Effects of Ampholyte Dissociation Constants on Protein Separation in On-Chip Isoelectric Focusing
Abstract:Numerical simulations are presented for ampholyte-based isoelectric focusing in 2D microgeometries. In this study, model proteins are focused in the presence of 25 biprotic ampholytes under an applied electric field. Each protein is considered as a simple polypeptide having ten charge states, while the biprotic ampholytes are selected to generate a shallow pH range of 6 to 9. Straight and contraction-expansion microchannels are considered here, and a nominal electric field of 300 V/cm is maintained for separation of proteins. Six distinct values of ΔpKs between 1 and 3.5 are investigated for ampholytes to form pH profiles in a 1 cm long microchannel. Simulation results show that relatively larger values of ΔpK (ΔpK > 3 are required to form stepless pH profiles in the system. The peak heights and differential resolutions of focused proteins are much higher for lower values of ΔpK for which a stepped pH profile is evident. For each protein, the time it takes for the two edges of a peak to merge increases linearly with ΔpK, while the focusing time goes up exponentially with increasing ΔpK. Both merging and focusing times of protein are higher for contraction-expansion microchannel than those of straight microchannel. For a particular value of ΔpK, the contracted "Zoom" region of contraction-expansion channel is able to form more tightly focused bands than the expanded region.
Document Type: Research Article
Publication date: 2008-07-01
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