PubMed · 16802458
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Nicole Branan. 2006-06-01. Microfluidic IEF without carrier molecules.. https://doi.org/10.1021/ac069414m
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We have developed a high-throughput, two-dimensional-mapping (isoelectric point [pI], mass-to-charge ratio [m/z]) method by combining a capillary isoelectric focusing chip sealed with removable resin tape and a matrix-assisted laser desorption/ionization time-of-flight mass spectrometer. Sample proteins are separated in a meandering channel on the chip and immediately frozen. The tape is then removed and the proteins are freeze-dried. The freeze-drying maintains the separation state of the proteins and prevents movement of the sample solution, which can reduce pI resolution. A matrix solution is then applied and mass spectrometry is carried out by laser irradiation. The whole process takes less than 70 min, more than 10 times faster than with two-dimensional, polyacrylamide gel electrophoresis.
Electric field gradient focusing (EFGF) is a relatively new separation technique with promising attributes, particularly for protein analysis. The fundamental fractionation mechanism in EFGF involves a gradient in electric field along the length of a separation column. The electrophoretic force that drives charged analytes in a region of changing electric field is opposed by a constant, pressure-driven bulk fluid flow. When the electrophoretic velocity of a particular moiety is equal and opposite to the velocity of the fluid flow, the analyte focuses into a stationary band. Thus, EFGF can both concentrate and separate charged species according to electrophoretic mobility. To date, the electric field gradients needed for EFGF have been established using a number of different approaches, including channels having changing cross-sectional areas, conductivity gradients caused by the diffusion of buffer ions across a membrane, electrode arrays, and temperature gradients in buffers whose conductivities change as a function of temperature. EFGF has proven particularly effective for sample enrichment, with concentration factors of 10,000 reported. In this article we review advances in EFGF technology and discuss prospects for further improving EFGF for chemical analysis.
The typically low aqueous solubilities of small, hydrophobic organic ampholytic molecules limit the production rates that can be achieved in their isoelectric trapping (IET) separations and call for the use of hydro-organic mixtures as solvents. The compatibility of methanol-water mixtures and poly(ethylene terephthalate) substrate-supported isoelectric polyacrylamide hydrogels, developed for binary IET separations in a Gradiflow BF200IET unit, was investigated. The isoelectric polyacrylamide-based hydrogels retained their functional and mechanical integrities when the methanol concentration in the hydro-organic solvent mixture was kept at or below 25% (v/v). The utility of the hydro-organic media was demonstrated in the purification of a hydrophobic ampholytic compound, technical grade 4-hydroxy-3-(morpholinomethyl) benzoic acid. Production rates as high as 7 mg/h were achieved using small, 15 cm2 active surface area isoelectric membranes.