The Investigations Of Free-Flow Electrophoresis On The Injection Technique And Its Applications

Free-Flow Electrophoresis （FFE） is a pure-liquid electrophoresis for bothpreparative and analytical purposes with the advantages of controllable process,gentle condition, high recovery, well resolution, complete preservation of activity, andwide applications. Until now, the development of FFE is to focus on how to narrowthe bandwidth, improve the resolution and increase the throughput. The currentsolutions are to （i） decrease the initial sample bandwidth;（ii） change the FFE runningmode （e.g. the introduction of interval FFE）;（iii） use the focusing method （such asIEF）. However, there are some problems remained.（i） To decrease the initial samplebandwidth can lower the sample throughput in no accordance with the preparativepurpose of FFE.（ii） The resolution of interval FFE mode has not been reported to bemore superior to that of continuous one at the same throughput.（iii） The focusingmethod does not only need a lot of ampholytes, making the cost higher, but also loseamphoteric substances （such as proteins） at their pIs. Based on the previous twoproblems, this paper has done some prelimilary works on the bandwidth controlling.Before experiment, we made a series of modifications on the previousconstructed FFE device with gratis gravity, including （i） the material of self-balancecollector （SBC） were changed,（ii） the lengths of electrode cavities were shortened;（iii） the dead volume of collection tubes were reduced;（iv） the up and down plateswere thickened; and （v） the cooling barriers were set on the up plate. Thus, the size ofwhole device （70cm×85cm×45cm） was greatly reduced in contrast to theprevious version of FFE apparatus （80cm×100cm×160cm）.After the device modifications, we carried out the following experiments:（1） It was studied on how to use the flux ratio between the sample andbackground solutions to control the sample bandwidth, resolution and throughput.Two injection methods were described herein. The first method was the one in whichsample injection flux were variable, whereas the background flux was a constantvalue. The second was the one in which the background flux were flexible, while the sample flux was stable. With the help of methyl green and crystal violet as twoviewable model compounds, we found that the bandwidth, resolution and throughputof mixed dyes could be under better control by the adjustment of flux ratios betweensample and background flux. The results were of significance to the future designs onour newly-developed FFE device with gratis gravity.（2） Under the same throughput, the resolution promotion of interval FFZE andcontinuous FFZE were compared quantitatively. In this paper, a commercial dye withmethyl green and crystal violet was well chosen as the model sample. Thecomparative experiments were conducted under the same sample loading of the modeldye （viz.3.49,1.75,1.17and0.88mg/h）, the same running time （viz.5,10,15and20min）, the same flux ratio between sample and background buffer (=10.64×10^-3) andthe same voltage （=200V）. Under the given conditions, the relevant experimentsdemonstrated that the band broadening was evidently caused by hydrodynamic factorin continuous FFZE mode, and the interval mode ccould clearly eliminate thehydrodynamic broadening existing in the continuous mode, greatly resulting in theresolution increase of dye separation in FFZE. Finally, the interval FFZE wassuccessfully used for the complete separation of two model antibiotics （hereinpyoluteorin and phenazine-1-carboxylic acid co-existing in fermentation broth of anew strain Pseudomonas aeruginosa M18）. These results might change FFE runningmode in the future.（3） The low concentration PCA （=0.3mM） extracted from fermentation brothof Pseudomonas sp. M18was selected to be isolated and prepared with a newly facileFFE device with gratis gravity. Three factors of pH value and concentration ofbackground buffer, and the cooling circle of FFE device were investigated for thepurification of PCA in the device. It was found that the pH value and concentration ofbackground buffer had mild influences on the separation of PCA whether withcooling circle or not. However, the cooling circle had a much greater impact on theseparation of PCA. The controlling of the band zone of PCA in FFE chamber wouldbe difficult if without cooling circle, while the controlling would become easy if withcooling circle. Under the optimal conditions （10mM pH5.5phosphate as background buffer,30mM pH5.5phosphate buffer as electrode solution,5.46mL/minbackground flux,10min residence time of injected sample, and500V）, PCA couldbe continuously prepared from its impurities with relative high purity. The flux ofsample injection was115μL/min, viz.7mL sample throughput per hour, and therecovery was up to85%. All of the experiments indicated that FFE was a goodalternative tool for the study on natural biological control agents.According to a series of experiments, we concluded that （i） the sample resolutionand throughput could be controlled by the flux ratio of sample and backgroundsolutions;（ii） the application of interval FFZE could increase largely the sampleresolution without the loss of the sample throughput;（iii） the substances difficult todissolve and ionize could be separated in our homemade device. All these resultswould be beneficial to the deep investigations and extension of applications in ourFFE in the future.