New Methods And Technologies Of Preparative Free-flow Electrophoresis

As a liquid-based,matrix-free separation technology,free-flow electrophoresis(FFE)is capable of separating bio-mixtures in a gentle circumstance,allowing high biological activity of analytes.FFE has been used to separate a range of bio-particles or bio-molecules,including proteins,peptides,cells,cell organelles and virus.Compared with other electrophoretic techniques,continuous processing capacity enabled it to process relatively large-volume samples for preparative applications.Besides its gentle separation circumstance and the continuous character,another advantage of FFE is that it has several operation modes,including zone electrophoresis,isoelectric focusing,isotachophoresis,field-step electrophoresis and moving reaction boundary electrophoresis.In this paper,we focus on new devices of free-flow isoelectric focusing(FFIEF)and its application.We also did some studies on free-flow zone electrophoresis(FFZE)and free-flow moving reaction boundary electrophoresis(FFMRB).FFIEF has the highest resolution in different FFE modes and was widely used in proteomics researches as a pre-fractionation technique.In this paper,two kinds of recycling free-flow isoelectric focusing(RFFIEF)devices and a reciprocating free-flow isoelectric focusing(ReFFIEF)were developed and applied for biologic sample separations.1)Self-balance recycling free-flow isoelectric focusing device.Based on our previous gravity-induced-balance FFE device,a novel RFFIEF was developed.The carrier buffer,driven by multi-channel pump,recycled between self-balance collector,separation chamber and multi-channel pump.At the begining,separation chamber was horizontally placed,and then was changed to vertically placed with the device was greatly simplified.Mitochondria from rabbit liver was separated with the newly designed device and several subpopulations were obtained.2)Novel reciprocating free-flow isoelectric focusing device.In this system,a reciprocating flow was for the first time introduced into FFE instead of the traditional unidirectional recycling flow.Carrier buffer reciprocated between transient self-balance collector,separation chamber and gas cushion chamber,forming a reciprocating flow.The major advantage is the complicated multi-channel pump was replaced by a one-channel pump,showing several merits of simpler structure,user-friendly operation and easy to extend.With the new device,model proteins and human serum proteins were well focused.In the second part,a continuous protein concentration method of FFMRB was studied experimentally and theoretically.First,the mechanism of FFMRB was discussed systematically and a series of experiments was performed to evaluate the new method.The prerequisites for protein concentration,the maximum processing capacity and optimizing strategy were firstly discussed.Proteins of Hb,Cyt C and C-PC were well concentrated with FFMRB.A maximum processing capacity of 125.7 ml/h and a maximum recovery ratio of 95.5% were obtained in Cyt C concentration,a maximum enrichment factor of 12.6 times was achieved in Hb concentration.It is well known that suitable carrier buffer could improve the resolution of FFZE.In our previous work,a strategy for preparation of suitable carrier buffer was developed.Carrier buffer was optimized by charge-to-mass ratio(C/M)analysis.However,band broadening which also highly affected by carrier buffer,was neglected.In this paper,C/M and band broadening analysis were combined to provide better guidance for the design of FFZE experiments:(1)the C/M was analysis under different pH conditions;(2)band dispersion due to the initial bandwidth,diffusion,and hydrodynamic broadening were discussed;(3)based on the analysis of the C/M and band broadening,the optimized separation conditions were obtained.Series of experiments were performed to validate the proposed method.Results showed that experimental data showed high accordance with our prediction.With the help of the newly developed strategy,C-PC was purified from crude extracts of Spirulina platensis,analytical grade product with purity up to 4.5(A620/A280)was obtained.