RESUMO
We report a new method of immobilization of gold nanoparticles (AuNPs) on a fused-silica capillary through covalent binding. The resulting modified capillary was applied to electrophoretic systems to improve the efficiency of separation and the selectivity of selected solutes. The immobilization of AuNPs on the capillary wall was performed in a very simple and fast way without requiring heating. The surface features of an AuNP-coated capillary column were determined using the scanning electron microscopy. The chromatographic properties of AuNP-coated capillaries were investigated through variation of the buffer pH and separation voltage. Effective separations of synthetic peptides mixture were obtained on the AuNP-coated capillaries. The method shows a remarkable stability since it was reused about 900 times. The capacity factor was duplicated. Therefore, this modification is stable and can be applied to different separation purposes. A complex mixture of tryptic peptide fragments of HSA was analyzed in both the bare- and the AuNP-coated capillaries. Better electrophoretic peptide profile was observed when using the AuNP-coated capillary.
Assuntos
Eletrocromatografia Capilar/instrumentação , Eletrocromatografia Capilar/métodos , Ouro/química , Nanopartículas Metálicas/química , Peptídeos/análise , Proteínas/análise , Humanos , Concentração de Íons de Hidrogênio , Microscopia Eletrônica de Varredura , Fragmentos de Peptídeos/análise , Fragmentos de Peptídeos/isolamento & purificação , Peptídeos/isolamento & purificação , Proteínas/isolamento & purificação , Espectrofotometria Ultravioleta , Tripsina/químicaRESUMO
The inner surface of fused silica capillaries has been covalently modified with different porphyrins (deuteroporphyrin, complexes of deuteroporphyrin with metal ions Fe(III), Cu(II), Zn(II), Ni(II), and Cu(II)-meso-tetra (carboxyphenyl) porphyrin) and it was applied for the separation of biologically active peptides by open-tubular capillary electrochromatography. Separations were performed in a mobile phase composed of 25 mM potassium phosphate, pH 4.0, 5% v/v ACN and 10 mM hydroquinone. Changes in the effective electrophoretic mobility of peptides were studied concerning porphyrin central metal atom, attachment geometry, and the presence of coordinating or aromatic amino acid residues in the peptide sequence. The results showed that differences in metal core on the porphyrin and the spatial conformation of attached porphyrin result in changes in the analyte interaction with the stationary phase.
Assuntos
Eletrocromatografia Capilar/instrumentação , Deuteroporfirinas/química , Mesoporfirinas/química , Metais Pesados/química , Neuropeptídeos/análise , Eletrocromatografia Capilar/métodos , Modelos Moleculares , Neuropeptídeos/química , Estatísticas não ParamétricasRESUMO
The separation of seven biologically active peptides was attempted by open-tubular capillary electrochromatography in fused-silica capillaries chemically modified with iron (III)-deuteroporphyrin using UV-absorption detection at 214 nm. The effect of BGE pH and content of organic solvent modifier was investigated. The best separations were obtained in 25 mM phosphate (BGE), pH 4.0, containing 5% v/v ACN and 10 mM hydroquinone, which was added to prevent gas bubble formation. Considering the method sensitivity, lower concentration LODs were obtained for all peptides in their open-tubular capillary electrochromatography separation as compared with their CZE separation in bare fused-silica capillary. The iron (III)-deuteroporphyrin column proved to be highly stable over time and showed acceptable precision of migration times and corrected peak areas (RSD in the range 2-4%).
Assuntos
Eletrocromatografia Capilar/métodos , Deuteroporfirinas/química , Compostos Férricos/química , Peptídeos/isolamento & purificação , Acetonitrilas/química , Eletrocromatografia Capilar/instrumentação , Eletrólitos/química , Humanos , Concentração de Íons de Hidrogênio , Modelos Lineares , Fragmentos de Peptídeos/análise , Fosfatos/química , Compostos de Potássio/química , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Albumina Sérica/análise , Tripsina/metabolismoRESUMO
An on-line preconcentration method using a polymeric monolithic support is proposed for the retention of the decapeptide angiotensin I and its subsequent analysis by CZE. Monolithic capillary columns were prepared in fused-silica (FS) capillaries of 150 microm id by ionizing radiation-initiated in situ polymerization and cross-linking of diethylene glycol dimethacrylate and glycidyl methacrylate, and chemically modified with iron protoporphyrin IX (Fe-ProP). Monolithic microcolumns (8 mm long) were coupled on-line to the inlet of the separation capillary (FS capillary, 75 microm id x10 cm from the inlet to the microcolumn and 27 cm from the microcolumn to the detector). Angiotensin I was released from the sorbent by a 50 mM sodium phosphate, pH 2.5/ACN, 75:25 v/v solution and then analyzed by CZE with UV absorption detection at 214 nm. The concentration LOQ (CLOQ) was 0.5 ng/mL. The Fe-ProP-derivatized monolithic microcolumn coupled to the separation capillary exhibited a high retention capacity for peptide angiotensin I, and showed as much as 10,000-fold improvement in concentration sensitivity.