Danilo Corradini

Electrophoresis of proteins in uncoated capillaries with amines and amino sugars as electrolyte additives

Abstract The effect of triethylamine and triethanolamine in the running electrolyte on the electroosmotic flow and the migration of four standard basic proteins in bare fused-silica capillaries was examined. At pH 2.5 the direction of the electroosmotic flow was anodal with both additives and at constant ionic strength its magnitude increased with increasing additive concentration. The observations are in qualitative agreement with a theoretical model which is based on the Gouy—Chapman—Stern theory and incorporates a Langmuir-type relationship and also the Von Smoluchowski expression for the electroosmotic mobility, with the approximation to identify the zeta potential with the potential at the Stern plane. From the independence of the electrophoretic mobilities of the additive concentration and type the absence of interactions between the four basic proteins and the two alkylamines is inferred. The utility of glucosamine and galactosamine as additives for the capillary electrophoresis of basic proteins is also demonstrated and their effectiveness is compared with that of the alkylamines.

Chemical and biochemical change of healthy phenolic fractions in winegrape by means of postharvest dehydration.

Clusters of Aleatico winegrape were picked at 18 degrees Brix and placed at 10, 20, or 30 degrees C, 45% relative humidity (RH) and 1.5 m/s of air flow to dehydrate the berries up to 40% of loss of initial fresh weight. Sampling was done at 0, 10, 20, 30, and 40% weight loss (wl). Selected polyphenols and sugar content (expressed as SSC = soluble solids content) both measured on dry weight basis, polyphenol oxidase (PPO), and phenylpropanoid pathway gene expression were analyzed. Phenolic acids increased significantly at 20% wl at 20 degrees C, while at 10 degrees C the increase was lower. Stilbenes (trans-resveratrol and trans-piceid) and catechins rose more than double to 100 mg/kg and more than 3-fold to 135 mg/kg at 20 degrees C and 10% wl. At 10 degrees C the increase of these compounds was less, but higher than initial values. At 30 degrees C, except for a significant rise at 10% wl for catechins and stilbenes, all the rest of the compounds diminished. Anthocyanins increased at 10 and 20 degrees C, but decreased at 30 degrees C. PPO rapidly increased at 20 and 30 degrees C at 10% wl and then declined, while at 10 degrees C the activity lasted longer. Relative gene expression of phenylalanine ammonia lyase (PAL), stilbene synthase (STS), chalcone isomerase (CHI), dihydroflavonol reductase (DFR) were upregulated at 10 degrees C more than at 20 degrees C, at 20% wl, while at 30 degrees C the gene expression was downregulated.

Effects of alkylamines on electroosmotic flow and protein migration behaviour in capillary electrophoresis

This paper reports the use of four closely related alkylamines as running electrolyte additives in capillary electrophoresis that permit the control of electroosmotic flow and protein migration behaviour in uncoated capillaries. At pH 2.5 the direction of the electroosmotic flow was anodic with all additives and at constant ionic strength its magnitude increased with increasing alkylamine concentration. The observations are in qualitative agreement with a previous reported theoretical model that correlates the electroosmotic mobility with the charge density in the Stern region of the electric double layer, arising from the adsorption of the additive, and the charge density at the capillary wall due to dissociation of silanols.

Resolution and identification of the protein components of the photosystem II antenna system of higher plants by reversed-phase liquid chromatography with electrospray-mass spectrometric detection.

Reversed-phase liquid chromatography (RPLC) was interfaced to mass spectrometry (MS) with an electrospray ion (ESI) source for the separation and accurate molecular mass determination of the individual intrinsic membrane proteins that comprise the photosystem II (PS II) major light-harvesting complex (LHC II) and minor (CP24, CP26 and CP29) antenna system, whose molecular masses range between 22,000 and 29,000. PS II is a supramolecular complex intrinsic of the thylacoid membrane, which plays the important role in photosynthesis of capturing solar energy, and transferring it to photochemical reaction centers where energy conversion occurs. The protein components of the PS II major and minor antenna systems were extracted from spinach thylacoid membranes and separated using a butyl-silica column eluted by an acetonitrile gradient in 0.05% (v/v) aqueous trifluoroacetic acid. On-line electrospray MS allowed accurate molecular mass determination and identification of the protein components of PS II major and minor antenna system. The proposed RPLC-ESI-MS method holds several advantages over sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the conventional technique for studying membrane proteins, including a better protein separation, mass accuracy, speed and efficiency.

Identification and Quantification of Phenolic Compounds in Grapes by HPLC-PDA-ESI-MS on a Semimicro Separation Scale

Reversed phase high performance liquid chromatography (RP-HPLC) on a semimicro separation scale was employed to develop a straightforward method for the simultaneous separation, identification, and quantification of phenolic compounds occurring in whole berries of Vitis vinifera, which comprise phenolic acids, flavonols, catechins, stilbenes, and anthocyanins. A C-18 narrow bore column of 150 x 2.0 mm I.D. and a semimicro photodiode array detector (PDA) cell of 2.5 microL, in conjunction with a mass spectrometry detector equipped with an electrospray ionization source (ESI-MS) to confirm peak identification, were employed. The C-18 narrow bore column was eluted by a multisegment gradient of increasing concentration of acetonitrile in water-formic acid solution that was optimized on the basis of the results of a study carried out to evaluate the influence of mobile phase composition and gradient shape on separation performance and detection sensitivity by ESI-MS. The identification of individual phenolic compounds was performed on the basis of their retention times and both UV-visible and mass spectra, acquired by a mass spectrometer (MS) equipped with an electrospray ionization (ESI) source, employed in conjunction with the PDA detector. Libraries comprising retention times, UV-visible, and mass spectra for major phenolic compounds expected in grape berries were made by subjecting solutions of each phenolic standard to the optimized RP-HPLC method. Quantification of individual compounds was performed by the external standard method using a six point regression graph of the UV-visible absorption data collected at the wavelength of maximum absorbance of each analyte determined by the PDA spectra. The RP-HPLC method was validated in terms of linearity of calibration graphs, limits of detection, limits of quantification, repeatability, and accuracy, which was evaluated by a recovery study. The developed method was successfully applied to identify the phenolic compounds occurring in the whole berries of nine red and one white grape of different varieties of Vitis vinifera, comprising some autochthonous varieties of south Italy such as Aglianico, Malvasia Nera, Uva di Troia, Negroamaro, Primitivo, and Susumaniello. Large differences in the content of phenolic compounds was found in the investigated grape varieties. As expected, only glycosilated flavonols were quantified, and the total amount of these compounds was higher in the whole berries of red grapes than in the white Moscato, where the most abundant phenolic compound was quercetin 3-O-glucoside. In almost all samples, the most and least abundant anthocyanins were malvidin 3-O-glucoside and cyanidin 3-O-glucoside, respectively, with the exception of Uva di Troia where the least abundant anthocyanin was delphinidin 3-O-glucoside.

Effect of mobile phase additives on peptide retention in reversed-phase chromatography with pellicular and totally porous sorbents

The effect of two mobile phase additives, trifluoroacetic acid and phosphoric acid, on the energetics of peptide retention in reversed-phase chromatography was investigated using Hy-Tach C18 micropellicular and Vydac C4 and C18 totally porous stationary phases. The effect of the relatively low phase ratio of columns packed with micropellicular sorbents was also examined. The logarithmic retention factors, of two model peptides, Ac-RGGGGLGLGK-amide and Ac-RGAGGLGLGK-amide, were evaluated with different columns and additives in a practical range of eluent strength. The dependence of the logarithmic retention factor on the concentration of acetonitrile in the mobile phase was linear in all cases. The higher sensitivity of the retention to the organic modifier concentration in the case of the Hy-Tach C18 column is attributed to the relatively low phase ratio of this column. Pairwise plots of the logarithmic retention factors were linear. The plots of data obtained with the two additives has unit slopes and thus reveal homoenergetic retention behavior. On the other hand data obtained on two different columns manifest homeoenergetic retention, the slopes of plots are different from unity. The analysis has yielded consistent results and validated the assumption that the retention free energy can be divided into two components arising from mobile phase and stationary phase contributions. The approach also allowed an estimation of the relative phase ratios of the columns and the Vydac C18 column was found to have an 3 and 8 times higher phase ratio than the Vydac C4 and the Hy-Tech C18 column, respectively.

Identification and Quantification of Soluble Free, Soluble Conjugated, and Insoluble Bound Phenolic Acids in Durum Wheat (Triticum turgidum L. var. durum) and Derived Products by RP-HPLC on a Semimicro Separation Scale

A straightforward semimicro separation scale RP-HPLC method was developed for the identification and quantification of phenolic acids (PAs) occurring as soluble free, soluble conjugated, and insoluble bound compounds, which were independently extracted from wholemeal of durum wheat and from its derived products coarse bran, semolina, and dried pasta. A narrow bore column and a semimicro photodiode array detector (PDA) cell, in conjunction with a single quadrupole mass spectrometer, equipped with an electrospray ionization source (ESI-MS), were employed. The method was validated in terms of linearity of calibration graphs, limits of detection, limits of quantification, repeatability, and accuracy, which was evaluated by a recovery study. In each sample (wholemeal, coarse bran, semolina, and dried pasta), the total amounts of the three different forms of PAs were in the order bound > conjugated > free, with bound PAs accounting for 61.0-83.6% of the total PAs. Ferulic acid was the most abundant PA in both soluble free and insoluble bound forms, whereas sinapic acid predominated in the conjugated ones. The highest PA content, calculated as the sum of total PAs quantified in the three forms, was found in coarse bran, followed by wholemeal, semolina, and dried pasta.

Co-electroosmotic capillary electrophoresis of basic proteins with 1-alkyl-3-methylimidazolium tetrafluoroborate ionic liquids as non-covalent coating agents of the fused-silica capillary and additives of the electrolyte solution

The paper reports the results of a study carried out to evaluate the use of three 1‐alkyl‐3‐methylimidazolium‐based ionic liquids as non‐covalent coating agents for bare fused‐silica capillaries and additives of the electrolyte solutions (BGE) for CE of basic proteins in the co‐EOF separation mode. The three ionic liquids are differentiated from each other by the length of the alkyl group on the imidazolium cation, consisting of either an ethyl, butyl or octyl substituent, whereas tetrafluoroborate is the common anionic component of the ionic liquids. Coating the capillary with the ionic liquid resulted in improved peak shape and protein separation, while the EOF was maintained cathodic. This indicates that each ionic liquid is effective at masking the protein interaction sites on the inner surface of the capillary, also when its adsorption onto the capillary wall has not completely neutralized all the negative charges arising from the ionization of the silanol groups and the ionic liquid is not incorporated into the BGE employed for separation. Using the coated capillaries with BGE containing the ionic liquid employed for the coating, at concentration low enough to maintaining the EOF cathodic, both peak shape and protein separation varied to different extents, based on the particular ionic liquid used and its concentration. Fast and efficient separation of the model basic protein mixture in co‐electroosmotic CE is obtained with the 1‐butyl‐3‐methylimidazolium tetrafluoroborate coated capillary and 100 mM acetate buffer (pH 4.0) containing 4.4 mM 1‐butyl‐3‐methylimidazolium tetrafluoroborate as the BGE.

Combined lectin-affinity and metal-interaction chromatography for the separation of glycophorins by high-performance liquid chromatography

Human erythrocyte sialoglycoproteins, or glycophorins, were chromatographed by lectin-affinity and metal-interaction chromatography on high-performance liquid chromatographic columns. Glycophorins A, B and C were separated from other proteins and from glycophorin E by using a column containing wheat germ agglutinin, immobilized on a microparticulate silica support. The glycophorins were adsorbed on the lectin column from a mobile phase containing 0.25 M sodium chloride and recovered by stepwise desorption with 0.2 M N-acetylglucosamine solution. Glycophorins A, B and C were separated into the individual components on a silica-bound iminodiacetic acid stationary phase in the copper(II) chelate form. The separation of the glycophorins by metal-interaction chromatography was accomplished by decreasing salt gradient elution. Retention times and resolution of the individual glycophorins were sensitive to the initial sodium chloride concentration and the pH of the eluent. Addition of methanol to the eluent increased the resolution. The effects of linear, decreasing gradients of pH and methanol in 25 mM phosphate buffer on the resolution of glycophorins were also investigated. In both types of chromatography the mobile phases contained 0.05% (w/v) sodium dodecyl sulfate. With octylglycoside or CHAPS in the eluent glycophorins A and C could not be eluted. Sodium dodecyl sulfate polyacrylamide gel electrophoresis was used to analyze all the chromatographic results.

High Performance Anion-Exchange Chromatography with Pulsed Amperometric Detection of Nutritionally Significant Carbohydrates

Abstract The separation of nutritionally significant saccharides in fruit juices was investigated by high-performance anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD}. The effect of sodium hydroxide concentration in the mobile phase on the retention and selectivity was investigated on three different pellicular anion-exchange columns (all from Dionex). The sugar alcohol sorbitol and the sugars glucose, fructose, and sucrose were well resolved in less then 10 minutes on the Carbopac PA 100 column by using simple isocratic elation with 150 mM sodium hydroxide solution, without column regeneration between runs. Under these conditions the relative standard deviations of the retention times were better than 0.98%. HPAEC-PAD was successfully applied to the determination of the major sugars in a fruit concentrate and in several varieties of commercial fruit juices by an internal standard method without any sample pretreatment.