Effect of the surface coverage of endcapped C18-silica on the excess adsorption isotherms of commonly used organic solvents from water in reversed phase liquid chromatography

The excess adsorption isotherms of methanol, ethanol, 2-propanol, acetonitrile, and tetrahydrofuran from water were measured on five different silica-based packing materials by the minor disturbance method. These materials were prepared with the same lot of 5-microm particles (average pore size 90 A), all endcapped with trimethylchlorosilane (TMS), and bonded to octadecyl chains with different surface coverages (0, 0.42, 1.01, 2.03, and 3.15 micromol/m2). The relative adsorption of one eluent by respect to a second one informs on the heterogeneity of the material (alkyl-bonded and bare silica regions) and on the accessibility of the unreacted silanol groups to the mobile phase. It is shown that the total surface area of the adsorbent decreases with increasing degree of surface coverage with octadecyl chains and that the relative surface area of the regions occupied by accessible silanol groups to the regions occupied by alkyl-bonded groups decreases. For the five columns, an average of 10% of the adsorbent surface area is covered of bare silica accessible to the liquid phase, with a minimum of 5% with tetrahydrofuran and a maximum of 12% with ethanol or 2-propanol. Increasing the surface coverage by the C18 chains causes a significant increase of the attraction potential of the hydrophobic surface toward the organic solvent. This result is confirmed by the increase of the number of adsorbate monolayers with increasing bonding density of the octadecyl chains. This number is twice larger for the 315C18 column than for the C1 column.     

Influence of the pressure on the properties of chromatographic columns. I. Measurement of the compressibility of methanol-water mixtures on a mesoporous silica adsorbent

The compressibilities of aqueous solutions of methanol or acetonitrile containing 0, 20, 40, 60, 80 and 100% (v/v) organic solvent were measured with a dynamic chromatographic method. The elution volumes of thiourea samples (2 microL) in these solutions were measured at different average column pressures, adjusted by placing suitable capillary restrictors on-line, after the detector. The reproducibility of the measurements was better than 0.2%. In the range of average pressures studied (10-350 bar), the maximum change in elution volume of thiourea is 1.3% (in pure water) and 4.0% (in pure methanol). This difference is due to the different compressibilities of these pure solvents. For mixtures, the plots of the elution volume of thiourea versus the pressure are convex downward, which is inconsistent with the opposite curvature predicted by the classical Tait model of liquid compressibility. This difference is explained by the variation of the amount of thiourea adsorbed with the pressure. The deconvolution of the two effects, adsorption of thiourea and solvent compressibility, allows a fair and consistent determination of the compressibilities of the methanol-water mixtures. A column packed with non-porous silica particles was also used to determine the compressibility of methanol-water and acetonitrile-water mixtures. A negative deviation by respect to ideal behavior was observed.     

Effect of the endcapping of reversed-phase high-performance liquid chromatography adsorbents on the adsorption isotherm

The retention mechanisms of n-propylbenzoate, 4-t ert-butylphenol, and caffeine on the endcapped Symmetry-C(18) and the non-endcapped Resolve-C(18) are compared. The adsorption isotherms were measured by frontal analysis (FA), using as the mobile phase mixtures of methanol or acetonitrile and water of various compositions. The isotherm data were modeled and the adsorption energy distributions calculated. The surface heterogeneity increases faster with decreasing methanol concentration on the non-endcapped than on the endcapped adsorbent. For instance, for methanol concentrations exceeding 30% (v/v), the adsorption of caffeine is accounted for by assuming three and two different types of adsorption sites on Resolve-C(18) and Symmetry-C(18), respectively. This is explained by the effect of the mobile phase composition on the structure of the C(18)-bonded layer. The bare surface of bonded silica appears more accessible to solute molecules at high water contents in the mobile phase. On the other hand, replacing methanol by a stronger organic modifier like acetonitrile dampens the differences between non-endcapped and endcapped stationary phase and decreases the degree of surface heterogeneity of the adsorbent. For instance, at acetonitrile concentrations exceeding 20%, the surface appears nearly homogeneous for the adsorption of caffeine.     

Thermodynamics of adsorption of binary aqueous organic liquid mixtures on a RPLC adsorbent

The surface excess adsorption isotherms of organic solvents commonly used in RPLC with water as co-eluent or organic modifiers (methanol, ethanol, 2-propanol, acetonitrile and tetrahydrofuran) were measured on a porous silica surface derivatized with chlorotrimethylsilane (C1-silica with 3.92 micromol C1 groups per m2 of SiO2), using the dynamic minor disturbance method. The 5 microm diameter particles were packed in a 150 mm x 4.6 mm column. The isotherm data were derived from signals resulting from small perturbations of the equilibrium between the aqueous-organic solutions and the adsorbent surface. The partial molar surface area of the adsorbed components were assumed to be the same as those of the pure components. The difference sigma-sigmai* between the surface tensions of the adsorbed mixtures and that of the pure liquids was measured as a function of the organic modifier molar fraction. A simple and unique convention for the position of the Gibbs dividing surface was proposed to delimit the Gibbs's adsorbed phase and the bulk liquid phase. The activity coefficients of the organic modifiers and of water and their thermodynamic equilibrium constants between the two phases were measured. The strong non-ideal behavior of the adsorbed phase is mostly accounted for by the surface heterogeneity. Some regions of the surface (bonded -Si(CH3)3 moieties) preferentially adsorb the organic compound while the regions close to unreacted silanols preferentially adsorb water.     

Retention of ionisable compounds on high-performance liquid chromatography XVIII: pH variation in mobile phases containing formic acid,piperazine, tris,boric acid or carbonate as buffering systems and acetonitrile as organic modifier

In the present work dissociation constants of commonly used buffering species, formic acid, piperazine, tris(hydroxymethyl)–aminomethane, boric acid and carbonate, have been determined for several acetonitrile–water mixtures. From these pK_a values a previous model has been successfully evaluated to estimate pH values in acetonitrile–aqueous buffer mobile phases from the aqueous pH and concentration of the above mentioned buffers up to 60% of acetonitrile, and aqueous buffer concentrations between 0.005 (0.001 mol L⁻¹ for formic acid–formate) and 0.1 mol L⁻¹. The relationships derived for the presently studied buffers, together with those established for previously considered buffering systems, allow a general prediction of the pH variation of the most commonly used HPLC buffers when the composition of the acetonitrile–water mobile phase changes during the chromatographic process, such as in gradient elution. Thus, they are an interesting tool that can be easily implemented in general retention models to predict retention of acid–base analytes and optimize chromatographic separations.     

Nanolayer liquid density on hydrophobic surfaces

 The excess volumes of dispersions of hydrophobic silica particles and hydrophobized layer silicates were studied in ethanol– cyclohexane mixtures. A plot of the excess volume vs. composition [V ^σ=f (x ₁)] indicates that the components, ethanol and cyclohexane, on hydrophobic SiO₂ are ordered to a considerable extent. The excess volumes which are positive in the pure solvent, i.e. without dispersed particles, take on negative values over the entire composition range. On the other hand, excess volumes for layer silicates the surface of which had been modified by long (n _c=18) alkyl chains were calculated by a combination of adsorption isotherms with X-ray diffraction data and were positive in the entire composition range. It is established that the density of the adsorption layer on the solid/liquid interface in colloid dispersions significantly differs from that of the bulk phase. The differences depend on the surface structure of the adsorbent. Received: 4 June 1998 Accepted: 7 July 1998     

Influence of temperature and pressure on the preferential adsorption of component of hydroorganic mobile phase in liquid chromatography

The excess isotherms of methanol and acetonitrile were measured on the series of C18 bonded phases. The measurements were done using the minor disturbance method. The goal of our work was to determine the influence of the temperature on the adsorption of two commonly used solvents. The influence on the mobile phase flow rate on the both organic solvent adsorption was also investigated. The effect of these two parameters was compared on the octadecyl packed columns with different coverage density and on the monolithic Chromolith column. Adsorption of both solvents decreases with the increase of the temperature. The increase of the pressure increases adsorption of methanol but decreases adsorption of acetonitrile.     

Comparison of the adsorption mechanisms of pyridine in hydrophilic interaction chromatography and in reversed-phase aqueous liquid chromatography

The adsorption isotherms of pyridine were measured by frontal analysis (FA) on a column packed with shell particles of neat porous silica (Halo), using water–acetonitrile mixtures as the mobile phase at 295 K. The isotherm data were measured for pyridine concentrations covering a dynamic range of four millions. The degree of heterogeneity of the surface was characterized by the adsorption energy distribution (AED) function calculated from the raw adsorption data, using the expectation-maximization (EM) procedure. The results showed that two different retention mechanisms dominate in Per aqueous liquid chromatography (PALC) at low acetonitrile concentrations and in hydrophilic interaction chromatography (HILIC) at high acetonitrile concentrations. In the PALC mode, the adsorption mechanism of pyridine on the silica surface is controlled by hydrophobic interactions that take place on very few and ultra-active adsorption sites, which might be pores on the irregular and rugose surface of the porous silica particles. The surface is seriously heterogeneous, with up to five distinct adsorption sites and five different energy peaks on the AED of the packing material. In contrast, in the HILIC mode, the adsorption behavior is quasi-homogeneous and pyridine retention is governed by its adsorption onto free silanol groups. For intermediate mobile phase compositions, the siloxane and the silanol groups are both significantly saturated with acetonitrile and water, respectively, causing a minimum of the retention factor of pyridine on the Halo column.     

Fast methods for screening of trichothecenes in fungal cultures using gas chromatography-tandem mass spectrometry

The excess adsorption isotherms of acetonitrile, methanol and tetrahydrofuran from water on reversed-phase packings were studied, using 10 different columns packed with C1-C6, C8, C10, C12, and C18 monomeric phases, bonded on the same type of silica. The interpretation of isotherms on the basis of the theory of excess adsorption shows significant accumulation of the organic eluent component on the adsorbent surface on the top of "collapsed" bonded layer. The accumulated amount was shown to be practically independent of the length of alkyl chains bonded to the silica surface. A model that describes analyte retention on a reversed-phase column from a binary mobile phase is developed. The retention mechanism involves a combination of analyte distribution between the eluent and organic adsorbed layer, followed by analyte adsorption on the surface of the bonded phase. A general retention equation for the model is derived and methods for independent measurements of the involved parameters are suggested. The theory was tested by direct measurement of analyte retention from the eluents of varied composition and comparison of the values obtained with those theoretically calculated values. Experimental and theoretically calculated values are in good agreement.     

3D structure-based protein retention prediction for ion-exchange chromatography

The interest in understanding fundamental mechanisms underlying chromatography drastically increased over the past decades resulting in a whole variety of mostly semi-empirical models describing protein retention. Experimental data about the molecular adsorption mechanisms of lysozyme on different chromatographic ion-exchange materials were used to develop a mechanistical model for the adsorption of lysozyme onto a SP Sepharose FF surface based on molecular dynamic simulations (temperature controlled NVT simulations) with the Amber software package using a force-field based approach with a continuum solvent model. The ligand spacing of the adsorbent surface was varied between 10 and 20 Å. With a 10 Å spacing it was possible to predict the elution order of lysozyme at different pH and to confirm in silico the pH-dependent orientation of lysozyme towards the surface that was reported earlier. The energies of adsorption at different pH values were correlated with isocratic and linear gradient elution experiments and this correlation was used to predict the retention volume of ribonuclease A in the same experimental setup only based on its 3D structure properties. The study presents a strong indication for the validity of the assumption, that the ligand density of the surface is one of the key parameters with regard to the selectivity of the adsorbent, suggesting that a high ligand density leads to a specific interaction with certain binding sites on the protein surface, while at low ligand densities the net charge of the protein is more important than the actual charge distribution.     

Fencing of Photoinduced Electron Transfer in Nonconjugated Bichromophoric System by β-cyclodextrin Nanocavity

²³Na NMR measurements were employed to monitor the stability of Na⁺ ion complexes with 18-crown-6 (18C6), dicycloxyl-18-crown-6 (DC18C6), dibenzo-18-crown-6 (DB18C6), 15-crown-5 (15C5) and benzo-15-crown-5 (B15C5) in binary acetonitrile–dimethylformamide mixtures of varying composition. In all cases, the variation of ²³Na chemical shift with [crown]/[Na⁺] mole ratios indicated the formation of 1:1 complexes. The formation constants of the resulting complexes were evaluated from computer fitting of the mole ratio data to an equation which relates the observed chemical shifts to the formation constants. It was found that, in pure acetonitrile, the stabilities of the resulting 1:1 complexes vary in the order 15C5>DC18C6>B15C5>18C6>DB18C6, while in pure dimethylformamide the stability order is DC18C6>18C6>15C5>B15C5>DB18C6. The observed changes in the stability order could be related to the specific interactions between some crown ethers and acetonitrile. It was found that, in the case of all complexes, an increase in the percentage of dimethylformamide in the solvent mixtures would significantly decrease the stability of the complexes.     

Comparative analysis of methods for determination of structural characteristics of carbon adsorbents

Experimental data on the equilibrium adsorption of sulfur hexafluoride, methane, carbon dioxide, and benzene on carbon adsorbents of different porosity obtained in a wide pressure range at 298–408 K were analyzed. The adsorption volumes, surface areas, and sizes of slit-shaped pores of the carbons were determined using several independent methods. A method for determination of the adsorption volume from the experimental isotherm of excessive adsorption of gases and the total content equation was proposed. The resulting values are similar to the adsorption volumes calculated from the data for vapors. A new method for the calculation of the adsorbent surface area is described. The method is based on the dependence of the adsorption volume of adsorbent pores on the effective size of adsorbate molecules. A possibility to determine the average size of narrow slit-shaped carbon pores from the difference of the initial heats of adsorption of the gas under study on the carbon black and porous carbon adsorbent is considered. Published in Russian in Izvestiya Akademii Nauk. Seriya Khimicheskaya, No. 10, pp. 2219–2227, October, 2005.     

Adsorption thin-layer chromatography and viscometry of polystyrenes in solvent mixtures

The effect of the thermodynamic quality of solvent mixtures on the adsorption behavior of macromolecules under dynamic conditions was investigated. The chromatographic behavior of polystyrenes (PSs) in various mixed eluents was studied under conditions of adsorption and size-exclusion thin-layer chromatography as well as at the exclusion-adsorption transition point with silica gel KSKG adsorbent. The thermodynamic quality of the solvent mixtures used in the chromatographic experiments was determined viscometrically. The dependences of the intrinsic viscosity of PSs on solvent composition for solvent-non-solvent, two solvents, and theta-solvent-solvent mixtures were obtained. A correlation was found between Snyder's polarity indices for the solvent mixtures used in polymer chromatography under "critical conditions" and the intrinsic viscosity values of PSs in the same mixtures.     

Adsorption of pure and mixed solvent solutions of spin probes onto stationary phases

Water, methanol (MeOH), acetonitrile (ACN), and binary MeOH-water and ACN-water solutions of different spin probes (nitroxides), selected to mimic the behavior of different pollutants, were adsorbed onto stationary phases usually used in reversed-phase high-performance liquid chromatography (RP-HPLC). These stationary phases are constituted by porous silica and differ from each other regarding the surface area, the pore size, the particle size, the surface functions (NH2, C8, and C18), and the percentage of functionalization. The electron paramagnetic resonance (EPR) spectra of the probe solutions adsorbed into the pores were analyzed by computer-aided computation of the spectral line shape, which provided structural and dynamical parameters of the probes and their environments. These parameters provided information on the surface properties of the stationary phases, such as alkyl chain density, solvent penetration, stationary-phase ordering, and residual silanol effects, which modify the retention times in HPLC. A different availability of polar surface groups in the pure and mixed solvents was found for the different stationary phases depending on (1) the different functionalization degree, (2) the surface-chain length, (3) the particle size, and (4) the polarity of both the probe and the solvent. The C8 functionalization rendered the surface more hydrophobic with respect to C18. The endcapping process of the residual silanols strongly enhanced the surface hydrophobicity tested by the probes. At the highest water content, the adsorption of the polar or charged probes onto the hydrophobic surface is the lowest and self-aggregation occurs. When the probes bear both hydrophilic and hydrophobic moieties, the adsorption is enhanced by a synergy between hydrophilic and hydrophobic bonds with the surface. A balance between the hydrophilic and hydrophobic forces leads to high adsorption and partial insertion of the surfactant probes in an ordered C18 chain layer at the solid surface which forms in the binary mixtures; this layer is ascertained between 40% and 70% of the less hydrophilic solvent, depending on the type of both the solid and the probe. This insertion and the response on the formation of the ordered layer were favored in ACN-water with respect to MeOH-water.     

Numerical simulation of rapid pressurization and depressurization of a zeolite column using nitrogen

The required durations of pressurization and depressurization steps of a rapid pressure swing adsorption process are primarily governed by adsorbent particle size, adsorption kinetics, column pressure drop, column length to diameter ratio, and the valve constant of the gas inlet and outlet control valve attached to the adsorbent column. A numerical model study of the influence of these variables for an adiabatic LiX zeolite column is presented using pure N₂ as an adsorbate gas. An adsorbent particle size range of 200–350 μm was found to minimize (<1 s) the times required for the pressurization and depressurization steps.     

Adsorption mechanism in reversed-phase liquid chromatography. Effect of the surface coverage of a monomeric C18-silica stationary phase

The effect of the bonding density of the octadecyl chains onto the same silica on the adsorption and retention properties of low molecular weight compounds (phenol, caffeine, and sodium 2-naphthalene sulfonate) was investigated. The same mobile phase (methanol:water, 20:80, v/v) and temperature (T = 298 K) were applied and two duplicate columns (A and B) from each batch of packing material (neat silica, simply endcapped or C1 phase, 0.42, 1.01, 2.03, and 3.15 micromol/m2 of C18 alkyl chains) were tested. Adsorption data of the three compounds were acquired by frontal analysis (FA) and the adsorption energy distributions (AEDs) were calculated using the expectation-maximization method. Results confirmed earlier findings in linear chromatography of a retention maximum at an intermediate bonding density. From a general point of view, the saturation capacity of the adsorbent tends to decrease with increasing bonding density, due to the vanishing space intercalated between the C18 bonded chains and to the decrease of the specific surface area of the stationary phase. The equilibrium constants are maximum for an intermediary bonding density (approximately 2 micromol/m2). An enthalpy-entropy compensation was found for the thermodynamic parameters of the isotherm data. Weak equilibrium constants (small deltaH) and high saturation capacities (large deltaS) were observed at low bonding densities, higher equilibrium constants and lower saturation capacities at high bonding densities, the combinations leading to similar apparent retention in RPLC. The use of a low surface coverage column is recommended for preparative purposes.     

Solvent effects on the retention of oligosaccharides in porous graphitic carbon liquid chromatography

Porous graphitic carbon (PGC) is known as well suited adsorbent for liquid chromatography of carbohydrates. In this work we report on systematic investigations of solvent effects on the retention mechanism of fluorescence labeled malto-oligosaccharides on PGC. The adsorption mechanism was found to depend on the type of organic modifier used in the mobile phase. Positive adsorption enthalpies and entropies, which have already been reported in the literature, were solely produced using acetonitrile. Both alternative solvents (tetrahydrofuran, 2-propanol) yielded in contrast negative enthalpies. As plausible retention mechanism for oligosaccharides on PGC applying acetonitrile as mobile phase component we propose the formation of a dense and highly ordered solvation layer of the PGC surface with the linear acetonitrile molecules. Adsorption of analyte molecules requires a displacement of numerous acetonitrile molecules, which explains the positive enthalpy and entropy values measured. The interplay of enthalpic and entropic contributions to the overall adsorption phenomena results in strongly temperature dependent chromatographic selectivity values.     

The adsorption mechanism of nortryptiline on C18-bonded Discovery

The adsorption isotherms of an ionizable compound, nortriptyline, were accurately measured by frontal analysis (FA) on a C(18)-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 x 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 micromol/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 C(18)-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.     

Excess isotherms as a new way for characterization of the columns for reversed-phase liquid chromatography

In this work, we present the excess isotherm of acetonitrile for stationary phases with different coverage density. Data obtained with the minor disturbance method were compared with (29)Si cross-polarization/magic-angle spinning NMR spectra to find dependence between acetonitrile adsorption on C18 chemically bonded stationary phases and coverage density of stationary phase. The preferential adsorption of acetonitrile on the bonded phase and the adsorption of water on the silica surface can be well correlated with the coverage density.     

Rapid analysis of opium alkaloids by ion-pair high performance liquid chromatography

A new procedure for the rapid analysis of four major opium alkaloids is described using ion-pair, reversedphase high performance liquid chromatography and recently developed radial compression column technology. The solvents were aqueous 10mM potassium perchlorate with 5.0mM n-butylamine (pH 3.0) and pure acetonitrile. A gradient was run for five minutes from 10% to 70% acetonitrile and held at that level for two minutes. Flow rates were 3 ml/min. Two column packing materials were compared with this solvent system: standard C-18 packing and a fully-end-capped C-18 material. Results obtained with the fully-end-capped C-18 packing and the two solvent ion-pair gradient showed elution times under six minutes, with greater sensitivity (<50 nanograms) than previous systems.