Effect of nature of support and impregnating agent on lipophilicity determination for nonionic surfactants by reversed-phase thin-layer chromatography

Summary The retention of 9 nonylphenyl ethylene oxide oligomers was determined in 15 reversed-phase chromatographic systems using silicone oils of various molecular mass as impregnating agents and silica, cellulose and alumina supports. The data were evaluated with principal component analysis carried out on the covariance and the correlation matrices. The R_M values did not follow the additivity rule and they did not change linearly with increasing length of the ethylene oxide chain. This is probably due to the folded state of the chain in the eluent. The molecular mass of the silicone oils had a negligible effect on the retention whereas the retention increased with increasing level of impregnation. The support considerably influenced the lipophilicity values of these nonionic surfactants and cellulose seemed to be the most appropriate support. Calculations proved that the application of a correlation matrix may cause data distortion; therefore, the use of a covariance matrix is strongly proposed.     

Concurrent adsorption and complex formation in reversed-phase liquid-liquid chromatography with tri-n-octylphosphine oxide

Summary Reversed-phase liquid-liquid chromatographic systems consisting of an aqueous mobile phase and an organic liquid stationary phase of the proton acceptor tri-n-octylphosphine oxide (TOPO) inn-decane, coated on LiChrosorb RP-8, have been studied. The solutes were hydrophilic aromatic carboxylic acids and phenol. The retention of the carboxylic acids shows a minimum at 10 mM of TOPO, whereas increasingly tailing peaks have been obtained with decreasing concentrations of TOPO. This behaviour is due to a concurrent complex formation by hydrogen bonding with TOPO in the liquid stationary phase and adsorption at the interface between the support and the liquid stationary phase. The adsorption of TOPO, ketones and aromatic acids from hexane on Li-Chrosorb RP-8 has been studied, and seems to be due to residual silanol groups. The adsorption isotherm of TOPO has been determined and can be described by a two-site Langmuir adsorption model. Non polar solutes are not adsorbed. The influence of TOPO on the retention and the peak symmetry of carboxylic acids in the liquid-liquid chromatographic system appears to be due to a competition between TOPO and the acids for the same adsorption sites. No competition was found for phenol.Presented at the 14th International Symposium on Chromatography London, September, 1982     

Determination of the enantiomeric composition of chiral carboxylic acids using chiral derivatization and HPLC

Summary The enantiomers of chiral carboxylic acids were separated as their diastereomeric amides with (1R,2R)-(−)-1-(4-nitrophenyl)-2-amino-1,3-propanediol (“levobase”) and with “dextrobase” (the enantiomer of levobase) by high-performance liquid chromatography using a conventional C-18 column and various solvent systems containing acetonitrile, methanol, water, and phosphoric acid.     

LC Determination of 4-Bromoaniline in Green Algae Chlamydomonas reinhardtii After Continuous-Flow Microextraction

In this study, the determination of 4-Bromoaniline (4-BA) in green algae Chlamydomonas reinhardtii (C. reinhardtii) was investigated by applying continuous-flow microextraction (CFME) combined with high-performance liquid chromatography (HPLC). Continuous-flow microextraction was conducted in a homemade glass chamber, i.e. the sample solution flowed through a constant volume drop of solvent in the chamber at a constant flow rate. The effects of different factors on extraction efficiencies were also investigated and these factors included the kind of extraction solvent, solvent drop volume, sample flow rate, extraction time and addition amount of salt. Under the optimum extraction conditions (extraction solvent, carbon tetrachloride; solvent drop volume, 3.5 μL; sample flow rate, 1.0 mL min⁻¹; extraction time, 10 min; no addition of salt), the calibration plot was set up by plotting peak area against a series of 4-Bromoaniline concentrations (0.01–10 μg mL⁻¹) in aqueous solution. The correlation coefficient (r) was 0.9990. The limit of detection (LOD) was 0.6 ng mL⁻¹. The precision of this method was obtained by successive five time analyses of 100-ng mL⁻¹ standard solution of 4-Bromoaniline, and the relative standard deviation (RSD) was 3.5%. The concentration factor was calculated by the ratio of peak area of the analyte obtained after and before extraction and found to be 10.6. 4-Bromoaniline residues in Chlamydomonas. reinhardtii cells and tap water samples were satisfactorily analyzed according to the method described above.     

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     

Concentration and characterization of dilute colloidal samples by potential-barrier field-flow fractionation

Summary Potential-barrier field-flow fractionation, which is a combination of potential-barrier chromatography and sedimentation field-flow fractionation, is shown to be a convenient and accurate method for the concentration and analysis (separation and characterization) ofdilute colloidal samples. Two sizes (0.158 and 0.271 μm) of haematite (α-Fe₂O₃) monodisperse colloidal samples diluted in volumes of up to 20 cm³ are used as model colloids. The particle diameters found by the present concentration procedure under various experimental conditions are in good agreement with those determined by conventional sedimentation field-flow fractionation, in which a small concentrated sample volume was injected directly into the column.     

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     

Online photocatalytic device for highly selective pre-column fluorescence derivatization of 5-hydroxyindoles with benzylamine

A fluorimetric liquid chromatographic method for the determination of 5-hydroxyindoles based on the benzylamine derivatization process mediated through an online photocatalytic oxidation has been developed. In this study, we used a photocatalytic column comprising tefzel tubing packed with TiO₂-coated glass beads, as a pre-column derivatization reactor. The fluorescence derivatization of 5-hydroxyindoles using benzylamine proceeded during their passage through the reaction column under near-UV irradiation. The 5-hydroxyindole derivatives were separated continuously on a reversed-phase liquid chromatography within 50 min, using 100 mM acetate buffer (pH 4.6)-acetonitrile (72:28, v/v; isocratic elution) containing 3 mM sodium octanesulfonate; the samples were detected fluorimetrically at 465 nm upon excitation at 350 nm. The detection limits (signal-to-noise ratio = 3) of the 5-hydroxyindoles were in the range from 160 to 360 fmol per 5 μL injection. We have applied this method, which requires minimal sample pre-treatment, to the determination of 5-hydroxyindole-3-acetic acid in human urine.     

The role of achiral sorbent matrix in chiral recognition of amino acid enantiomers in ligand-exchange chromatography

Summary As an alternative to the known three-point interaction model describing recognition of optical isomers by a chiral resolving agent, a new concept has been developed stating that two interaction points between the resolving agent and the enantiomers are also sufficient for achieving chiral recognition of the latter, provided that the diastereomeric adducts formed by the resolving agent with the enantiomers additionally interact with a non-chiral chromatographic sorbent. This concept is based on the results of ligand-exchange chromatography of -amino acid enantiomers with copper(II) complexes of chiral bifunctional ligands as the resolving agents in chromatographic systems.     

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.