Retention and selectivity tests of silica-based and metal-oxide bonded stationary phases for RP-HPLC

Chromatographic properties of silica-, zirconia- and alumina-based columns with octadecyl-, polyethylene glycol- and pentafluorophenylpropyl-bonded stationary phases were tested. Selectivities of nine columns for LC were characterized using chromatographic methods including Walters, Engelhardt, Tanaka and Galushko hydrophobicity and silanol activity tests, measurements of methylene selectivity in various aqueous-methanol and aqueous-acetonitrile mobile phases and of gradient lipophilic capacity as a measure of the effect of the sample hydrophobicity on gradient-elution separations. A semi-empirical interaction indices model, assuming a predominant role of the solvophobic interactions of test compounds with different polarities, was compared with the linear free energy relationships approach taking into account selective polar interactions. The interaction indices model was applied to both non-polar stationary phases bonded on silica, alumina and zirconia supports, and to the non-modified adsorbents in the normal-phase LC. The retention data of isomeric naphthalene disulfonic acids were used to compare the attractive and repulsive ionic interactions of the columns in purely aqueous mobile phases. The results of the hydrophobicity and polarity tests were consistent, and allowed column characterization and classification. Silanol activity was important with octadecyl silica columns, but was relatively insignificant with bonded polyethylene glycol and pentafluorophenylpropyl phases on silica gel support. Polar interactions with the alumina and zirconia support materials significantly affect the retention.     

Rapid separation of microcystins and nodularin using a monolithic silica C18 column

A monolithic C18-bonded silica rod column (Merck Chromolith) was compared to particle-based C18 and amide C16 sorbents in the HPLC separation of eight microcystins and nodularin-R. Two gradient mobile phases of aqueous trifluoroacetic acid modified with acetonitrile or methanol, different flow-rates and different gradient lengths were tested. The performance of the Chromolith column measured as the resolution of some microcystin pairs, the selectivity, efficiency (peak width) and peak asymmetry equalled, or exceeded, the performance of traditional particle-based columns. The Chromolith column allowed a shortening of the total analysis time to 4.3 min with a flow-rate 4 ml min(-1).     

Optimization of separation in two-dimensional high-performance liquid chromatography by adjusting phase system selectivity and using programmed elution techniques

The overall peak capacity in comprehensive two-dimensional liquid chromatographic (LC x LC) separation can be considerably increased using efficient columns and carefully optimized mobile phases providing large differences in the retention mechanisms and separation selectivity between the first and the second dimension. Gradient-elution operation and fraction-transfer modulation by matching the retention and the elution strength of the mobile phases in the two dimensions are useful means to suppress the band broadening in the second dimension and to increase the number of sample compounds separated in LC x LC. Matching parallel gradients in the first and second dimension eliminate the necessity of second-dimension column re-equilibration after the independent gradient runs for each fraction, increase the use of the available second-dimension separation time and can significantly improve the regularity of the coverage of the available retention space in LC x LC separations, especially with the first- and second-dimension systems showing partial selectivity correlations. Systematic development of an LC x LC method with parallel two-dimensional gradients was applied for separation of phenolic acids and flavone compounds. Several types of bonded C18, amide, phenyl, pentafluorophenyl and poly(ethylene glycol) columns were compared using the linear free energy relationship method to find suitable column combination with low correlation of retention of representative standards. The phase systems were optimized step-by-step to find the mobile phases and gradients providing best separation selectivity for phenolic compounds. The optimization of simultaneous parallel gradients in the first and second dimension resulted in significant improvement in the utilization of the available two-dimensional retention space.     

Naphthalene sulphonic acids--new test compounds for characterization of the columns for reversed-phase chromatography

Non-substituted naphthalene sulphonic acids are strong acids, which are completely ionised in aqueous and aqueous-organic solutions. Because of repulsive electrostatic interactions, they are more or less excluded from the pores of the column packing materials commonly used in reversed-phase chromatography. The ionic exclusion can be suppressed by increasing the ionic strength of the mobile phase. In aqueous sodium sulphate solutions, very good selectivity was observed for isomeric naphthalene di- and tri-sulphonic acids, allowing reversed-phase separations of these strongly ionic compounds without addition of ion-pairing reagents to the mobile phase. The retention of the isomeric acids increases proportionally to the dipole moment, which can be explained by its effect on increasing exposure of the naphthalene ring to hydrophobic interactions with the non-polar stationary phases. Chromatographic behaviour of isomeric naphthalene di- and trisulphonic acids was investigated on 25 different columns for reversed-phase chromatography. The elution order of the isomers is the same on all the columns, but very strong stationary phase effects were observed on the retention and on the band asymmetry, depending on polar interactions with residual silanol groups and other polar adsorption centres in the stationary phases. These effects are independent of the organic solvents, as the tests are performed in purely aqueous mobile phases and allow classification of the columns into several groups.     

Effects of stationary-phase polarity on retention in reversed bonded phase HPLC columns

Summary Variations in retention and selectivity have been studied in cyano, phenyl and octyl reversed bonded phase HPLC columns. The retention of toluene, phenol, aniline and nitrobenzene in these columns has been measured using binary mixtures of water and methanol, acetonitrile or tetrahydrofuran mobile phases in order to determine the relative contributions of proton donor-proton acceptor and dipole-dipole interactions in the retention process. Retention and selectivity in these columns was correlated with polar group selectivities of mobile phase organic modifiers and the polarity of the bonded stationary phases. In spite of the prominent role of bonded phase volume and residual silanols in the retention process, each column exhibited some unique selectivities when used with different organic modifiers.     

Synthesis and characterization of novel polar-embedded silica stationary phases for use in reversed-phase high-performance liquid chromatography

We have developed a series of new C₁₀ dipeptide stationary phases via a simple and effective synthetic method. The preparation of the new phases involves the synthesis of silanes and the surface modification of silica. Chromatographic evaluations of these columns were performed using the Engelhardt, Tanaka, and Neue test mixtures. The applicability of these new stationary phases was also evaluated using a series of diagnostic probes including acids, bases or neutral compounds and several generic applications. These new C₁₀ dipeptide stationary phases showed excellent hydrolytic stability over a wide pH range. Like other existing amide-embedded columns, these new stationary phases exhibit higher retention for polar and hydrophilic compounds and different selectivity as compared to conventional C₁₈ columns. These new phases are compatible with 100% aqueous mobile phases, and also provide high column efficiency and good peak shapes for both acidic and basic compounds.     

Stationary-phase effects in gradient high-performance liquid chromatography

The type of the stationary phase for reversed-phase liquid chromatography significantly affects the sample polarity range that can be covered using gradients of organic solvents in water. The polarity range available for gradient separations of samples containing compounds differing in the lipophilic parts of the molecules can be characterized by "gradient lipophilic capacity", Pl, based on the retention of standard compounds with a repeat lipophilic structural unit, such as a methylene group. The gradient lipophilic capacity is also suitable to characterize the separation possibilities of the columns in non-aqueous reversed-phase gradient elution of strongly non-polar compounds, such as triacylglycerols. In the same way, the suitability of various columns for reversed-phase gradient separations of oligomers can be characterized by "gradient oligomer capacity", as demonstrated in the example of oligo(ethylene glycols). To enable a comparison of the properties of stationary phases independent of column efficiency and dimensions, the gradient lipophilic capacity or the gradient oligomer capacity should be normalized for a "standard" column plate number, gradient range and volume (in column hold-up volume units). The gradient lipophilic capacity or the gradient oligomer capacity and the number of compounds that can be resolved during a gradient run decrease as the initial concentration of the strong solvent in the mobile phase increases and (or) the gradient time decreases. These quantities can be used to select a suitable column and to adjust the optimum gradient profile (the initial composition of the mobile phase and the gradient steepness) with respect to the time of analysis and the number of oligomers or other compounds with regular repeat structural groups that can be resolved during the gradient run.     

Comparison of the performance of Chromolith Performance RP-18e, 1.8-μm Zorbax Eclipse XDB-C18 and XTerra MS C18, based on modelling approaches

Achievement of the highest separation efficiency and quick delivery of results are key requirements in liquid chromatography for enhancing productivity and reducing analysis cost, especially in the pharmaceutical industry. This work concerns two of the most popular current solutions to get fast separations: the use of a silica-based monolithic column (Chromolith Performance RP-18e) and a small-particle packed column (1.8-μm Zorbax Eclipse XDB-C18, which needs dedicated instruments allowing higher backpressures). Both columns succeeded in the full separation of phenols and β-adrenolytic drugs, which are compounds that interact with residual silanols, giving rise to wider peaks. The results were compared with those obtained with a special column designed to avoid silanol interaction, containing 5 μm particles (XTerra MS C18). Chromolith gave the shortest times at the expense of higher solvent consumption at the high flow rates needed. In contrast to other studies, comprehensive conclusions on the chromatographic performance, in terms of selectivity, peak shape, resolution, and analysis time, are derived from the inspection of the whole experimental domain using retention and peak shape modelling. In the literature, column comparison is usually carried out based on the performance for selected mobile phases (very often a single one), which offers deceiving results.     

The influence of the mobile phase pH and the stationary phase type on the selectivity tuning in high performance liquid chromatography nucleosides separation

Analysis of the modified nucleosides is particularly important in the medical area because of a possibility of cancerogenic processes studies. The aim of this work was to study the selectivity tuning of modified nucleosides through the investigations of interactions analyte (modified nucleoside) <==> stationary phase <==> mobile phase. A series of homemade stationary phases with different surface properties has been utilized. All of them contain various interaction sites such as: cholesterol (SG-CHOL); n-acylamide (SG-CHOL, SG-AP); aminopropyl (SG-CHOL, SG-AP, SG-NH2, SG-MIX); cyanopropyl, phenyl, octyl (SG-MIX), octadecyl (SG-MIX, SG-C18) and silanols localized on the silica gel surface of all packings. The attempt to predict the main interactions responsible for the retention between nucleosides and stationary phase ligands was done on the basis of the elemental analysis, and proportional part of an individual ligand bonded to silica surface results. In order to study the influence of different packing types on the analyzed nucleosides retention, the relationship between pH of the mobile phase buffer and the selectivity of a stationary phase was investigated.     

Comprehensive two-dimensional liquid chromatography with parallel gradients for separation of phenolic and flavone antioxidants

Various combinations of PEG-silica, phenyl-silica and C18 columns in a single-column or serial (tandem) arrangement in the first dimension and a monolithic Chromolith column in the second dimension were tested for comprehensive two-dimensional (2D) LCxLC separation of phenolic and flavone natural antioxidants. The combinations of different stationary phase chemistries provided low selectivity correlations between the first-dimension and the second-dimension separation systems. Improvement in system orthogonality, bandwidths suppression, more regular band distribution over the whole 2D retention plane and increased peak capacity in different 2D setups was achieved by using gradients with matching profiles running in parallel in the two dimensions over the whole 2D separation time range. Instead of two sampling loops, two alternating trapping XTerra columns were used for sample fraction transfer from the first-dimension column to the second dimension. Stronger retention on the XTerra columns in comparison to PEG-silica or phenyl-silica columns in the first dimension allowed using focusing of sample fractions in narrow zones on the top of a trapping column and back-flushing into the second dimension in a very low volume of the mobile phase. This fraction transfer modulation provided significant bandwidth suppression in the second dimension. 2D systems with optimized stationary phase selectivity, parallel gradients and fraction transfer modulation using two trapping columns were applied for the analysis of natural antioxidants in beer and wine samples.     

High-performance liquid chromatography of sialooligosaccharides and gangliosides

Glycans were cleaved from gangliosides and separated by high-performance liquid chromatography (HPLC). The columns were packed with bonded stationary phases made of microparticulate, macroporous silica with serotonin, phenylpropanolamine or tryptamine as the biogenic amine ligate. The ganglioside oligosaccharides were eluted in the order of increasing number of sialic acid residues in the molecule and their retention decreased with the ionic strength of the mobile phase. Best selectivity was obtained in the pH range from 3.0 to 4.0. The two major sialic acids, N-acetylneuraminic and N-glycolylneuraminic acids, were separated by lectin affinity chromatography using an HPLC column packed with silica-bound wheat germ agglutinin and 10 mM phosphate buffer, pH 4.0, as the eluent. Throughout this study, isocratic elution was used and the column effluent was monitored at 195 nm.     

Comparison of a C30 Bonded Silica Column and Columns with Shorter Bonded Ligands in Reversed-Phase LC

The Carotenoid S is a new C₃₀ bonded silica stationary phase, intended for reversed-phase chromatographic applications, which is more hydrophobic and consequently shows stronger retention in comparison to conventionally used C₁₈ stationary phases. We compared the non-polar selectivities of the columns for homologous alkylbenzenes in acetonitrile—water and methanol–water mobile phases and polar reversed-phase selectivities employing the interaction indices and the Linear Free Energy Relationship models. Further, we investigated possibilities of separations of structurally closely related compounds in the groups of phenolic acids, flavones, phthalic acids and related compounds and of acylglycerols on the new C₃₀ column and with different types of columns for reversed-phase chromatography, including shorter alkyl C₄, C₈, C₁₈ and phenyl bonded stationary phases. The C₃₀ column has in some aspects properties similar to the non-endcapped Nova-Pak column for separation of some acylglycerols with equal equivalent carbon numbers, but enables separations of longer chain triacylglycerols in a single gradient run.

     

Effect of water on the retention on diol and amide columns in hydrophilic interaction liquid chromatography

The amount of water adsorbed on polar columns plays important role in hydrophilic interaction liquid chromatography. It may strongly differ for the individual types of polar columns used in this separation mode. We measured adsorption isotherms of water on an amide and three diol‐bonded stationary phases that differ in the chemistry of the bonded ligands and properties of the silica gel support. We studied the effects of the adsorbed water on the retention of aromatic carboxylic acids, flavonoids, benzoic acid derivatives, nucleic bases, and nucleosides in aqueous‐acetonitrile mobile phases over the full composition range. The graphs of the retention factors versus the volume fraction of water in mobile phase show “U‐profile” characteristic of a dual hydrophilic interaction–reversed phase retention mechanism. The minimum on the graph that marks the changing retention mechanism depends on the amount of adsorbed water. The linear solvation energy relationship model suggests that the retention in the hydrophilic interaction liquid chromatography mode is controlled mainly by proton–donor interactions in the stationary phase, depending on the column type. Finally, the accuracy of hydrophilic interaction liquid chromatography gradient prediction improves for columns that show a high water adsorption.     

System maps for retention of neutral organic compounds under isocratic conditions on a reversed-phase monolithic column

The solvation parameter model is used to create systems maps for the separation of neutral organic compounds on a Chromolith Performance RP-18e octadecylsiloxane-bonded silica-based monolithic column for water-acetonitrile and water-methanol mobile phase compositions from 10 to 70% (v/v) organic solvent. These results demonstrate that the retention properties of the monolithic column are similar to those of conventional octadecylsiloxane-bonded silica particle-packed columns. It is further shown that the selectivity for the monolithic column falls within the selectivity range for typical particle-packed columns at two mobile phase compositions for which a direct comparison is possible.     

Mass spectrometry-compatible ICH (International Conference on Harmonization) impurity analysis with a high-pH mobile phase advantages and pitfalls

Recent advances in bonding chemistry and novel silica synthesis have significantly extended the pH range of silica-based HPLC columns. This extended range now enables the analysis of water-soluble basic drugs at high pH without ion-pairing reagents, thus offering an alternative approach to assay or impurity analyses. This paper describes the many advantages and potential pitfalls of using high-pH mobile phases in the development of MS-friendly LC gradient impurity analytical methods for water-soluble basic drugs under International Conference on Harmonization (ICH) guidelines. Operating at high-pH provides excellent peak shapes and retention, and accentuates selectivity differences of structurally similar impurities and degradants. However, several problems unique to the use of high-pH mobile phases, such as column lifetime, robustness of pH adjustments, peak fronting, and on-column dimerization, were encountered. Each of these problems is discussed with its respective remedy.     

Study of the retention and selectivity of cholesterol bonded phases with different linkage spacers

The chromatographic properties of four cholesterol bonded phases with different structures were studied. The columns used were packed with a stationary phase containing a cholesterol molecule attached to the silica surface using different types of linkage molecules. As a basic characteristic of the bonded phases the hydrophobicity and silanol activity (polarity) were investigated. The presence of the polar amino and carboxyl groups in the structure of the bonded ligand strongly influences the polarity of the bonded phase. Columns were compared according to methylene selectivity using a series of benzene homologues and according to their shape and size selectivity using polycyclic aromatic hydrocarbons (PAHs). The measurements were done using MeOH–water and ACN–water mobile phases. The presented results show that the coverage density of the bonded ligands and length of the linkage strongly influence the retention and selectivity of cholesterol bonded phases.     

Characterization of calixarene‐bonded stationary phases

Calixarene‐bonded stationary phases received growing interest in HPLC as stationary phases with special retention characteristics and selectivity. The commercially available unsubstituted and p‐tert‐butyl‐substituted Caltrex^® columns have been intensively studied and characterized in our workgroup. They can be used as reversed phases, yet they support additional interactions. Especially, their steric, polar and ionic properties differ from conventional alkyl‐bonded phases. However, also the hydrophobic interaction shows differences since adsorption and partition interactions on or in a bonded layer of calixarenes are not similar to those of alkyl‐bonded layers. The relative strength of the hydrophobic properties of the stationary phases has been found depending on the methanol concentration of the mobile phase. Generally, the dependencies of their interaction strengths on mobile‐phase conditions, e.g. the change of the intensity of the hydrogen‐bonding abilities with decreasing methanol content, are not similar from phase to phase either. This probably gives calixarene‐bonded stationary phases enhanced suitability for analyses at extreme compositions of the mobile phase. An overview about the synthesis, retention and selectivity properties of Caltrex^® columns is given here.     

Characterization of a highly stable zwitterionic hydrophilic interaction chromatography stationary phase based on hybrid organic–inorganic particles

We have characterized a sulfobetaine stationary phase based on 1.7 μm ethylene-bridged hybrid organic–inorganic particles, which is intended for use in hydrophilic interaction chromatography. The efficiency of a column packed with this material was determined as a function of flow rate, demonstrating a minimum reduced plate height of 2.4. The batch-to-batch reproducibility was assessed using the separation of a mixture of acids, bases, and neutrals. We compared the retention and selectivity of the hybrid sulfobetaine stationary phase to that of several benchmark materials. The hybrid sulfobetaine material gave strong retention for polar neutrals and high selectivity for methyl groups, hydroxy groups, and configurational isomers. Large differences in cation and anion retention were observed among the columns. We characterized the acid and base stability of the hybrid sulfobetaine stationary phase, using accelerated tests at pH 1.3 and 11.0, both at 70°C. The results support a recommended pH range of 2–10. We also investigated the performance of columns packed with this material for metal-sensitive analytes, comparing conventional stainless steel column hardware to hardware that incorporates hybrid surface technology to mitigate interactions with metal surfaces. Compared to the conventional columns, the hybrid surface technology columns showed a greatly improved peak shape.     

Synthesis and evaluation of silica hydride‐based fluorinated stationary phases

Two novel silica hydride‐based fluorinated bonded phases have been synthesized using a hydrosilation procedure to test combined fluorine and hydride selectivity. The bonded moieties were characterized by elemental and spectral analysis. Chromatographic testing was done using hydrophilic analytes in the aqueous normal phase mode. At higher amounts of the nonpolar solvent in the mobile phase, there should be increased retention for solutes such as acids, bases and other polar compounds, whereas nonpolar solutes can be retained when water is increased as in RP chromatography. The synergistic effects of the fluorinated phase selectivity and aqueous normal phase retention on a hydride surface have been explored for small polar molecules. The stability and repeatability of the hydride‐based fluorinated stationary phases were evaluated. The use of acetone as the organic component in the mobile phase was also tested.     

Effect of high-temperature on high-performance liquid chromatography column stability and performance under temperature-programmed conditions

Six commercially available analytical (4.1 or 4.6 mm i.d.) columns were evaluated under temperature-programmed high-temperature liquid chromatography (HTLC) conditions to access their stability and performance at extreme temperatures. Seven components consisting of acidic, basic and neutral compounds were analyzed under temperature-programmed conditions and solvent gradient conditions using three different mobile phase compositions (acidic, basic and neutral). Each column was checked with a two-component test mix at various stages of the evaluation to look for signs of stationary phase collapse. Three zirconia based stationary phases studied exhibited column bleed under temperature-programmed conditions. The other three columns, a polydentate silica column, a polystyrene-divinylbenzene (PS-DVB) polymeric column, and a graphitic carbon column performed well with no evidence of stationary phase degradation. The R.S.D. for the retention times and efficiencies were less than 10% for most conditions, and not more than 15% during the course of the evaluation for each column. The polydentate silica stationary phase was temperature programmed to 100 degrees C, the PS-DVB stationary phase was temperature programmed up to 150 degrees C, and the graphitic carbon column was used with temperature programming up to 200 degrees C. Comparable peak capacities and similar retention behaviors were observed under solvent gradient and temperature-programmed conditions. Temperature programming with dynamic mobile phase preheating can replace solvent gradient analysis without a loss of peak capacity when used with 4.1 or 4.6 mm columns.