Physical origin of peak tailing on C18-bonded silica in reversed-phase liquid chromatography

Single component isotherm data of caffeine and phenol were acquired on two different stationary phases for RPLC, using a methanol/water solution (25%, v/v, methanol) as the mobile phase. The columns were the non-endcapped Waters Resolve-C18, and the Waters XTerra MS C18. Both columns exhibit similar C18 -chain densities (2.45 and 2.50 micromol/m2) and differ essentially by the nature of the underivatized solid support (a conventional, highly polar silica made from water glass, hence containing metal impurities, versus a silica-methylsilane hybrid surface with a lower density of less acidic free silanols). Thirty-two adsorption data points were acquired by FA, for caffeine, between 10(-3) and 24 g/l, a dynamic range of 24,000. Twenty-eigth adsorption data points were acquired for phenol, from 0.025 to 75 g/l, a dynamic range of 3000. The expectation-maximization procedure was used to derive the affinity energy distribution (AED) from the raw FA data points, assuming a local Langmuir isotherm. For caffeine, the AEDs converge to a bimodal and a quadrimodal distribution on XTerra MS-C18 and Resolve-C18, respectively. The values of the saturation capacity (q(s,1) approximately equal to 0.80 mol/l and q(s,2) approximately equal to 0.10 mol/l) and the adsorption constant (b1 approximately equal to 3.11/mol and b2 approximately equal to 29.1 l/mol) measured on the two columns for the lowest two energy modes 1 and 2, are comparable. These data are consistent with those previously measured on an endcapped Kromasil-C18 in a 30/70 (v/v), methanol/water solution (q(s,1) = 0.9 mol/l and q(s,2) = 0.10 mol/l, b1 = 2.4 l/mol and b2 = 16.1 l/mol). The presence of two higher energy modes on the Waters Resolve-C18 column (q(s,3) approximately equal to 0.013 mol/l and q(s,4) approximately equal to 2.6 10(-4) mol/l, b3 approximately equal to 252 l/mol and b4 = 13,200 l/mol) and the strong peak tailing of caffeine are explained by the existence of adsorption sites buried inside the C18-bonded layer. It is demonstrated that strong interactions between caffeine and the water protected bare silica surface cannot explain these high-energy sites because the retention of caffeine on an underivatized Resolve silica column is almost zero. Possible hydrogen-bond interactions between caffeine and the non-protected isolated silanol groups remaining after synthesis amidst the C18-chain network cannot explain these high energy interactions because, then, the smaller phenol molecule should exhibit similarly strong interactions with these isolated silanols on the same Resolve-C18 column and, yet, the consequences of such interactions are not observed. These sites are more consistent with the heterogeneity of the local structure of the C18-bonded layer. Regarding the adsorption of phenol, no matter whether the column is endcapped or not, its molecular interactions with the bare silica were negligible. For both columns, the best adsorption isotherm was the Bilangmuir model (with q(s,1) approximately equal to 2 and q(s,2) approximately equal to 0.67 mol/l, b1 0.61 and b2 approximately equal to 10.3 l/mol). These parameters are consistent with those measured previously on an endcapped Kromasil-C18 column under the same conditions (q(s,1) = 1.5 and q(s,2) = 0.71 mol/l, b1 = 1.4 l/mol and b2 = 11.3 l/mol). As for caffeine, the high-energy sites are definitely located within the C18-bonded layer, not on the bare surface of the adsorbent.     

Effect of the ionic strength of salts on retention and overloading behavior of ionizable compounds in reversed-phase liquid chromatography II. Symmetry-C18

In a companion paper, we describe the influence of the concentration and the nature of salts dissolved in the mobile phase (methanol:water, 40:60, v/v) on the adsorption behavior of propranolol (R'-NH2+ -R, Cl-) on XTerra-C18. The same experiments were repeated on a Symmetry-C18 column to compare the adsorption mechanisms of this ionic compound on these two very different RPLC systems. Frontal analysis (FA) measurements were first carried out to determine the best isotherm model accounting for the adsorption behavior of propranolol hydrochloride on Symmetry with a mobile phase without salt (and only 25% methanol to compensate for the low retention in the absence of salt). The adsorption data were best modeled by the bi-Moreau model. Large concentration band profiles of propranolol were recorded with mobile phases having increasing KCl concentrations (0, 0.002, 0.005, 0.01, 0.05, 0.1 and 0.2 M) and the best values of the isotherm coefficients were determined by the inverse method (IM) of chromatography. The general effect of a dissociated salt in the mobile phase was the same as the one observed earlier with XTerra-C18. Increasing the salt concentration increases the two saturation capacities of the adsorbent and the adsorption constant on the low-energy sites. The adsorption constant on the high-energy sites decreases and the adsorbate-adsorbate interactions tend to vanish with increasing salt concentration of the mobile phase. The saturation capacities decrease with increasing radius of the monovalent cation (Na+, K+, Cs+, etc.). Using sulfate as a bivalent anion (Na2SO4) affects markedly the adsorption equilibrium: the saturation capacities are drastically reduced, the high-energy sites nearly disappear while the adsorption constant and the adsorbate-adsorbate interactions on the low-energy sites increase strongly. The complexity of the thermodynamics in solution might explain the different influences of these salts on the adsorption behavior.     

Effect of the ionic strength of salts on retention and overloading behavior of ionizable compounds in reversed-phase liquid chromatography I. XTerra-C18

The influence of the salt concentration (potassium chloride) on the retention and overloading behavior of the propranolol cation (R'-NH2+ -R) on an XTerra-C18 column, in a methanol:water solution, was investigated. The adsorption isotherm data were first determined by frontal analysis (FA) for a mobile phase without salt (25% methanol, v/v). It was shown that the adsorption energy distribution calculated from these raw adsorption data is bimodal and that the isotherm model that best accounts for these data is the bi-Moreau model. Assuming that the addition of a salt into the mobile phase changes the numerical values of the parameters of the isotherm model, not its mathematical form, we used the inverse method (IM) of chromatography to determine the isotherm with seven salt concentrations in the mobile phase (40% methanol, v/v; 0, 0.002, 0.005, 0.01, 0.05, 0.1 and 0.2 M). The saturation capacities of the model increase, q(s,1) by a factor two and q(s,2) by a factor four, with increasing salt concentration in the range studied while the adsorption constant b1 increases four times and b2 decreases four times. Adsorbate-adsorbate interactions vanish in the presence of salt, consistent with results obtained previously on a C18-Kromasil column. Finally, besides the ionic strength of the solution, the size, valence, and nature of the salt ions affect the thermodynamic as well as the mass transfer kinetics of the adsorption mechanism of propranolol on the XTerra column.     

Comparison between the adsorption behaviors of an organic cation and an organic anion on several reversed-phase liquid chromatography adsorbents

Adsorption data of an organic cation (propranololium chloride) and an organic anion (sodium 1-naphthalene sulfonate) were measured by frontal analysis on two RPLC adsorbents, Symmetry-C18 and XTerra-C18, with aqueous solutions of methanol as the mobile phases. The influence of supporting neutral salts on the adsorption behavior of these two ions are compared. The Henry constants are close (H approximately 5). The four sets of isotherm data are all well accounted for using the bi-Moreau model. However, the isotherms of the two ions behave differently at high concentrations. The initial behaviors of all the isotherms are antilangmuirian but remain so in a much wider concentration range for the cation than for the anion, due to its stronger adsorbate-adsorbate interactions on the low-energy adsorption sites. The retention times of both ions increase with increasing concentration of neutral salt in the mobile phase, suggesting the formation of ion-pair complexes, with Cl- for the cation and with Na+ for the anion. The adsorbate-adsorbate interactions vanish in the presence of salt and the bi-Moreau isotherm model tends toward a bi-Langmuir model. Differences in adsorption behavior are also observed between the cation and the anion when bivalent inorganic anions and cations, respectively, are dissolved in the mobile phase. High concentration band profiles of 1-naphthalene sulfonic acid are langmuirian, except in the presence of a trivalent cation, while those of propranolol are antilangmuirian under certain conditions even with uni- or divalent cations.     

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.     

Band splitting in overloaded isocratic elution chromatography II. New competitive adsorption isotherms

The equations of two new binary competitive isotherms models are derived. The first of these models assumes that the isotherms of the two pure, single compounds have distinct monolayer capacities. Its derivation is based on kinetic arguments. The ideal adsorbed solution (IAS) framework was applied to derive the second model that is a thermodynamically consistent competitive isotherm. This second model predicts the competitive adsorption isotherm behavior of a mixture of two compounds that have single-component adsorption behavior following a BET and/or a Langmuir isotherms. Both models apply well to the binary adsorption of ethylbenzoate and 4-tert.-butylphenol on a Kromasil-C18 column (with methanol-water, 62:38, v/v, as the mobile phase). The best single-solute adsorption isotherms of these two compounds are the liquid-solid extended multilayer BET and the Langmuir isotherms, respectively. The kinetic and thermodynamic new competitive models were compared, regarding the accuracy of their prediction of the elution band profiles of mixtures of these two compounds. A better agreement between experimental and calculated profiles was observed with the kinetic model. The IAS model failed because the behavior of the ethylbenzoate/4-tert.-butylphenol adsorbed phase mixture is probably non-ideal. The most striking result is the qualitative prediction by these models of the peak splitting of 4-tert.-butylphenol during its elution in presence of ethylbenzoate.     

Effect of the ionic strength of the solution and the nature of its ions on the adsorption mechanism of ionic species in RPLC III. Equilibrium isotherms and overloaded band profiles on Kromasil-C18

In two companion papers, we have described the influence of the concentration and the nature of completely dissociated salts dissolved in the mobile phase (methanol:water, 40:60, v/v) on the adsorption behavior of propranolol (R'-NH2+-R, Cl-) on XTerra-C18 and on Symmetry-C18. The same experiments were repeated on a Kromasil-C18 column to compare the adsorption behavior of this ionic compound on these three different RPLC systems. The adsorption data of propranolol hydrochloride were first measured by frontal analysis (FA) using a mobile phase without salt. These data fit best to the Bi-Moreau model. Large concentration band profiles of propranolol were recorded with mobile phases containing increasing KCl concentrations (0, 0.002, 0.005, 0.01, 0.05, 0.1 and 0.2 M) and the best values of the isotherm coefficients were determined using the numerical solution of the inverse problem of chromatography. The general effect of a dissociated salt in the mobile phase was the same as the one observed earlier with XTerra-C18 and Symmetry-C18. However, obvious differences were observed for the shape of the band profiles recorded at low column loading (1.5 g/L, 250 microL injected). A long shoulder is visible at all salt concentrations and the band broadening is maximum at low salt concentrations. A slow mass transfer kinetics on the high-energy sites of the bi-Moreau model might explain this original shape. Five other salts (NaCl, CsCl, KNO3, CaCl2 and Na2SO4) were also used at the same ionic strength (J = 0.2 M). As many different band profiles were observed, suggesting that specific solute-salt interactions take place in the adsorbed phase.     

Correlation between retention and the C18 silica surface coverage in reversed-phase liquid chromatography

Summary Members of three homologous series and five non-homologous solutes with various functional groups were chromatographed on a series of well-characterized C₁₈ reversed stationary phases with a range of methanol-water mobile phases. Measured capacity factors of solutes were correlated with the concentration of C₁₈ ligands on the stationary phase. A linear relationship with the slope depending on a solute molecular structure and the volume fraction of methanol in the mobile phase was obtained. A method for the evaluation of phase ratio is also proposed. Presented at the 17th International Symposium on Chromatography, September 25–30, 1988, Vienna, Austria.     

Influence of the solvation process on solute adsorption in reversed phase liquid chromatography

The adsorption isotherms of phenol were acquired by frontal analysis on six different reversed phase adsorbents from five different organic solvent solutions. The end-capped octadecyl columns only differed in the bonding density of the C(18) ligands. The inverse method was used to confirm the estimated isotherm parameters derived from the frontal experiments. The effect of the bonding density of the end-capped octadecyl bonded phase on the adsorption properties of phenol from different mobile phase compositions was investigated. The adsorption behavior of phenol has changed from Langmuir type to BET type with the change of the organic modifier and the bonding density of the adsorbent.     

Kinetic performance of reversed-phase C18 high-performance liquid chromatography columns compared by means of the Kinetic Plot Method in pharmaceutically relevant applications

Four fully porous C18 columns (Hypersil Gold, ACE3, Xbridge and Gemini NX), widely employed in the pharmaceutical industry, were compared in terms of efficiency and analysis speed with the Kinetic Plot Method. Weakly basic, medium-sized, N-containing pharmaceutical compounds from GlaxoSmithKline Research and Development were used as test molecules. Isocratic elution was carried out at pH 4.5 and pH 8.0 with acetonitrile as organic modifier. The columns under evaluation included highly pure silica supports (Hypersil Gold, ACE3) and hybrid polymer-silica supports (XBridge, Gemini NX). Both types of columns claim for nearly absent secondary interactions with ionized silanol groups and are therefore applicable in a wide pH range. This is an important feature for method development purposes in pharmaceutical industries. The Kinetic Plot Method was used to compare the support characteristics and assess the kinetic performance of the columns in different experimental conditions. Although the evaluated columns have roughly identical particle diameters (from 3.0 to 3.5 μm) according to their manufacturers, large differences in kinetic performance were observed at pH 4.5 that can be accounted for by different flow resistances, porosities and average particle diameters, experimentally determined from scanning electron microscopy and laser light scattering experiments on loose stationary phase material. The ACE3 column was the best performing support among the evaluated columns, due to its excellent efficiency and average flow resistance. The better performance of the ACE3 column was due to its better packing quality, as could be derived from its impedance plot. Kinetic plots of resolution of a critical pair versus analysis time and column length were established at pH 8.0. These plots can be used as a method development tool to tailor the separation conditions to the required resolution of a given critical pair, combining efficiency and selectivity features of the column.     

Effect of the flow rate on the measurement of adsorption data by dynamic frontal analysis

The adsorption data of propyl benzoate were acquired by frontal analysis (FA) on a Symmetry-C18 column, using a mixture of methanol (65%, v/v) and water as the mobile phase, at three different flow rates, 0.5, 1.0 and 2.0 mL/min. The exact flow rates Fv were measured by collecting the mobile phase in volumetric glasses (deltaFv / Fv < or = 0.2%). The extra-column volumes and the column hold-up volume were accurately measured at each flow rate by tracer injections. The detailed effect of the flow rate on the value of the amount adsorbed was investigated. The best isotherm model accounting for the adsorption data was the same BET isotherm model at all three flow rates. Only slight differences (always less than 5%) were found between the three different sets of isotherm parameters (saturation capacity, q(s), equilibrium constant on the adsorbent, b(s) and equilibrium constant on successive layers of propyl benzoate, bL). The reproducibility of the same isotherm parameters measured by the inverse method (IM) is less satisfactory, leading to R.S.D.s of up to 10%. A flow rate increase is systematically accompanied by a slight increase of the amount adsorbed. This phenomenon is consistent with the influence of the pressure on the equilibrium constant of adsorption due to the difference between the partial molar volumes of the solute and the adsorbate. The larger average pressure along the column that is required to achieve a larger flow rate causes a larger amount of solute to be adsorbed on the column at equilibrium. This result comforts the high sensitivity and versatility of the FA method for isotherm determination under any kind of situation.     

Comprehensive study of the macropore and mesopore size distributions in polymer monoliths using complementary physical characterization techniques and liquid chromatography

Poly(styrene‐co‐divinylbenzene) monolithic stationary phases with two different domain sizes were synthesized by a thermally initiated free‐radical copolymerization in capillary columns. The morphology was investigated at the meso‐ and macroscopic level using complementary physical characterization techniques aiming at better understanding the effect of column structure on separation performance. Varying the porogenic solvent ratio yielded materials with a mode pore size of 200 nm and 1.5 μm, respectively. Subsequently, nano‐liquid chromatography experiments were performed on 200 μm id × 200 mm columns using unretained markers, linking structure inhomogeneity to eddy dispersion. Although small‐domain‐size monoliths feature a relatively narrow macropore‐size distribution, their homogeneity is compromised by the presence of a small number of large macropores, which induces a significant eddy‐dispersion contribution to band broadening. The small‐domain size monolith also has a relatively steep mass‐transfer term, compared to a monolith containing larger globules and macropores. Structural inhomogeneity was also studied at the mesoscopic level using gas‐adsorption techniques combined with the non‐local‐density‐function‐theory. This model allows to accurately determine the mesopore properties in the dry state. The styrene‐based monolith with small domain size has a distinctive trimodal mesopore distribution with pores of 5, 15, and 25 nm, whereas the monolith with larger feature sizes only contains mesopores around 5 nm in size.     

Practical aspects of fast reversed-phase high-performance liquid chromatography using 3 microm particle packed columns and monolithic columns in pharmaceutical development and production working under current good manufacturing practice

The potential and limitations of fast reversed-phase high-performance liquid chromatographic separations for assay and purity of drug substances and drug products were investigated in the pharmaceutical industry working under current good manufacturing practice using particle packed columns and monolithic columns. On particle packed columns, the pressure limitation of commercially available HPLC systems was found to be the limiting factor for fast separations. On 3 microm particle packed columns, HPLC run times (run to run) for assay and purity of pharmaceutical products of 20 min could be achieved. As an interesting alternative, monolithic columns were investigated. Monolithic columns can be operated at much higher flow rates, thus allowing for much shorter run times compared to particle packed columns. Compared to particle packed columns, the analysis time could be reduced by a factor up to 6. However, some compounds investigated showed a dramatic loss of efficiency at higher flow rates. This phenomenon was observed for some larger molecules supporting the theory that mass transfer is critical for applications on monolithic columns. At flow rates above 3 ml/min some HPLC instruments showed a dramatic increase in noise, making quantifications at low levels impossible. For very fast separations on monolithic columns, the maximum data acquisition rate of the detector is the limiting factor.     

Influence of the eluent composition on column efficiency in reversed-phase high-performance liquid chromatography

Summary The dependence of column efficiency on the eluent (MeOH/H₂O) composition in a reversed-phase liquid chromatography system within a wide concentration range has been systematically examined. It is shown that when the intracolumn effect of mass transfer and diffusion is the main factor controlling band broadening the column efficiency decreases with the increase of the viscosity of the MeOH/H₂O mixture; on the other hand, when the extra-column effect is the main factor an increase in the viscosity of the eluents will help in improving column efficiency. The column efficiency is also related to the properties of the sample.     

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.