University of North Florida
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Stuart Chalk, Ph.D.
Department of Chemistry
University of North Florida
Phone: 1-904-620-1938
Fax: 1-904-620-3535
Email: schalk@unf.edu
Website: @unf

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Emilia Bramanti

Abbrev:
Bramanti, E.
Other Names:
Address:
Laboratory of Instrumental Analytical Chemistry, Italian National Research Council-Istituto per i Processi Chimico-Fisici, Via G. Moruzzi 1, 56124 Pisa, ItalybLaboratory of Gene and Molecular Therapy, Italian National Research Council-Istituto di Fisiolo
Phone:
+39 050 3152293
Fax:
+39 050 3152555

Citations 10

"Mercury Speciation By Liquid Chromatography Coupled With On-line Chemical Vapour Generation And Atomic Fluorescence Spectrometric Detection (LC-CVGAFS)"
Talanta 2005 Volume 66, Issue 3 Pages 762-768
Emilia Bramanti, Cristina Lomonte, Massimo Onor, Roberto Zamboni, Alessandro D'Ulivo and Giorgio Raspi

Abstract: Reverse phase chromatography (RPC) coupled on-line with UV-vis diode array detector (DAD) and cold vapor generation atomic fluorescence spectrometry (CVGAFS) is proposed for the speciation and determination of inorganic and organic mercury (methylmercury, ethylmercury and phenylmercury) in the form of cysteine, penicillamine and glutathione complexes. The mercury-thiol complexes are separated on a C18 Reverse Phase column and oxidized on-line with bromine, generated in situ by KBr/KBrO3 in HCl medium, in order to fast convert organic mercury species to inorganic mercury in less than 2.5 s, at room temperature, in a 30 cm knitted coil. Hg(II) is selectively detected by AFS in a Ar/H2 miniaturized flame after sodium borohydride reduction to Hg(0). Under optimized conditions, on-line bromine treatment gives recoveries of thiol-complexed methylmercury, ethylmercury and phenylmercury with respect to inorganic mercury ranging between 79 and 85%, 80 and 85%, 63 and 76%, respectively, depending on the complexing thiol employed. Optimized elution conditions were provided in the three complexing agents. The detection limits (LODc) for inorganic mercury, methylmercury, ethylmercury and phenylmercury, in the optimized conditions complexed with thiols were about 16, 18, 18 and 20 pg (as mercury), respectively, a relative standard deviation (R.S.D) ranging between 1.5 and 2.0%, and a linear dynamic range between 0.1 and 100 ng injected. LC-DAD-CVGAFS method has been validated by analyzing two certificate reference material, DORM-2 and NIES CRM 13, obtaining 98 ± 6 and 97 ± 5% of methylmercury recovered, respectively.

"Hydrophobic Interaction Chromatography Coupled With Atomic Fluorescence Spectrometric Detection: Effect Of The Denaturation On The Determination Of Thiolic Proteins"
Talanta 2004 Volume 63, Issue 2 Pages 383-389
Emilia Bramanti, Chandra Sortino, Cristina Lomonte, Massimo Onor, Roberto Zamboni, Giorgio Raspi and Alessandro D Ulivo

Abstract: Hydrophobic interaction chromatography coupled online with chemical vapor atomic fluorescence spectrometry (HIC-CVGAFS) has been optimized for the analysis of thiolic proteins in denaturing conditions. Proteins are pre-column simultaneously denatured and derivatized in phosphate buffer solution containing 8.0 mol L-1 urea and p-hydroxymercurybenzoate (PHMB) and the derivatized denatured proteins are separated on a silica HIC Eichrom Propyl column in the presence of 8.0 M urea in the mobile phase. Post-column online reaction of derivatized denatured proteins with bromine, generated in situ by KBr/KBrO3 in HCl medium, allowed the fast conversion of the uncomplexed PHMB and of the PHMB bound to proteins to inorganic mercury also in presence of urea. Hg2+, present in solution as Hg2+-urea complex, is selectively detected by AFS in a Ar/H2 miniaturized flame after sodium borohydride reduction to Hg. Under optimized conditions, online bromine treatment gives a 100±2% recovery of both free and protein-complexed PHMB. Denatured glyceraldehyde-3-phosphate dehydrogenase, aldolase, lactate dehydrogenase, trioso phosphate isomerase and β-lactoglobulin have been examined. As the sensitivity and limit of detection of proteins in the HIC-CVGAFS apparatus depends on number of ---SH groups reacting with PHMB, the denaturation process, which increases the number of PHMB-reactive thiolic groups in proteins, improves the analytical performances of the described system in protein analysis. The detection limit for the denatured proteins examined was found in the range of 10^-10-10-12 mol L-1, depending on the considered protein, with linear calibration curves spanning over four decades of concentration.

"Determination And Characterization Of Phytochelatins By Liquid Chromatography Coupled With On Line Chemical Vapour Generation And Atomic Fluorescence Spectrometric Detection"
J. Chromatogr. A 2006 Volume 1133, Issue 1-2 Pages 195-203
Emilia Bramanti, Daniel Toncelli, Elisabetta Morelli, Leonardo Lampugnani, Roberto Zamboni, Keith E. Miller, Joseph Zemetra and Alessandro D'Ulivo

Abstract: Liquid chromatography (LC) coupled on line with UV/visible diode array detector (DAD) and cold vapor generation atomic fluorescence spectrometry (CVGAFS) has been developed for the speciation, determination and characterization of phytochelatins (PCs). The method is based on a bidimensional approach, e.g. on the analysis of synthetic PC solutions (apo-PCs and Cd2+-complexed PCs) (i) by size exclusion chromatography coupled to UV diode array detector (SEC-DAD); (ii) by the derivatization of PC -SH groups in SEC fractions by p-hydroxymercurybenzoate (PHMB) and the indirect detection of PC-PHMB complexes by reversed phase liquid chromatography coupled to atomic fluorescence detector (RPLC-CVGAFS). MALDI-TOF/MS (matrix assisted laser desorption ionization time of flight mass spectrometry) analysis of underivatized synthetic PC samples was performed in order have a qualitative information of their composition. Quantitative analysis of synthetic PC solutions has been performed on the basis of peak area of PC-PHMB complexes of the mercury specific chromatogram and calibration curve of standard solution of glutathione (GSH) complexed to PHMB (GS-PHMB). The limit of quantitation (LOQ) in terms of GS-PHMB complex was 90 nM (CV 5%) with an injection volume of 35 µL, corresponding to 3.2 pmol (0.97 ng) of GSH. The method has been applied to analysis of extracts of cell cultures from Phaeodactylum tricornutum grown in Cd-containing nutrient solutions, analyzed by SEC-DAD-CVGAFS and RPLC-DAD-CVGAFS. © 2006 Elsevier B.V. All rights reserved.

"Characterization Of Denatured Metallothioneins By Reversed Phase Coupled With On-line Chemical Vapour Generation And Atomic Fluorescence Spectrometric Detection"
J. Chromatogr. A 2004 Volume 1054, Issue 1-2 Pages 285-291
Emilia Bramanti, Cristina Lomonte, Alvaro Galli, Massimo Onor, Roberto Zamboni, Giorgio Raspi and Alessandro D Ulivo

Abstract: A new analytical hyphenated technique is proposed for determination and characterization of thiolic proteins, based on reverse phase chromatography (RPC) coupled on-line with cold vapor generation atomic fluorescence spectrometry (CVGAFS). Proteins are pre-column simultaneously denatured and derivatized in phosphate buffer solution containing 8.0 mol L-1 urea and p-hydroxymercurybenzoate (PHMB). The derivatized proteins are separated on a C4 Vydac Reverse Phase column. Post-column on-line reaction of derivatized denatured proteins with bromine, generated in situ by KBr/KBrO3 in HCl medium, allowed the fast conversion of both the uncomplexed PHMB and of the PHMB bound to proteins to inorganic mercury, also in the presence of methanol in the RPC eluent phase. Hg(II) is selectively detected by AFS in a Ar/H2 miniaturized flame after sodium borohydride reduction to Hg(0). Under optimized conditions, on-line bromine treatment gives a 98±2% recovery of both free and protein-complexed PHMB. The effect of methanol on the sensitivity of Hg(II) detection was studied and controlled. RPC-CVGAFS system has been applied to the analysis of metallothioneins from rabbit liver (MTRL) standard solutions, and their commercial isoforms MT-1 and MT-2. The analysis of denatured, PHMB-complexed MTs allowed the determination of the number of thiolic groups complexed by PHMB. It was found that MTs from rabbit liver have 10.0±0.3 (MT-1) and 6.7±0.3 (MT-2 and MTRL) -SH groups complexed by PHMB. The detection limit (LODc) for PHMB in 95% methanol in the optimized conditions was about 9.3 x 10^-9 mol L-1 and for the denatured MTs LODc was about 8.6 x 10^-10 mol l-1, taking into account an approximate complexating ratio PHMB:MTs of 7:1.

"Multidimensional Analysis Of Denatured Milk Proteins By Hydrophobic Interaction Chromatography Coupled To A Dynamic Surface Tension Detector"
J. Chromatogr. A 2004 Volume 1023, Issue 1 Pages 79-91
Emilia Bramanti, Wes W. C. Quigley, Chandra Sortino, Francesca Beni, Massimo Onor, Giorgio Raspi and Robert E. Synovec

Abstract: Multidimensional analysis of denatured milk proteins is reported using high-performance liquid chromatography (HPLC) combined with dynamic surface tension detection (DSTD). A hydrophobic interaction chromatography (HIC) column (a TSK-Gel(R) Phenyl-5PW column, TosoBiosep), in the presence of 3.0 M guanidine hydrochloride (GdmHCl) as denaturing agent is employed as the mobile phase. Dynamic surface tension is measured through the differential pressure across the liquid-air interface of repeatedly growing and detaching drops. Continuous surface tension measurement throughout the entire drop growth (50 ms to 4 s) is achieved, for each eluting drop of 4 s length, providing insight into both the kinetic and thermodynamic behavior of molecular orientation processes at the liquid-air interface. An automated calibration procedure and data analysis method is applied with the DSTD system, which allows two unique solvents to be used, the HIC mobile phase for the sample and a second solvent (water for example) for the standard, permitting real-time dynamic surface tension data to be obtained. Three-dimensional data is obtained, with surface tension as a function of drop time first converted to surface pressure, which is plotted as a function of the chromatographic elution time axis. Experiments were initially performed using flow injection analysis (FIA) with the DSTD system for investigating commercial single standard milk proteins (α-lactalbumin, β-lactoglobulin, α-, β-, [kappa]-casein and a casein mixture) denatured by GdmHCl. These FIA-DSTD experiments allowed the separation and detection conditions to be optimized for the HIC-DSTD experiments. Thus, the HIC-DSTD system has been optimized and successfully applied to the selective analysis of surface-active casein fractions (αs1- and β-casein) in a commercial casein mixture, raw milk samples (cow's, ewe's and goat's milk) and other diary products (yogurt, stracchino, mozzarella, parmesan cheese and chocolate cream). The different samples were readily distinguished based upon the selectivity provided by the HIC-DSTD method. The selectivity advantage of using DSTD relative to absorbance detection is also demonstrated.

"Selective Determination Of Thiolic Proteins By Hydrophobic Interaction Chromatography Coupled With On-line Cold Vapor Atomic Fluorescence Spectrometry"
J. Anal. At. Spectrom. 2001 Volume 16, Issue 2 Pages 166-171
Emilia Bramanti, Serena Lucchesini, Alessandro D'Ulivo, Leonardo Lampugnani, Roberto Zamboni, Maria Carla Spinetti and Giorgio Raspi

Abstract: A new analytical method is proposed for the determination and characterization of thiolic proteins, based on hydrophobic interaction chromatography (HIC) coupled on-line with cold vapor atomic fluorescence spectrometry (CVAFS). Thiolic groups are derivatized pre-column by p-hydroxymercurybenzoate (PHMB) and the derivatized proteins are separated on a TSKgel Ether-5PW column. Post-column on-line reaction of derivatized proteins with bromine, generated in situ by KBr/KBrO3 in HCl medium, allowed the fast conversion of protein-bound PHMB to inorganic mercury, Hg(ii), which is selectively detected by AFS after sodium borohydride reduction to Hg-0. Under optimized conditions, on-line bromine treatment gives a 85±2% recovery of both free and protein-complexed PHMB in less than 2.5 s and at room temperature. Glyceraldehyde-3-phosphate dehydrogenase, aldolase, pyruvate kinase, trioso phosphate isomerase and phospho-glucose isomerase have been examined. Sensitivity and limit of detection of proteins depends on the number of -SH groups reacting with PHMB and are in the range of 10^-8-10^-9 mol L-1 with calibration curves spanning over four decades of concentration.
Speciation

"Speciation And Quantification Of Thiols By Reversed-phase Chromatography Coupled With On-line Chemical Vapor Generation And Atomic Fluorescence Spectrometric Detection: Method Validation And Preliminary Application For Glutathione Measurements In Human Wh"
Clin. Chem. 2005 Volume 51, Issue 6 Pages 1007-1013
Emilia Bramanti1,a, Cecilia Vecoli2, Danilo Neglia2, Maria Paola Pellegrini2, Giorgio Raspi1 and Renata Barsacchi

Abstract: Background: We developed a sensitive, specific method for the low-molecular-mass thiols cysteine, cysteinylglycine, glutathione, and homocysteine and validated the method for measurement of glutathione in blood. Methods: The technique was based on reversed-phase chromatography (RPC) coupled on line with cold vapor generation atomic fluorescence spectrometry (CVGAFS). Thiols were derivatized before introduction on the column by use of a p-hydroxymercuribenzoate (PHMB) mercurial probe and separated as thiol-PHMB complexes on a Vydac C4 column. Post-column on-line reaction of derivatized thiols with bromine allowed rapid conversion of the thiol-PHMB complexes to inorganic mercury with recovery of 100 (2)% of the sample. Hg(II) was selectively detected by atomic fluorescence spectrometry in an Ar/H2 miniaturized flame after sodium borohydride reduction to Hg0. Results: The relationship between thiol-PHMB complex concentration and peak area (CVGAFS signal) was linear over the concentration range 0.01-1400 ?mol/L (injected). The detection limits were 1, 1, 0.6, and 0.8 nmol/L for cysteine, cysteinylglycine, homocysteine, and glutathione in the injected sample, respectively. The CVs for thiols were 1.5%-2.2% for calibrator solutions and 2.1% and 3.0% for real samples. The RPC-CVGAFS method allowed speciation of glutathione (reduced and oxidized) in human whole blood from healthy donors and from the coronary sinus of patients with idiopathic dilated cardiomyopathy during and after chronotropic stress. Conclusion: The RPC-CVGAFS method could be used to measure reduced and oxidized glutathione in human whole blood as disease biomarkers.

"Determination Of Hydrogen Sulfide And Volatile Thiols In Air Samples By Mercury Probe Derivatization Coupled With Liquid Chromatography-atomic Fluorescence Spectrometry"
Anal. Chim. Acta 2006 Volume 579, Issue 1 Pages 38-46
Emilia Bramanti, Lucia D'Ulivo, Cristina Lomonte, Massimo Onor, Roberto Zamboni, Giorgio Raspi and Alessandro D'Ulivo

Abstract: A new procedure is proposed for the sampling and storage of hydrogen sulphide (H2S) and volatile thiols (methanethiol or methyl mercaptan, ethanethiol and propanethiol) for their determination by liquid chromatography. The sampling procedure is based on the trapping/pre-concentration of the analytes in alkaline aqueous solution containing an organic mercurial probe p-hydroxymercurybenzoate, HO-Hg-C6H4-COO- (PHMB), where they are derivatized to stable PHMB complexes based on mercury-sulfur covalent bonds. PHMB complexes are separated on a C18 reverse phase column, allowing their determination by liquid chromatography coupled with sequential non-selective UV-vis (DAD) and mercury specific (chemical vapor generation atomic fluorescence spectrometry, CVGAFS) on-line detectors. PHMB complexes, S(PHMB)2CH3S-PHMB, C2H5S-PHMB and C3H7S-PHMB, are stable alt least for 12 h at room temperature and for 3 months if stored frozen (-20°C). The best analytical figures of merits in the optimized conditions were obtained by CVGAFS detection, with detection limits (LODc) of 9.7 µg L-1 for H2S, 13.7 µg L-1 for CH3SH, 17.7 µg L-1 for C2H5SH and 21.7 µg L-1 for C3H7SH in the trapping solution in form of RS-PHMB complexes, the relative standard deviation (RSD) ranging between 1.0 and 1.5%, and a linear dynamic range (LDR) between 10 and 9700 µg L-1. Conventional UV absorbance detectors tuned at 254 nm can be employed as well with comparable RSD and LDR, but with LODc one order of magnitude higher than AFS detector and lower specificity. The sampling procedure followed by LC-DAD-CVGAFS analysis has been validated, as example, for H2S determination by a certified gas permeation tube as a source of 3.071±0.154 µg min-1 of H2S, giving a recovery of 99.8±7% and it has been applied to the determination of sulfur compounds in real gas samples (biogas and the air of a plant for fractional distillation of crude oil).

"Flow Injection Analysis With Diode Array Absorbance Detection And Dynamic Surface Tension Detection For Studying Denaturation And Surface Activity Of Globular Proteins"
Anal. Biochem. 2006 Volume 351, Issue 1 Pages 100-113
Emilia Bramanti, Chiara Allegrini, Massimo Onor, Giorgio Raspi, Kristen J. Skogerboe and Robert E. Synovec

Abstract: In this article, a multidimensional dynamic surface tension detector (DSTD), in a parallel configuration with a UV-visible diode array absorbance detector, is presented in a novel flow injection analysis (FIA) application to study the effects of chemical denaturants urea, guanidinium hydrochloride (GdmHCl), and guanidinium thyocyanate (GdmSCN) on the surface activity of globular proteins at the liquid-air interface. The DSTD signal is obtained by measuring the changing pressure across the liquid-air interface of 4-?l drops repeatedly forming at the end of a capillary using FIA. The sensitivity and selectivity of the DSTD signal is related to the surface-active protein concentration in aqueous solution combined with the thermodynamics and kinetics of protein interaction at a liquid-air drop interface. Rapid on-line calibration and measurement of dynamic surface tension is applied, with the surface tension converted into surface pressure results. Continuous surface tension measurement throughout the entire drop growth is achieved, providing insight into kinetic behavior of protein interactive processes at the liquid-air drop interface. Specifically, chemical denaturation of 12 commercial globular proteins-chicken egg albumin, bovine serum albumin, human serum albumin, ?-lactalbumin (?-Lac), myoglobin, cytochrome c, hemoglobin, carbonic anhydrase, ?-chymotrypsinogen A, ?-lactoglobulin (?-LG), lysozyme, and glyceraldehyde-3-phosphate-dehydrogenase-is studied in terms of surface pressure (i.e., surface activity) after treatment with increasing concentrations of urea, GdmHCl, and GdmSCN in the 0-8, 0-6, and 0-5 M ranges, respectively. For several of these proteins, the spectroscopic absorbance changes are monitored simultaneously to provide additional information prior to drop formation. Results show that surface pressure of proteins generally increases as the denaturant concentration increases and that effectiveness is GdmSCN > GdmHCl > urea. Protein unfolding curves obtained by plotting surface pressure as a function of denaturant concentration are presented and compared with respect to unfolding curves obtained by using UV absorbance and literature data. Kinetic information relative to the protein adsorption to the air-liquid interface of two proteins, ?-Lac and ?-LG (chosen as representative proteins for comparison), denatured by the three denaturants is also studied and discussed.

"Study Of The Disulfide Reduction Of Denatured Proteins By Liquid Chromatography Coupled With On-line Cold-vapor-generation Atomic-fluorescence Spectrometry (LC?CVGAFS)"
Anal. Bioanal. Chem. 2004 Volume 380, Issue 2 Pages 310-318
Emilia Bramanti, Cristina Lomonte, Massimo Onor, Roberto Zamboni, Giorgio Raspi and Alessandro D'Ulivo

Abstract: Hydrophobic-interaction chromatography coupled on-line with chemical-vapor-generation atomic-fluorescence spectrometry (HIC-CVGAFS), optimized recently for the analysis of thiol-containing proteins under denaturing conditions, has been used to study the chemical reduction of denatured proteins. Four proteins chosen as models (human serum albumin (HSA), bovine serum albumin (BSA), α-lactalbumin (α-Lac) from bovine milk, and lysozyme from chicken egg (Lys)) were denatured with urea and reduced with dithiothreitol (DTT), with selenol as catalyst. The method is based on derivatization of the -SH groups of proteins with p-hydroxymercurybenzoate (PHMB), followed by HIC separation and post-column on-line reaction of the derivatized reduced, denatured proteins with bromine generated in situ. Hg(II), derived from rapid conversion of uncomplexed and protein-complexed PHMB, is selectively detected by AFS in an Ar/H2 miniaturized flame after sodium borohydride (NaBH4) reduction to Hg(0). The yield of the reduction was studied as a function of reductant concentration, reduction time (tred), and urea concentration. Results showed that the optimum values for DTT and selenol concentrations and for tred were between 1 and 100 mmol L-1 and between 1 and 20 min, respectively, depending on the protein studied. The percentage disulfide bond reduction increases as the urea concentration used for protein denaturation increases, giving a single-step sigmoid increment for single-domain, low-MW proteins (<α-Lac and Lys), and a two-step sigmoid increment for multi-domain, high MW proteins (HSA and BSA). The shapes of plots of percentage reduced disulfide against urea concentration are characteristic of each protein and are correlated with the location of S-S in the protein. Under the adopted conditions complete protein denaturation is the conditio sine qua non for obtaining 100% S-S reduction. The detection limit for denatured, reduced proteins examined under the optimized conditions was found to be in the range 1-5?0^-12 mol L-1 (10-30 pg), depending on the protein considered.