Isoelectric Split-Flow Thin (SPLITT) Fractionation of Proteins

Abstract Electric split-flow thin (SPLITT) fractionation permits the continuous separation of charged species, particularly proteins, at gram and subgram levels. We characterized this system and separated protein mixtures based on the difference between protein isoelectric points (pI). For characterization, we examined seven variables. Buffer stability was determined by measuring pH changes per hour and the UV spectrum before and after an electrical potential of 50 V was applied. The electrical field across the channel was determined by measuring the buffer conductivity and the current passed through it. The experimental and theoretical (calculated) fractional retrieval of proteins was determined by the relative magnitude of field-induced and outlet flow rates. The protein response at various electrical fields (0, 10, 20, 30 V) and solution pHs (4.85, 5.60, 6.87, and 7.80) was examined, as were the effects of the ionic strength of the buffer, protein recovery, and protein separation with pulsed sample inj...

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