Enhancing Separation Of Histidine From Amino Acids Via Free-Flow Affinity Electrophoresis With Gravity-Induced Uniform Hydrodynamic Flow

Two fundamental issues of scarcity of separation modes and much low separation resolution have been present in free-flow electrophoresis (FFE) for decades. Herein, a novel mode of free-flow affinity electrophoresis (FFAE) was developed to enrich the modes of FFE and indirectly enhance the resolution of FFE. In the mode of FFAE, a Ni(II) with high electric charge density was used as a model ligand and histidine (His) as a target solute. Through the controlling of experimental conditions, 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 as compared with those of uninterested amino acids. With the mode, we developed the procedure of FFAE and performed the relevant experiments.Experiments were divided into two parts, the first part was the design of capillary zone electrophoresis simulation software for predicting the separation efficiency of twenty amino acids under different conditions, which was used to determine the valuable target amino acids to be separated for the following research. The introduction of this simple, practical, time-saving modern technique is one of the highlight of this article. Then the following work was focused on the separation conditions in capillary electrophoresis to optimize the conditions of free-flow affinity electrophoresis.The second part was the verification of the feasibility of free-flow affinity electrophoresis and application in purity of amino acids. In this section, two kinds of free-flow electrophoresis, capillary electrophoresis detection, amino acid analysis and other experiments have been conducted. At last, through the comparison of the separation efficiency between the common free flow zone electrophoresis and free-flow affinity electrophoresis affinity, it is undifficult to obtain the advantages and potential applications of this novel mode of free-flow electrophoresis.The experiments demonstrated that (1) the technique could greatly enhance the separation between the target solute of His and uninterested amino acids; (2) the technique could selectively separate His from its complex matrix; (3) the FFAE technique was used for the continuous separation of His from its complex sample, and the purity of His was near to 100%. The technique of FFAE developed herein has obvious application potential for the separation of important target biological molecules (e.g., protein, peptide and nucleic acid).