Haitao Mo

Jiajiong Lu

Liya Liu

Shan Chen

Yuanjin Xu

Zhong Han

Comptes Rendus Chimie

Available online at www.sciencedirect.comC. R. Chimie 11 (2008) 80e83http://france.elsevier.com/direct/CRAS2C/Preliminary communication / CommunicationClinical dextran purified by electric ultrafiltrationcoupling with solvent crystallizationShan Chen*, Liya Liu, Jiajiong Lu, Zhong Han, Yuanjin Xu, Haitao MoSugar Engineering and Technology Research Center, Guangxi University, Nanning 530004, PR ChinaReceived 15 December 2006; accepted after revision 6 June 2007Available online 30 July 2007AbstractA new separation method was used to control the molecular weight distribution and improve the quality of clinical dextran.Crystalline solid of dextran was precipitated in the charging basket during the membrane filtration, coupled with solvent crystal-lization and enhanced by a DC electric field. It was found that the permeate flux of ultrafiltration could be distinctly improved by theapplied DC electric field, and the permeate flux of it could be preserved at a high and stable level. Molecular weight distribution ofdextran was controlled effectively by ultrafiltration coupled with solvent crystallization. Moreover, organic solvent consumptionand processing time can be saved markedly by this new technology. To cite this article: S. Chen et al., C. R. Chimie 11 (2008). 2007 Published by Elsevier Masson SAS on behalf of Académie des sciences.Keywords: Ultrafiltration; Solvent crystallization; Electric-field intensity; Dextran1. IntroductionDextran is a naturally occurring non-toxic, biode-gradable and microbial polysaccharide. Dextran andits derivatives are used as plasma expanders, blood sub-stitutes, bone healing promoters, and also for the dermaland subcutaneous augmentations [1]. Furthermore, dex-tran is widely under investigation as a polymer carrier innovel drug delivery systems [2]. Dextran is usually pro-duced from sugar by fermentation of extracellular dex-transucrases, which contain different percentages of thebranched a-1,3 linkages. However, since highmolecular weight dextran could generate toxicity forthe human body, so the fermented rough dextran cannotbe used directly in a clinical context until it is* Corresponding author.E-mail address: chenshan@gxu.edu.cn (S. Chen).1631-0748/$ - see front matter  2007 Published by Elsevier Masson SASdoi:10.1016/j.crci.2007.06.006hydrolyzed into that with low molecular weight [3].Thus, studying the method of improving the purity ofdextran with certain molecular weight is of technologi-cal importance, especially for clinical applications.Certain organic solvents could influence the solubil-ity and the crystallization kinetics (crystal nucleationand growth rate) of crystalline materials in solution,so they are widely used in extraction and refining of nat-ural products. Ethanol grading sediment is a method ofsolvent crystallization, in which ethanol dissolved withmoisture but unsolvable with solute makes solute de-hydrated and crystals are formed while the solute con-centration in water increases up to its saturatedconcentration [4e6]. Dextran can also be separated bythis way from enzymic fluid.However, the products are mixtures with differentmolecular weights, and quality problems induced bythe disproportion of molecular weight were reportedon behalf of Académie des sciences.81S. Chen et al. / C. R. Chimie 11 (2008) 80e83[7,8]. At the same time, the role of solventesurface in-teractions in enhancing or inhibiting crystal growth isstill not completely resolved [9].In recent years, ultrafiltration, a well-establishedseparation process in the biotechnology and fermenta-tion industries [10], was used to separate polysaccha-rides [11e13]. The control of membrane fouling bythe application of external DC (direct current) electricfields has been studied in several applications, and insome cases a clear enhancement has been achieved.This treatment technology has been found to be capableof reducing the formation of filter cake and increasingthe flux of filtrate [14,15].In this study, electric ultrafiltration-solvent crystalli-zation (EUSC), which coupled electro-ultrafiltration(EU) with solvent crystallization (SC), is a new separa-tion method proposed for the separation and purifica-tion of dextran. Our object is to separate dextran andcontrol its molecular distribution at the same time,and prepare dextran with high purity.2. Experiment2.1. EUSC systemThe five sections within the EUSC system werepump, charging basket, ultrafiltration, direct currentsource and crystallizer. Feed liquid could be concen-trated and separated by the EU, as the organic solventis added into the charging basket. The dynamic forceof liquid flow is supplied by the pump. The experimen-tal set-up used in this work is shown in Fig. 1.Within the EUSC system, the area of P10 membraneis 15 cm2 6 cm2 and the height of membrane cavitycan be adjusted from 0.2 cm to 1.0 cm by replacing fill-ing pieces. The membrane is made of polyethersulfonewith 10-kDa molecular weight cut-off (MWCO) pro-vided by Sunstar Membrane Technology Company,UFPumpPermeate flux(Solvent + Water)Retentive fluxSolvent + WaterSolute + Water+DCOrganic solvent CrystallizerCharging BasketFig. 1. Schematic sketch of the EUSC process (total-reflux operation).China. The pressure of EUSC system was provided bya WATSON323 constant-flow pump (Amersham Bio-sciences Company, US) with an entrance pressure of0.10 MPa, and the DC electric field was provided bya WYI-24 electrical power unit (Golink TechnologyCompany, China).2.2. MaterialsThe commercial dextran D20, with an average molec-ular weight of about 20 kDa, was obtained from Shang-hai Reagent Co. Ltd. (Shanghai, China). Ethanol was ofreagent grade and was used without further purification.The dextran solution was prepared by dissolving30.00 g of dextran D20 in 300 mL of distilled water atroom temperature, and agitated by an electric stirrerat 3000 rpm for 60 min in order to make dextran dis-solve sufficiently. After that, dextran solutions were fil-trated by a UF membrane with 30 kDa MWCO, and thepermeated fluid was taken as specimen solution for theEUSC process.2.3. MethodsRegurgitation operation was opted to increase theconcentration of retention fluid quickly. The addedquantity of ethanol was controlled at 5.0%, 10.0%,15.0%, 20.0%, 25.0%, 30.0%, 40.0% and 60.0% (v/v)according to the capacity of the specimen solution.The proper addition quantity of ethanol was confirmedbased on the permeate flux, and selected for the exper-iment of EUSC.The mixed fluid of dextran and ethanol prepared withthe proper addition quantity of ethanol was taken as thefeed fluid for the EUSC process. Intensity of DC electri-cal field was exerted in the membrane separation withdifferent voltages, such as 5 V, 10 V, 15 V, 20 V, 25 Vand 30 V. The proper intensity of the DC electrical fieldwas confirmed based on the permeate flux.3. Results and discussion3.1. The effect of ethanol concentrationIt was reported that the presence of ethanol plays animportant role during ultrafiltration of macromolecules[16]. Experiments were performed to determine theeffects of different ethanol concentrations on the perme-ate flux of dextran. The permeate flux (J) was calculatedby the time-consumption of getting 10 mL permeatedfluid, and the average membrane flux (average J) basedon the interval of getting 100 mL permeated fluid. Fig. 20.000.100.200.300.400.500.600.700.800 20 40 60 80 100Volume of permeate/mLPermeate flux/Lm-2min-10.000.100.200.300.400.500.600.700.80Average J/Lm-2min-1Dosage of ethanol (V/V)Fig. 2. Permeate flux and average J of different dosages of ethanol.82 S. Chen et al. / C. R. Chimie 11 (2008) 80e83shows permeate flux as a function of solvent volume fordifferent ethanol concentrations (0%e60% (v/v)). Asexpected, the membrane flux was obviously affectedby the volume of added ethanol. As the dosage of etha-nol increased, membrane permeate flux increased at thebeginning, and then, decreased as the dosage of addedethanol was above 25% (v/v). It seems that properdosage of ethanol could reduce the viscosity of thefeed liquid, and expand the polarization time whilegel formation was delayed. However, too much addedethanol could destroy the stability of dextran solution,and induce dextran crystallization on the face of mem-brane and block the membrane’s holes.3.2. The effect of electric fieldThe mixture fluid used for EUSC process was pre-pared by dextran and ethanol until ethanol dosage wasabout 25% (v/v). The intensity of DC electrical fieldwas exerted in the membrane separation with different0 20 40 60 80 100Volume of permeate/mL0V 5V 10V15V 20V 25V30VPermeate flux/Lm-2min-10.000.200.400.600.801.001.201.401.60Fig. 3. Permeate flux and averagevoltages between 5 V to 30 V. The effects of electricfield on permeate flux are more clearly shown inFig. 3. It was clear that DC electric field could distinctlyimprove the permeate flux of the Ultrafiltration-SolventCrystallization (USC) process. The permeate flux in-creased with the increase in the DC voltage. Whatshould be noted is that with increasing the voltage,the average permeate flux is increasing linearly at thebeginning and then there is no obvious enhancement.It means that there is an optimal voltage of DC electricfield while dealing with different consistencies of dex-tran solution. For 10% (w/v) dextran solution, the opti-mum voltage should be about 25 V. It seems that therewas a critical voltage in EUSC processing, and it mightbe caused of the charges on the surface of dextran mo-lecular. As the voltage increased, the electrophoresistransportation strengthened, thereby avoiding the exac-erbation of the polarization concentration when the re-tentive flux is enriched. The critical electric voltageindicated that there was a definitive point, by which0 5 10 15 20 25 30Intensity of DC electric filed/VAverage J/Lm-2min-10.000.200.400.600.801.001.201.40J of different electric fields.Fig. 4. HPLC chromatogram of specimen dextran D20.Fig. 5. HPLC chromatogram of separated dextran by EUSC.83S. Chen et al. / C. R. Chimie 11 (2008) 80e83the diffusion and adsorption of the concentration polar-ization layer would be in equilibrium.3.3. HPLC chromatographyDextran specimen D20 and separated dextran ofEUSC (electric field 25 V, ethanol concentration25%) were measured by the Waters HPLC system,which consists of a pump 515, a column heater CH-30, and a differential refractometer detector 996. Anal-ysis was performed by TSK G2000 column, 5mm,7.8 mm 300 mm. Detector output was processedand manipulated by TL9900 software. The mobilephase used 0.7% Na2SO4 solution. Consistency of sam-ple solution is 10 mg/mL, which was prepared withmobile phase. The conditions of chromatography in-clude column temperature 35 C, velocity of flow1 mL/min and injection magnitude 20 mL.According to the peak widths shown in Figs. 4 and 5,the molecular weight distribution of separated dextranwas restrained into a small range by comparing withthe original dextran specimen. It indicated that the sep-aration and purification of rough dextran on EUSC waseffective.4. ConclusionIt was observed that the presence of ethanol plays animportant role during ultrafiltration of clinical dextran,and an external DC electric field is effective to maintainthe permeate flux of UF at a stable level. Moreover, theservice life of EUSC system can be prolonged. As theviscosity of solution is not improved quickly, low mo-lecular weight species can pass through the membrane,and higher molecular weight dextran could be concen-trated and purified.It was concluded that ultrafiltration of dextran isinfluenced by the formation of gel at higher dextranconcentration, and electric filtration coupled with sol-vent crystallization can delay the concentration polari-zation time of the ultrafiltration process. However,excess ethanol could reduced the dissolvability of dex-tran in solvent, inducing dextran crystallization on theface of the membrane, thereby blocking the holes ofthe membrane.References[1] M.G. Cascone, G. Polacco, L. Lazzeri, N. Barbani, J. Appl.Polym. Sci. 66 (1997) 2089.[2] R. Mehvar, J. Controlled Release 69 (2000) 47.[3] H. Yuuki, A. Mitsuo, K. Hidekii, Y. Hiroyuki, Biomaterials 20(1999) 303.[4] S. Mosseri, Z.B. Alfassi, J. Sep. Sci. Technol. 18 (1983) 165.[5] Z.B. Alfassi, J. Mosseri, AIChE J. 30 (1984) 874.[6] D.W. Gao, Q. Zhao, S.J. Yu, SCUT Transaction 11 (1998) 16.[7] V.B. Veljkovie, M.L. Lazic, M.D. Cakie, Pharmazie 43(1988) 840.[8] T.R. Shamala, M.S. Prasad, Proc. Biochem. 30 (1995) 237.[9] H.G. Hao, B.H. Hou, J.K. Wang, G.G. Lin, J. Cryst. Growth 290(2006) 192.[10] A.S. Grandison, A.K. Goulas, R.A. Rastall, SongklanakarinJ. Sci. Technol. 24 (2002) 915.[11] M.R. Freixo, P.M. Norberta, Desalination 149 (2002) 237.[12] S. Kim, W. Kim, I.K. Hwang, Int. J. Food. Sci. Technol. 38(2003) 337.[13] F.T. Moerman, M.B. Leeuwen, J.A. Delcour, J. Agric. Food.Chem. 52 (2004) 3780.[14] J.O. Kim, J.T. Junga, I.T. Yeomb, G.H. Aoh, Desalination 202(2007) 31.[15] C.C. Tarazaga, M.E. Campderros, A.P. Padilla, J. Membr. Sci.278 (2006) 219.[16] S.K. Zaidi, A. Kumar, J. Membr. Sci. 237 (2004) 189.

Clinical dextran purified by electric ultrafiltration coupling with solvent crystallization

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Clinical dextran purified by electric ultrafiltration coupling with solvent crystallization

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