Charge-based separation of proteins and peptides by electrically induced dynamic pH profiles

A new method for generating complex, dynamic pH profiles in an ampholyte-free separation channel is presented together with the theory behind its operation. The pH is modulated by an array of proton and hydroxide ion injectors placed along the separation channel. The ions generated in-situ by electrically driven water splitting across a bipolar membrane are injected to the channel in the presence of a longitudinal electric field, leading to the formation of a multi-step pH profile. Real-time control over the pH profile along the channel facilitates new dynamic separation strategies as well as steering and harvesting of focused molecules, which are both impossible with conventional separation methods. These freedoms are particularly attractive for Lab-on-a-Chip applications. The pH step-like profile alleviates one of the main hurdles of conventional isoelectric separation methods, namely, the slowing down of focused molecules as they approach their focusing spot. As a result, separation is completed within minutes for both peptides and proteins, even with low applied electric fields. We demonstrate protein and peptide separation within minutes, and resolution of ΔpI = 0.2. Novel separation strategies based on spatio-temporal pH control are demonstrated as well.