Dominant Factors Affecting the Chromatographic Behaviour of Bile Acids

With the aim of optimizing the chromatographic process by avoiding any preliminary derivatizing step, we examined the chromatographic behaviour of a selected set of unconjugated bile acids looking at the dominant factors that affect the performances of three different stationary phases: RP-8, RP-18 and RP-18 Base Deactivated (RP-18-BD). Accordingly to its structural peculiarity, the RP-18-BD column combined with a specific mobile phase has proved to be the most suitable one, in enhancing both separation factor α and resolution R _S within the selected set of analytes. Pronounced changes in the chromatographic profiles by only slightly changing the mobile phase composition (pH, buffer concentration, percentage and kind of organic modifier) prompted us to achieve satisfactory results in the separation and resolution of the selected set of bile acids.Presented at: CE in the Biotechnology & Pharmaceutical Industries: 7th symposium on the practical applications for the analysis of proteins, nucleotides and small molecules, Montreal, Canada, August 12–16, 2005.An erratum to this article can be found at     

Comparative study on separation of diastereomers by HPLC

Summary Reversed (RP-HPLC) and normal phase chromatographic (NP-HPLC) separations have been developed for diastereomers ofN-acyl-1-methyl-1,2,3,4-tetrahydo-β-carbolines which are acylated derivatives of simple natural β-carboline alkaloids. Separations of derivatives having different acyl moieties in theO,O-diacyl-tartaric acid ester subtituent differed remarkably. Little or no resolution in either NP-HPLC or RP-HPLC could be achieved with the diacetyl-tartrate derivative. Base-line separation by RP-HPLC but no separation by NP-HPLC was possible with the bulkier and more apolar dipivaloyl derivative. Retention order of the bis-benzoylated diastereomers was reversed and separation time increased dramatically by RP-HPLC. Good separation of the medium polarity, but rigid,N-camphanyl derivative by NP-HPLC has been achieved, whereas RP-HPLC could not be used for separation of these diastereomers. Separability of different diastereomers was highly dependent on polarity and rigidity of the derivatizingN-acyl moieties. Conformational analysis by molecular mechanics and comparison of the lowest energy conformational states of the diastereomers was applied to rationalise separation-retention behaviour of stereoisomers by RP-HPLC. Presented at Balaton Symposium '01 on High-Performance Separation Methods, Siófok, Hungary, September 2–4, 2001     

N-Nosyl-α-amino acids in solution phase peptide synthesis

A highly efficient and practical synthesis of peptides in solution phase has been developed. The procedure is based on the use of p-nitrobenzenesulfonyl (nosyl) group for the protection of the amino function of α-amino acids. Every step of the procedure, protection of the amino function by the nosyl group, formation of the peptide bond, and removal of the sulfonamide group, is characterized by high yields and excellent purity of the final products. The described strategy allows the preparation of short peptide sequences keeping the chiral integrity of amino acid precursors. Compatibility of nosyl group with the side-chain protecting groups used in Fmoc-based strategy is demonstrated. The method here presented is an alternative strategy that could provide advantages for future peptide synthesis.     

Cadmium colloids from non-aqueous solvents

Cadmium colloids have been prepared by Chemical Liquid Deposition (CLD). The metal is evaporated to yield atoms which are solvated at liquid nitrogen temperature, and upon warming, stable liquid colloids are formed with particle size ranging between 25–100 Å. Zeta potentials were calculated according to the conversion of Hunter and the Hückel equation, for ethanol and dimethyl sulphoxide. UV/VIS measurement of most of the black colloids showed absorption band around 280 nm. For comparison, we prepared CdS colloid with size 400–625 Å. The colloids are stable to oxidation in air and/or oxygen bubbling. The synthesis of colloids and films from Cd with acetone, 2-butanone, ethanol, 2-propanol, 2-methoxyethanol, DMF and DMSO is reported. Transmission Electron Microscopy (TEM) allows us to determine particle size.     

Preparation of trout liver microsomes for iron speciation in P-450 enzymes by AE–FPLC with ICP–(ORS)MS detection

Cytochromes P-450 are members of a superfamily of hemoproteins involved in the oxidative metabolism of various physiological and xenobiotic compounds in eukaryotes and prokaryotes. The multiplicity of this group of enzymes has been widely studied by chromatographic techniques, mainly high-performance liquid chromatography (HPLC). Because these enzymes are membrane-bound proteins, sample preparation for chromatographic separation of P-450 enzymes requires a solubilization step. The sample-preparation procedures are critical, because detergents affect not only the efficiency of protein solubilization but also their further chromatographic resolution. Trout liver microsomes have been taken here as a model sample to investigate iron speciation in cytochrome P-450. Trouts were treated intraperitoneally with -naphthoflavone, a potent inducer of some P-450 enzymes, and a microsomal suspension containing 7.4±0.1 nmol mL^–1 P-450 enzymes was obtained by ultracentrifugation. Lubrol PX was selected as detergent for solubilization, resulting in about 90% solubilization recovery. The solubilized cytochromes P-450 were further separated by AE–FPLC, with UV detection, or coupled to ICP–MS with an octapole reaction system, ICP–(ORS)MS (monitoring Fe signals at masses 54, 56, and 57). A sampling procedure and chromatographic conditions are developed and were successfully applied to iron speciation in trout liver P-450 enzymes. ICP–(ORS)MS detection of P-450 enzymes is Fe-specific and so will give accurate information on the prosthetic group of the protein, which can constitute an advantageous alternative to classical methods for detection of these hemoproteins.     

HPLC Analysis of Cotinine in Urine After SPE with a Cholesterol-Modified Adsorbent

A one-step solid-phase extraction procedure, based on a new silica gel adsorbent modified with cholesterol groups, has been investigated for measurement of cotinine in urine. Cotinine is the main metabolite of nicotine in the human body and is analyzed as a biomarker for assessment of direct or passive exposure to tobacco smoke. New cholesterol-modified adsorbents have been obtained by chemical modification of silica gel of different porosity with cholesterol ligands. Although recovery by this extraction procedure were optimum over a relatively broad range of sample pH (3.1–8.0), analytical conditions such as sample loading, washing and elution conditions, concentration of cotinine to be extracted, and the type of adsorbent used for extraction were found to affect the efficiency of the procedure and had to be controlled for optimum recovery. When these conditions were controlled, recovery of cotinine from spiked human urine was reproducible and depended on compound ionization. Quantitative analysis of cotinine was performed by reversed-phase high-performance liquid chromatography with UV detection. Presented at: Conference of the Hyphenation of Liquid Chromatography–Nuclear Magnetic Resonance Spectroscopy, Liquid Chromatography–Mass Spectrometry and Related Topics, Tuebingen, Germany, March 25–29, 2006.     

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.     

Color Displays with Gel-Glass Dispersed Liquid Crystals

Liquid crystal microdroplets trapped into silica gel-glasses by sol-gel processes may be used for display applications. Gel-glass dispersed liquid crystals (GDLC) are switched between opaque and transparent states by applying external AC-voltage signals; no polarizers are required. The feasibility of applying these materials to colored displays has been explored. A comparative study of different dyes for GDLC color displays, which were either embedded in the sol-gel matrix or dissolved in the liquid crystal microdroplets is presented.     

Modular design of icosahedral metal clusters

The concept of crystalline module, that is, an unambiguously isolated, repeated quasi-molecular element, is introduced. This concept is more general than the concept of crystal lattice. The generalized modular approach allows extension of the methods and principles of crystallography to quasi-crystals, clusters, amorphous solids, and periodic biological structures. Principles of construction of aperiodic, nonequilibrium regular modular structures are formulated. Limitations on the size of icosahedral clusters are due to the presence of spherical shells with non-Euclidean tetrahedral tiling in their structure. A parametric relationship between the structures of icosahedral fullerenes and metal clusters of the Chini series was found.     

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.