Separation of structurally related peptides by open-tubular capillary electrochromatography using (metallo)porphyrins as the adsorbed stationary phase

Several (metallo)porphyrins, particularly the porphyrin derivative tetraphenylporphyrin, and complexes of porphyrin derivatives with metal ions (Zn2+, Cu2+, Ni2+, Co2+, Co3+) have been employed as the stationary phase physically adsorbed onto the inner fused-silica capillary surface for open-tubular capillary electrochromatography, and applied for the separation of structurally related peptides. Four octapeptides, derivatives of the B23-B30 fragment of the B-chain of human insulin with minor changes in their sequences (presence of lysine or ornithine in position B-29, presence or absence of phenylacetyl protecting group on the amino group of lysine/ornithine or N-terminal amino group of glycine), were studied as model analytes. Separations were performed both in alkaline (pH 9.0) and in acidic (pH 2.25) background electrolytes, and the changes in the migration/retention behaviour of the model set of peptides were investigated with respect to the porphyrin periphery/central metal atom and the charge of the octapeptides modified. The key moment of successful separation of these peptides seems to be the accessibility of functional groups of the peptides to the interaction with the modifiers tested herein.     

Pre-column chelation liquid chromatographic separation and determination of trace V,Cu, Co,Pd, Fe and Ni

Conditions for the separation by reversed-phase liquid chromatography (LC) of V(V), Cu(II), Co(III), Pd(II), Fe(III) and Ni(II) chelates with 2-(5-bromopyridylazo)-5-diethylaminophenol (5-Br-PADAP) were studied. Six species of metal chelates were separated successfully with methanol-acetonitrile-water (72:12:16, v/v/v) containing 0.13 M NaCl and 0.29 mM cetyltrimethylammonium bromide (pH 5.0) as the mobile phase on a Nucleosil C₁₈ (5 μm) column (250 × 4 mm i.d.).The conditions of the determination of these metal chelates are discussed. A simple and rapid method for the determination of trace amounts of V(V), Cu(II), Co(III), Pd(II) and Ni(II) simultaneously by reversed-phase LC has been developed. The detection limits are 5 × 10^?12, 1 × 10^?10, 3 × 10^?11, 5.3 × 10^?9 and 2 × 10^?10 g, respectively. The method is applied to the determination of these metals in natural waters and mineral samples.     

Dynamic study of metal-ion incorporation into porphyrins based on the dynamic characterization of metal ions and on sitting-atop complex formation.

We succeeded in the detection of the sitting-atop (SAT) copper(II) complex of TPP (5,10,15,20-tetraphenylporphyrin) in acetonitrile (AN) as a solvent with a very low Br?nsted basicity, where two pyrrolenine nitrogens in the Cu(II)-SAT complex coordinate to the metal ion and two protons still remain on the pyrrole nitrogens. The structure parameters around the copper(II) ion in the Cu(II)-SAT complex, as determined by a fluorescent EXAFS method, suggest an axially elongated and equatorially distorted six-coordinate geometry. We measured the rates of the formation reaction of the SAT complexes for a series of transition metal(II) ions in AN using the stopped-flow technique. We propose the mechanism where there is a rapid deformation equilibrium of the porphyrin ring prior to the rate-determining step of the bond rupture of a coordinated solvent molecule on the metal(II) ion. Furthermore, we measured the rates of the deprotonation reaction of the Cu(II)-SAT complex by some Br?nsted bases and indicated that the rate-determining step is the attack of the base on the proton of the pyrrole nitrogen in the SAT complex. Finally, a unified mechanism relevant to the porphyrin metalation mechanism has been proposed.     

Immobilized metal-ion affinity chromatography of peptides on metalloporphyrin stationary phases

The retention of selected dipeptides and tripeptides containing tyrosine was examined. As stationary phase an aminopropylated silica gel loaded with covalently linked tetraphenylporphyrin was used. The effect of metalization of porphyrin with Cu(II) and Zn(II) on retention was investigated. The observed separation is based on a mixed mechanism involving π-π and hydrophobic interactions as well as complex formation between immobilized metal ions and peptides. A satisfactory separation was demonstrated for C-peptide and bovine insulin. The possibility of separation of various insulins was also investigated. Received: 10 August 1998 / Revised: 21 December 1998 / Accepted: 28 December 1998     

CZE of Sulfur-Containing Amino Acids and Peptides and Its Application to the Quantitative Study of Heavy Metal-Caused Thiol Oxidations

The redox-active, sulfur-containing amino acids cysteine and methionine, the tripeptide glutathione and their oxidized counterparts cystine, methionine sulfoxide, and glutathione disulfide were separated as anions by capillary zone electrophoresis (CZE) in a 72?cm long fused silica capillary filled with 100?mM phosphate buffer, pH 8.0, at a voltage of +30?kV in 20?min. The optimized CZE method was suited for the implementation of quantitative metal interaction studies of the biomolecules in a biologically relevant concentration range (μM–mM). Decreasing peak areas of the reduced forms of cysteine and glutathione and simultaneously increasing peak areas of the oxidized forms after incubation of the reduced biomolecules with divalent heavy metal cations indicated redox reactions which could be responsible for toxic metal actions in biological systems. CZE measurements revealed that a 50?% oxidation grade of cysteine was achieved at a molar metal:cysteine ratio of 0.85 in case of Zn(II) addition and of 0.11 in case of Cu(II) addition, respectively. Cu(II) oxidized 50?% of the initial glutathione at a molar Cu:peptide ratio of 0.036, whereas the 50?% oxidation grade was not reached after incubation with Co(II) up to a molar ratio of Co:peptide of 0.25.     

Solid phase extraction of trace metals in water and leafy vegetables using a resin functionalized with potassium 2-benzoylhydrazinecarbodithioate and determination by ICP–AES

A solid phase extraction method for the determination of Cu(II), Mn(II) and Zn(II) metal ions in natural water and leafy vegetable samples by ICP-AES was developed. The method was based on the sorption of metal ions onto Amberlite XAD-16 functionalized with a new chelating ligand potassium 2-benzoylhydrazinecarbodithioate (Amberlite XAD-16-PBHCD) and elution with nitric acid. The optimum experimental conditions for the quantitative sorption of the three metal ions, namely, effect of pH, sample volume, flow rate, concentration of eluent, sorption capacity, kinetics of sorption, and the effect of diverse ions on the sorption of analytes have been investigated. All the metal ions were quantitatively retained by the functionalized resin at pH 5.0 and sorbed metals could be eluted with 2.0?M HNO₃. The detection limits were 5.6, 4.5 and 1.8?µg?L^?1 for Cu(II), Mn(II) and Zn(II), respectively. The developed method was applied for the determination of Cu(II), Mn(II) and Zn(II) in water and leafy vegetable samples.     

Study of immobilized metal affinity chromatography sorbents for the analysis of peptides by on‐line solid‐phase extraction capillary electrophoresis‐mass spectrometry

Several commercial immobilized metal affinity chromatography sorbents were evaluated in this study for the analysis of two small peptide fragments of the amyloid β‐protein (Aβ) (Aβ(1–15) and Aβ(10–20) peptides) by on‐line immobilized metal affinity SPE‐CE (IMA‐SPE‐CE). The performance of a nickel metal ion (Ni(II)) sorbent based on nitrilotriacetic acid as a chelating agent was significantly better than two copper metal ion (Cu(II)) sorbents based on iminodiacetic acid. A BGE of 25 mM phosphate (pH 7.4) and an eluent of 50 mM imidazole (in BGE) yielded a 25‐fold and 5‐fold decrease in the LODs by IMA‐SPE‐CE‐UV for Aβ(1–15) and Aβ(10–20) peptides (0.1 and 0.5 μg/mL, respectively) with regard to CE‐UV (2.5 μg/mL for both peptides). The phosphate BGE was also used in IMA‐SPE‐CE‐MS, but the eluent needed to be substituted by a 0.5% HAc v/v solution. Under optimum preconcentration and detection conditions, reproducibility of peak areas and migration times was acceptable (23.2 and 12.0%RSD, respectively). The method was more sensitive for Aβ(10–20) peptide, which could be detected until 0.25 μg/mL. Linearity for Aβ(10–20) peptide was good in a narrow concentration range (0.25–2.5 μg/mL, R² = 0.93). Lastly, the potential of the optimized Ni(II)‐IMA‐SPE‐CE‐MS method for the analysis of amyloid peptides in biological fluids was evaluated by analyzing spiked plasma and serum samples.     

Preparation and characterization of monoliths covalently bonded chelating groups for capillary electrochromatographic separation of metal ions

In this work, a novel polymer-based monolithic column was prepared using an o-phthalaldehyde-l-phenylalanine Schiff base complex as the reactive center and a mixture of methanol and n-propanol as the porogen. The monolithic column was employed for the separation of a metal ion mixture including Pb(II), Mn(II), Cu(II), Ni(II), Cr(III), Fe(III) and Cr(VI). Tetrabutylammonium bromide (TBAB) was used as a mobile phase additive to enhance the separation efficiency of metal ions by EDTA precomplexation. Using a phosphate buffer (20 mM, pH 3.0), TBAB (10 mM), MeOH (15%, v/v), an applied voltage of −15 kV, and detection at 220 nm, the metal ion mixture was satisfactorily resolved. The average theoretical plate number was 17,900 plates/m. The separation was also carried out in the absence of TBAB, leading to dissimilar elution order and shorter retention time. The separation behavior of the monolithic column was also compared with that of the blank polymer. The unique properties of the monolithic column might be mediated by a combination of electrophoretic behavior and chromatographic retention involving hydrophobic and hydrophilic interactions, as well as ligand exchange.     

Ion-interaction chromatography of several metallocyanide complexes stabilized by adding cyanide in a neutral mobile phase.

Standard solutions (at 10(-5) M levels) of Cu(I)- and Fe(II)-cyanide complexes were stabilized for at least 5 h using 0.5 mM cyanide solution (around pH 9) as a medium. Complexes of Cu(I)- and Fe(III)-cyanide also could be stabilized without any dissociation by adding 1 mM cyanide to an acetonitrile-water (18:82, v/v) mobile phase (pH 7.0) containing 10 mM tetra-n-propylammonium salt (TPA). Under the optimal conditions, the six complexes of Cu(I)-, Ag(I)-, Ni(II)-, Fe(II)-, Fe(III)- and Au(I)-cyanides were resolved from their mixtures within about 45 min, with well-shaped chromatographic peaks.     

Solubility of Tetra(pyrid-3- and 4-yl)porphines and Their Complexes with <Emphasis Type="Italic">d</Emphasis>-Metals in Chloroform and Ethanol

The solubility of 5,10,15,20-tetra(pyrid-3- and 4-yl)porphine isomers and their coordination compounds with d-metals in the temperature range of 298–318 K is studied. Patterns of the dissolving of porphyrin ligands and the metal complexes they form with Co(II), Cu(II), and Zn(II) in chloroform and ethanol depending on the nature of the metal and the position of the nitrogen atom in the pyridyl substituent of the porphyrin molecule are discussed. The thermodynamic parameters of dissolution are calculated for the investigated compounds.     

N-confused porphyrin-bearing meso-perfluorophenyl groups: a potential agent that forms stable square-planar complexes with Cu(II) and Ag(III)

[reaction: see text] N-Confused porphyrin (NCP) bearing pentafluorophenyl groups at meso-positions, which were obtained from N-confused dipyrromethane in ca. 20% yield, can form Cu(II) complex as well as Ag(III), Ni(II), and Pd(II) complexes. The square-planar structures of all these metal complexes were elucidated by X-ray single-crystal analyses.     

Core modified meso-aryl corrole: first examples of CuII, NiII, CoII and RhI complexes

A variety of metal complexes of 5,10,15-triphenyl-21-monooxa-corrole 4 have been investigated. This monooxa corrole, where one of the pyrrole ring is replaced by a furan moiety, is synthesized by the alpha-alpha coupling reaction of 16-oxa tripyrrane and dipyrromethane. The single crystal X-ray structure of 4 indicates only small deviation of the inner-core heteroatoms from planarity and this macrocycle arrange themselves into a columnar structure. Insertion of metals further flattens the corrole framework. Specifically, oxacorrole 4 binds to Nil(II), Cu(II), and Co(II) with the participation of all heteroatoms in the coordination. However, Rh(I) ion binds to only one imino and one amino nitrogen of the macrocycle. The bond angles at the metal center in the Ni(II) and Rh(I) complexes reveal square planar geometry completed by two CO molecules for Rh(I). The EPR spectra of the paramagnetic that Cu(II) and Col(II) complexes display significant decreases in the metal hyperfine couplings compared with the corresponding porphyrin complexes. The presence of superhyperfine coupling in the Cu(II) complex suggests delocalization of unpaired electron density into the ligand orbitals. Electrochemical studies reveal easier oxidations and harder reductions relative to the corresponding porphyrin derivatives while, the metallated derivatives did not show their characteristic metal reductions due to the high energy of their LUMO.     

Absorption spectroscopic and ESR studies on one-electron reduction products of copper(II) and oxovanadium(IV) tetraphenylporphyrins produced by γ-radiolysis in 2-methyltetrahydrofuran solutions at 77 K

Optical absorption spectra of one-electron reduced species of copper(II) and oxovanadium(IV) tetraphenylporphyrins. Cu(II)TPP and V(IV)OTPP, in 2-methyltetrahydrofuran at 77 K reveal that not the central metal but the porphyrin ligand is reduced by an excess electron. The triplet ESR spectrum resulting from the spin-spin interaction between two odd electrons located on the porphyrin ligand and the central metal is observed for the one-electron reduced species of V(IV)OTPP while not for that of Cu(II)TPP.     

Studies on the Complexation of Transition Metal Ions with Macrocyclic Compounds in Mixed Solvents by Competitive Potentiometry and Polarography

Complexation of Cu(II), Zn(II), Ni(II), Co(II), Pb(II), Cd(II), Cr(III) and Fe(III) ions with 15-crown-5, benzo-15-crown-5, 18-crown-6, dibenzo-18-crown-6, dicyclohexano-18-crown-6, dibenzo-24-crown-8, 5,6,14,15-dibenzo-1,4-dioxa-8,12-diazacyclopentadeca-5,14-diene, 1,4,10-trioxa-7,13-diazacyclopentadecane and cyclam has been investigated in 75% (v/v) DMF+water using the silver(I) ion as an auxiliary cation at 0.05?M ionic strength adjusted with tetrabutylammonium perchlorate (TBAP) by competitive potentiometry. Stability constant values obtained for the metal ion–aza macrocyclic complexes are higher than those for the oxa crowns. Shifts in the peak potential and reduction in the peak current in the differential pulse polarography (DPP) method were also used to determine the stability constants of some of the metal ions with aza and tosylated-aza macrocycles using TBAP as supporting electrolyte in 75% (v/v) DMF+water and 90% (v/v) DMSO+water media. Stability constant values determined both by shifts in the potential and by reduction in the peak current were found to be in good agreement with each other.     

Histidine-rich branched peptides as Cu(II) and Zn(II) chelators with potential therapeutic application in Alzheimer's disease

Two histidine-rich branched peptides with one lysine as a branching unit have been designed and synthesized by solid-phase peptide synthesis. Their complex formation with Cu(II) and Zn(II) as well as their ability to attenuate the metal-ion induced amyloid aggregation has been characterized. Both peptides can keep Cu(II) and Zn(II) in complexed forms at pH 7.4 and can bind two equivalents of metal ions in solutions with excess metal. The stoichiometry, stability and structure of the complexes formed have been determined by pH potentiometry, UV-Vis spectrophotometry, circular dichroism, EPR and NMR spectroscopy and ESI-MS. Both mono- and bimetallic species have been detected over the whole pH range studied. The basic binding mode is either a tridentate {N(amino), N(amide), N(im)} or a histamine-type of coordination which is complemented by the binding of far imidazole or amino groups leading to macrochelate formation. The peptides were able to prevent Cu(II)-induced Aβ(1-40) aggregation but could not effectively compete for Zn(II) in vitro. Our results suggest that branched peptides containing potential metal-binding sites may be suitable metal chelators for reducing the risk of amyloid plaque formation in Alzheimer's disease.     

Solid phase extraction and diffusive gradients in thin films techniques for determination of total and labile concentrations of Cd(II), Cu(II), Ni(II) and Pb(II) in Black Sea water

Total dissolved and labile concentrations of Cd(II), Cu(II), Ni(II) and Pb(II) were determined at six locations of the Bourgas Gulf of the Bulgarian Black Sea coast. Solid phase extraction procedure based on monodisperse, submicrometer silica spheres modified with 3-aminopropyltrimethoxysilane followed by the electrothermal atomic absorption spectrometry (ETAAS) was developed and applied to quantify the total dissolved metal concentrations in sea water. Quantitative sorption of Cd, Cu, Ni and Pb was achieved in the pH range 7.5–8, for 30?min, adsorbed elements were easily eluted with 2?mL 2?mol?L^?1 HNO₃. Since the optimal pH for quantitative sorption coincides with typical pH of Black Sea water (7.9–8.2), on-site pre-concentration of the analytes without any additional treatment was possible. Detection limits achieved for total dissolved metal quantification were: Cd 0.002?µg?L^?1, Cu 0.005?µg?L^?1, Ni 0.03?µg?L^?1, Pb 0.02?µg?L^?1 and relative standard deviations varied from 5–13% for all studied elements (for typical Cd, Cu, Ni and Pb concentrations in Black Sea water). Open pore diffusive gradients in thin films (DGT) technique was employed for in-situ sampling and pre-concentration of the sea water and in combination with ETAAS was used to determine the proportion of dynamic (mobile and kinetically labile) species of Cd(II), Cu(II), Ni(II) and Pb(II) in the sea water. Obtained results showed strong complexation for Cu and Pb with sea water dissolved organic matter. The ratios between DGT-labile and total dissolved concentrations found for Cu(II) and Pb(II) were in the range 0.2–0.4. For Cd and Ni, these ratios varied from 0.6 to 0.8, suggesting higher degree of free and kinetically labile species of these metals in sea water.     

Use of tube radial distribution of ternary mixed carrier solvents for introduction of absorption reagent for metal ion separation and online detection into capillary

When ternary mixed solvents consisting of water-hydrophilic/hydrophobic organic solvents are fed into a micro-space under laminar flow conditions, the solvent molecules are radially distributed in the micro-space. The specific fluidic behavior of the solvents is called the "tube radial distribution phenomenon (TRDP)". A novel capillary chromatography method was developed based on the TRDP that creates the inner major and outer minor phases in a tube, where the outer phase acts as a pseudo-stationary phase. This is called "tube radial distribution chromatography (TRDC)". In this study, Chrome Azurol S as an absorption reagent was introduced into the TRDC system for metal ion separation and online detection. The fused-silica capillary tube (75 μm id and 110 cm length) and water-acetonitrile-ethyl acetate mixture (3:8:4 volume ratio) including 20 mM Chrome Azurol S as a carrier solution were used. Metal ions, i.e. Co(II), Cu(II), Ni(II), Al(III), and Fe(III), as models were injected into the present TRDC system. Characteristic individual absorption characteristics and elution times were obtained as the result of complex formation between the metal ions and Chrome Azurol S in the water-acetonitrile-ethyl acetate mixture solution. The elution times of the metal ions were examined based on their absorption behavior; Co(II), Ni(II), Al(III), Fe(III), and Cu(II) were eluted in this order over the elution times of 4.7-6.8 min. The elution orders were determined from the molar ratios of metal ion to Chrome Azurol S and Irving-Williams series for bivalent metal ions.     

Cloud point extraction preconcentration for capillary electrophoresis of metal ions

The application of the cloud point extraction (CPE) technique for capillary electrophoresis (CE) determination of metal ions was demonstrated using Cu(II) and Co(II) as model metal ions. The preconcentration of Cu(II) and Co(II) in aqueous solution was achieved by CPE with 1-(2-pyridylazo)-2-naphthol (PAN) as the chelating agent and Triton X-114 as the extractant. Baseline separation of the PAN chelates of Cu(II) and Co(II) was realized by CE with a photodiaode array detector in a  μm i.d. fused-silica capillary at 17 kV. A 50 mM NH₄Ac buffer solution (pH 8.0) containing 0.2 mM of PAN in 80% (v/v) of acetonitrile and 20% (v/v) doubly deionized water (DDW) was used as the separation medium to avoid the adsorption of hydrophobic substances and nonionic surfactant Triton X-114 onto the inner surface of the separation capillary, ensuring the separation efficiency and reproducibility. The precision (relative standard deviation (R.S.D.), n=5) for five replicate injections of a mixture of 20 μg/l of Co(II) and Cu(II) were 0.74 and 1.8% for the migration time, 3.1 and 0.64% for the peak area measurement, respectively. The apparent concentration factor, which is defined as the concentration ratio of the analyte in the final diluted surfactant-rich extract ready for CE separation and in the initial solution, was 15.9 for Co(II) and 16.3 for Cu(II). The linear concentration range was from 3 to 100 μg/l for both Co(II) and Cu(II). The detection limits of Co(II) and Cu(II) were 0.12 and 0.26 μg/l, respectively. The developed method was successfully applied to the determination of Co(II) and Cu(II) in tap water, snow water, and flavor wines.     

Coordination polymers of glutaraldehyde with glycine metal complexes: synthesis,spectral characterization,and their biological evaluation

Glycine metal complexes were prepared by the reaction of glycine with Mn(II), Co(II), Ni(II), Cu(II), and Zn(II) in 1?:?2 molar ratio. Thereafter their condensation polymerization was done with glutaraldehyde to obtain polymer metal complexes. All the synthesized polymer metal complexes were characterized by elemental analysis, FT-IR, ¹H-NMR, and UV-Vis spectrometry, magnetic susceptibility, and thermogravimetric studies. The analytical data of all the polymers agreed with 1?:?1 molar ratio of metal complex to glutaraldehyde and magnetic moment data suggest that PGG–Mn(II), PGG–Co(II), PGG–Ni(II), and PGG–Cu(II) have an octahedral geometry around the metal atom, whereas the tetrahedral geometry was proposed for PGG–Zn(II) polymer. The PGG–Mn(II) and PGG–Cu(II) showed octahedral geometry. Thermal behavior of the polymer metal complexes was obtained at a heating rate of 10°C?min^?1 under nitrogen atmosphere from 0°C to 800°C. The antimicrobial activities of synthesized polymers were investigated against Streptococcus aureus, Escherichia coli, Bacillus sphaericus, Salmonella sp. (Bacteria), Fusarium oryzae, Candida albicans, and Aspergillus niger (Yeast).     

Capillary isotachophoretic determinations of metal ions by use of complexation equilibria in acetone-water medium

The determination of metal ions by capillary isotachophoresis and the complexation equilibria between metal ions and polyaminopolycarboxylic acids has been investigated. A seven-component mixture of metal ions can be separated in 45% v/v acetone-water medium when EDTA or DCTA is used as the terminating ion. Linear calibration graphs are obtained for a standard mixture of Mn(+), Cu(2+), Zn(2+), Cd(2+), Pb(2+) and Fe(3+) in the range 0.5-5.0 nmole, with relative standard deviations of 1.0% or better. The effective mobilities of the Mn(II), Co(II), Ni(II), Cu(II) and Zn(II) complexes increase in parallel with the stability constants, except for the Cu(II) complexes. It is concluded that the abnormal behaviour of the Cu(II) complexes may be attributed to a difference in steric configuration.