A simple subcritical chromatographic test for an extended ODS high performance liquid chromatography column classification

This paper describes a new test designed in subcritical fluid chromatography (SFC) to compare the commercial C18 stationary phase properties. This test provides, from a single analysis of carotenoid pigments, the absolute hydrophobicity, the silanol activity and the steric separation factor of the ODS stationary phases. Both the choice of the analytical conditions and the validation of the information obtained from the chromatographic measurements are detailed. Correlations of the carotenoid test results with results obtained from other tests (Tanaka, Engelhard, Sander and Wise) performed both in SFC and HPLC are discussed. Two separation factors, calculated from the retention of carotenoid pigments used as probe, allowed to draw a first classification diagram. Columns, which present identical chromatographic behaviors are located in the same area on this diagram. This location can be related to the stationary phase properties: endcapping treatments, bonding density, linkage functionality, specific area or silica pore diameter. From the first classification, eight groups of columns are distinguished. One group of polymer coated silica, three groups of polymeric octadecyl phases, depending on the pore size and the endcapping treatment, and four groups of monomeric stationary phases. An additional classification of the four monomeric groups allows the comparison of these stationary phases inside each group by using the total hydrophobicity. One hundred and twenty-nine columns were analysed by this simple and rapid test, which allows a comparison of columns with the aim of helping along their choice in HPLC.     

Characterization of chromatographic supports for the analysis of basic compounds

Reversed‐phase liquid chromatography (RP‐HPLC) has become a powerful and widely employed technique for the analysis of a great variety of substances and, in particular, of basic compounds. These compounds are present in various areas. In pharmacy, 80% of drugs are estimated to possess a basic function. Basic compounds can strongly interact with free silanol groups on the surface of the silica particles. These ion exchange interactions produce peak tailing which affects resolution, sensitivity, and reproducibility. For these reasons, many new stationary phases have been designed to reduce access to silanol groups. The main problem facing the analyst is to effectively select the best column for a particular type of separation. In order to characterize and evaluate the properties of these packings, several tests are proposed in the literature, which can be divided into two main categories: general tests and particular tests. In this work, a particular test was developed for the characterization of base deactivated RP‐HPLC stationary phases. For this purpose, a set of 14 basic test substances was selected and five different chromatographic supports were tested with three isocratic mobile phases. Furthermore, in order to undertake a complete characterization of these supports, batch and column reproducibility were also studied. Principal Component Analysis was applied to evaluate both the performance of the test compounds and of the stationary phases.     

Optimisation and characterisation of silica-based reversed-phase liquid chromatographic systems for the analysis of basic pharmaceuticals

Reversed-phase liquid chromatography using silica-based columns is successfully applied in many separations. However, also some drawbacks exist, i.e. the analysis of basic compounds is often hampered by ionic interaction of the basic analytes with residual silanols present on the silica surface, which results in asymmetrical peaks and irreproducible retention. In this review, options to optimise the LC analysis of basic pharmaceutical compounds are discussed, i.e. eluent optimisation (pH, silanol blockers) and stationary phase optimisation (development of new columns with minimised ionic interactions). The applicability of empirical based, thermodynamically based and test methods based on a retention model to characterise silica-based reversed phase stationary phases, as well as the influence of the eluent composition on the LC analysis of basic substances is described. Finally, the applicability of chemometrical techniques in column classification is shown.     

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.     

Coating and fusing cell membranes onto a silica surface and their chromatographic characteristics

Summary A new cell membrane stationary phase (CMSP) consisting of porous silica coated with active cell membranes is presented for affinity chromatography. By immersing, silica into a suspension of cell membranes, the whole surface of silica was covered by the cell membranes due to the irreversible adsorption of silanol groups (Si−OH) on the silica surface and the self-fusion of the cell membranes. CMSP can be used directly as a chromatographic packing material without any additional chemical modification. The surface characteristics, enzymatic activity, and chromatographic behavior of CMSP were investigated. The results obtained from scanning electron microscope, surface energy spectrometer, enzyme assay, and liquid chromatography showed that the surface characteristics of CMSP were very different from that of normal and reversed stationary phases. CMSP was found to have the characteristics of both cell membrane activity and chromatographic separation. Moreover, CMSP, as a chiral stationary phase, could be used for the enantiomeric separation of (±) Bay-K8644. The capacity factor of some calcium antagonists on CMSP was found to have a good correlation with their pharmacological actions. It is concluded that CMSP may be used not only as a kind of packing material in bio-affinity chromatography, but also as a tool for studying the interactions between a drug and its receptor.     

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.     

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.     

Evaluation of silanol-deactivated silica-based reversed phases for liquid chromatography of erythromycin

The suitability of eleven silanol-deactivated reversed phases for the liquid chromatography of erythromycin was investigated. The selectivity and efficiency of each stationary phase were examined. The performance was compared to that of a non-deactivated C₁₈ silica-based reversed-phase material, Hypersil C₁₈ (5 μm). Two types of mobile phases were used, one containing no tetrabutylammonium (TBA) and the other containing TBA. Addition of TBA as a silanol-blocking agent improved the theoretical plate number and symmetry factor of the peaks corresponding to erythromycin A (EA) and erythromycin A enol ether for all the deactivated reversed phases. These results are an indication of the presence of some residual silanol activity in these phases. Separation of erythromycin E and EA was achieved on only two of the eleven phases. The selectivity was always poorer than that obtained in a previously described method using a poly(styrene-divinylbenzene) stationary phase.     

Retention mechanism of the multifunctional solute on columns with different coverage densities using highly aqueous reversed‐phase conditions

A series of 11 homemade octadecyl bonded phases with different coverage densities were tested to determine the influence of the stationary phase on the retention in highly aqueous mobile phases. The concentrations of the organic modifiers (methanol and ACN) were in the range of 0–20%_v/v. The coverage density of bonded ligands and the presence of the end‐capping have strong influence on the solute retention. Amoxicillin (AMO) was chosen as the test compound. Dual properties of AMO, which contain hydrophobic skeleton and polar groups (amino, hydroxyl and carbonyl), cause irregular changes of the retention over the stationary phase hydrophobicity and silanol activity at given mobile phase composition. Presented data show that application of non‐standard low coverage density C18 phases allow to determine AMO in the RPLC condition with high retention.     

The effect of stationary phase on lipophilicity determination of beta-blockers using reverse-phase chromatographic systems

Evaluation of lipophilicity parameters for basic compounds using different chromatographic stationary phases is presented. An HPLC method for determination of lipophilic molecule-stationary phase interactions was based on gradient analysis. Differences in correlation between the lipophilicity of compounds and experimental chromatographic results obtained in pseudo-membrane systems showed a strong influence of stationary phase structure and physico-chemical properties. beta-Blocker drugs with varying lipophilicity and bio-activity were chosen as test compounds. The stationary phases used for the study were monolithic rod-structure C18 and silica gel octadecyl phase SG-C18 as reference material. The second group was silica gel-based polar-embedded alkylamide and cholesterolic phases. The mobile phase was composed of acetonitrile or methanol with ammonium acetate, and a linear gradient of methanol and acetonitrile in mobile phase was performed. A linear correlation of plots of log k(g) = f(log P) was observed, especially for polar-embedded phases, and this allowed log P(HPLC) to be calculated. The behavior of stationary phases in methanol and acetonitrile buffer showed differences between obtained log P(HPLC) values.     

Correlations between conformational isomerism and chromatographic diastereoselectivity of bis amino amide s-triazine derivatives

NMR spectroscopy was used to probe the conformational behavior of diastereomeric s-triazine derivatives containing two chiral amino amide substituents, in order to shed light onto the mechanism of chromatographic diastereoselectivity. Utilizing the amino hydrogen signals in the proton NMR spectrum, the population of the conformations caused by rotation about the bond between the amino nitrogen and aromatic carbon atoms could be observed. The population distribution between the three possible conformations was similar but not identical between the two diastereomers, with similar trends being observed for both bis alanine amide and bis valine amide derivatives. Based on a simple model in which it is assumed that adsorption to the hydrophobic RP-LC stationary phase occurs only for the conformations having both amino amide R-groups on the same side of the triazine ring plane, the different conformation populations between the two diastereomers obtained by NMR was consistent with the observed RP-LC elution order (L-L diastereomer followed by L-D). The predicted diastereoselectivity values from NMR data were compared to RP-LC diastereoselectivity values obtained using both C18 and polymeric columns, with both acetonitrile/water and DMSO/water mobile phases. Values obtained with the polymeric column were in better agreement with calculated values than those obtained with the C18 column, suggesting that the simple adsorption model used to calculate the diastereoselectivity is more relevant towards a simple hydrophobic polymeric surface rather than a more complex C18 stationary phase. This study indicates that proton NMR is a useful tool for studying the diastereoselective mechanism of these derivatives, due to the relatively slow C-N bond rotation caused by the significant sp(2) character of the amino nitrogen atoms.     

Effect of ionic liquid additives to mobile phase on separation and system efficiency for HPLC of selected alkaloids on different stationary phases

The silica-based stationary phases with favorable physical characteristics are the most popular in liquid chromatography. However, there are several problems with silica-based materials: severe peak tailing in the chromatography of basic compounds, non-reproducibility for the same chemistry columns, and limited pH stability. Ionic liquids (ILs) as mobile phase components can reduce peak tailing by masking residual free silanol groups. The chromatographic behavior of some alkaloids from different classes was studied on C18, phenyl, and pentafluorophenyl columns with different kinds and concentrations of ionic liquids as additives to aqueous mobile phases. Ionic liquids with different alkyl substituents on different cations or with different counterions as eluent additives were investigated. The addition of ionic liquids has great effects on the separation of alkaloids: decrease in band tailing, increase in system efficiency, and improved resolution. The retention, separation selectivity, and sequence of alkaloid elution were different when using eluents containing various ILs. The increase of IL concentration caused an increase in silanol blocking, thus conducted to decrease the interaction between alkaloid cations and free silanol groups, and caused a decrease of alkaloids retention, improvement of peak symmetry, and increase of theoretical plate number in most cases. The effect of ILs on stationary phases with different properties was also examined. The different properties of stationary phases resulted in differences in analyte retention, separation selectivity, peak shape, and system efficiency. The best shape of peaks and the highest theoretical plate number for most investigated alkaloids in mobile phases containing IL was obtained on pentafluorophenyl (PFP) phase.     

Correlation between the chromatographic poperties of silica phenyl packing materials and the retention behaviour of methylated β-cyclodextrins

Summary The chromatographic properties of five columns packed with phenyl-bonded phases were characterized by an approach based on Tanaka's method for column to column comparisons in order to develop an LC analysis of partially methylated β-cyclodextrins. The retention behaviour of β-cyclodextrin, heptakis(2,6-di-O-methyl)-β-cyclodextrin and heptakis(2,3,6-tri-O-methyl)-β-cyclodextrin was examined in order to evaluate the different interactions between phenyl stationary phase and this family of compound. Chromatograms show that the residual silanol activity of the packing materials plays a beneficial role in the partially methylated β-cyclodextrins separation process. It is concluded that knowledge of the interactions involved allows one to make a reasoned choice of the stationary phase in order to obtain the best possible analysis of three different commercial samples of partially methylated β-cyclodextrins.     

Characterization of a mixed-mode reversed-phase/cation-exchange stationary phase prepared by thermal immobilization of poly(dimethylsiloxane) onto the surface of silica

A novel stationary phase prepared by the thermal immobilization of poly(dimethylsiloxane) onto the surface of silica (PDMS–SiO₂) has been described, evaluated and compared with 229 commercially available RP-LC stationary phases using the Tanaka column classification protocol. The phase exhibited many unique chromatographic properties and, based on the phases in the database, was most similar to the fluoroalkylated phases (aside from the obvious lack of fluoro selectivity imposed by the C–F dipole). The phase exhibited classic reversed-phase behaviour in acid mobile phase conditions and mixed-mode reversed-phase/cation-exchange retention behaviour in neutral mobile phase conditions. The phase exhibited acceptable stability at both low and intermediate pH, conditions which should impart optimum chromatographic selectivity to the phase. Retention of basic analytes was shown to occur by a “three site model” as proposed by Neue. This new PDMS–SiO₂ stationary phase is extremely interesting in that the dominancy of its hydrophobic and ion-exchange interactions can be controlled by the influence of mobile phase pH, buffer type and concentration. The PDMS–SiO₂ stationary phase may provide a complementary tool to reversed-phase and HILIC stationary phases. The present results highlight the fact that the type of buffer, its concentration and pH can not only affect peak shape but also retention, selectivity and hence chromatographic resolution. Therefore, in method development and optimization strategies it is suggested that more emphasis should be given to the evaluation of these mobile phase operating parameters especially when basic solutes are involved.     

Polar-copolymerized approach based on horizontal polymerization on silica surface for preparation of polar-modified stationary phases

A new approach for preparation of polar-modified reversed-phase liquid chromatography stationary phases was developed by using horizontal polymerization technique on silica surface, which was defined as “polar-copolymerized” approach. Based on this new approach, a representative polar-copolymerized stationary phase composed of mixed n-octadecyl and chloropropyl (C18–C3Cl) was synthesized. The resulting stationary phase named C18HCE was characterized with elemental analysis and solid phase ¹³C and ²⁹Si NMR, which proved the chemistry of polar-copolymerized stationary phases. Chromatographic evaluation and application of the C18HCE were also investigated. The results of preliminary chromatographic evaluation demonstrated that the C18HCE stationary phase exhibited 100% aqueous mobile phase compatibility, low silanol activity. In addition, the application results demonstrated that the C18HCE had superior separation performance in alkaloids separation at acidic conditions compared to some commercial stationary phases.