Residual silanols at reversed-phase silica in HPLC--a contribution for a better understanding

As porous silica gel is the most common adsorbent and support for bonded stationary phase synthesis, residual silanol groups are a recurring problem in the field of liquid chromatography and other separation techniques. Residual silanols most often have a negative effect on the separation process by causing peak tailing. Therefore, there was an attempt to remove residual silanols during stationary-bonded phase synthesis. The type and surface concentration of residual silanols were measured using different instrumental techniques such as NMR and infrared spectroscopy, calorimetry, and various chromatographic methods. Residual silanols exhibit acidic characteristic and they can ionize depending on the environment. Thus, they posses cation-exchange properties and cause the zeta potential of silica particle in liquid environment. Presented review discusses the influence of the residual silanol groups on the solvation process and retention of polar compounds. The novel methodology of residual silanols determination is presented as well as the influence of the silanols on the zeta potential of the stationary-bonded phases in chromatographic conditions.     

Deactivation of silica surfaces with a silanol-terminated polysiloxane; structural sharacterization by inverse gas chromatography and solid-state NMR

Retention gape deactivated with Silicone OV-1701-OH show good chromatographic performance and remarkable stability against water induced stationary phase degradrdation. In an attempt to better understand the findamentals off the deactivation process using silanol terminated polysiloxanes, a fumed silica was deactivated with Silicon OV-1701-OH. In contrast to fused silic capillaries, fumed silica (Aerosil A-200) can be studied by ²⁹Si cross-polarization magic-angle-spinning (CPMAS) NMR, thus serving as a model substrate for fused silica. Retention data from inverse gas chromatography at infinite dilurion and ²⁹Si CP MAS NMR data of five Aerosil phases, differing in residual silanol surface concentration, are correlated with the aim of validating this approach for stationary phase characterization. A comparatively detailed model of the deactivating polymer layer that explains the observed absorption activities is deduced. Surface silanols are shown to play a key role in the polymer layer, the structure of which is of primary importance for the absorption behavior after deactivation. Contrary to common belief, the absolute silanol surface concentration after deativation is only of secondary importance for the overall absorption activity. High silanol surface concentrations enhance degradation of the polysiloxane chains into small cyclic fragments as well as subsequent absorption and immobolization to the silica substrate surface. The mobility of linear polysiloxane chains in the kHz regime (as determined bby NMR cross-polarization dynamics) appears to determine the extent which the residual silanols are accessible for analytes. It is therefore anticipated that there is an optimum silanol surface concentration of fused silica surfaces to be deactivated with silanol terminated polysiloxanes; it should be lazrge enough to adsord polymer fragments, but not large to avoid excessive residual silanol activity.     

DFT study of the adsorption of microsolvated glycine on a hydrophilic amorphous silica surface

Density functional theory (DFT) periodic ab initio molecular dynamics calculations are used to study the adsorption of gaseous and microsolvated glycine on a hydroxylated, hydrophilic silica surface. The silica model is presented and the interaction of water with surface silanols is studied. The heat of interaction of water is higher with the associated silanols (be they terminal or geminal ones) studied here than with isolated silanols presented in past works. Glycine is stabilized in a parallel mode on the hydroxylated surface. Terminal silanols do not allow the stabilization of the zwitterionic form, whereas geminal silanols do. Molecular dynamics (MD) first-principle calculations show that microsolvated zwitterion glycine directly binds through the carboxylate function to a surface silanol rather than through water molecules. The adsorption mode, whether with or without additional water molecules, is parallel to the surface. The ammonium function does not interact directly with the silanol groups but rather through water molecules. Thus, the carboxylate and ammonium functions exhibit two different reactivities towards silanols. The calculated free energies, taking into account the chemical potentials of water and glycine in the gas phase, suggest the existence of a thermodynamic domain in which the glycine is present in the gas phase as well as strongly adsorbed on specific sites of the surface.     

A Mixed Retention Model of Reversed Phase HPLC

The retention behavior of solutes in reversed phase chromatographic system, especially containing hydrophobic ions in mobile phase, has been studied by many authors, but the silanol effect on alkyl-modified silica surface was neglected. Actually, because of stereo hindrance, numerous unreacted (residual) silanol groups, which can interact with some solutes, are left within the bonded phase after the silica surface has be modified. A mixed retention model, which considers adsorbed hydrophobic ions on the bonded phase can decrease the hydrophobicity and mask residual silanol groups, is proposed, based on hydrophobic distribution of neutral solutes, ion-pair distribution of ionic solutes and coulombic attraction between cationic solutes and the dissociated silanols.     

The role of the dual nature of ionic liquids in the reversed-phase liquid chromatographic separation of basic drugs

The cationic nature of basic drugs gives rise to broad asymmetrical chromatographic peaks with conventional C18 columns and hydro-organic mixtures, due to the ionic interaction of the positively charged solutes with the free silanol groups on the alkyl-bonded reversed-phase packing. Ionic liquids (ILs) have recently attracted some attention to reduce this undesirable silanol activity. ILs are dual modifiers (with a cationic and anionic character), which means that both cation and anion can be adsorbed on the stationary phase, giving rise to interesting interactions with the anionic free silanols and the cationic basic drugs. A comparative study of the performance of four imidazolium-based ILs as modifiers of the chromatographic behaviour of a group of β-blockers is shown. The ILs differed in the adsorption capability of the cation and anion on C18 columns. Mobile phases without additive and containing a cationic (triethylamine, TEA) or anionic (sodium dodecyl sulphate, SDS) additive were used as references for the interpretation of the behaviours. The changes in the nature of the chromatographic system, at increasing concentration of the additives, were followed based on the changes in retention and peak shape of the β-blockers. The silanol suppressing potency of the additives, and the association constants between the solutes and modified stationary phase or additive in the mobile phase, were estimated. The study revealed that SDS and the ionic liquid 1-hexyl-3-methylimidazolium tetrafluoroborate are the best enhancers of chromatographic peak shape among those studied.     

Comparison of D,L-Mandelic Acid Resolution on Zeolite and Silica Supported Pirkle-Type Chiral Stationary Phases

 Zeolite A, a material of crystalline character, and Hypersil silica have been used as support for the preparation of chiral stationary phases. On the amorphous silica support surface the silanol groups are randomly dispersed. The crystalline zeolite secondary building units consisting primarily of SiO₄, AlO₄ ⁻ tetrahedra determine the regularity of surface silanol groups. Owing to the crystal lattice structure, the location of silanols is well determined and hence the dispersion of chiral selector molecules chemically bonded onto the zeolite surface silanol groups is fundamentally arranged. Amides of DNB-L-Leu, DNB-L-Phe, B-L-Leu chiral selector molecules were anchored onto the zeolite silanols and B-L-Leu onto the silica support silanols. Lipophilic buffer in RP conditions has dynamically modified the residual silanols of each support. The enantioseparation of ion paired D,L-mandelic acid from aqueous solution on the zeolite and silica supported chiral stationary phases prove a superior enantioseparation on the zeolite supported phases. Revision February 18, 2000.     

Annealing of silica to reduce the concentration of isolated silanols and peak tailing in reverse phase liquid chromatography

Non-porous, colloidal silica particles were annealed at three different temperatures, 800, 900 and 1050 °C. The adsorption of lysozyme, a probe of surface roughness, was consistent with progressively reduced surface roughness as temperature increased. The heat treated silica particles were rehydroxylated and then used to pack UHPLC columns. The cationic protein lysozyme was used to probe silanol activity, which exhibited progressively less tailing as the annealing temperature increased. FTIR spectroscopy confirmed that the abundance of isolated silanols on the surface was reduced by annealing at 900 °C or 1050 °C. FTIR also revealed that there was markedly increased hydrogen bonding of the isolated silanols to neighbors after rehydroxylation. These results combine to support the hypothesis that (a) isolated silanols on silica cause tailing in RP-LC and (b) nonplanar topography gives rise to isolated silanols.     

Effect of End-Capping and Surface Coverage on the Mechanism of Solvent Adsorption

Adsorption of acetonitrile and methanol was measured on non-endcapped and endcapped octadecyl stationary phases. The results enabled us to characterize the effect of end-capping on the adsorption of the organic modifiers. Our results clearly demonstrate how the end-capping groups modify the chromatographic properties of adsorbents. Differences between solvent adsorption mechanisms are also discussed. Adsorption of acetonitrile is governed by hydrophobic effects whereas methanol is adsorbed by the organic ligand by a hydrophobic interaction and by the residual silanols by hydrogen-bond formation and dipole–dipole interactions.     

Suppression of deleterious effects of free silanols in liquid chromatography by imidazolium tetrafluoroborate ionic liquids

Silica-based stationary phases are commonly used in liquid chromatography, but their surface acidity causes known problems, especially when separating basic compounds. Deleterious effects of free silanols are not fully removed by standard prevention procedures consisting in adding alkylamines or other amino quenchers to the eluents. We found that ionic liquids of the imidazolium tetrafluoroborate class, added to mobile phases at concentrations of 0.5-1.5% (v/v), blocked silanols and provided excellent thin-layer chromatographic separations of strongly basic drugs which were otherwise not eluted, even with neat acetonitrile as the mobile phase. The silanol suppressing potency of imidazolium tetrafluoroborates was demonstrated to markedly exceed that of the standard mobile phase additives, like triethylamine, dimethyloctylamine and ammonia. The proposed new mobile phase additives were also demonstrated to provide reliable lipophilicity parameters of base drug analytes as determined by gradient mode of high-performance liquid chromatography. By applying the readily available and environmentally friendly imidazolium tetrafluoroborate ionic liquids, simple and efficient means of improvement of liquid chromatographic analysis of organic bases were elaborated.     

Comparison of the chromatography of octadecyl silane bonded silica and polybutadiene-coated zirconia phases based on a diverse set of cationic drugs

In this study, we compare the separation of basic drugs on several octadecyl silane bonded silica (ODS) phases and a polybutadiene-coated zirconia (PBD-ZrO2) phase. The retention characteristics were investigated in detail using a variety of cationic drugs as probe solutes. The ODS phases were selected to cover a relatively wide range in silanol activity and were studied with ammonium phosphate eluents at pH 3.0 and 6.0. Compared to any of the ODS phases, the PBD-ZrO2 phase showed very significant differences in selectivities towards these drugs. Due to the presence of both reversed-phase and ion-exchange interactions between the stationary phase and the basic analyte on ODS and PBD-ZrO2, mixed-mode retention takes place to some extent on both types of phases. However, very large differences in the relative contributions from ion-exchange and reversed-phase interactions on the two types of phases led to quite different selectivities. When phosphate is present in the eluent and adsorbs on the surface, the PBD-ZrO2 phase takes on a high negative charge over a wide pH range due to phosphate adsorption on its surface. On ODS phases, ion-exchange interactions result from the interactions between protonated basic compounds and ionized residual silanol groups. Since the pH of the eluent influences the charge state of the silanol groups, the ion-exchange interactions vary in strength depending on pH. At pH 6.0, the ion-exchange interactions are strong. However, at pH 3.0 the ion-exchange interactions on ODS are significantly smaller because the silanol groups are less dissociated at the lower pH. Thus, not only are the selectivities of the ODS and PBD-ZrO2 phases different but quite different trends in retention are observed on these two types of phases as the pH of the eluent is varied. More importantly, by using the large set of "real" basic analytes we show the extreme complexity of the chromatographic processes on the reversed stationary phases. Both the test condition and solute property influence the column performance. Therefore, use of only one or two probe solutes is not sufficient for column ranking.     

Mobile phase effects in reversed-phase liquid chromatography: a comparison of acetonitrile/water and methanol/water solvents as studied by molecular simulation

Molecular simulations of water/acetonitrile and water/methanol mobile phases in contact with a C(18) stationary phase were carried out to examine the molecular-level effects of mobile phase composition on structure and retention in reversed-phase liquid chromatography. The simulations indicate that increases in the fraction of organic modifier increase the amount of solvent penetration into the stationary phase and that this intercalated solvent increases chain alignment. This effect is slightly more apparent for acetonitrile containing solvents. The retention mechanism of alkane solutes showed contributions from both partitioning and adsorption. Despite changes in chain structure and solvation, the molecular mechanism of retention for alkane solutes was not affected by solvent composition. The mechanism of retention for alcohol solutes was primarily adsorption at the interface between the mobile and stationary phase, but there were also contributions from interactions with surface silanols. The interaction between the solute and surface silanols become very important at high concentrations of acetonitrile.     

Reducing residual silanol interactions in reversed-phase liquid chromatography. Thermal treatment of silica before derivatization

This study describes the thermal pretreatment of a silica gel between 150 and 800 degrees C before derivatization with dimethyloctadecylchlorosilane as a means of reducing residual silanol activity in HPLC bonded stationary phases. A time study was done from 12 to 48 h to find the optimum time needed for dehydroxylation. With increasing pretreatment temperatures, the number of reactive silanols is reduced from 8 micromol/m2 to essentially zero at 1000 degrees C (where sintering occurs). The effects of the thermal pretreatments were observed with diffuse reflectance infrared Fourier transform spectroscopy (DRIFTS) and solid state cross polarization magic angle spinning (CP-MAS) 29Si NMR. Following derivatization, residual silanol activity and pH stability were tested by packing columns with the derivatized silica and carrying out a series of reversed-phase liquid chromatography (RPLC) experiments. Residual silanol activity was greatly reduced when the silica was pretreated at 800 degrees C, leading to less peak tailing for basic solutes. In a strongly basic mobile phase (pH 11.5) the pretreated silica was surprisingly stable, although bond cleavage of C18 groups from the surface was observed.     

A study of the adsorption activities of silanol surface structures on a fused silica model substrate by combining 29Si CP MAS NMR and inverse gas chromatographic data

The possibilities of inverse gas-solid chromatography (IGC) in obtaining chromatographic data on fumed silica were examined. Aerosil A-200, a fused silica model substrate in ²⁹Si nuclear magnetic resonance analysis, was trimethylsilylated to different degrees. IGC was used to very reproducibly determine the free specific energies of adsorption of several functionalized probe solutes. Hydrogen bonding solutes have a free specific energy of adsorption that is at least about 50% higher than that of non-hydrogen bonding probe solutes. NMR was used in combination with elemental analysis to calculate surface concentrations of the different chemical surface structures. IGC data and surface concentrations were combined in order to determine the contribution of each type of surface structure to the total free specific adsorption energy. It could be concluded that residual silanols from the reaction of dihydroxydi-siloxysiloxane (Q₂ groups) with trimethylchlorosilane possess a higher adsorption activity than the silanols initially present.     

Reversed Phase Chromatography – the Mystery of Surface Silanols

The nature of surface silanols is reviewed and their influence on retention of basic solutes in reversed phase chromatography is demonstrated and evaluated. The influence of the type of organic modifier and/or pH of buffer on retention of basic analytes with silica based RP columns is evaluated, and means for characterization of RP columns are discussed. Attempts are described to determine the surface silanol concentration of RP columns by chromatographic methods. Surprisingly the experimentally measured silanol concentrations with bare silica and with RP are by one order of magnitude lower than discussed in literature. At pH 7.6 values of 0.12 µmol m^?2 have been measured for benzylamine with RP columns, corresponding to about 43% of the measurable silanols of plain silica. The break-through curves of amines let suggest that even at low pH values unprotonated species are present within the pores of the RP stationary phases and their interaction with the bonded alkyl groups contribute to retention.     

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.     

Study of solvation processes on cholesterol bonded phases

Four cholesterol bonded phases with different structures were investigated. The columns studied were packed with stationary phase containing cholesterol attached to the silica surface using different types of linkage molecules. The presence of the polar amino and carboxyl groups in the structure of the bonded ligand strongly influence on the solvation process. The possibility of hydrogen bonding, dipole-dipole and π-π electron interactions lead to preferential solvation of bonded ligands. The coverage density of bonded ligands and length of the linkage strongly influence the adsorption of solvent from the mobile phase. The removal of residual silanols during the hydrosilation procedure significantly influences solvation of the bonded phase. Excess isotherms of the commonly used solvents in RP HPLC (methanol and acetonitrile) were obtained using the minor disturbance method. For comparison of the stationary phases prepared on different silica gels the excess adsorbed amounts were calculated per volume of the stationary phase in the column. The hydrosilated UDC Cholesterol bonded phase is preferentially solvated by methanol whereas the highest coverage Cosmosil Cholester phase exhibit high adsorption of acetonitrile. Polar groups in the Amino-cholesterol type bonded phase are solvated with both solvent but the mechanisms of these processes are different.     

Analysis of basic compounds at high pH values by reversed-phase liquid chromatography

Reversed phase high performance liquid chromatography (RPLC) is currently the method of choice for the analysis of basic compounds. However, with traditional silica materials, secondary interactions between the analyte and residual silanols produce peak tailing which can negatively affect resolution, sensitivity, and reproducibility. In order to reduce these secondary interactions, which comprise ion exchange, hydrogen bonding, and London forces interactions, chromatographic analyses can be carried out at low or high pH values where silanol groups and basic compounds are mostly uncharged. The chromatographic behaviour of a particular bidentate stationary phase, Zorbax Extend C18, was studied with a set of basic and neutral compounds. Thanks to a higher chemical stability than traditional silica based supports, analyses were carried out with a high pH mobile phase, which represents a good alternative to the acidic mobile phases generally used to reduce ion exchange interactions. The performance of this bidentate stationary phase was also compared with that of other supports and it was proved that it is advantageous to work with high pH mobile phases when analyzing basic compounds.     

Characterization of the acidity of residual silanol groups in microparticulate and monolithic reversed-phase columns

The residual silanol acidity and activity of several microparticulate and monolithic C18 columns has been measured from the retention of LiNO3 in the columns with a methanol/buffer (1 mM in Na+) (60:40 v/v) mobile phase buffered to different pH values. For Luna C18 (2) and LiChrospher RP-18 columns, at least two different types of silanols with different acidity for each packing, were observed. Purospher RP-18e and Chromolith RP-18e packings present evidence of some active silanols only at pH values close to their basic pH stability limit or higher. The results obtained have been compared with those obtained previously for Resolve C18, Resolve Silica, Symmetry C18, Symmetry Silica, XTerra MSC18 and Underivatized XTerra. A modification of an equation previously proposed has been applied to all columns studied and the results obtained have been used to classify the columns according to their silanol acidity and activity. The method allows the prediction of the extent of the silanol activity of the columns studied at a particular mobile phase pH.     

Reversed Phase High-Performance Liquid Chromatography of Basic Drugs on a Silanol Deactivated Support

Abstract

When polar and non-polar basic drugs were separated by reversed phase HPLC, a short-chain silanol deactivated (SCD-100) reversed phase column gave superior results over a standard C18 reversed phase column. The nature of silane, the type of silica and the chemistry of endcapping influenced chromatographic behavior. The peaks in the chromatogram obtained from this column had excellent peak shapes and eluted at predictable retention times, indicating that no silanols remained on the surface. The mobile phase was composed of phosphate buffer at pH 3.5, with variable amounts of methanol. All applications were isocratic without the addition of silanol suppressing reagents.