2.1. pH gradient chromatofocusing

Figure ​Figure1A1A shows the principle of pH gradient chromatofocusing of proteins. When protein mixture flows through a column where positively charged particles are packed, proteins bind onto the particles because they are negatively charged under the chosen pH conditions (Fig. ​(Fig.1A1A (a)) 21, 39, 40. The bound proteins are released from the particles when there is no net electrical force between proteins and particles, which depends on their isoelectric point in combination with the pH at the location of the particles. Since the isoelectric points of different proteins are different, proteins bound to the particles can be separately eluted by controlling the pH of elution buffer (Fig. ​(Fig.1A(b)).1A(b)). The selective elution of pH gradient chromatofocusing starts from the high pH, and the pH value of elution buffer decreases linearly (linear gradient elution) or gradually (step gradient elution). Therefore the protein which has the highest isoelectric point will be eluted at first and selective elution of proteins based on their pI will continue. Figure ​Figure1B1B shows a conventional experimental setup for pH gradient chromatofocusing. The setup includes an anion exchange column, pumping systems for a crude sample and two pH buffer solutions, an a fractionation unit. The process flows through sample loading, pH gradient generation by mixing high pH buffer and low pH buffer, and elution. The microfluidic pH gradient chromatofocusing device adapts and scales down the complete process, and consists of a pH gradient generator, a micro‐sized anion exchange column, and fraction collectors. The microcolumn is created by sieving anion exchange particles from a particle suspension. Since the volume of the column is nanoliter‐scale (20 nL), the separation process is fast and sample consumption is extremely low compared with the conventional method.

(A) Principle of pH gradient chromatofocusing. (B) Conventional experimental setup for pH gradient chromatofocusing. (C) A microfluidic method to separate proteins based on pH gradient chromatofocusing.