Effect of chromatographic conditions on the separation and system efficiency for HPLC of selected alkaloids on different stationary phases

Retention parameters of alkaloid standards were determined on different stationary phases, i.e., octadecyl silica, base-deactivated octadecyl silica, cyanopropyl silica, preconditioned cyanopropyl silica, and pentafluorophenyl, using different aqueous eluant systems: acetonitrile-water mixtures; buffered aqueous mobile phases at pH 3 or 7.8; and aqueous eluants containing ion-pairing reagents (octane-1-sulfonic acid sodium salt and pentane-1-sulfonic acid sodium salt) or silanol blockers (tetrabutyl ammonium chloride and diethylamine). Improved peak symmetry and separation selectivity for basic solutes was observed when basic buffer, ion-pairing reagents, and, especially, silanol blockers as mobile phase additives were applied. The best separation selectivity and most symmetric peaks for the investigated alkaloids were obtained in systems containing diethylamine in the mobile phase. The influence of acetonitrile concentration and kind and concentration of ion-pairing reagents or silanol blockers on retention, peak symmetry, and system efficiency was also examined. The most efficient and selective systems were used for separation of the investigated alkaloids and analysis of Fumaria officinalis and Glaucium flavum plant extracts.     

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.     

Modeling of chromatographic retention of the selected antiarrhythmics and structurally related compounds in the hydrophilic interactions under the TLC and HPLC conditions

Abstract

High-performance liquid chromatography (HPLC) plays an important role in testing the pharmaceutically active compounds. In despite of the advantages of HPLC, thin-layer chromatography (TLC) retains its applicability to the different experimental tasks. The experimental conditions which allow hydrophilic interactions in the chromatographic system were tested in the HPLC and TLC systems for ivabradine, its related compounds, diltiazem and verapamil. Under the TLC conditions, retention behavior of the investigated compounds was tested on silica gel modified with cyanopropyl ligands as stationary phase and acetonitrile?+?methanol containing 25% v/v formic acid. Under the HPLC conditions, we used silica gel modified with cyanopropyl ligands as a column packing and the acetonitrile + 0.25% aqueous solution of formic acid as mobile phase. Retention behavior of the investigated analytes depending on the changing volume fractions of the mobile phase modifier was characterized both for TLC and HPLC data sets by the Soczewiński–Wachtmeister equation. Linear relationships were established between the retention coefficients characterizing the retention mechanism (R_M⁰/m, logk₀/m) and molecular properties of the investigated compounds. The Quantitative Structure Retention Relationship (QSRR) modeling was performed with the use of the stepwise multiple linear regression, in order to select molecular properties which influence retention.     

Effect of Chromatographic Conditions on Separation and System Efficiency in HPTLC of Selected Quinoline Standards on Cyanopropyl Stationary Phases

JPC – Journal of Planar Chromatography – Modern TLC - The retention behavior of thirteen quinolines on cyanopropyl stationary phase was studied using different mobile phases prepared...     

Dependence of the retention volumes of additional streaming current responses and of the column void volume on mobile phase composition

Summary The influence of the alcohol content of the mobile phase and water, acetic acid and aniline as mobile phase additives on the generation and shape of two additional changes of the streaming current, generated inside the liquid chromatography column by injection of any sample and recorded before the responses of retained solutes, was studied in a normal-phase system using silica gel as the stationary phase. The mobile phases were based on a n-heptane-1-propanol mixtures. Under the same conditions the relationships between the column interparticle volume, the column void volume and the total liquid volume in the column and the retention volumes of these two streaming current responses, having the form of chromatographic peaks, were studied. The column void volume was identified with the retention volume of n-octane. The total liquid volume in the column (column hold-up) was calculated from the weight loss of the column wetted with water at first and then dried in nitrogen stream. The retention volume of the first streaming current response equals the column interparticle volume disregarding the mobile phase composition. If the 95∶5 n-heptane-1-propanol mobile phase contains water up to 80% of its saturated concentration (up to 0.114% by vol.), the retention volume of the second response agrees with the total volume of the liquid in the silica gel column, with a precision better than 2%. At a higher relative water saturation the retention volume of the second response increases, while the column void volume decreases. Both changes are explained by the spontaneous formation of a highly polar stagnant liquid in the pores of the silica gel.     

Characterization of stationary phases in subcritical fluid chromatography by the solvation parameter model. I. Alkylsiloxane-bonded stationary phases

Varied types of alkylsiloxane-bonded and fluoroalkylsiloxane-bonded stationary phases, all commercially available, were investigated with subcritical fluid mobile phase. The effect of the alkyl chain length (from C4 to C18) and of the nature of the bonding (fluorodecylsiloxane, phenyl-C18 and polar-embedded-C18) on the chromatographic behaviour was investigated by the use of a linear solvation energy relationship (LSER), the solvation parameter model. A large set of test compounds provides precise and reliable information on the intermolecular interactions responsible for retention on these stationary phases used with a subcritical mobile phase. First of all, the results underline the close properties between subcritical fluid and organic liquid. The use of non aqueous mobile phases reduces the cavity energy and the mobile phase acidity generally encountered with aqueous liquid phases, allowing other interactions to take a part in retention. As expected, an increase in the alkyl chain length favours the dispersive interactions between the solutes and the stationary phases. Changes in basicity and acidity of the stationary phases are also related to the chain length, but, in this case, mobile phase adsorption onto the stationary phase is supposed to explain these behaviours. The addition of a phenyl group at the bottom of the C18 chain, near the silica, does not induce great modifications in the retentive properties. The fluorodecylsiloxane and the polar-embedded alkylsiloxane phases display very different properties, and can be complementary to the classical alkylsiloxane-bonded phases. In particular, the fluorinated phase does not favour the dispersive interactions, in comparison to hydrogenated stationary phases, when the basicity of the polar-embedded phase is obviously greater than the one of classical alkylsiloxane-bonded phases, due to the amide function. Finally, logk-logk curves plotted between the different phases illustrate the effect of the interaction properties on the retention of different classes of compounds.     

The mobile-phase effects on stationary-phase properties in capillary supercritical fluid chromatography

A significant uptake of supercritical mobile phases into immobilized stationary phases has been observed. The phase swelling effect has been evaluated hydrodynamically for SE-30, SE-54, and cyanopropyl silicone phases with respect to the mobile-phase density of supercritical butane, column temperature, and the presence of a polar retention moderator. Some kinetic and retention consequences of the swelling phenomena are discussed.     

Effect of chemically bonded stationary phases and mobile phase composition on beta-blockers retention in RP-HPLC

The effects of stationary and mobile phase on retention of 18 beta-adrenolytic drugs (beta-blockers) have been studied. Four 'deactivated surface' stationary phases (polar-embedded or end-capped) were examined. Special attention was drawn to the cholesterolic (SG-CHOL) and alkylamide (SG-AP) stationary phases, and their application for analysis of the compounds. The retention of analyzed substances was also examined in terms of mobile phase composition. Sixteen different configurations of mobile phases were prepared, all based on methanol and acetonitrile with ammonium acetate and ammonium formate. The difference in retention between ammonium formate and acetate water solutions, and peak shape changes related to the addition of triethylamine (TEA), were investigated. Principal component analysis was used to find the similarities between stationary phases. Polar-embedded phases synthesized on the same sorbent possess very similar properties. All phases based on silica gel compared with the monolithic column also showed similarities in retention of beta-blockers. The addition of TEA to the mobile phase did not influence strongly the retention, and analysis of asymmetry factors showed only a little peak broadening for a few compounds on the monolithic column.     

Insights into the Retention Mechanism of Small Neutral Compounds on Octylsiloxane-Bonded and Diisobutyloctadecylsiloxane-Bonded Silica Stationary Phases in Reversed-Phase Liquid Chromatography

The system constants of the solvation parameter model are used to prepare system maps for the retention of small neutral compounds on an octylsiloxane-bonded (Kinetex C8) and diisobutyloctadecylsiloxane-bonded (Kinetex XB-C18) superficially porous silica stationary phases for aqueous mobile phases containing 10–70% (v/v) methanol or acetonitrile. Electrostatic interactions (cation-exchange) are important for the retention of weak bases with acetonitrile–water but not for methanol–water mobile phases. Compared with an octadecylsiloxane-bonded silica stationary phase (Kinetex C18) retention is reduced due to a less favorable phase ratio for both the octylsiloxane-bonded and diisobutyloctadecylsiloxane-bonded silica stationary phases while selectivity differences are small and solvent dependent. Selectivity differences for neutral compounds are larger for methanol–water but significantly suppressed for acetonitrile–water mobile phases. The selectivity differences arise from small changes in all system constants with solute size and hydrogen-bond basicity being the most important due to their dominant contribution to the retention mechanism. Exchanging the octadecylsiloxane-bonded silica column for either the octylsiloxane-bonded or diisobutyloctadecylsiloxane-bonded silica column affords little scope for extending the selectivity space and is restricted to fine tuning of separations, and in some cases, to obtain faster separations due to a more favorable phase ratio. For weak bases larger differences in relative retention are expected with acetonitrile–water mobile phases on account of the additional cation exchange interactions possible that are absent for the octadecylsiloxane-bonded silica stationary phase.     

Characterisation of stationary phases in subcritical fluid chromatography with the solvation parameter model. III. Polar stationary phases

In this third paper, varied types of polar stationary phases, namely silica gel (SI), cyano (CN)- and amino-propyl (NH2)-bonded silica, propanediol-bonded silica (DIOL), poly(ethylene glycol) (PEG) and poly(vinyl alcohol) (PVA), were investigated in subcritical fluid mobile phase. This study was performed to provide a greater knowledge of the properties of these phases in SFC, and to allow a more rapid and efficient choice of polar stationary phase in regard of the chemical nature of the solutes to be separated. The effect of the nature of the stationary phase on interactions between solute and stationary phases and between solute and carbon dioxide-modifier mobile phases was studied by the use of a linear solvation energy relationship (LSER), the solvation parameter model. The retention behaviour observed with sub/supercritical fluid with carbon dioxide-methanol is close to the one reported in normal-phase liquid chromatography with hexane. The hydrogen bond acidity and basicity, and the polarity/polarizability favour the solute retention when the molar volume of the solute reduces it. As with non-polar phases, the absence of water in the subcritical fluid allows the solute/stationary phase interactions to play a greater part in the retention behaviour. As expected, the DIOL phase and the bare silica display a similar behaviour towards acidic and basic solutes, when interactions with basic compounds are lower with the NH2 phase. On the CN phase, all interactions (hydrogen bonding, dipole-dipole and charge transfer) have a nearly equivalent weight on the retention. The polymeric phases, PEG and PVA, provide the most accurate models, possibly due to their better surface homogeneity.     

Optimization of chromatographic systems for the high-performance thin-layer chromatography of flavonoids

Summary To characterize the retention and selectivity of separations of 23 flavonoids (aglycones and glycosides) relationships betweenR _F and modifier concentration were determined for silica and diol adsorbents (with mixtures of ethyl acetate and methanol as mobile phases), for cyanopropyl silica (with mixtures of ethyl acetate and dichloromethane as mobile phases), for aminopropyl silica (with mixtures of ethyl acetate, methanol and water as mobile phases) and for octadecyl silica (with mixtures of methanol and water as mobile phases). Owing to large polarity differences between aglycones and glycosides, these groups of compounds cannot be separated other than by use of reversed-phase systems, for which the selectivity is lower. It follows from correlation plots ofR _F1 againstR _F2 that for some pairs of adsorbents (e. g. silica and diol) selectivity differences are small; for others the points in the plot are widely dispersed, indicating selectivity differences. The chemometric database obtained can be used to choose optimum chromatographic systems for the separation of given sets of flavonoids and for planning gradient elution programs for separation of flavonoid aglycones and glycosides in a single TLC experiment.     

Stationary and mobile phases in hydrophilic interaction chromatography: a review

Hydrophilic interaction chromatography (HILIC) is valuable alternative to reversed-phase liquid chromatography separations of polar, weakly acidic or basic samples. In principle, this separation mode can be characterized as normal-phase chromatography on polar columns in aqueous-organic mobile phases rich in organic solvents (usually acetonitrile). Highly organic HILIC mobile phases usually enhance ionization in the electrospray ion source of a mass spectrometer, in comparison to mobile phases with higher concentrations of water generally used in reversed-phase (RP) LC separations of polar or ionic compounds, which is another reason for increasing popularity of this technique. Various columns can be used in the HILIC mode for separations of peptides, proteins, oligosaccharides, drugs, metabolites and various natural compounds: bare silica gel, silica-based amino-, amido-, cyano-, carbamate-, diol-, polyol-, zwitterionic sulfobetaine, or poly(2-sulphoethyl aspartamide) and other polar stationary phases chemically bonded on silica gel support, but also ion exchangers or zwitterionic materials showing combined HILIC-ion interaction retention mechanism. Some stationary phases are designed to enhance the mixed-mode retention character. Many polar columns show some contributions of reversed phase (hydrophobic) separation mechanism, depending on the composition of the mobile phase, which can be tuned to suit specific separation problems. Because the separation selectivity in the HILIC mode is complementary to that in reversed-phase and other modes, combinations of the HILIC, RP and other systems are attractive for two-dimensional applications. This review deals with recent advances in the development of HILIC phase separation systems with special attention to the properties of stationary phases. The effects of the mobile phase, of sample structure and of temperature on separation are addressed, too.     

Study of the elution mechanism of sodium aryl sulfonates on bare silica and a cyano bonded phase with methanol-modified carbon dioxide containing an ionic additive

The elution mechanism of sodium sulfonates on both Deltabond cyanopropyl and bare silica stationary phases with an isocratic mobile phase composed of methanol-modified CO2 wherein an ammonium salt additive was dissolved in the methanol has been studied. The presence of the additive was crucial concerning elution of the sulfonate salts. Solid state 29silicon nuclear magnetic resonance spectroscopy provided some insight concerning the interaction of the mobile phase additive with the silica-based stationary phase. Computational calculations concerning the charge distribution on various ammonium salts were performed in an effort to explain the elution behavior. Ammonium ions are believed to deactivate available silanol sites on both phases. In addition, ammonium ion is speculated to interact with the cyano groups on the bonded phase. For concentrations of additive greater than 2 mM, stationary phase coverage of ammonium ion is anticipated to exceed one monolayer for both bare and bonded silica. The acetate counter-ion is thought to facilitate elution of the anionic sulfonates from the positively charged stationary phase in a pseudo ion exchange mechanism.     

Insights into the Retention Mechanism on a Pentafluorophenylpropylsiloxane-Bonded Silica Stationary Phase (Discovery HS F5) in RP-LC

The solvation parameter model is used to elucidate the retention mechanism of neutral compounds on the pentafluorophenylpropylsiloxane-bonded silica stationary phase (Discovery HS F5) with methanol-water and acetonitrile-water mobile phases containing from 10 to 70% (v/v) organic solvent. The dominant factors that increase retention are solute size and electron lone pair interactions while polar interactions reduce retention. A comparison of the retention mechanism with an octadecylsiloxane-bonded silica stationary phase based on the same silica substrate and with a similar bonding density (Discovery HS C18) provides additional insights into selectivity differences for the two types of stationary phase. The methanol-water solvated pentafluorophenylpropylsiloxane-bonded silica stationary phase is more cohesive and/or has weaker dispersion interactions and is more dipolar/polarizable than the octadecylsiloxane-bonded silica stationary phase. Differences in hydrogen-bonding interactions contribute little to relative retention differences. For mobile phases containing more than 30% (v/v) acetonitrile selectivity differences for the pentafluorophenylpropylsiloxane-bonded and octadecylsiloxane-bonded silica stationary phases are no more than modest with differences in hydrogen-bond acidity of greater importance than observed for methanol-water. Below 30% (v/v) acetonitrile selectivity differences are more marked owing to incomplete wetting of the octadecylsiloxane-bonded silica stationary phase at low volume fractions of acetonitrile that are not apparent for the pentafluorophenylpropylsiloxane-bonded silica stationary phase. Steric repulsion affects a wider range of compounds on the octadecylsiloxane-bonded than pentafluorophenylpropylsiloxane-bonded silica stationary phase with methanol mobile phases resulting in additional selectivity differences than predicted by the solvation parameter model. Electrostatic interactions with weak bases were unimportant for methanol-water mobile phase compositions in contrast to acetonitrile-water where ion-exchange behavior is enhanced, especially for the pentafluorophenylpropylsiloxane-bonded silica stationary phase. The above results are compatible with a phenomenological interpretation of stationary phase conformations using the haystack, surface accessibility, and hydro-linked proton conduit models.     

Column selectivity in reversed-phase liquid chromatography. VII. Cyanopropyl columns

Eleven cyanopropyl ("cyano") columns were characterized by means of a relationship developed originally for alkyl-silica columns. Compared to type-B alkyl-silica columns (i.e., made from pure silica), cyano columns are much less hydrophobic (smaller H), less sterically restricted (smaller S*), and have lower hydrogen-bond acidity (smaller A). Because sample retention is generally much weaker on cyano versus other columns (e.g., C8, C18), a change to a cyano column usually requires a significantly weaker mobile phase in order to maintain comparable values of k for both columns. For this reason, practical comparisons of selectivity between cyano and other columns (i.e., involving different mobile phases for each column) must take into account possible changes in separation due to the change in mobile phase, as well as change in the column.     

Development and evaluation of new zwitterionic hydrophilic interaction liquid chromatography stationary phases based on 3-P,P-diphenylphosphonium-propylsulfonate

New zwitterionic stationary phases were synthesized by covalently bonding 3-P,P-diphenylphosphonium-propylsulfonate to silica gel. The resulting materials possess both a negatively charged sulfonate group and a positively charged quaternary phosphonium group, which means that there is no net charge over a wide pH range. The retention mechanism and chromatographic behavior of polar solutes under HILIC conditions were studied on these zwitterionic phases. Compared to the commercial ZIC-HILIC column and a bare silica gel stationary phase, the newly synthesized zwitterionic stationary phases provided greater retention, higher peak efficiency and better peak symmetry in the HILIC mode. The analytes examined included: β-blockers, nucleic acid bases and nucleosides, salicylic acid and its analogues, and water soluble vitamins. Factors, such as the type of organic modifiers, solvent composition, pH and the buffer concentration of the mobile phase, have been considered as potential variables for controlling the chromatographic retention of polar analytes.     

On the polarity of stationary phases in straight-phase LC

Summary The LC retention strength of non-bonded silica gel is compared with that of seventeen derivatized silica gels and polystyrenes in straight-phase conditions with hexane-dioxan and hexane-tetrahydrofuran mobile phases. For this purpose a polarity mixture containing UV-active polar compounds is used. Larger retention with dioxan containing eluents is observed. This is an indication that non-displacement H-bond and dipole interactions with adsorbed dioxan contribute to the retention of polar solutes. The linear correlation between log k′ and lox Xs was verified for all phases.     

The use of derivatized cyclodextrins as solubilizing agents in the preparation of macroporous polymers employed as amphiphilic continuous beds in capillary electrochromatography

Employing solubilization by complexation with CDs, new mixed-mode monolithic stationary phases for CEC and micro-LC were synthesized. Free radical copolymerization was performed in aqueous solution with a CD-solubilized hydrophobic monomer, a water-soluble crosslinker (piperazinediacrylamide), and a charged monomer (vinylsulfonic acid). Different hydrophobic methacrylate monomers (isobornyl, adamantyl, cyclohexyl, and phenyl methacrylate) were investigated. Chromatographic properties of the synthesized monoliths were studied with aqueous and nonaqueous mobile phases with hydrophobic and polar analytes. Due to the amphiphilic nature of the polymers synthesized, the elution orders obtained correspond to the RP mode and to the normal-phase mode dependent on the polarity of the mobile phase. However, observations made with polar solutes and polar mobile phase can only be explained by a mixed-mode retention mechanism. The influence of the total monomer concentration (%T) on the chromatographic properties and on the specific permeability was elucidated. Run-to-run, day-to-day, and capillary-to-capillary reproducibility of electroosmotic mobility and retention factors were determined. Comparison of retention data with those of a commercial octadecyl silica gel HPLC column reveals that the methylene selectivity of the monolithic capillaries prepared in this study is very similar to that of routinely used octadecyl silica gels.     

Effect of the mobile phase on the retention behaviour of optical isomers of carboxylic acids and amino acids in liquid chromatography on bonded Teicoplanin columns

Conditions for separation of enantiomers of underivatized amino acids phenyl glycine and tryptophan and of mandelic acid as test compounds were studied on a Chirobiotic T column packed with amphoteric glycopeptide Teicoplanin covalently bonded to the surface of silica gel. The effects of the mobile phase composition on the retention and selectivity under analytical conditions, on the profile of the adsorption isotherms of the enantiomers and on the overloaded separation were investigated. The concentration of ethanol or of methanol in aqueous-organic mobile phases and the pH of the mobile phase affect not only the retention and selectivity, the saturation capacity and the isotherm profile, but also the solubility of the acids, which should be taken into account in development of preparative separations. A compromise between the separation selectivity and the solubility should be made in selecting the mobile phase suitable to accomplish preparative separations at acceptable production rate and throughput of the operation.