| Free-flow electrophoresis (FFE) is a continuous flow electrophoretic technique for biological separation of solutes in free-solution background environment enveloped between two parallel plates. On use of an electric field being perpendicular to the background buffer flow, the components were separated in accordance with their electrophoretic mobilities. FFE has the following advantages: (1) good preservation of biological activity due to mild adjustable aqueous circumstance matching to requirement of enzyme as shown herein; (2) continuous sample preparation; (3) near 100% field; and (4) low use cost (except for isoelectric focusing, IEF) because of use of simple electrolytic buffer, rather than expensive organic solvents used in LC. These merits bequeath FFE with widespread application potential in biological separation. However, up to now normal FFE has rarely been used for the micropreparation and/or micropurification of biological sample thanks to its large dimension. Among these four FFE mode(sFFZE,FFIEF,FFFSE,FFITP), FFZE was the most popular one used for the isolation of enzyme. A detailed description of the principles can be found elsewhere. However, these works with regard to enzyme's separating via the large-scale preparative FFE, rather than micropreparation of enzyme byμl-scale FFE apparatus. Chip FFE has been given great attentions in the field of separative science in the last two decades. Nonetheless, rare work has been shown on the micropurification and/or micropreparation of biosample via chip FFE due to its weak preparation ability and easily blockage of protein precipitation. Furthermore, few of works have been reported on the (micro-)preparation and/or (micro-) purification of enzymes via FFE.To meet to the rapidly accelerant need of micropreparation and/or micropurification of biologic matrix in lab, we developed a quasi-chip FFE device with size of 45 mm×30 mm×100-500μm (135-654μl chamber volume) slightly higher than the size of chip FFE (20-40 mm×20-56 mm×20-60μm) due to thicker separative chamber, but extremely less than that of commercial FFE. In the FFE device, the gravity including a single 16-channel pump was successfully used for uniform flows of background buffer and sample solution as well as sample collection, and a simple adjuster originally used for clinical intravenous injection was well applied for the control of sample band width. The quasi-chip FFE was used for the micropurification of instable trypsin in crude pancreatin. It was observed that the separation efficiency strongly depends on the pH and conductivity of background buffer, residence time, flow rate and applied voltage. Under the optimized conditions, the purification factor was 11.68, the specific activity reached 6236 BAEE U/mg fitting to a commercial grade, the yield was 58%, the separative (or resident) time for ~20μL sample was 48 sec and the throughput of the trypsin was 3.34 mg/h implying great rapid micropurification. To our knowledge, it was the first report on enzyme micropurification via quasi-chip FFE. The adoption of gravity in the quasi-chip FFE was evidently distinct from that in the large-scale one. As will be shown below, the developed FFE device had numerous advantages: (1) no interference of blockage caused by protein precipitation and strong electrodynamic phenomenon existing in chip FFE chamber, (2) no interruption of electric current due to air bubble induced by electrolysis on two electrodes chip FFE, (3) easily complete clearing and reintegration of FFE chamber unperformed in chip one, and (4) proper enzyme amount of micropurification with short time for further activity analysis. Below are the descriptions of the quasi-chip FFE device, the brief evaluation of FFE system and the experimental results on enzyme micropreparation as well as the illumination on those advantages.
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