Enhancing separation of histidine from amino acids via free-flow affinity electrophoresis with gravity-induced uniform hydrodynamic flow

In this paper, a novel mode of free-flow affinity electrophoresis (FFAE) was developed to indirectly enhance the separation of free-flow electrophoresis (FFE). In the mode of FFAE, a Ni(II) with high electric charge density and histidine (His) is chosen as a model ligand and target solute, respectively. Through the controlling of experimental conditions (10 mM pH 6.0 Na 2 HPO 4 –NaH 2 PO 4 with 2.0 mM NiCl 2 ·6H 2 O background buffer), Ni(II) can combine with His and the combination leads to the high electric charge density of affinity complex of His-Ni(II) in contrast to the low density of free His molecule. But the ligand has weak interaction with uninterested amino acids. Thus, the mobility of His existing as His-Ni(II) is greatly increased from 14.5×10 −8 m 2 V −1 s −1 to 30.2 × 10 −8 m 2 V −1 s −1 , while those mobilities of uninterested amino acids are almost constant. By virtue of the mode, we developed the FFAE procedure and conducted the relevant experiments. The experiments demonstrated the following merits of the FFAE technique: (i) clear enhancement of separation between the target solute of His and uninterested amino acids; (ii) simplicity, and (iii) low cost. Furthermore, the technique was used for the continuous separation of His from its complex sample, and the purity of His was near to 100%. All of the results demonstrate the feasibility of affinity separation in FFE. The developed FFAE may be used in the separation and pretreatment of some biological molecules (e.g. peptides).