Development Of A New Preparative Free-flow Electrophoresis Apparatus With Self-balance Collector Based On Gratis Gravity And Investigation Of Its Application

Free flow electrophoresis (FFE) is an electrophoresis method without using solidmedium which separates analytes according to their charge differences. In FFE,analytes driven into the chamber by carrier buffer are separated continuouly in anelectrical field perpendicular to the background buffer flow direction. Analytes withdifferent charges deflect to different outlets where they are collected. FFE is acontinuous and gentle separation technology which can be applied in many modes.Both preparation and analysis can be achieved using large scale or micro FFE. SinceSevsson developed the first simple FFE in1949, the research related to FFE has neverstopped up to now. At the same time, separation of small or macro molecules,peptides or proteins and cells or subcellular structures is successfully and widelyperformed using FFE.With the development of technology, many commercial FFE instruments haveentered into market in the past years. In1994, Elphor Vap21FFE was developed byBender&Hobein and was used for preparation of many biology samples later. Then,Gerhard Weber improved the instrument and commercialized it by FFE ServiceCompany. In addition, BD company also pushed its BDTMFFE into market.However, import instruments priced at RMB12~14million are very expensivewhich limits the application of FFE. In China, very little research related to FFE is reported for lack of national commercial FFE instrument. The first FFE prototype wasdeveloped in1980s used for microgravity separation experiments.Since2009, Bio-separation and Bio-analysis Lab in Shanghai Jiao TongUniversity developed the first FFE device based on gratis gravity collection. The FFEworked well. Although that device is successfully used in form of free flow movingreaction boundary electrophoresis (FFMRBE) and for phenazine-1-carboxylic acid(PCA) purification, protein separation and bacteria separation, there are still manydrawbacks coherent in the device. Firstly, it is labor-intensive. Secondly, leakage iseasily seen after assemble. Lastly, the device looks ugly. So, this article is aimed atimproving and commercializing the national FFE instrument.The new FFE can be divided into four parts:(1) automated integrated circuitsystem (including liquid pump system, cooling system, temperature control systemand power supply system);(2) gas cushion injector (GCI)(with function of bubbleselimination);(3) separation unit (a size of45mm*120mm*(0.6-0.8) mm, with12inlets and24outlets);(4) self balance collector (SBC).The nearly commercialized FFE shows good performance which is confirmed bylarge number of results. plot and bubble generation experiments show that thedevice’s maximum voltage can be applied at400V in10mM CB (conductivity:720μS·cm-1),50mM electrode buffer (EB)(conductivity:2530μS·cm-1). Withoutcooling system, the current increased by-5.3%~70.26%compared to the current withsemiconductor cooling system. With cooling system, optimized voltage is300V in20mM CB,100mM EB. Under this experiment condition, this new FFE instrument canseparate dyes mixture (amino black, reactive red and hematoxylin) or proteins mixture(BSA, Mb and Cyt C) successfully. Besides, Escherichia coli andStreptococcus thermophilus can be separated from each other at370V in TBE CB,Streptococcus thermophiles and Saccharomyces cerevisiae can be partly separated.