RESUMEN
A preparative scale free-flow IEF device is developed and characterized with the aim of addressing needs of molecular biologists working with protein samples on the milligrams and milliliters scale. A triangular-shape separation channel facilitates the establishment of the pH gradient with a corresponding increase in separation efficiency and decrease in focusing time compared with that in a regular rectangular channel. Functionalized, ion-permeable poly(acrylamide) gel membranes are sandwiched between PDMS and glass layers to both isolate the electrode buffers from the central separation channel and also to selectively adjust the voltage efficiency across the separation channel to achieve high electric field separation. The 50 x 70 mm device is fabricated by soft lithography and has 24 outlets evenly spaced across a pH gradient between pH 4 and 10. This preparative free-flow IEF system is investigated and optimized for both aqueous and denaturing conditions with respect to the electric field and potential efficiency and with consideration of Joule-heating removal. Energy distribution across the functionalized polyacrylamide gel is investigated and controlled to adjust the potential efficiency between 15 and 80% across the triangular separation channel. The device is able to achieve constant electric fields high as 370+/-20 V/cm through the entire triangular channel given the separation voltage of 1800 V, enabling separation of five fluorescent pI markers as a demonstration example.
Asunto(s)
Focalización Isoeléctrica , Técnicas Analíticas Microfluídicas , Resinas Acrílicas , Compuestos Azo/química , Compuestos Azo/aislamiento & purificación , Azul de Bromofenol/química , Azul de Bromofenol/aislamiento & purificación , Dimetilpolisiloxanos/química , Diseño de Equipo , Colorantes Fluorescentes/química , Colorantes Fluorescentes/aislamiento & purificación , Concentración de Iones de Hidrógeno , Focalización Isoeléctrica/instrumentación , Focalización Isoeléctrica/métodos , Técnicas Analíticas Microfluídicas/instrumentación , Técnicas Analíticas Microfluídicas/métodos , Nylons/química , Presión , Urea/químicaRESUMEN
Dynamic field gradient focusing uses an electric field gradient generated by controlling the voltage profile of an electrode array to separate and concentrate charged analytes according to their individual electrophoretic mobilities. This study describes a new instrument in which the electrodes have been placed within the separation channel. The major challenge faced with this device is that when applied voltages to the electrodes are larger than the redox potential of water, electrolysis will occur, producing hydrogen ions (H+) plus oxygen gas on the anodes and hydroxide (OH(-)) plus hydrogen gas on the cathodes. The resulting gas bubbles and pH excursions can cause problems with system performance and reproducibility. An on-column, degassing system that can remove gas bubbles "on-the-fly" is described. In addition, the use of a high capacity, low-conductivity buffer to address the problem of the pH shift that occurs due to the production of H+ on the anodes is illustrated. Finally, the successful separation of three, low-molecular-weight dyes (amaranth, bromophenol blue and methyl red) is described.
Asunto(s)
Focalización Isoeléctrica/instrumentación , Focalización Isoeléctrica/métodos , Colorante de Amaranto/aislamiento & purificación , Compuestos Azo/aislamiento & purificación , Azul de Bromofenol/aislamiento & purificación , Electrodos , Colorantes Fluorescentes/química , Gases , Concentración de Iones de Hidrógeno , Peso Molecular , AguaRESUMEN
Electrophoretic field gradient focusing has been used to separate the two oxidation states of myoglobin (Mb), and to separate Mb from bromophenol blue (BPB). Polyacrylamide and Sephadex were shown to be suitable packing materials whilst silica led to band broadening with Mb. BPB and Mb could be simultaneously focused apart using either a fixed 21-electrode setup or a dynamic 6-electrode setup. Using a dynamic three-electrode setup either analyte could be focused but not both simultaneously. It was shown that a higher ionic strength buffer in the separation channel compared to the coolant channel enhanced focusing between electrodes due to a conductivity gradient. Different running buffers were investigated and it was found that using a pH 8.6 buffer containing N,N,N-tris(hydroxymethyl)aminomethane (Tris) and phosphate ions the oxidation states of Mb could be separated but the separation of Mb from BPB was not as good as would be hoped for. Using a pH 8.6 buffer containing Tris, N-2-hydroxyethylpiperazine-N'-3-propanesulphonate and chloride ions as running buffer, BPB and Mb could be well separated but the two oxidation states of Mb merged.
Asunto(s)
Azul de Bromofenol/aislamiento & purificación , Focalización Isoeléctrica/métodos , Mioglobina/aislamiento & purificación , Focalización Isoeléctrica/instrumentación , Mioglobina/química , Oxidación-ReducciónRESUMEN
Laboratory investigations of the potential use of dried biomasses of Rhizopus stolonifer, Fusarium sp., Geotrichum sp., and Aspergillus fumigatus as biosorbents for the removal of bromophenol blue (BPB) dye from aqueous solutions were conducted. Kinetics studies indicated that the BPB dye uptake processes can be well described by the pseudo-second-order model. The fungal biomasses exhibited the highest dye biosorption at pH 2.0. The Langmuir adsorption model appears to fit the dye biosorption better than the Freundlich model, with maximum dye uptake capacities ranging from 526 to 1,111 mg/g, depending on the biomass used.