Retention of ionizable compounds on HPLC. Modelling retention for neutral and ionizable compounds by linear solvation energy relationships

Summary A global LSER model that relates HPLC retention to mobile phase composition and pH is tested for a varied group of solutes, both neutral and ionizable, in a polymeric column and methanol-water mobile phases. It is compared to the local LSER model developed only for a given mobile phase, i.e., a fixed organic modifier content, and to the global LSER model set only for neutral solutes. The global LSER model for neutral and ionizable solutes requires a few supplementary parameters over the other models tested, but it accounts for retention under any experimental conditions for a given column and methanol-water mobile phases, describing properly the interactions established in the HPLC system (hydrophobicity, hydrogen-bond acidity and basicity, dipolarity/polarizability…). This paper is number 13 of a series with the same general title: “Retention of Ionizable Compounds on HPLC” published in various journals.     

On the mechanism of solute retention in RP-HPLC systems with B+AB1+AB2 type eluents

Summary Intermolecular interactions between the solute and the moieties constituting the mobile phase significantly contribute to the overall retention pattern of a given solute in a given chromatographic system. In this paper retention of solute is discussed in the case of the B+AB₁+AB₂ type mobile phase, which, in a quasithermodynamic way, can be divided into seven individual moieties. One evaluates the influence of each moiety on solute retention, and refers these regularities to the polarity of the solutes.     

LC-ESI-MS Determination of Bilobalide and Ginkgolides in Canine Plasma

A sensitive and selective method using liquid chromatography with electrospray ionization mass spectrometric detection was developed for the quantification of bilobalide and ginkgolides in canine plasma. The analytes were extracted with diethyl ether-dichloromethane-isopropanol (6:3:1, v/v) after spiking the samples with daidzein (internal standard). The lower limit of quantification (LLOQ) of the method was 2.5 μg L⁻¹ for ginkgolide B and 10.0 μg L⁻¹ for bilabolide, ginkgolide A and ginkgolide C. The accuracy of the method was within 15% of the actual values over a wide range of plasma concentrations. The intra-day and inter-day precision was better than 15% (R.S.D.). Finally, the LC-ESI-MS method was successfully applied to study the pharmacokinetics of ginkgolides and bilabolide after administration of Ginkgo biloba extracts to dogs.     

Potentiometric determination of fluoride in geological and biological samples following pyrohydrolytic decomposition

A method for the determination of fluoride in coal, coal fly ash, phosphate rock, limestone, mineral clay, fossilised materials, oyster tissue and vegetation using pyrohydrolysis for sample decomposition is proposed. A specific apparatus was constructed and the influence of vanadium pentoxide (V₂O₅) as a catalyst for the pyrohydrolysis reaction was investigated. It was verified that V₂O₅ does not influence the release of fluoride from the vegetation, oyster tissue, coal and coal fly ash matrices analysed. However, the catalyst was necessary for the phosphate rock, fossil bone, mineral clay and limestone samples. Certified and noncertified samples were analysed using an ion selective electrode (ISE) for the analyte detection. Precise (relative standard deviation—R.S.D.<7%) and accurate (recovery in accordance to certified values) results were obtained. The limit of quantification (LOQ) of the method was 5.0 μg g⁻¹ of fluoride using 20 mg of sample and a final dilution to 10 ml. The sample frequency was five samples per hour.     

Mixed hybrid bilayer lipid membrane incorporating valinomycin: improvements in preparation and functioning

The paper deals with the reconstruction of lipid bilayer membranes on a Au-covered polycarbonate membrane. Such a kind of like-biomembranes (namely mixed hybrid bilayer lipid membrane (MHBLM)) are characterised by appreciable long-term stability. Here we describe changes that have been made in the geometry of the experimental device in order to avoid artefacts and render membrane reproduction easier. Incorporation of valinomycin was performed to check the membrane and its stability: conductance and membrane potential following the changes of ion concentration were recorded. This new approach permits increase of successful trials and renders possible, when it breaks, easily formation of a new MHBLM on the same Au-covered polycarbonate membrane support. Finally, the stability shown by the MHBLM renders this system a promising tool for use under flowing conditions.     

Optimization of alkali-alkalkine earth cation separation on weak cation-exchangers

Summary The elution behavior of alkali and earth alkaline cations has been studied on a newly synthesized weak cationexchanger (WCX). Applying this stationary phase isocratic separation of these cations is possible. The dependence of retention on competing ion concentration is described via a simple model. The influence of crown ethers as well as organic eluent additives on selectivity and peak efficiency is demonstrated. With optimized separation such cations can be determined with detection limits of about 0.5 ppm using a conductivity detector without ion suppression.     

Chiral stationary phases for LC and SFC obtained by “Polymer coating”

Summary Chirally substituted Si–H-containing polysiloxanes were synthesized, which can be immobilized on small particle silica gel as well as on the smooth surfaces of fused silica capillaries. Immobilization is achieved either by crosslinking or by chemical bonding to the surfaces via silanol groups; both reactions can only be performed by addition of H₂PtCl₆, which acts as catalyst for hydrosilylation and as stoichiometric reagent for crosslinking. Chiral substituents of systematically varied chemical structure were introduced into the polysiloxanes by hydrosilylation. The mechanism of immobilization was investigated by spectroscopic methods, notably²⁹Si-NMR. Homogenous stationary-phase coatings of variable film thickness and corresponding retentivity can easily be achieved. The enantioselectivity of the phase systems was characterized in dependency on the chemical structure of the chiral selectors attached to the polysiloxane chain of the chiral stationary phases and also in terms of the functional groups introduced into the solutes by derivatisation.     

HPLC determination of urinary 2,4- and 2,6-toluendiamines as potential degradation products of polyurethane breast implants

Summary A reversed-phase HPLC method has been developed for the urinary determination of mutagenic 2,4- and 2,6-toluendiamines. These amines are degradation products of polyurethane, a material used to cover textured silicone breast implants. FMOC-Cl was used as fluorescent derivatising agent in order to obtain a limit of detection of 15 ng/ml in urine. Pre-treatment of urine samples was by liquid/liquid extraction and urine specimens of patients after surgury were analysed.     

High-Performance Liquid Chromatographic Analysis of Amino Acids in Edible Seaweeds after Derivatization with Phenyl Isothiocyanate

Summary A reversed-phase high-performance liquid-chromatographic method has been used for analysis of the amino acids in edible seaweed. Sample proteins were hydrolysed with hydrochloric acid and the amino acids produced were derivatized with phenyl isothiocyanate. The resulting phenylthiocarbamyl amino acids were chromatographed on an ODS2 column with UV detection at 254 nm. The mobile phase was a mixture of 0.14 M ammonium acetate buffer, pH 6.4, containing 0.05% triethylamine (A) and 60:40 (v/v) acetonitrile–water (B), at a flow rate of 1.1 mL min^–1; the elution gradient (min:A%) was: 0:90, 8:90, 10:70, 12:70, 18:52, 20:0, 25:0, 28:90, 35:90. Method precision for the different amino acids was between 1.33 and 3.88% (relative standard deviation); detection limits were between 6.9 and 14.3 ng mL^–1. The amino acid content of the algae analysed ranged from 22.4 ± 1.9 to 138.0 ± 5.6 mg g^–1 d.w. The amino acids present at highest concentrations were glutamic acid, alanine, and phenylalanine.     

Determination by LC of Polyphenols in Italian Red Wine

A reversed-phase liquid chromatographic method, optimised for the separation of trans-, and cis-resveratrol, catechin, epicatechin, quercetin and rutin, is reported. Separation was achieved using a C18 column and a gradient elution with acetonitrile and 5% formic acid aqueous solution. The analyses required an equilibration period of 10 min and a run time of 25 min for completion. Identification was based on retention characteristics and by relative UV spectra, obtained by photodiode array detector and were compared with commercial standards. Analyses were performed without any sample pre-treatment. Detection was carried out by UV–Vis detector at three different wavelengths. The detection limit ranged from 0.16 μgm L⁻¹ (cis-resveratrol) to 1.5 μgm L⁻¹ (+)-catechin. Investigation was extended to quantitative determination of phenol compounds in Italian red wine and to investigate the stability of the six antioxidants.