Effect of mobile phase composition on the retention of selected alkaloids in reversed-phase liquid chromatography with chaotropic salts

Sodium hexafluorophosphate, perchlorate and trifluoroacetate were applied as ion-ion interaction reagents in reversed-phase liquid chromatography. The separation of chosen alkaloids was performed by changing the kind of the organic modifier (methanol, acetonitrile, tetrahydrofuran), concentration of the ion-ion-interaction reagents and the concentration of phosphate buffer at constant pH (2.7) in the mobile phase. Obtained results were analyzed in connection to a dynamic ion-exchange model of retention and ion-ion interaction effects. The perturbation method was applied to test proposed retention theories. The formation of ion-complexes controlling the retention in chaotropic systems was confirmed. On the basis of the relationships of capacity factors (k) versus salt concentrations derived experimentally, absolute increases in capacity factors, the desolvation parameters and the limiting retention factors were calculated and compared for all the investigated compounds in eluent systems studied. The selectivity of the proposed mobile phases was compared on the basis of the separation of alkaloid mixture.     

Comparison of chaotropic salt and ionic liquid as mobile phase additives in reversed-phase high-performance liquid chromatography of biogenic amines

Highly hydrophilic compounds belonging to biogenic amines were analysed in the reversed-phase system, modified with the addition of ionic liquids: 1-ethyl-3-methyl-imidazolium hexafluorophosphate (EMIM PF(6)) and chaotropic salt NaPF(6) on Discovery HS C18 column at acidic conditions. The effect of the additives concentration and the presence of organic solvent on the analytes' chromatographic parameters such as retention factor, tailing factor and theoretical plates number were all determined. On the basis of k versus ionic liquid concentration in aqueous-organic mobile phase with constant amount of phosphate buffer, retention mechanism was studied. It was established that the presence of organic solvent with low dielectric constant and ionic liquid with both chaotropic ions allows achieving typical Langmuir shape of this relationship. Investigated mobile phase additives are comparable according to efficiency and selectivity towards biogenic amines analysis. However, the sensitivity was found to be better for the eluent system modified with chaotropic salt.     

Regioselective separation of isomeric triacylglycerols by reversed-phase high-performance liquid chromatography: stationary phase and mobile phase effects

Complete regioselective separation of five pairs of isomeric dipalmitoyl polyalkenoyl glycerols with two to six double bonds in the unsaturated acyl residues has been achieved by RP-HPLC on a single ODS column. Four ODS columns with stationary phases containing different percentages of free silanol groups have been tested. Binary mobile phases of ACN admixed with dichloromethane, tetrahydrofuran, 1-propanol, 2-propanol, ethanol, or acetone have been examined. The choice of modifier depended on the nature of the stationary phase. The more polar solvents were better suited for stationary phases with higher percentage of free silanol groups. Isomeric species were eluted according to chain length, number of double bonds, and the position of the unsaturated acyl chain in the glycerol molecule. Retention increases in the order 20:5 < 22:6 < 18:3 < 20:4 < 18:2. Within each isomeric pair, the species with unsaturated acyl chain occupying either the sn-1- or the 3-position were retained preferentially. Complete simultaneous regioselective separation of 10 isomeric triacylglycerols in a single isocratic run on a single ODS column was demonstrated.     

Isocratic reversed-phase high-performance liquid chromatographic separation of tetracyclines and flumequine controlled by a chaotropic effect

Fast isocratic reversed-phase high-performance liquid chromatographic (RP-HPLC) separation of four tetracyclines and flumequine was obtained using a Zorbax SB-C18 column. Baseline resolution was achieved in 11 min. The peaks were narrow, well separated and without any tails although there was no chelating agents added to the mobile phase. Due to the chaotropic effect, the addition of potassium perchlorate allowed controlling the tetracyclines retention while the retention of flumequine was almost constant.     

Isolation of teratogenic alkaloids by reversed-phase high-performance liquid chromatography

Reversed-phase high-performance liquid chromatography was used for both analytical and preparative separations of several steroidal alkaloids which occur in extracts of Veratrum californicum. The inclusion of 0.1% trifluoroacetic acid in the mobile phase improved the efficiency of the chromatography and the solubility of the compounds in aqueous acetonitrile. Nuclear magnetic resonance was used to assist the identification of the isolated steroidal alkaloids. The effect of the interaction of trifluoroacetic acid with the alkaloids could be clearly seen by changes in the chemical shifts in the nuclear magnetic resonance spectra.     

The separation of nonapeptides by reversed-phase high-performance liquid chromatography.

The separation properties of five nonapeptides on commercial reversed-phase materials have been investigated and the effects of pH, salt concentration and solvent composition have been studied. With appropriate variation of the pH and salt concentration in the mobile phase, it is possible to resolve all of the peptides investigated and their by-products. Mixtures of water and organic solvents (acetonitrile, dioxan, methanol and n-propanol) have been used. The choice of the organic solvent does not strongly influence the separation pattern. The simplicity, speed and quality of the separations and the favourable detection limits (ca. 30 ng) at 220 nm render this technique suitable to routine quantitative analysis.     

Polysiloxane-encapsulated stationary phase for reversed-phase high-performance liquid chromatography

Summary Silica beads of 6-μm average diameter were silanized with methylvinyldiethoxysilane and then subjected to encapsulation with poly(methylvinylsiloxane). The resulting product is a new stationary phase for reversed-phase high performance liquid chromatography (RP-HPLC) which has superior ability for the separation of polar, non-polar and basic compounds. The chromatographic peaks are symmetric. Its stability has been studied; after continuous use for three months the carbon content and chromatographic behaviour of the phase were unchanged. on to the silica surface to given an uniform organic film. Material prepared in this way has both good chromatographic behaviour and superior selectivity. Because contact of the silica matrix with the mobile phase is avoided, the alkali-resisting ability of the stationary phase is increased. The non-specific adsorption of alkaline solutes on to the silica surface is also avoided because of the complete coverage of surface silanol groups. Reports of stationary phases encapsulated with polystyrene [6], polybutadiene [I] and octadecylsiloxane polymers have recently appeared in the literature [3]. In this paper we report the encapsulation of poly-(methylvinylsiloxane) (analogous to the phase SE-31 often used in GC) on to a silica matrix previously modified with methylvinyldiethoxysilane. The resulting phase has superior performance in reversed-phase HPLC.     

Determination of the five major opium alkaloids by reversed-phase high-performance liquid chromatography on a base-deactivated stationary phase

Summary A reversed phase HPLC method for the separation of the five major alkaloids fromPapaver somniferum L., morphine, codeine, thebaine, papaverine and noscapine, has been developed and validated. By use of a basedeactivated silica-based stationary phase excellent peak shape was achieved for each substance. The five alkaloids were quantified by internal standardization within 20 min and with good precision. The method is applicable to opium and to poppy straw.     

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.     

Stereoselective determination of p-hydroxyphenyl-phenylhydantoin enantiomers in rat liver microsomal incubates by reversed-phase high-performance liquid chromatography using beta-cyclodextrin as chiral mobile phase additives

An analytical method was developed for determination of p-hydroxyphenylphenylhydantoin enantiomers in rat liver microsome by using reversed-phase high-performance liquid chromatography. A 50 mm C(8) column was used as the analytical column. The mobile phase was made up of 8.8 mmol/L beta-cyclodextrin, 0.25 mol/L urea and 0.05 mol/L ammonium acetate in water. The assay was linear from 2.05 to 410.0 micromol/L for each enantiomer. The limits of detection and of quantitation for the method were 0.90 and 2.05 micromol/L for each enantiomer, respectively. The analytical method afforded average recoveries of 93.59 +/- 2.75% and 94.72 +/- 1.78% for S- and R-p-hydroxyphenylphenylhydantoin, respectively. The method allowed study of the in vitro glucuronidation of p-hydroxyphenylphenylhydantoin in rat liver microsomal incubates. The stereoselectivity of p-hydroxyphenylphenylhydantoin phase II metabolism was observed.     

Preparation and separation of metallobacteriochlorophylla by reversed-phase high-performance liquid chromatography

Summary Semi-preparative high-performance liquid chromatography has been used for the preparation of copper(II) bacteriochlorophylla [Cu(II)-BChl-a] and zinc(II) bacteriochlorophylla [Zn(II)-BChl-a]. Both compounds are separated on a reversed-phase Inertsil ODS-2 column using a mobile phase of acetone-methanol (2575, v/v). The fractionated metallobacteriochlorophylls (M-Bchl-a) are identified by fast atom bombardment mass spectrometry. The spectroscopic parameters such as the wavelength of absorption maxima and the molar extinction coefficients are determined using pure M-Bchl-a obtained by preparative HPLC. The HPLC method proposed here has been demonstrated to be useful for the purification and determination of components of M-Bchl-a.     

Microwave-immobilized polybutadiene stationary phase for reversed-phase high-performance liquid chromatography

Polybutadiene (PBD) has been immobilized on high-performance liquid chromatography (HPLC) silica by microwave radiation at various power levels (52-663 W) and actuation times (3-60 min). Columns prepared from these reversed-phase HPLC materials, as well as from similar non-irradiated materials, were tested with standard sample mixtures and characterized by elemental analysis (%C) and infrared spectroscopy. A microwave irradiation of 20 min at 663 W gives a layer of immobilized PBD that presented good performance. Longer irradiation times give thicker immobilized layers having less favorable chromatographic properties.     

Determination of lipophilicity by reversed-phase high-performance liquid chromatography. Influence of 1-octanol in the mobile phase

Lipophilicity was evaluated using a novel RP-HPLC stationary phase (Discovery-RP-Amide-C16) with and without 1-octanol added to the mobile phase. A set of 46 drugs and flavonoids characterized by a broad structural diversity and a wide log Poct range (-0.69 to 5.70) was selected for this study. This set consists of neutral solutes and solutes with acidic or ampholytic functionalities which were maintained neutral at pH 2.5 or 4. In our conditions, the addition of 1-octanol in the mobile phase proved a key factor to derive a lipophilicity index log k(w) highly correlated with log Poct for all investigated solutes. 1-Octanol improved the correlation between log Poct and log k(w) mainly by influencing the retention behavior of the solutes with log Poct values below +3. This study brings additional evidence that under proper experimental conditions of stationary and mobile phases, RP-HPLC is a very useful method to obtain log Poct values.     

Influence of the presence of methyl cyclodextrins in high-performance liquid chromatography mobile phases on the separation of beta-carboline alkaloids

The presence of cyclodextrins (CDs) in the mobile phase alters the chromatographic equilibria and induces a secondary chemical equilibrium associated to the chromatographic separation by HPLC. In this study the influence of the presence of CDs in the mobile phase as chemically modified beta-CDs, i.e. 2,3-di-O-methyl-beta-cyclodextrin (DMbeta-CD) and 2,3,6-tri-O-methyl-beta-cyclodextrin (TMbeta-CD) on the separation of the alkaloids norharmane, harmane and harmine is described. These beta-carboline alkaloids are chemically and structurally related and their quantitation by RP-HPLC is of interest due to their biological and pharmacological properties. Two stationary phases (methyl-, C(1) and octadecyl-, C(18)) were employed, with methanol:buffered aqueous solution and ethanol:buffered aqueous solution as mobile phases. The role of tert-butyl alcohol as a mobile phase modifier and also as an inclusion complex stabiliser was also considered. The concentrations of DMbeta-CD and TMbeta-CD vary from 0 to 17 mM. The presence of increasing amounts of CDs in the mobile phase reduces the retention factor. The changes observed in the retention factor allow the determination of the alkaloid/CD apparent association constants, whose magnitude is influenced by the chemical and structural properties of the guest molecules but also by the composition of the mobile phase. Assuming a 1:1 stoichiometry for the inclusion complexes, the apparent association constants obtained were higher for norharmane and for both DMbeta-CD and TMbeta-CD. The strength of the complexes is higher for DMbeta-CD than for TMbeta-CD and this behaviour can be explained considering the steric problems associated to the permethylated-beta-CDs. Besides significant differences in the magnitude of the apparent association constants were observed for the two stationary phases employed and thus can be related to the adsorption of CDs on the stationary phase. A significant reduction in the proportion of organic solvent in the mobile phase (50%) without a decrease in the selectivity or resolution of the separation is a favourable consequence of the presence of the CDs in the mobile phase.     

On the use of ionic liquids as mobile phase additives in high-performance liquid chromatography. A review

The popularity of ionic liquids (ILs) has grown during the last decades in several analytical separation techniques. Consequently, the number of reports devoted to the applications of ILs is still increasing. This review is focused on the use of ILs (mainly imidazolium-based associated to chloride and tetrafluoroborate) as mobile phase additives in high-performance liquid chromatography (HPLC). In this approach, ILs just function as salts, but keep several kinds of intermolecular interactions, which are useful for chromatographic separations. Both cation and anion can be adsorbed on the stationary phase, creating a bilayer. This gives rise to hydrophobic, electrostatic and other specific interactions with the stationary phase and solutes, which modify the retention behaviour and peak shape. This review updates the advances in this field, with emphasis on topics not always deeply considered in the literature, such as the mechanisms of retention, the estimation of the suppressing potency of silanols, modelling and optimisation of the chromatographic performance, and the comparison with other additives traditionally used to avoid the silanol problem.     

Influence of mobile phase composition on surface diffusion in reversed-phase liquid chromatography

Influence of column temperature and mobile phase composition (phi) on surface diffusion was studied in reversed-phase liquid chromatography using a C(18)-silica gel column and methanol-water mixtures. The temperature dependence of surface diffusion coefficient (D(s)) was explained by considering that of the molecular diffusivity, suggesting the presence of a kind of correlation between surface and molecular diffusions. The influence of phi on D(s) was accounted for by considering the restriction energy (E(r)) for surface diffusion. Physical meanings of the linear correlation between E(r) and the enthalpy change due to sample retention are discussed in connection with the mechanism of surface diffusion.