Anna Maria Girelli

Oxidation of ferulic acid by laccase: identification of the products and inhibitory effects of some dipeptides

The laccase activity, in the presence of ferulic acid as substrate, was tested with high performance liquid chromatography (HPLC). The two products initially formed were identified by HPLC-mass spectrometry and HPLC-diode array as trans-5-[(E)-2-carboxyvinyl]-2-(4-hydroxy-3-methoxy-phenyl)-7-methoxy-2,3-dihydrobenzofuran-3-carboxylic acid and (Z)-beta-{4-[(E)-2-carboxyvinyl]-2-methoxyphenoxy}-4-hydroxy-3-methoxy cinnamic acid. The inhibitory effects of some dipeptides due to the formation of copper complexes have also been reported. The results obtained confirm that the inhibitory effects on laccase activity are strongly related to the structure of the dipeptides and confirm the important role of copper(II) in the reaction mechanism.

Liquid membranes for chiral separations. Application of cinchonidine as a chiral carrier

Enantiomeric enrichment of mandelic acid and phenylglycine racemics is achieved by using a liquid membrane containing cinchonidine as a chiral carrier (a = 1.5 and 0.9, respectively). Transport of the respective enantiomers through a liquid membrane was characterized by using mandelic acid as target analyte. To this end, the influence on this transport of different parameters was determined, for instance the amount of decanol added as modifier to the cinchonidine in dodecane solution (constituting the liquid membrane), the initial carrier:analyte ratio in the feed phase, the pH of the aqueous solutions (both feed and receiving), and the effect of varying the membrane solvent from dodecane-decanol to the more solvating chloroform. Additionally, and for the sake of comparison with the mandelic acid results, the transport of phenylglycine and phenylalanine was studied. The membrane showed a slight selectivity only for the phenylglycine transport, as phenylalanine was transported as a 50% racemic mixture under all the conditions examined. The observed results may perhaps be accounted for by hydrogen bonding interactions between carrier and analytes.

High-performance liquid chromatographic resolution of enantiomers on chiral amine-bonded silica gel

SummaryA chiral stationary phase with an immobilized, optically active diamine was prepared for the separation of enantiomers. The synthesis of the phase was carried out by bonding (−)trans-1,2-cyclohexanediamine to microparticulate silica gel through the coupling agent 3-glycidoxypropylsilane. The resolution of the racemic compounds catechin, 2,2′-dihydroxy-1,1′binaphthyl and trans-1,2-cyclohexandiol, is reported.

Enantioselective transport of D,L-phenylalanine and D,L-phenylglycine through a bulk liquid membrane containing cinchona alkaloid derivatives as chiral selectors

Different cinchona derivatives were tested as chiral mobile carriers for enantioselective transport of D,L-phenylglycine and D,L-phenylalanine through a bulk organic liquid membrane (BLM). The effects of several parameters such as carrier nature, membrane solvent, buffer, solute and carrier concentrations on BLM enrichment were evaluated. Only D,L-phenylglycine is subjected to enantioselective transport at a certain degree; the maximum enantioselectivity ratio (the initial transport rates of the L-enantiomer relative to the antipode) was reached by employing O-allyl-N-(9-anthracenylmethyl) cinchonidinium bromide as carrier. In all cases the highest selectivity was observed during the initial stages of the process, indicating the rate of amino acid release from the source phase (SP) to the membrane organic phase (MP) as the driving factor. Chiral enrichment appeared to depend on thermodynamic factors more than on kinetic ones, since the complex formation was observed at (SP)/MP) interface, while complex decomposition was evidenced at (RP)/(MP) interface, where RP is receiving phase. The last phenomenon was promoted by H+ ions present in the RP.

Determination of polycyclic aromatic hydrocarbons in Italian milk by HPLC with fluorescence detection

The presence of polycyclic aromatic hydrocarbons (PAHs) in Italian commercial milk samples is reported. The study was carried out on lactating (cow and goat) and plant (rice, soya, oat) milk. The quantitative determination involved liquid–liquid extraction of PAHs, a pre-concentration and determination by HPLC using a fluorescence detector. The recovery of analytes was in the range of 70–115%. The precision of the method was found to be between 6% and 24%. The detection limit ranged from 0.66 to 33.3 µg l–1 corresponding to 0.03–1.66 µg kg–1 milk (wet weight), at a signal-to-noise ratio of 3, depending on the compound. By this procedure, the levels of more volatile PAHs (two to three aromatic rings) were confirmed in 34 commercial milk and three plant milk samples, whereas benzo[a]pyrene was found only in five pasteurised milk samples at a mean concentration of 0.17 µg kg–1 milk. These results provide evidence that PAH levels are influenced by heat treatments and skimming processes of milk production. Graphical Abstract

Variations of hepatic dolichols during rat development

The content and the percent distribution of dolichol and dolichyl phosphate homologues were measured by high-performance liquid chromatography in perinatal rat livers. Short dolichol chains and no dolichyl phosphate are detectable in the liver at foetal stages; dolichol content progressively increases during liver development. A good correlation is observable between the changes of the dolichol, dolichyl phosphate and the activity of dolichyl-phosphate phosphatase.

Separation of dansylamino acid enantiomers by thin-layer chromatography

A simple thin-layer chromatographic method for the separation of D- and L-dansylamino acids using a new resolving agent is described. The enantiomeric resolution of racemates was accomplished on reversed-phase plates pre-treated with a poly-L-phenylalaninamide-copper(II) complex using water-acetonitrile mixtures as eluent under isocratic conditions. The polymeric ligand was easily prepared by reaction of the optically active amide with ethylene glycol diglycidyl ether at room temperature. The method is rapid and sensitive, and was successfully employed to resolve various dansylamino acids.

Chitosan-coated silica gel as a new support in high-performance liquid chromatography

SummaryA new stationary phase was prepared by immobilizing the chitosan, a natural hydrophilic polymer, on microparticolate silica gel. The effect of the polarity, pH and ionic strength of the mobile phase has been studied in order to find optimal conditions for the separation of nucleotides and aminoacids. The influence of the properties of the mobile phase on the retention was examined, allowing to employ the chitosan-coated silica gel both to anion exchange and adsorption, depending on the pH and the polarity of the eluent used.

Determination of aspartate aminotransferase activity by high-performance liquid chromatography

A sensitive and reproducible assay of aspartate aminotransferase activity based on UV detection of the reaction products after their separation by HPLC is described. The main advantage is the direct measurement of the enzyme activity as micromoles of product (glutamate) formed within a known period of time without any coupled reaction. Further, with the chromatographic method, all components of the reaction mixture are identified, allowing the reaction course to be controlled and the possible presence of side-reactions to be monitored.

Enzymatic hydrolysis of pectic substances by gel-permeation chromatography

SummaryA rapid liquid chromatographic (HPLC) method for the determination of pectinolytic activity of enzymes produced by Aspergillus Niger and Rhizopus species is reported.Compared with more conventional methods, HPLC is more reliable and has a much improved maximum sensitivity. The limit of quantitation of galacturonic acid is 0.1μg.