Biography:

In the past Fabrice Gritti has collaborated on articles with Wojciech Piątkowski and Wojciech Piatkowski. One of their most recent publications is Comparison of heat friction effects in narrow-bore columns packed with core–shell and totally porous particles. Which was published in journal Chemical Engineering Science.

More information about Fabrice Gritti research including statistics on their citations can be found on their Copernicus Academic profile page.

Fabrice Gritti's Articles: (62)

Comparison of heat friction effects in narrow-bore columns packed with core–shell and totally porous particles

AbstractThe mass transfer kinetic properties of 2.1 mm I.D. columns packed with core–shell (Kinetex) and totally porous (BEH) sub-2μm particles, operated at high flow rates, under high pressure gradient, are investigated and discussed. The differences between their kinetic performance at low, intermediate, and high velocities are explained by the Kinetex column having a smaller longitudinal diffusion than the BEH column (due to the lower internal porous volume), a larger eddy diffusion (due to a less well-packed bed, with significant trans-column velocity biases), and a smaller amplitude of the radial temperature gradient across the column diameter generated by friction of the mobile phase percolating the bed at high flow rates. This last observation is accounted for by the heat conductivity of the Kinetex bed being three times larger than that of the BEH bed.

Non-invasive measurement of eddy diffusion in very efficient liquid chromatography columns packed with sub-3 μm shell particles

AbstractMass transfer kinetics in a particular column packed with 2.6μm Kinetex-C18 particles was measured using a series of non-invasive methods. According to the manufacturer, this column was the most efficient one in a lot of 133 columns of the same dimensions, packed with particles of the same batch, with an efficiency about 6% larger than the lot average. The total eddy diffusion term of this column was determined by subtracting from the experimental HETP data (1) the longitudinal diffusion term (given by the peak parking method); (2) the trans-particle solid–liquid mass transfer resistance term (given by the peak parking method and an axial diffusion model); and (3) the external film mass transfer resistance term (derived from the Wilson and Geankoplis correlation). The results are analyzed based on the general coupling theory of eddy diffusion of Giddings and are compared to those previously obtained with a column packed with 2.7μm Halo-C18 column, which also has a low eddy diffusion term. The particular column investigated shows no trans-column velocity bias and a smaller reduced short-range inter-channel eddy diffusion term than the Halo column. This result explains the unusual efficiency of this exceptionally well packed column.

Diffusion models in chromatographic columns packed with fully and superficially porous particles

AbstractMost theoretical analyzes of molecular diffusion in chromatographic columns are based on more or less approximate models. Macroscopic sample diffusion along packed columns results from a complex combination of the local sample diffusivity in the external porosity of the bed (Dm) and in the porous particles (ΩDm). A further complication arise from the use of the new superficially porous (or core–shell) particles. The obstruction to axial diffusion caused by the presence of their impermeable core has to be quantified.Two original models of longitudinal diffusion in packed beds are derived for these ternary composite materials. They account for the actual 3D micro-structure of the packed column bed. The micro-structure results from the presence of (1) the impermeable spherical cores; (2) the porous shells surrounding these cores; and (3) the eluent within which the particles are randomly dispersed. The theoretical approach is based on the combination of the effective medium theory of Garnett for core–shell spherical inclusions and of the probabilistic theory of Torquato for randomly dispersed spheres in a continuous matrix. The impacts of the core to the shell diameter, ρ, and of the porous shell to the bulk diffusivity, Ω, on the longitudinal diffusion B coefficient of chromatographic columns packed with core–shell particles are analyzed from a theoretical point of view.

Determination of single component isotherms and affinity energy distribution by chromatography

AbstractAdsorption isotherm data were acquired by frontal analysis (FA) and large sample-size band profiles were recorded for phenol and caffeine. For both compounds, the isotherm data fit well to the Langmuir, Toth, and Bi-Langmuir models of adsorption. The Langmuir model must be dismissed because it does not predict accurately the overloaded band profiles. However, profiles calculated using the unimodal Toth and the bimodal Bi-Langmuir models are indistinguishable. The expectation-maximization procedure was used to calculate directly the affinity energy distribution (AED) from the raw FA data points. For both compounds, the AED converges to a bimodal distribution at high numbers of iterations. This result, which shows the high sensitivity of the EM method, suggest that the Bi-Langmuir model makes better physical sense than the Toth model. This model also permits a detailed investigation of the properties of active sites, a feature often evoked in chromatography but so far rarely the topic of a quantitative investigation.

Influence of the particle porosity on chromatographic band profiles

AbstractThe mass transfer kinetics of butyl benzoate, eluted on a monolithic RPLC column with methanol–water (65:35, v/v) as the mobile phase was investigated, using the perturbation method to acquire isotherm data and the mobile phase velocity dependence of the height equivalent to a theoretical plate of perturbation peaks to acquire kinetics data. The equilibrium isotherm of butyl benzoate is accounted for by the liquid–solid extended multilayer BET isotherm model. The total porosity of the column varies much with the butyl benzoate concentration, influencing strongly the parameters of its mass transfer kinetics and the profiles of the breakthrough curves. Using all these parameters, the general rate model of chromatography predicts band profiles and Van Deemter curves that are in excellent agreement with experimental results provided the influence of concentration on the porosity is properly taken into account. This agreement confirms the validity of the models selected for the isotherm and for the mass transfer kinetics.

Study of the competitive isotherm model and the mass transfer kinetics for a BET binary system

AbstractThe competitive adsorption behavior of the binary mixture of phenetole (ethoxy-benzene) and propyl benzoate in a reversed-phase system was investigated. The adsorption equilibrium data of the single-component systems were acquired by frontal analysis. The same data for binary mixtures were acquired by the perturbation method. For both compounds, the single-component isotherm data fit best to the multilayer BET model. The experimental overloaded band profiles are in excellent agreement with the profiles calculated with either the general rate model or the modified transport-dispersive models. The competitive adsorption data were modeled using the ideal adsorbed solution (IAS) theory. The numerical values of the coefficients were derived by fitting the retention times of the perturbation pulses to those calculated using the IAS theory compiled with the coherence conditions. Finally, the elution profiles of binary mixtures were recorded. They compared very well with those calculated. As a characteristic feature of this case, an unusual retainment effect of the chromatographic band of the more retained component by the less retained one was observed. The combination of the General Rate Model and the adsorption isotherm model allowed an accurate prediction of the band profiles.

Adsorption–desorption isotherm hysteresis of phenol on a C18-bonded surface

AbstractSingle component adsorption and desorption isotherms of phenol were measured on a high-efficiency Kromasil-C18 column (N=15 000 theoretical plates) with pure water as the mobile phase. Adsorption isotherm data were acquired by frontal analysis (FA) for seven plateau concentrations distributed over the whole accessible range of phenol concentration in pure water (5, 10, 15, 20, 25, 40, and 60 g/l). Desorption isotherm data were derived from the corresponding rear boundaries, using frontal analysis by characteristic points (FACP). A strong adsorption hysteresis was observed. The adsorption of phenol is apparently modeled by a S-shaped isotherm of the first kind while the desorption isotherm is described by a convex upward isotherm. The adsorption breakthrough curves could not be modeled correctly using the adsorption isotherm because of a strong dependence of the accessible free column volume on the phenol concentration in the mobile phase. It seems that retention in water depends on the extent to which the surface is wetted by the mobile phase, extent which is a function of the phenol concentration, and of the local pressure rate, which varies along the column, and on the initial state of the column. By contrast, the desorption profiles agree well with those calculated with the desorption isotherms using the ideal model, due to the high column efficiency. The isotherm model accounting best for the desorption isotherm data and the desorption profiles is the bi-Langmuir model. Its coefficients were calculated using appropriate weights in the fitting procedure. The evolution of the bi-Langmuir isotherm parameters with the initial equilibrium plateau concentration of phenol is discussed. The FACP results reported here are fully consistent with the adsorption data of phenol previously reported and measured by FA with various aqueous solutions of methanol as the mobile phase. They provide a general, empirical adsorption model of phenol that is valid between 0 and 65% of methanol in water.

Overloaded gradient elution chromatography on heterogeneous adsorbents in reversed-phase liquid chromatography

AbstractOverloaded band profiles of phenol were measured on a C18-Kromasil column in gradient elution conditions. The mobile phase used was a mixture of methanol and water. The volume fraction of methanol was allowed to vary between 0 and 0.5. A general adsorption model, which expresses the amount of phenol adsorbed q∗ as a function of both its concentration C and the composition ϕ of the organic modifier (methanol) in the mobile phase, was empirically derived from previous independent adsorption experiments based on frontal analysis (FA) and frontal analysis by the characteristic point (FACP). Accordingly, the general model was an extension of the simplest heterogeneous model, the Bilangmuir model, to non-isocratic conditions. The low-energy sites followed the classical linear solvent strength model (LSSM), but not the high-energy sites whose saturation capacity linearly decreased with ϕ. The general model was validated by comparing the experimental and simulated band profiles in gradient elution conditions, in linear and non-linear conditions, as well. The band profiles were calculated by means of the equilibrium-dispersive model of chromatography with a finite difference algorithm. A very good agreement was observed using steps gradient (Δϕ) from 0 to 50% methanol and gradient times tg of 20, 25, 30, 40, 60, 80 and 100 min. The agreement was still excellent for steps gradient from 5 to 45% (tg=25 min), 5 to 35% (tg=50 min), 5 to 25% (tg=50 min) and 5 to 15% (tg=50 min). Significative differences appeared between experience and simulation when the slope of the gradient (Δϕ/tg) became too strong beyond 3.3% methanol per minute. This threshold value probably mirrored the kinetic of arrangement of the G18-bonded chains when the methanol content increased in the mobile phase. It suggested that the chromatographic system was not in a full thermodynamic equilibrium state when very steep mobile phase gradients were applied.

Repeatability and reproducibility of high-concentration data in reversed-phase liquid chromatography: III. Isotherm reproducibility on Kromasil C18

AbstractSingle component equilibrium isotherms of six compounds (aniline, caffeine, ethylbenzene, phenol, propranolol, and theophylline) were determined by the inverse method on 10 Kromasil-C18 columns, using water–methanol solutions as the mobile phase. This method offers an economic and fast isotherm determination by means of the overloaded band profiles of the compounds. Five out of the ten columns used in this test come from the same batch whilst the other five columns represent five additional batches. Statistical evaluation was used to assess the reproducibility of the isotherm parameters. We found that the column-to-column reproducibility of the isotherm parameters is of the same magnitude as the batch-to-batch reproducibility (with the exception of one outlier column). In most of the cases, the reproducibilities of the saturation capacities and that of the retention factors are excellent, they are typically between 1.2 and 3%, and very often below 2%. Within the limits of the experimental precision, these results agree with those obtained earlier, using a conventional method of isotherm determination.

Influence of a buffered solution on the adsorption isotherm and overloaded band profiles of an ionizable compound

AbstractThe overloaded band profiles and the adsorption isotherms of propranolol were acquired at 23 °C, on the endcapped C18-Kromasil stationary phase, using two aqueous solutions of methanol as the mobile phase. The first solution contained 40% methanol and no buffer. The second contained an aqueous acetate buffer at Cbuffer=0.20 M and pH=5.9. In both cases, 33 isotherm data points were acquired by frontal analysis (FA), to achieve an accurate description of the isotherms in the concentration range between 1.54×10−3 and 1.54×10−1 mol/l of propranolol. The isotherms obtained were best described by a bi-Langmuir and a bi-Moreau isotherm model, depending on whether the mobile phase was buffered or not. This shows that the adsorption of propranolol takes place on two different types of sites, a behavior similar to the one already observed with phenol and caffeine on the same column. The presence of the buffer in the mobile phase drastically changes the adsorption mechanism of propranolol. Weak adsorbate–adsorbate interactions (two and three times RT on the low- and the high-energy sites, respectively) take place in the absence of buffer but vanish when the mobile phase is buffered. As expected, the adsorption constants on the abundant low-energy sites with or without buffer are comparable because the mobile phase composition was adjusted to give similar retention times in the two cases. On the other hand, the adsorption of propranolol on the high-energy sites is stronger in presence of the buffer. The difference probably comes from ion-pair formation in the adsorbed phase between the propranolol cation and the acetate anion. The change in total saturation capacity of the adsorbent (22%) compared to that for phenol is explained by the difference in methanol content of the mobile phase.

The adsorption mechanism of nortryptiline on C18-bonded Discovery

AbstractThe adsorption isotherms of an ionizable compound, nortriptyline, were accurately measured by frontal analysis (FA) on a C18-Discovery column, first without buffer (in an aqueous solution of acetonitrile at 15%, v/v of ACN), then with a buffer (in 28%, v/v ACN solution). The buffers were aqueous solutions containing 20 mM of formic acid or a phosphate buffer at pH 2.70. The linear range of the isotherm could not be reached with the non-buffered mobile phase using a dynamic range larger than 40 000 (from 1.2× 10−3 g/L to 50 g/L). With a 20 mM buffer in the liquid phase, the isotherm is linear for concentrations of nortriptyline inferior to 10−3 g/L (or 3 μ mol/L). The adsorption energy distribution (AED) was calculated to determine the heterogeneity of the adsorption process. AED and FA were consistent and lead to a trimodal distribution. A tri-Moreau and a tri-Langmuir isotherm models accounted the best for the adsorption of nortriptyline without and with buffer, respectively. The nature of the buffer affects significantly the middle-energy sites while the properties of the lowest and highest of the three types of energy sites are almost unchanged. The desorption profiles of nortriptyline show some anomalies in relation with the formation of a complex multilayer adsorbed phase of acetonitrile whose excess isotherm was measured by the minor disturbance method. The C18-Discovery column has about the same total saturation capacity, around 200 g of nortriptyline per liter of adsorbent (or 116 mg/g), with or without buffer. About 98–99% of the available surface consists in low energy sites. The coexistence of these different types of sites on the surface solves the McCalley’s enigma, that the column efficiency begins to drop rapidly when the analyte concentration reaches values that are almost one hundred times lower than those that could be predicted from the isotherm data acquired under the same experimental conditions. Due to the presence of some relatively rare high energy sites, the largest part of the saturation capacity is not practically useful.

A chromatographic estimate of the degree of surface heterogeneity of reversed-phase liquid chromatography packing materials: II-Endcapped monomeric C18-bonded stationary phase

AbstractIn a previous report, the heterogeneity of a non-endcapped C30-bonded stationary phase was investigated, based on the results of the measurements of the adsorption isotherms of two neutral compounds (phenol and caffeine) and two ionizable compounds (sodium naphthalene sulfonate and propranololium chloride) by frontal analysis (FA). The same method is applied here for the characterization of the surface heterogeneity of two new brands of endcapped C18-bonded stationary phases (Gemini and Sunfire). The adsorption isotherms of the same four chemicals were measured by FA and the results confirmed by the independent calculation of the adsorption energy distribution (AED), using the expectation-maximization (EM) method. The effect of the length of the bonded alkyl chain was investigated. Shorter alkyl-bonded-chains (C18 versus C30) and the end-capping of the silica surface contribute to decrease the surface heterogeneity under the same experimental conditions (30% methanol, 25 mM NaCl). The AEDs of phenol and caffeine are bimodal with the C18-bonded columns while they are trimodal and quadrimodal, respectively, with a non-endcapped C30-bonded column. The “supersites” (adsorption energy >20 kJ/mol) found on the C30-Prontosil column and attributed to a cation exchange mechanism completely disappear on the C18-Gemini and C18-Sunfire, probably because the end-capping of the silica surface eliminates most if not all the ionic interactions.

Effect of the density of the C18 surface coverage on the adsorption mechanism of a cationic compound and on the silanol activity of the stationary phase in reversed phase liquid chromatography

AbstractRPLC columns with different surface coverages (a C1 endcapped column with a bonding density of 3.92 μmol/m2 and four C18-bonded, endcapped columns, with octadecyl chain densities of 0.42, 1.01, 2.03, and 3.15 μmol/m2) were used to investigate the effects of the density of the surface coverage of RPLC columns on the adsorption mechanism of a cationic compound, amitriptyline chloride, and on the silanol activity of these columns. The mobile phases used were acetonitrile–water (30/70, v/v) solutions, buffered at either pH 2.7 or pH 6.9. At pH 2.7, the residual silanol groups are not ionized. At pH 6.9, some of these groups are ionized and these surface anions can strongly interact with the cationic compound. The adsorption isotherms were measured by frontal analysis (FA) at pH 2.7 and by frontal analysis by characteristic points (FACP) at pH 6.9, because the very high retention observed at neutral pH made FA measurements excessively long and poorly accurate. The adsorption energy distributions (AEDs) were calculated when possible, according to the expectation-maximization (EM) algorithm. A bimodal and a trimodal energy distribution were found for all the columns at pH 2.7 and 6.9, respectively. The third site measured at pH 6.9 was attributed to the strong ion-exchange interactions between the ionized silanol groups and the amitriptylinium cation. The contribution of the ionized silanol groups to the overall retention is maximum for the phases with intermediary bonding densities (1.01 and 2.03 μmol/m2). The peak tailing is most pronounced for the lowest (C1 column) and the highest (3.15 μmol/m2) surface coverages.

Influence of the errors made in the measurement of the extra-column volume on the accuracies of estimates of the column efficiency and the mass transfer kinetics parameters

AbstractThe influences of the errors made in the measurement of the extra-column volume of an instrument on the accuracies of the estimates made of the column efficiency and of the parameters of the mass transfer kinetics were investigated from an experimental point of view. A standard HP1090 apparatus (extra-column volume, ≈ 50 μ L) was used to measure the efficiency of a Sunfire-C18 RPLC column (column hold-up volume, ≈ 1.50 mL). The first and second moments of the peaks of phenol (a retained compound) and of thiourea (a practically non-retained compound) were measured at six different temperatures between 22 and 78 °C, for flow rates between 0.10 and 4.70 mL/min (i.e., for linear velocities between 0.025 and 1.179 cm/s). Each series of measurements was successively made with the instrument being fitted with and without the column. The experimental HETP data must be corrected for the solute dispersion in the connected tubes in order properly to assess the true column efficiency. Even with a modern, high performance instrument, the dispersion of a non-retained compound is essentially due to the band broadening phenomena that take place in the extra-column volumes, the sum of all these extra-column band broadening contributions accounting for more than 80% of the total band broadening measured. The contribution of the sampling device is particularly deleterious since, for a 2 μ L injection, the maximum solute concentration in the peak that enters into the column is nearly ten-fold lower than that of the sample. Nevertheless, the impact of the extra-column volumes on the estimates of the kinetic parameters (e.g., molecular diffusion coefficient Dm and effective particle diffusivity De) remains negligible. Obviously, the relative error made on the column efficiency of a retained compound depends much on its retention factor. It decreases from 8 to 1% when the retention factor increases from 5 to 17.

The bandwidth in gradient elution chromatography with a retained organic modifier

AbstractThe variance of a chromatographic band is derived in the case of RPLC gradient elution when the organic modifier is significantly retained onto the stationary phase. This derivation is based on the extension of a model due to Poppe et al. [H. Poppe, J. Paanakker, M. Bronckhorst, J. Chromatogr., 204 (1981) 77] which assumes that the gradient front remains unchanged and propagates along the column at the same speed as the mobile phase, following piston flow. Theoretical and experimental results are compared in the case of caffeine on a C1-silica stationary phase eluted with an acetonitrile gradient. The actual retention behaviors of caffeine and acetonitrile were implemented in the theoretical calculations. The model predicts compression factors between 0.71 and 0.34 for relatively smooth gradient steepness, βt0, between 0.009 and 0.054 while the corresponding experimental band compression factors vary between 1.01 and 0.43 for the very same gradient steepness. The model underestimation of these factors arises likely from the strong deviation of the actual retention behavior from the prediction of the Linear Solvent Strength Model (LSSM).

Limits of the numerical estimation of the adsorption energy distribution from adsorption isotherm data using the expectation-maximization method

AbstractThe limits of the use of the expectation-maximization (EM) method for the study of the heterogeneity of adsorbent surfaces were tested by calculating the adsorption energy distribution of systems having known degrees of heterogeneity. Connecting on-line two different columns allows the simulation of a heterogeneous system. The two columns used were endcapped, C18-bonded silica used as stationary phases and having different degrees of C18 chain coverages (0.42 and 2.03 μmol/m2). The adsorption constants of phenol measured by frontal analysis (FA) are significantly different on these two columns. On each column, the adsorption behavior was best accounted for by a bi-Langmuir isotherm model, corresponding to a heterogeneous surface with a bimodal energy distribution. The difference between the adsorption energies on the weak adsorption sites of the two columns is 1.5 kJ/mol. The energy difference of their high energy sites is 2.2 kJ/mol. The EM method can readily distinguish between adsorption sites having energies that differ by more than 5 kJ/mol after more than 10 million iterations, but it cannot distinguish between adsorption sites for which this energy difference is less than 2 kJ/mol, even after 100 million iterations. For highly heterogeneous systems, (e.g., those with more than three different types of adsorption sites), the EM program does not converge necessarily towards the actual energy distribution function but toward a simpler one, having fewer adsorption sites that are almost equally spaced in the energy space. This failure of the EM program is related to the fact that, despite the excellent precision of the FA measurements (<1%), any series of adsorption data can be represented by several distinct AEDs. Thus, the degree of heterogeneity of RPLC adsorbents determined with the EM method might often be minimized, resulting in erroneous values of the isotherm parameters.

Comparison between the efficiencies of columns packed with fully and partially porous C18-bonded silica materials

AbstractThe chromatographic performance of a new brand of shell particles is compared to that of a conventional brand of totally porous silica particles having a similar size. The new material (Halo, Advanced Materials Technology, Wilmington, DE) is made of 2.7 μm particles that consist in a 1.7 μm solid core covered with a 0.5 μm thick shell of porous silica. The other material consists of the porous particles of a conventional 3 μm commercial silica-B material. These two columns have the same dimensions, 150 mm × 4.6 mm. The reduced plate heights of two low molecular weight compounds, naphthalene and anthracene, two peptides (lys-bradykinin and bradykinin), and four proteins, insulin, lysozyme, β-lactoglobulin, and bovine serum albumin were measured in a wide flow rate range and analyzed on the basis of the Van Deemter equation and of modern models for its terms. The Halo column provides a smaller axial diffusion coefficient B and a smaller eddy dispersion term A than the other column, a result consistent with its lower internal porosity (∈p = 0.19 versus 0.42) and with the narrower size distribution of its particles (σ = 5% versus 13%). The two columns have similar C terms for the two low molecular weight compounds and for the two peptides. However, the C term of the proteins that are not excluded is markedly lower on the column packed with the Halo particles than on the other column. A recent theoretical analysis of the mass transfer kinetics in shell particles predicts a C term for moderately retained proteins (3 < k′ < 5) that is about 35% lower for shell than for fully porous particles while the experimental data show a value nearly 45% lower, an excellent agreement considering that the internal tortuosity of the particles might be different, affecting the ratio of the effective diffusivities (Deff) of the proteins in the two materials. Surprisingly, the Kozeny–Carman constant of the Halo packed column is 50% larger than that of the other column, in spite of which the permeability of the Halo column is slightly larger, due to its larger external porosity.

Consequences of the radial heterogeneity of the column temperature at high mobile phase velocity

AbstractWhen a high velocity stream of mobile phase percolates through a chromatographic column, the bed cannot remain isothermal. Due to the mobile phase decompression, heat is generated along the column. Longitudinal and radial temperature gradients take place along and across its bed. The various consequences of this thermal heterogeneity are calculated and their effects on the column efficiency investigated for a 0.46cm×25 cm stainless steel column packed with 5 μm particles. The maximum pressure drop applied was varied from 0.1 to 2 kbar. The amplitude of the longitudinal temperature gradient can be estimated on the basis of the integral heat balance equation applied to the whole column and of measurements of the eluent temperature at the column exit. Assuming that the radial gradient is parabolic and the longitudinal gradient linear, the amplitude of the radial gradient can be determined on the basis of the energy balance across the column and of direct measurements of the radial gradient at high inlet pressures. A radial temperature gradient causes a radial distribution of the eluent viscosity, hence of its local velocity. The result is that bands move faster in their center than along the wall, become warped, hence a radial concentration gradient, similar in origin to the one observed in open cylindrical tubes. Diffusion relaxes this gradient. If there is only a longitudinal temperature gradient, the column efficiency would be 30% smaller for a 2 kbar pressure drop than if there is no longitudinal temperature gradient. However, when both a longitudinal and a radial temperature gradient coexist, there is a large loss of efficiency. If the influence of the diffusive relaxation of the radial concentration gradient is neglected, the peak shape would be broad and exhibit a marked shoulder.

Comparison between the loading capacities of columns packed with partially and totally porous fine particles: What is the effective surface area available for adsorption?

AbstractThe adsorption isotherms of phenol, caffeine, insulin, and lysozyme were measured on two C18-bonded silica columns. The first one was packed with classical totally porous particles (3 μm Luna(2)-C18from Phenomenex, Torrance, CA, USA), the second one with shell particles (2.7μm Halo-C18 from Advanced Materials Technology, Wilmington, DE, USA). The measurements were made at room temperature (T=295±1 K), using mainly frontal analysis (FA) and also elution by characteristic points (FACP) when necessary. The adsorption energy distributions (AEDs) were estimated by the iterative numerical expectation-maximization (EM) procedure and served to justify the choice of the best adsorption isotherm model for each compound. The best isotherm parameters were derived from either the best fit of the experimental data to a multi-Langmuir isotherm model (MLRA) or from the AED results (equilibrium constants and saturation capacities), when the convergence of the EM program was achieved. The experiments show than the loading capacity of the Luna column is more than twice that of the Halo column for low-molecular-weight compounds. This result was expected; it is in good agreement with the values of the accessible surface area of these two materials, which were calculated from the pore size volume distributions. The pore size volume distributions are validated by the excellent agreement between the calculated and measured exclusion volumes of polystyrene standards by inverse size exclusion chromatography (ISEC). In contrast, the loading capacity ratio of the two columns is 1.5 or less with insulin and lysozyme. This is due to a significant exclusion of these two proteins from the internal pore volumes of the two packing materials. This result raises the problem of the determination of the effective surface area of the packing material, particularly in the case of proteins. This area is about 40 and 30% of the total surface area for insulin and for lysozyme, respectively, based on the pore size volume distribution validated by the ISEC method. The ISEC experiments showed that the largest and the smallest mesopores have rather a cylindrical and a spherical shape, respectively, for both packing materials.

Experimental band compression factor of a neutral compound under high pressure gradient elution

AbstractTo measure the gradient compression factor for a low molecular weight compound (caffeine, MW=194 g/L), 1 μL samples of solution were injected into a 2.1mm×100 mm column packed with bridged ethylsiloxane BEH-silica. These samples were successively run under isocratic and gradient elution modes. Both chromatograms were recorded with either a low (<250 bar) or a high pressure drop (>650 bar), corresponding to flow rates of 0.10 and 0.35 mL/min, respectively. Caffeine was eluted with a mixture of methanol and water at room temperature. The efficiency and the retention factors of caffeine on the BEH-C18column were measured as a function of the mobile phase composition under isocratic conditions. During the gradient elution, the methanol concentration was increased from 10 to 25% (v/v). The experimental compression factors measured are in excellent agreement with those predicted with an equation previously derived, which is valid at low flow rates and for smooth gradients. The negative relative difference observed between experimental and theoretical values at high flow rates originates from the compressibility of the eluent. Using a high gradient steepness to perform fast gradient elution limits the degree of band compression that can be achieved, even when the radial temperature gradient across the column is as small as 0.2 K.

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