide ES-C18) and sub-2μm (1.6μm prototype) core-shell particles. It was validated by observing the linear behavior of the plot of the apparent column plate height versus the reciprocal of (1+k')(2) for at least three homologous compounds, with a linear regression coefficient R(2) larger than 0.999. Irrespective of the contribution of the several, different instruments used to the total band broadening, the same column HETP value was obtained within 5%. This new protocol outperform the classical one in which the chromatographic column is replaced with a zero dead volume (ZDV) union connector to measure the extra-column volume variance, which is subtracted from the variance measured with the column to measure the intrinsic HETP. This protocol fails because it significantly underestimates the system volume variance., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Five methods for controlling the mobile phase flow rate for gradient elution analyses using very high pressure liquid chromatography (VHPLC) were tested to determine thermal stability of the column during rapid gradient separations. To obtain rapid separations, instruments are operated at high flow rates and high inlet pressure leading to uneven thermal effects across columns and additional time needed to restore thermal equilibrium between successive analyses. The purpose of this study is to investigate means to minimize thermal instability and obtain reliable results by measuring the reproducibility of the results of six replicate gradient separations of a nine component RPLC standard mixture under various experimental conditions with no post-run times. Gradient separations under different conditions were performed: constant flow rates, two sets of constant pressure operation, programmed flow constant pressure operation, and conditions which theoretically should yield a constant net heat loss at the column's wall. The results show that using constant flow rates, programmed flow constant pressures, and constant heat loss at the column's wall all provide reproducible separations. However, performing separations using a high constant pressure with programmed flow reduces the analysis time by 16% compared to constant flow rate methods. For the constant flow rate, programmed flow constant pressure, and constant wall heat experiments no equilibration time (post-run time) was required to obtain highly reproducible data., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Using a column packed with fully porous particles, four methods for controlling the flow rates at which gradient elution runs are conducted in very high pressure liquid chromatography (VHPLC) were tested to determine whether reproducible thermal conditions could be achieved, such that subsequent analyses would proceed at nearly the same initial temperature. In VHPLC high flow rates are achieved, producing fast analyses but requiring high inlet pressures. The combination of high flow rates and high inlet pressures generates local heat, leading to temperature changes in the column. Usually in this case a post-run time is input into the analytical method to allow the return of the column temperature to its initial state. An alternative strategy involves operating the column without a post-run equilibration period and maintaining constant temperature variations for subsequent analysis after conducting one or a few separations to bring the column to a reproducible starting temperature. A liquid chromatography instrument equipped with a pressure controller was used to perform constant pressure and constant flow rate VHPLC separations. Six replicate gradient separations of a nine component mixture consisting of acetophenone, propiophenone, butyrophenone, valerophenone, hexanophenone, heptanophenone, octanophenone, benzophenone, and acetanilide dissolved in water/acetonitrile (65:35, v/v) were performed under various experimental conditions: constant flow rate, two sets of constant pressure, and constant pressure operation with a programmed flow rate. The relative standard deviations of the response factors for all the analytes are lower than 5% across the methods. Programming the flow rate to maintain a fairly constant pressure instead of using instrument controlled constant pressure improves the reproducibility of the retention times by a factor of 5, when plotting the chromatograms in time., (Copyright © 2013 Elsevier B.V. All rights reserved.)

The pressure, temperature and density drops along SFC columns eluted with a CO2/methanol mobile phase were measured and compared with theoretical values. For columns packed with 3- and 5-μm particles the pressure and temperature drops were measured using a mobile phase of 95% CO2 and 5% methanol at a flow rate of 5mL/min, at temperatures from 20 to 100°C, and outlet pressures from 80 to 300bar. The density drop was calculated based on the temperature and pressure at the column inlet and outlet. The columns were suspended in a circulating air bath, either bare or covered with foam insulation. The experimental measurements were compared to theoretical results obtained by numerical simulation. For the convective air condition at outlet pressures above 100bar the average difference between the experimental and calculated temperature drops and pressure drops were 0.1°C and 0.7% for the bare 3-μm column, respectively, and were 0.6°C and 4.1% for the insulated column. The observed temperature drops for the insulated columns are consistent with those predicted by the Joule-Thomson coefficients for isenthalpic expansion. The dependence of the temperature and the pressure drops on the Joule-Thomson coefficient and kinematic viscosity are described for carbon dioxide mobile phases containing up to 20% methanol., (Copyright © 2013 Elsevier B.V. All rights reserved.)

This work demonstrates the possible advantages and the challenges of accurate on-line measurements of the CO2 mass flow rate during supercritical fluid chromatography (SFC) operations. Only the mass flow rate is constant along the column in SFC. The volume flow rate is not. The critical importance of accurate measurements of mass flow rates for the achievement of reproducible data and the serious difficulties encountered in supercritical fluid chromatography for its assessment were discussed earlier based on the physical properties of carbon dioxide. In this report, we experimentally demonstrate the problems encountered when performing mass flow rate measurements and the gain that can possibly be achieved by acquiring reproducible data using a Coriolis flow meter. The results obtained show how the use of a highly accurate mass flow meter permits, besides the determination of accurate values of the mass flow rate, a systematic, constant diagnosis of the correct operation of the instrument and the monitoring of the condition of the carbon dioxide pump., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Uwe Neue developed a theoretical treatment to account for the adsorption of ions on mesopores of packing materials the walls of which are bonded to ionic ligands but left this work unfinished. We elaborated upon this treatment and refined it, based on the equivalence that he suggested between charged surface particles and a membrane that separates two ionic solutions but is impermeable to one specification. He had written that the electro-chemical potentials in both ionic solutions are equal (Donnan equilibrium). The equilibrium between the surface and the pore concentrations is accounted for by an homogeneous electrostatically modified Langmuir (EML) isotherm model. The theoretical results are presented for four different charge surface concentrations σ0=0, 0.001, 0.002, and 0.003C/m(2), using a phosphate buffer (W(S)pH=2.65) of ionic strength I=10mM. The average pore size, the specific surface area, and the specific pore volume of the stationary phase were Dp=140Å, Sp=182m(2)/g, and Vp=0.70cm(3)/g, respectively. The theoretical results provide the quantitative difference between the ionic strength, the pH, and the concentrations of all the ions in the pores and in the bulk eluent. The theory predicts (1) that the retention times of cations under linear conditions is lower and (2) that their band widths under overloaded conditions for a given retention factor shrinks when the surface charge density σ0 is increased. These theoretical results are in good agreement with experimental results published previously and explain them., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Five methods were used to implement fast gradient separations: constant flow rate, constant column-wall temperature, constant inlet pressure at moderate and high pressures (controlled by a pressure controller),and programmed flow constant pressure. For programmed flow constant pressure, the flow rates and gradient compositions are controlled using input into the method instead of the pressure controller. Minor fluctuations in the inlet pressure do not affect the mobile phase flow rate in programmed flow. There producibilities of the retention times, the response factors, and the eluted band width of six successive separations of the same sample (9 components) were measured with different equilibration times between 0 and 15 min. The influence of the length of the equilibration time on these reproducibilities is discussed. The results show that the average column temperature may increase from one separation to the next and that this contributes to fluctuation of the results.

The adsorption isotherms of the naproxen enantiomers were measured on a Kromasil Whelk-O1 column, eluted with mixtures of supercritical carbon dioxide and methanol or ethanol. Five chromatographic methods were used: frontal analysis, frontal analysis by characteristic points, elution by characteristic points, retention time method and the inverse method. In all methods, the effects of the two modifiers were compared. The use of these methods suffers from limitations due to supercritical fluid chromatographic instruments. These problems and drawbacks are discussed. The accurate and precise determination of the isotherm parameters was not possible with the instrument currently used. In contrast, the different methods allow to show qualitatively that the adsorption of the naproxen enantiomers show heterogeneous mechanism on the adsorbent surface common in chiral chromatography. Finally, the experimental high concentration elution band profiles of naproxen are compared with those calculated from the isotherm parameters provided by the five different methods., (Copyright © 2013 Elsevier B.V. All rights reserved.)

The reduced trans-column (or long-range eddy dispersion) height equivalent to a theoretical plate (HETP) of short and wide 4.6mm×30mm columns packed with 2.6μm Accucore-C18 superficially porous particles was measured under conventional (no split flow) and parallel segmented (outlet and inlet) flow chromatography. The overall reduced HETP was derived from the true moments of the recorded concentration profiles. The longitudinal diffusion HETP term was measured at a very small flow rate (0.05mL/min). The solid-liquid mass transfer resistance was derived from the shell diffusivity, using the composite Garnett-Torquato model of effective diffusion in a heterogeneous system made of a dense packing of core-shell particles immersed in a continuous matrix (the eluent). The trans-channel and short-range interchannel eddy dispersion HETP terms were assumed to be equal to the calculated h data after solving the Navier-Stokes equation and simulating the advection-diffusion transport process. Experimental results confirmed that the optimum efficiency of these short columns was increased by a factor of about two. The ratio of the detection sensitivities on the PSFC stream and on a regular stream increased from 1 to 1.45 when the retention factor decreases from about 10 to 0.5. These phenomena are due to a strong reduction of the trans-column eddy dispersion HETP term. The system loses about 60% of the sample mass when only outlet skimming is carried out when the flow rate ratio of 55% is applied, as was done in this work. It loses about 50% of the sample when inlet/outlet segmentation is applied. In gradient elution, the peak capacity is increased by only 15%, due to post-column band spreading, which should imperatively be minimized when the outlet flow is split., (Copyright © 2013 Elsevier B.V. All rights reserved.)

The influence of the modifier concentration in supercritical mixtures of carbon dioxide and methanol on the speed-resolution properties of columns packed with 1.7μm core-shell particles was investigated from a theoretical viewpoint. Molar fractions of methanol up to 30% were considered. The column was assumed to be operated under strict adiabatic conditions in order to maximize its efficiency. Four inlet temperatures were tested, between 297 and 337K. Four different pressure drops along the column were considered, between 25 and 200bar. The physico-chemical properties (density, viscosity, and heat capacity) of the mixtures of carbon dioxide and methanol were derived from the NIST REFPROP program for temperatures between 287 and 337K and pressures between 150 and 390bar. The axial heterogeneity of the column was taken into account by segmenting it into 500 hundreds slices in each of which all physical and chromatographic properties were assumed to be uniform. The apparent kinetic Poppe plots were built from the apparent column efficiency calculated from the sum of the increments of the retention times and time variances from the column inlet to its outlet. The numerical results showed that the axial heterogeneity of the column due to axial variations of the temperature and the equilibrium constant decreases with increasing molar fraction of methanol in the eluent when the pressure drop is increased from 25 to 200bar. The methanol content decreases the speed-resolution of the column more particularly when the analysis is done at low pressure drops. The results demonstrate also that under adiabatic conditions, for pressure drops larger than 100bar, an increase of methanol concentration does not cause a dramatic loss of speed-resolution. For example, at 310K and with a pressure drop of 25bar, the longest column is expected to deliver 17,000 plates; increasing the molar fractions of methanol from 0 to 30% decreases this efficiency by about 15% and increases the analysis time by +140%. In contrast, under the same experimental conditions except for a pressure drop of 200bar, the maximum efficiency (130,000 plates) would remain unchanged and the analysis time increase by only 40%., (Copyright © 2013 Elsevier B.V. All rights reserved.)

The excess adsorption isotherm of ethanol from liquid carbon dioxide on a silicagel column was determined in a range of back pressures and temperatures. The retention patterns of minor disturbance peaks were mapped and their dependences on the applied back pressure and column temperature measured. The analysis of the linear decreasing part of the excess isotherm around its inflection point provided the finite layer thickness of the adsorbed phase. Our results show that increasing the temperature and decreasing the back pressure result in a thicker layer of the layer of enriched organic solvent above the silicagel surface., (Copyright © 2013 Elsevier B.V. All rights reserved.)

This study introduces a new tracer that is useful for the determination of the hold-up time or volume of packed columns, particularly of those used in supercritical fluid chromatography. The thermodynamic void volume of three columns packed with different adsorbents were determined using the weight difference method. These void volumes were used as the reference point in the further discussion. The hold-up volumes of these columns were determined under dynamic conditions, using nitrous oxide dissolved in methanol as the hold-up time marker. Changes in the hold-up volumes of these columns were monitored during changes of the volumetric flow rate of pure supercritical carbon dioxide and of dilute mixtures of organic modifier and supercritical carbon dioxide. The results suggest significant methanol enrichment on the adsorbent surface., (Copyright © 2013 Elsevier B.V. All rights reserved.)

An algorithm was developed to recognize peaks in a multidimensional separation and calculate their cumulative peak area. To find the retention times of peaks in a one dimensional chromatogram, the Savitzky-Golay smoothing filter was used to smooth and find the first through third derivatives of the experimental profiles. Close examination of the shape of these curves informs on the number of peaks that are present and provides starting values for fitting theoretical profiles. Due to the nature of comprehensive multidimensional HPLC, adjacent cut fractions may contain compounds common to more than one cut fraction. The algorithm determines which components were common in adjacent cuts and subsequently calculates the area of a two-dimensional peak profile by interpolating the surface of the 2D peaks between adjacent peaks. This algorithm was tested by calculating the cumulative peak area of a series of 2D-HPLC separations of alkylbenzenes, phenol and caffeine with varied concentrations. A good relationship was found between the concentration and the cumulative peak area., (Copyright © 2013 Elsevier B.V. All rights reserved.)

The surface excess isotherms of methanol, ethanol, 2-propanol and acetonitrile from liquid carbon dioxide on a silica adsorbent were measured, using the minor disturbance method. The minor disturbance peaks - or system peaks - of each organic modifier were recorded using UV-detection in the whole composition range of the organic/liquid carbon dioxide mixtures, the whole composition range was completed by injecting the modifiers into pure liquid carbon dioxide as well. The excess isotherms were calculated based on the retention of the organic solvent signals. Our results show an enrichment of the organic modifier at the adsorbent surface. The alcohols show multilayer adsorption with an extremely high retention on the silica surface while acetonitrile shows weaker interactions and only a slight trend toward the formation of a multilayer above the surface., (Copyright © 2013 Elsevier B.V. All rights reserved.)

This paper compares two methods used for the preparative purification of a mixture of (S)-, and (R)-naproxen on a Whelk-O1 column, using either high performance liquid chromatography or supercritical fluid chromatography. The adsorption properties of both enantiomers were measured by frontal analysis, using methanol-water and methanol-supercritical carbon dioxide mixtures as the mobile phases. The measured adsorption data were modeled, providing the adsorption isotherms and their parameters, which were derived from the nonlinear fit of the isotherm models to the experimental data points. The model used was a Bi-Langmuir isotherm, similar to the model used in many enantiomeric separations. These isotherms were used to calculate the elution profiles of overloaded elution bands, assuming competitive Bi-Langmuir behavior of the two enantiomers. The analysis of these profiles provides the basis for a comparison between supercritical fluid chromatographic and high performance liquid chromatographic preparative scale separations. It permits an illustration of the advantages and disadvantages of these methods and a discussion of their potential performance., (Copyright © 2013 Elsevier B.V. All rights reserved.)

The retention behavior of carbamazepine eluted in SFC was studied by changing the back pressure between 100 and 200bar and the volumetric flow rate between 0.5 and 5mL/min. Carbamazepine was eluted under linear conditions from a silica column with 90:10% (V/V) mixtures of supercritical carbon dioxide and methanol at 310K. The retention factor of carbamazepine decreases by 20% when the pressure drop along the column increases from 1 to 28bar and the outlet pressure increases from 100 to 200bar. The variation of the retention factor at low pressure drops (below 15bar) is negligible (a few percent) but it is significant (exceeding 20%) for pressure drops larger than 15bar. Under these latter conditions, the variation of the equilibrium constant along the column is significant at high pressure drops. Because the adsorption parameters depend on the local pressure, the extension of dynamic methods from LC to SFC should be made cautiously for the determination of adsorption isotherms. Our results suggest that the flow rate applied during the course of dynamic method measurements should be kept small. The determination of the adsorption parameters from measurements carried out at high flow rates might not be as accurate as it is when the column is axially uniform., (Copyright © 2013 Elsevier B.V. All rights reserved.)

The adsorption of methanol, ethanol and isopropyl alcohol on a silicagel adsorbent from supercritical carbon dioxide was investigated using nonlinear chromatographic techniques. Series of adsorption isotherm data points of these alcohols were determined by frontal analysis. Their nonlinear adsorption behavior were modeled using a modified BET equation extended for heterogeneous adsorption surfaces. Our results suggest the formation of an adsorbed multilayer of alcohols above the silica surface. This organic solvent enriched layer governs the separation of the analytes in supercritical fluid chromatographic separations, both under analytical and preparative conditions., (Copyright © 2013 Elsevier B.V. All rights reserved.)

The mass transfer mechanism in HILIC was investigated in depth. The reduced heights equivalent to a theoretical plate (HETP) of five low molecular weigh compounds with retention factors of -0.05 (slight exclusion from the surface due to the presence of a water-rich layer in which naphthalene is insoluble) to 3.64 were measured at room temperature for a 4.6mm×100mm column packed with 3.5μm 140Å XBridge HILIC particles in a wide range of flow velocities. The mobile phase was a buffered acetonitrile-water mixture (92.5/7.5, v/v). Using a physically reliable model of effective diffusion in binary composite media (Torquato's model), the longitudinal diffusion and solid-liquid mass transfer resistance reduced HETP terms were measured. The reduced short-range eddy dispersion HETP was taken from the literature data. The long-range reduced HETP was directly measured from the subtraction of these HETP terms to the overall HETP measured from moment analysis. In contrast to RPLC, the plots of the reduced HETP versus the reduced velocity depend weakly on the retention factor, due to the constant, low intra-particle diffusivity observed in HILIC. So, the reduced longitudinal diffusion HETP is smaller and the reduced solid-liquid mass transfer resistance HETP is larger in HILIC than in RPLC. Whereas border effects can be concealed in RPLC for retained analytes due to fast radial equilibration across the column diameter, a residual long-range eddy dispersion term persists in 4.6mm I.D. HILIC columns, even at very slow flow rates. Experiments show that the minor differences in the long-range eddy dispersion term between analytes having different retention factors is directly correlated to the reciprocal of their bulk diffusion coefficient. The performance of HILIC columns packed with fine particles is then more sensitive to the inlet sample distribution and to the outlet sample collection than RPLC columns due to the relatively poor radial mixing controlled by lateral diffusion., (Copyright © 2013 Elsevier B.V. All rights reserved.)

The fundamental assumptions of the van Deemter height equivalent to a theoretical plate (HETP) equation were formulated nearly 60 years ago in its rigorous final mathematical derivation in 1956. The limit of applicability of this classical theory of band broadening in chromatographic columns is discussed on the basis of accurate measurements of diffusion coefficients (in the bulk, in particles, and in column beds), of peak moments in both RPLC and HILIC, on the recent numerical solution of the Navier-Stokes equation and on the results of the simulation of the advection-diffusion transport in the bulk region of computer-generated random packed beds. A result of this discussion is that serious errors are made in the interpretations of the mass transfer mechanism in HILIC and RPLC that are based on the use of the original van Deemter expressions of the longitudinal diffusion coefficient through packed bed, of the mass transfer resistance in the mobile phase, and of the mass transfer resistance in the stationary phase. These errors are discussed and quantitatively assessed. Physically acceptable and relevant expressions are proposed to account for the true mass transfer mechanism in packed columns., (Copyright © 2013 Elsevier B.V. All rights reserved.)

The effective diffusion coefficients of five low molecular weigh compounds (naphthalene, uracil, uridine, adenosine, and cytosine) were measured at room temperature in a 4.6mm×100mm column packed with 3.5μm XBridge HILIC particles. The mobile phase was an acetonitrile-water mixture (92.5/7.5, v/v) containing 10mM ammonium acetate and 0.02% acetic acid. Using a physically reliable model of effective diffusion in binary composite media (Torquato's model), accurate estimates of the intra-particle diffusivities in the HILIC particles were obtained as a function of the retention of these analytes. The HILIC diffusion coefficients were compared to those previously obtained for endcapped RPLC-C18 particles (5.0μm Gemini-C18). The experimental results confirm that adsorption sites are not localized in RPLC whereas they are so in the HILIC mode. In contrast to RPLC columns, HILIC columns provide longitudinal diffusion B/u terms that increase very little with increasing retention factors. This confirms the absence of surface diffusion in HILIC. The impact of intra-particle diffusivity on the column efficiency was projected in HILIC and RPLC on the basis of the measured intra-particle diffusivities and on the well established theory of band broadening in particulate columns. Accordingly, RPLC columns generate short-range eddy dispersion and solid-liquid mass transfer resistance Cu terms that increase less than do HILIC column with increasing retention factors. The HETP contribution caused by the trans-column structure heterogeneity is smaller in the HILIC than in the RPLC modes because the transverse excursion length is smaller in HILIC. Even though the overall column efficiencies are comparable in HILIC and RPLC, this study shows that the individual mass transfer phenomena are inherently different in the HILIC and the RPLC retention modes., (Copyright © 2013 Elsevier B.V. All rights reserved.)

The mass transfer kinetics in short and wide 4.6mm×30mm columns packed with 3.0μm Hypurity-C18 fully porous particles were measured for three different configurations of the inlet sample distribution and outlet sample collection: (1) both the inlet and outlet column endfittings are standard, (2) the inlet endfitting is standard while the outlet endfitting allows parallel segmentation of the exiting flow between a central and a peripheral region across the column diameter, and (3) both the inlet and outlet endfittings allow a parallel segmentation of the flow entering and exiting the column, respectively. The total reduced heights equivalent to a theoretical plate (HETP) were carefully measured, using the first and second central moments of the elution band profiles, obtained by its accurate numerical integration. The longitudinal diffusion term was measured at the lowest experimental reduced velocity applied. The solid-liquid mass transfer resistance was estimated from measurements of the intra-particle diffusivity using the Torquato's model of effective diffusion in packed beds. The trans-channel and short-range interchannel eddy diffusion HETP terms were taken from data obtained by solving numerically the Navier-Stokes equations and simulating advective-diffusive transport in computer-generated random sphere packings. The results clearly show that the trans-column eddy diffusion HETP term can account for up to 85% of the total intrinsic HETP (corrected for extra-column contributions) of the standard columns. Parallel segmented flow chromatography can reduce this contribution by half at high velocities, by eliminating most of the baseline peak tailing. This holds true irrespective of the retention factor of the analyte. It was found also that segmenting the inlet/outlet flow increases detection sensitivity by 25-50% for peaks with large to small retention factors, respectively. In practice, the advantage of parallel segmented flow chromatography in gradient elution (thin peak widths) is essentially limited by post-column bandspreading and diffusion in the dwell volume of the instrument used. Analyst should minimize post-column bandspreading (caused by connectors and detection cell volume) and synchronize the eluent composition in the peripheral and central inlet ports of the column, by using two separate pumps with appropriate dwell volumes., (Copyright © 2013 Elsevier B.V. All rights reserved.)