Chemical Engineering Science 64 (2009) 1462 -- 1473

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Chemical Engineering Science

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Chitosan chiral ligand exchangemembranes for sorption resolution of amino acids

Hai-DongWanga, Rui Xiea, Catherine Hui Niub, Hang Songa, Mei Yanga, Shuai Liua, Liang-Yin Chua,aSchool of Chemical Engineering, Sichuan University, Chengdu, Sichuan 610065, ChinabDepartment of Chemical Engineering, University of Saskatchewan, 57 Campus Drive, Saskatoon, Canada SK S7N 5A9

A R T I C L E I N F O A B S T R A C T

Article history:Received 12 August 2008Received in revised form 15 September 2008Accepted 7 December 2008Available online 16 December 2008

Keywords:MembranesSeparationsAdsorptionChiral resolutionChiral ligand exchange

The concept of chiral ligand exchange is employed in the present study to achieve the chiral resolutionof tryptophan (Trp) enantiomers by using chitosan (CS) membrane in a sorption resolution mode andcopper(II) ion as the complexing ion. CS porous membranes are prepared by freeze-drying method (CS-LT)and solgel process at high temperature (CS-HT), respectively, to investigate their sorption resolutioncharacteristics. The proposed CS chiral ligand exchange membranes exhibit good chiral resolution capabil-ity. Meanwhile the sorption selectivity of the CS membranes is found to be reversed from L-selectivity atlow copper(II) ion concentration to D-selectivity at high copper(II) ion concentration, which is attributableto the stability difference between the copper(II)L-Trp and copper(II)D-Trp complexes. Moreover, theCS-HT membrane shows better performance with respect to both sorption selectivity and sorption ca-pability than the CS-LT membrane, which mainly results from its more amorphous structures comparedwith the more crystalline structures of the CS-LT membrane. The superiority of sorption capability of theCS-HT membrane is also attributable to its larger specific surface area than that of the CS-LT membrane.The results obtained in this study are conducive to the design and fabrication of chiral ligand exchangemembranes for enantiomer separation in sorption mode.

2008 Elsevier Ltd. All rights reserved.

1. Introduction

Chiral resolution technology has received increasing attentionover the last two decades (Armstrong and Jin, 1987; Belogi et al.,1997; Wang et al., 2007; Yang et al., 2008), which is the result of thehigh demand of single enantiomers nowadays (Maier et al., 2001).Many conventional chiral resolution methods, such as crystalliza-tion, chromatography, and microbiological methods, have difficul-ties in the scaling-up though they could produce high selectivity(Yoshikawa et al., 1996). Membrane-based chiral resolution process,a newly emerging technique, features many advantages over tradi-tional methods, for instance large process capability, continuous op-eration mode, low-energy consumption (Maier et al., 2001; Ulbricht,2006), etc. Accordingly, in recent years membrane-based chiral reso-lution technology has been extensively studied in view of achievingboth high chiral selectivity and high flux (Afonso and Crespo, 2004;Breccia et al., 2003; Lakshmi andMartin, 1997; Lee et al., 2002;Wanget al., 2007; Yang et al., 2008). For permeation-selective chiral res-olution membranes, which are currently given the most attentionas far as chiral resolution membrane is concerned, the top concern

Corresponding author. Tel.: +862885460682; fax: +862885404976.E-mail address: chuly@scu.edu.cn (L.-Y. Chu).

0009-2509/$ - see front matter 2008 Elsevier Ltd. All rights reserved.doi:10.1016/j.ces.2008.12.007

always consists in the selectivity and the permeability. However, theinherent disadvantage of these membranes lies in the fact that it isdifficult to retain both high selectivity and permeability (Teraguchiet al., 2005; van der Ent et al., 2001), as the over-increase in thepermeability would definitely give rise to the nonselective perme-ation of enantiomers. Consequently, there is usually a trade-offphenomenon between the selectivity and the permeability, whichpartly limits the membrane technique to be applied in large-scaleindustrial process (Teraguchi et al., 2005).

Recently adsorptive membranes (or affinity membranes), withfunctional groups such as amino and hydroxyl groups to bond withadsorbates to achieve separation, have become an attractive meansof separation (Avramescu et al., 2003; Klein, 2000). Compared withthe conventional membrane processes (permeation, filtration and re-verse osmosis), it has advantages of high flux, low energy consump-tion (Bai and Liu, 2006), and easy operation. Adsorptive membranescould be widely applied to clarification, concentration, fractionationand purification (Roper and Lightfoot, 1995).

The technology of ligand exchange chromatography was first pro-posed by Helfferich in 1960s (Helfferich, 1961), which was then suc-cessfully developed to have good performance in the field of chiralresolution. Due to incomparable preponderance over traditionalchromatography methods, for instance high complexation capability(high affinity with the adsorbates), high selectivity and flexibility(the resolution of different solutes could be achieved by using dif-ferent central metal ions), the ligand exchange chromatography

H.-D. Wang et al. / Chemical Engineering Science 64 (2009) 1462 -- 1473 1463

Scheme 1. The proposed chiral resolution of Trp utilizing the stability difference in the complexes formed by CS membrane, copper(II) ion and D-Trp (a) or L-Trp (b).

has attracted great attention (Davankov, 1994; Kurganov, 2001).Chitosan (CS) is a kind of abundant natural polysaccharide. Due torich functional group content such as amino and hydroxyl groups,polysaccharides, including chitin, chitosan, cellulose, etc., havebeen widely used in separation and sorption processes (de CastroDantas et al., 2001; Kim et al., 2003; Niu, 2007; Niu et al., 2007;Piron et al., 1997). The use of CS as stationary phase in ligand ex-change chromatography gave rise to good resolution performance(Liu et al., 2006).

As above-mentioned, permeation-selective chiral resolutionmembranes, though having potentials to be applied to large-scalechiral resolution process, usually could not get rid of the trade-off between the selectivity and the permeability; whereas ligandexchange chromatography, though possessing desirable chiral se-lectivity and flexibility, has low process capability. Therefore, thecombination of membrane and ligand exchange chromatographymight be a better choice.

In the present research, the concept of CS chiral ligand exchangemembrane is proposed to achieve the chiral resolution of trypto-phan (Trp) enantiomers in the sorption mode, which is schemati-cally illustrated in Scheme 1. Flat CS membranes with porous struc-tures are applied to the sorption resolution process. The resolutioncould be achieved utilizing the complex stability difference betweenD- and L-form complexes, with copper(II) ion as the central ion. Dueto the steric hindrance, D-form complex is less stable than L-form(Liu et al., 2006), and hence the resolution of two enantiomers canbe achieved. The merit of the resolution by the sorption mode con-sists in that the selectivity only results from the complex stabilitydifference which is determined by the inherent property of the com-plex under given operation conditions, while the process capabilitymainly depends on the physical properties of the adsorbent such asporosity and surface area. Consequently, it is possible to manipu-late the selectivity and the process capability, respectively, withoutenhancing one at the cost of another. Moreover, the use of chiralligand exchange in the sorption mode makes it easier to adjust theselectivity by simply changing the concentration or the speciationof the central metal ion. The residual metal ion in the final product(either on the CS membrane or in the bulk solution) could be easilyremoved by the cation-exchange resin, if necessary.

Considering the significance of metal ion in the formation of thecomplex, special attention is given to the role of copper(II) ion in thechiral resolution process. Meanwhile the influence of the membranepreparation conditions is another top concern. The results obtainedin chiral resolution process are discussed in detail.

2. Experimental

2.1. Materials

Chitosan (CS, viscosity average molecular weight 105 g/mol anddegree of N-deacetylation 85%) is purchased from Ji'nan Haidebei

Marine Bioengineering Co., Ltd. and tryptophan (Trp) racemate(D,L-Trp, with purity higher than 98%) from Shijiazhuang Shixingamino acid Co., Ltd. D- and L-Trp enantiomers (with both purityhigher than 98%) are from Alfa Aesar and Sigma, respectively.Polyethylene glycol (PEG20,000), copper sulfate and other chemicalreagents are all of analytical grade. The deionized water (DI wa-ter) used in the experiment is from a Millipore Milli-Q purificationsystem.

2.2. Preparation of porous chitosan membranes by freeze-inducedphase separation

In the present study, both freeze-induced phase separation anddissolution of porogen are employed to fabricate porous CS mem-branes. The adsorption selectivity and capability of membranesprepared by different methods are investigated in detail. For thefreeze-induced phase separation method, 2.0 g of CS is dissolvedin 50ml of 4wt% aqueous acetic acid solution and the solution isstirred till uniform. After removal of impurities by sand filter, thesolution is degassed through centrifugation (3000 rpm, 5min). Thelight-yellow solution is cast onto a glass plate and frozen at 25 Cfor 8h, which is then subjected to a freeze-drying process at 35 Cfor 48h to form porous structure. The prepared membrane is treatedwith alkaline (1.0mol/L aqueous NaOH solution) and DI water suc-cessively, and then subjected to freeze-drying at 35 C for 48hagain to get the final product which is termed as CS-LT membrane.

2.3. Preparation of porous chitosan membranes by dissolution ofporogen

CS of 2.0 g and PEG20,000 of 2.0 g are dissolved in 50ml of 4wt%aqueous acetic acid solution and the solution is stirred till uniform.After removal of impurities by sand filter, the solution is degassedthrough centrifugation (3000 rpm, 5min). The light-yellow solutionis cast onto a glass plate and then dried at 60 C for 8h to form theblend membrane. Following the alkaline (1.0mol/L aqueous NaOHsolution) and DI water treatment to remove residual acetic acid, themembrane is then subjected to rinsing with DI water at 90 C in awater bath for 12h (the DI water is refreshed every 3h) to generateporous structure. Since high temperature during the drying processmight lead to shrinkage, the membrane is freeze-dried at 35 C for48h and the final product is designated as CS-HT membrane.

2.4. Chemical structure and morphological characterizations

Before subsequent tests on the chiral resolution performance ofmembranes, the prepared CS-HT and CS-LT membranes are firstcharacterized by Fourier transform infrared (FT-IR) spectroscopy toascertain whether the chemical structure of the two membranes

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are identical, namely whether PEG is removed thoroughly from theCS-HT membrane. The FT-IR spectra of membranes are obtained bya NICOLET-380 Fourier transform infrared spectrometer using ATR(attenuated total reflection) method. Scanning electron microscopy(SEM, JSM-5900LV, JEOL, Japan) is employed to observe the morpho-logical structures of the membranes. The membrane specimen forSEM, being immersed in liquid nitrogen in advance, is cut into mem-brane strips, and then the cross-section as well as surface is gilt andsubjected to morphological analysis.

2.5. Sorption resolution of Trp

Sorption resolution experiment is carried out to determine thesorption selectivity and capability of CS-HT and CS-LT membranes,respectively. Dry membrane specimen is cut into small pieces (5mgfor each, flat and square) and then immersed in 5ml of aqueous Trpracemate/copper sulfate solution with different Trp and copper(II)ion concentrations at 25 C for 48h sufficient for reaching sorptionequilibrium. After that, the D- and L-Trp concentrations in the so-lutions are analyzed by high performance liquid chromatography(HPLC).

2.6. HPLC analysis

HPLC analysis is carried out using a chiral ligand exchange chro-matography method on a HPLC apparatus equipped with a Waters515 HPLC pump, a SymmetryShieldTM RP18 column (150mm 3.9mm) and a Waters 2487 UV detector (278nm). Chiral analy-sis is performed at 45 C using a mobile phase containing 6.0mML-phenylalanine and 4.0mM copper sulfate as chiral ligand ex-change reagent as well as methanol/water (5:95, v/v). The flow rateis constant at 1.0ml/min. All the chiral analyses are performed intriplicate to guarantee accuracy. Sorption selectivity (expressed inenantiomeric excess, e.e.) and uptake (qTrp) of CS membranes couldbe expressed by Eqs. (1) and (2), respectively (Wang et al., 2007):

e.e.= CD CLCD + CL

100% (1)

qTrp =(C0 C) V

m(2)

where CD and CL denote equilibrium D- and L-Trp concentrationsin the bulk solution (mg/ml), respectively. C0 and C represent initialand equilibrium Trp concentrations in the bulk solution (mg/ml),and V as well as m the solution volume (ml) and the weight of drymembrane specimen (mg).

2.7. X-ray photoelectron spectroscopy (XPS) analysis

XPS (XSAM, KRATOS., UK) analysis is carried out to investigate themechanism of Trp adsorption on CS membranes. Dry CS-HT mem-brane specimen is cut into small pieces (5mg for each) and thenimmersed in 5ml of aqueous Trp racemate/copper sulfate solution(1 and 0.8mg/ml for Trp and copper(II) ion, respectively) or cop-per sulfate solution (0.8mg/ml for copper(II) ion) at 25 C for 48h toreach sorption equilibrium. Afterward the membrane is rinsed withDI water to remove residual Trp or copper(II) and then dried in air.Virgin CS-HT membrane, CS-HT membrane with adsorbed copper(II)ion and CS-HT membrane with adsorbed copper(II)-Trp complex areunder investigation. The changes in binding energy (BE) of differentelements before and after adsorption process are discussed throughdata comparison. The change less than 0.5 eV in BE could be regardedas insignificant (Dambies et al., 2002).

2.8. Fluorescence titration

To make clear the chelating capability of D- and L-Trp withcopper(II) ion, fluorescence titration is conducted on a Hitachi 4500fluorescence spectrophotometer. The excitation wavelength of de-tector is set at 280nm and the emission wavelength ranges from 300to 500nm. All the measurements are done at 25 C. The initial Trpconcentration is fixed at 5mM, and aqueous copper sulfate solutionis added stepwise to observe the degree of fluorescence quenchingupon copper(II) ion addition. Fluorescence intensities are given inarbitrary units.

2.9. X-ray diffraction (XRD) analysis

To make clear the influence of preparation conditions on thecrystallinity of the porous CS membrane, dry virgin CS-HT and CS-LTmembraneswith similar size and shape are subjected to XRD analysisand the diffractograms are recorded on a diffractometer (D/max-rA,Rigaku Corp., Japan) with the Cu K ray source (=1.5421010 m).Scanning diffraction angle range and scanning rate are 340 and3/min (2), respectively. Crystallinity index (CrI, %) is determinedby using (Kumar et al., 2007)

CrI = I110 IamI110

100 (3)

where I110 is the maximum intensity (2, 20) of the (110) latticediffraction and Iam is the intensity of amorphous diffraction (2, 16).

3. Results and discussion

3.1. Chemical structure and morphological characterizations

The FT-IR spectra of CS-HT and CS-LT membranes are given inFig. 1. The CS-LT membrane shows typical IR spectrum of CS which isconsistent with previous reports (Kim et al., 2003;Wang et al., 2007).The CS-HT membrane almost has identical IR spectrum with the CS-LT, and has no characteristic peaks of PEG at 1240 and 1280 cm1

(Zeng and Fang, 2004; Zeng et al., 2004a,b), which indicates that PEGin the CS-HT membrane has been removed thoroughly.

From SEM images (Figs. 2 and 3), it is evident that CS-LT andCS-HT membranes exhibit quite different porous structures (macro-porous and microporous structure for CS-LT and CS-HT membranes,respectively). In the present study, two methods, namely freeze-induced phase separation and dissolution of porogen, are employedto fabricate porous CS membranes. As to the freeze-induced phaseseparation method, the porous structure is formed in the process ofsublimation of solidified solvent (acetic acid) during freeze-dryingprocess. For the dissolution of porogen, PEG is chosen as the porogensince its interaction with CS is relatively weak and therefore phaseseparation occurs in the membrane-forming process (Zeng and Fang,2004; Zeng et al., 2004a,b). In the following hot water extraction,PEG is dissolved and removed from blend membrane, and hence theporous structure is formed.

For the CS-LT membrane (Fig. 2), it features irregular pores ofseveral tens of microns and the distribution of pores is also irregular.Meanwhile there is no morphological distinction either between thetop and bottom surfaces or throughout the cross-section. The surfaceattached to the glass plate during the membrane-forming process isdefined as the bottom surface and the other side is therefore thetop surface. The formation of such irregular macroporous structureis attributable to the solubility of CS in the aqueous acetic acid solu-tion. The CSmembrane casting solution is a polymer distribution sys-tem, in which CS polymeric network is distributed through chargedamino groups. CS, though hydrophilic, cannot effectively dissolvein water without the aid of acids. When the CS membrane casting

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Figure 1. FT-IR spectra of CS-HT membrane (a) and CS-LT membrane (b) (ATR method).

Figure 2. SEM images of cross-section (a,b) and surface (c,d) of CS-LT membrane.

solution is subjected to freezing before freeze-drying, with the rapiddecrease of temperature, the acetic acid in the solution is liable toexist in the form of molecule rather than acetate group and hydrogenion. Therefore the concentration of hydrogen ion is greatly reducedand CS polymer chain may partly precipitate from the solutiondue to the lack of hydrophilic group (protonized amino group). Con-sequently, the CS solution is not uniform before it totally becomesfrozen up. The non-uniformly distributed CS polymer chains in thesolidified solvent (acetic acid) at low temperature gives rise to thenon-uniformly distributed CS phase when phase separation occurs,and as a result irregular macroporous structure is produced.

According to Fig. 3, the CS-HT membrane exhibits relativelyregular microporous structure of several microns throughout the

Figure 3. SEM images of cross-section and surface of CS-HT membrane. (a,e) topand bottom surfaces; (b,d,f) upper, mid and bottom cross-sections; (c) the overallcross-section. The surface attached to the glass plate during the membrane-formingprocess is defined as the bottom surface and the other one is therefore the topsurface.

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Figure 4. Equilibrium Trp concentrations in the bulk solution (CTrp) and on the CS membrane (qTrp) with the change of initial copper(II) ion concentration (CCopper,0). (a,c)CS-HT membrane; (b,d) CS-LT membrane. The initial Trp concentration is fixed at 1.0mg/ml.

overall membrane thickness. However, there are distinct differencesbetween the upper and the lower cross-sections as well as thetop and the bottom surfaces. The definition of the top and bottomsurfaces of the CS-HT membrane is the same as that of CS-LT mem-brane. The upper cross-section possesses pore structure of largersize while the lower cross-section has smaller and more regularpore structures. For surface structures, the top surface of the CS-HTmembrane features inter-connected pores and the bottom surfaceis relatively dense with detached pores. The distinct differencesbetween the top and the bottom could be mainly attributed to therelative position of different parts of membrane casting solutionduring the membrane-forming process. As the CS/PEG solution issubjected to drying process to form blend membrane, the solventnearby the top surface evaporates rapidly and CS/PEG precipitatesdownward. Accordingly, the CS/PEG layer formed on the surfacefirst which hinders the solvent evaporation from below, and thusthe hindered solvent evaporation gives rise to slower and more ad-equate polymer precipitation in the lower part. On the other hand,the solvent evaporation from the bottom of the membrane castingsolution would interfere with the membrane-forming process onthe top. Therefore the upper part of membrane is loose while thelower part is relatively densely organized. Following the removalof PEG by hot water extraction, large pores are generated from theloose part and small pores from dense part.

The thickness of CS-LT and CS-HT membranes, measured bySEM images, are 250300 and 100125m, respectively, though allthe membrane preparation processes are identical except for the

membrane-forming conditions (freeze-drying for CS-LT membraneand solgel process at high temperature for CS-HT membrane). Thefact that the CS-LT membrane is thicker consists in that the solgelprocess of the CS-LT membrane is induced by the precipitation ofpolymeric network of CS at low temperature which is surroundedby the solidified solvent, and consequently the thickness of themembrane is larger.

3.2. Sorption resolution of Trp

Since the chiral resolution is based on the stability difference ofCS-copper(II)-Trp complexes, the effects of both initial copper(II) ionconcentration and initial Trp concentration are investigated, respec-tively. All the sorption resolution data are given in Figs. 47, whereCTrp, CCopper,0 and CTrp,0 denote equilibrium Trp concentration in thebulk solution (mg/ml), initial copper(II) ion and Trp concentrations(mg/ml), respectively; e.e. and qTrp represent sorption selectivity andequilibrium Trp concentration on the CS membrane (mg/mg), whichare defined in Eqs. (1) and (2).

Fig. 4 gives the equilibrium Trp concentrations in the bulk solu-tion and on the CS-HT and CS-LT membranes with the initial cop-per(II) ion concentration ranging from 0 to 0.8mg/ml when theinitial Trp concentration is fixed at 1.0mg/ml. It is clear that thesorption capability and selectivity are not evident without the pres-ence of copper(II) ion which indicates that the adsorption of Trpon the CS membrane is achieved through binding the copper(II)-Trp

H.-D. Wang et al. / Chemical Engineering Science 64 (2009) 1462 -- 1473 1467

Figure 5. Equilibrium Trp concentrations in the bulk solution (CTrp) and on the CS membrane (qTrp) with the change of initial Trp concentration (CTrp,0). (a,c) CS-HT membrane;(b,d): CS-LT membrane. The initial copper(II) ion concentration is fixed at 0.8mg/ml.

Figure 6. Sorption capability (q, the overall D- and L-Trp uptake) of CS-HT and CS-LT membranes with the change of initial copper(II) ion concentration CCopper,0 (a) andinitial Trp concentration CTrp,0 (b). (a) the initial Trp concentration is fixed at 1.0mg/ml; (b) the initial copper(II) ion concentration is fixed at 0.8mg/ml.

complex but not the single Trp molecule. The sorption capability ofthe CS membranes with respect to D- and L-Trp varies significantlyas the initial copper(II) ion concentration increases. In Figs. 4(a) and(b), both D- and L-Trp concentrations in the bulk solution decreasesto a low level at first when the initial copper(II) ion concentration

increases within a low range, and then the Trp concentration risesup slowly. It is interesting and noteworthy to give special attentionto the concentration changes of D- and L-Trp when the initial cop-per(II) ion concentration goes up across a certain value (0.16mg/ml).D-Trp is in excess in low copper(II) ion concentration range while

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Figure 7. Sorption selectivity (e.e.) of CS-HT and CS-LT membranes with the change of initial Trp concentration CTrp,0 (a) and initial copper(II) ion concentration CCopper,0 (b).(a) the initial copper(II) ion concentration is fixed at 0.8mg/ml; (b) the initial Trp concentration is fixed at 1.0mg/ml.

the situation is gradually reversed with the increase of initial cop-per(II) ion concentration, which definitely indicates a selectivity re-version of the CS membranes toward Trp in the sorption resolutionprocess. Figs. 4(c) and (d) clearly shows the trend of selectivity re-version of the CS membranes with increasing the initial copper(II)ion concentration. In low copper(II) ion concentration range the ad-sorbed L-Trp content is more than that of D-Trp, while more D-Trpis adsorbed in the case of high copper(II) ion concentration.

The equilibrium Trp concentrations in the bulk solution and onthe CS-HT and CS-LT membranes with the initial Trp concentrationranging from 0.1 to 1.0mg/ml (when the copper(II) ion concentra-tion was fixed at 0.8mg/ml) are shown in Fig. 5. The D- and L-Trpcontent in the bulk solution is reduced rapidly following the signifi-cant initial increase, and then shows a trend to rise up (see Figs. 5(a)and (b)). When the initial Trp concentration is low, the D-Trp con-tent in the bulk solution is slightly more than L-Trp which is liter-ally due to the fact that D-Trp content in the Trp racemate is a bitmore than that of L-Trp (D-Trp is about 5% in excess). As the initialTrp concentration rises across a certain value, the L-Trp content inthe bulk solution gradually surpasses the D-Trp, indicating that theselectivity of the CS membrane is not brought into full play unlessat higher Trp concentration. According to Figs. 5(c) and (d), there isnearly no Trp adsorption onto the CS membranes when the initialTrp concentration remains at low level, whereas the adsorbed Trpcontent rises steadily with the Trp concentration rising up across0.3mg/ml. Meanwhile, the CS membranes show more affinity forD-Trp regardless of the initial Trp concentration.

Figs. 6 and 7 summarize the sorption capability (the overallD- and L-Trp uptake) and sorption selectivity with respect to CS-HTand CS-LT membranes. The CS-HT membrane shows by far the bet-ter sorption selectivity than the CS-LT membrane, and meanwhilethe sorption capability of the CS-HT membrane is also superior tothat of the CS-LT membrane (however, the difference in sorption ca-pability is not as significant as that in sorption selectivity). The hugedifference in membrane performance is clearly attributable to themembrane preparation conditions, indicating that the membrane-forming temperature and use of the porogen are of great importanceto the membrane performance in the current research. Higher otherthan lower membrane preparation temperature may enhance themembrane performance.

In the sorption resolution of Trp enantiomers, two interestingand noteworthy phenomena have to be elucidated: (1) the sharp re-version of the sorption selectivity with copper(II) ion concentration

increasing from low to high, and (2) the superior membrane perfor-mance exhibited by the CS-HT membrane over the CS-LT.

3.3. Investigations on the mechanism of sorption resolution

To ascertain the adsorption mechanism of copper(II)-Trp com-plex on CS membrane, XPS analyses are carried out and the corre-sponding results are shown in Fig. 8 and Table 1. There are majorchanges in the N 1s spectra of the CS-HT membrane before andafter the adsorption of copper(II) ion or copper(II)-Trp complex,which indicates that the amino group on the membrane takes animportant part in the copper(II) ion adsorption and the complexformation. Clearly, the spectrum of the virgin CS-HT membraneexhibits a single N 1s peak at 399.30 eV which is in agreement withprevious literature reports (Miyama and Yonezawa, 2004; Zhang andBai, 2003). After adsorption of copper(II) ion on the CS membrane,the spectrum of the membrane does not only shows the peak at399.75 eV corresponding to the peak at 399.30 eV on the virginCS-HT membrane, but also a new peak at 401.31 eV which suggestsan oxidation state of N atoms. This new peak is attributable to thecomplexation of amino groups on the CS membrane with copper(II)ion (Zhang and Bai, 2003). It is interesting to find that the N 1s spec-trum reverts to a single peak after the adsorption of copper(II)-Trpcomplex onto the membrane. However, the BE shifted to 400.12 eVwhich is 0.83 eV higher than that of the virgin CS membrane andnearly 1.2 eV lower than that of the copper(II) ion-adsorbing CSmembrane. The significant change in the BE of N 1s indicates dif-ferent copper(II)-amino group complexation modes with respect tosingle copper(II) ion adsorption and copper(II)-Trp complex adsorp-tion onto the CS membrane. In the case of copper(II) ion adsorption,due to the fact that the size of copper(II) ion is relatively small, thecopper(II)-amino group complex is close-knit and therefore the elec-tron density of nitrogen atom is greatly reduced by the copper(II)ion. As a result, the BE of N 1s increases considerably. However, thesituation is different with respect to the adsorption of copper(II)-Trpcomplex. The ternary complex formed by CS-copper(II)-Trp maynot be as close-knit and stable as single copper(II)-Trp complex.Accordingly, the electron density of nitrogen atom is not reducedsignificantly and therefore the BE of N 1s does not show a majorincrease as in the case of single copper(II) ion adsorption. In Table 1,it is obvious that apart from N 1s, there are no major changes in C 1sand O 1s spectra. From the XPS results, amino group should account

H.-D. Wang et al. / Chemical Engineering Science 64 (2009) 1462 -- 1473 1469

Figure 8. N 1s XPS spectra of virgin CS-HT membrane (a), CS-HT membrane with copper(II) ion adsorption (b) and CS-HT membrane with copper(II)/Trp adsorption (c).

Table 1XPS results of virgin CS-HT membrane, CS-HT membrane with copper(II) adsorptionand CS-HT membrane with copper(II)/Trp adsorption.

Element Binding energy (eV)

Virgin CS-HTmembrane

CS-HT membranewith copper(II)adsorption

CS-HT membranewith copper(II)/Trpadsorption

N 1s 399.30 399.75 400.12401.31

C 1s 284.63 284.74 284.84286.08 286.16 285.98287.28 287.65 287.33 288.93a

O 1s 532.81 531.03b 531.68b

532.52 532.69

aC= O from Trp.bSO24 from copper sulfate.

for the copper(II) ion adsorption and complex formation on the CSmembrane.

As can be seen in the above XPS analyses, the sorption chiral res-olution is actually closely related to the formation of the complexbetween the amino groups of CS and Trp as well as copper(II) ion.Accordingly, the amino groups of CS and Trp take an important rolein the chiral resolution process. Moreover, the Trp itself could form

copper(II)(Trp)2ternary complex in the solution (Shen et al., 2005),which means that the solution behavior of Trp enantiomers mighthave influence on the sorption selectivity and capability of the CSmembrane. Therefore the final resolution behavior of the CS mem-brane actually results from the distribution of copper(II)-Trp com-plexes within the overall system (including both CS membrane andbulk solution). Correspondingly, to clarify the selectivity reversion itis of importance to make clear the stabilities of complexes formedby copper(II) and different Trp enantiomers.

The fluorescence titration experiment is carried out to investi-gate the stability of the complex formed by Trp and copper(II) ion inthe aqueous solution (see Fig. 9). The phenomenon of fluorescencequenching induced by metal ions (especially copper(II) ion) is welldocumented in the previous reports (Corradini et al., 1997; Shenet al., 2005; Stockel et al., 1998). In the current research, the de-gree of fluorescence quenching is employed to determine the rel-ative stability of different copper(II)(Trp)2 complexes. In the morestable complex, the copper(II) ion would have stronger influence toreduce the electron density of the phenyl group on its neighboringTrp within the same complex, and thus induce a more significantdecrease in the fluorescence intensity. As presented in Fig. 9, withthe addition of copper(II) ion, the fluorescence spectra of D-, L- andD,L-Trp exhibit a decrease in intensity to different extent. In addi-tion, there is a general trend that the decrease of fluorescence in-tensity is rapid when the copper(II) ion concentration is low; while

1470 H.-D. Wang et al. / Chemical Engineering Science 64 (2009) 1462 -- 1473

Figure 9. Fluorescence quenching of D-Trp (a), L-Trp (b) and D,L-Trp (c) aqueous solutions (concentration: 5mM) upon the addition of copper(II) ion at 25 C. Copper(II)ion concentrations (mM, from the top to the bottom): 0, 0.625, 1.25, 1.875, 2.5, 3.125, 3.75, 4.375, 5, 5.625, and 6.25.

the decrease rate gradually slows down as the copper(II) ion con-centration goes up to a higher level. This trend could be explainedby the following: in low copper(II) ion concentration range (in thiscase Trp content is in excess), copper(II) ion could sufficiently com-plex with Trp to form copper(II)-Trp complex while with the furtherincrease of the copper(II) ion concentration, the complex concentra-tion in the solution is saturated, therefore the addition of copper(II)ion could not give rise to evident decrease in the fluorescence inten-sity as in the case of low copper(II) ion concentration. Accordingly,the spectra in the low copper(II) ion concentration range reflect thestability of copper(II)-Trp complex better, therefore the compari-son among copper(II)-Trp complex stabilities is based on the changeof fluorescence spectra upon the addition of copper(II) ion at lowcopper(II) concentration. It is clear that in the low copper(II) con-centration range, the fluorescence intensity of D,L-Trp racematesdecreases the most rapid while that of D-Trp enantiomers the leastrapid, suggesting the Trp conformation would have major influenceon the stability of its corresponding copper(II)-Trp complex. The pro-posed complex conformations in the current research are given inScheme 2, which shows basic ternary complex form copper(II)(Trp)2and the complex might also be in a copper(II)(Trp)n form accordingto the previous report (Weckhuysen et al., 1996). From the spectraof the fluorescence titration, the fluorescence intensity of D,L-Trpracemates shows the most significant decrease upon the addition ofcopper(II) ion in the low copper(II) ion concentration range, indicat-ing the complex formed by copper(II) ion and D,L-Trp racemates isthemost stable. On the other hand, the copper(II)-D-Trp complex has

Scheme 2. The proposed ternary complex copper(II)(Trp)2 formed by D-Trp (a),L-Trp (b), and D,L-Trp (c).

the least stability. Namely the complex stability, from the strongestto the weakest, is in the order of copper(II)-D,L-Trp, copper(II)-L-Trp and copper(II)-D-Trp. The difference in the complex stability isattributable to the difference of conformation and intramolecular in-teractions of complexes (Zuilhof and Morokuma, 2003). It is obvious,in the present fluorescence titration experiments, that the complexformed with L-Trp has relatively higher stability.

H.-D. Wang et al. / Chemical Engineering Science 64 (2009) 1462 -- 1473 1471

Based on the above-mentioned analyses of the fluorescence titra-tion, the selectivity reversion of the CS membrane (as shown inFigs. 4 and 7(b)) could be elucidated. At low copper(II) ion concen-tration (lower than 0.16 mg/ml), copper(II) ion could not complexsufficiently with both D- and L-Trp due to its very small amount. Insuch condition, copper(II) ion tends to form copper(II)-L-Trp com-plex with relatively high stability leading to more copper(II)-L-Trpthan copper(II)-D-Trp complex in the bulk solution, which eventu-ally leads to higher equilibrium uptake of L-Trp on the CSmembrane.Therefore the CS membrane naturally shows L-selectivity.

As the initial copper(II) ion concentration increases to higher than0.16mg/ml, the amount of copper(II) ion in the solution is enough toform complexes with both D- and L-Trp in the system. As a result,the total concentration of copper(II)-D-Trp complexes (tertiary ormultiple D-Trp complexes) is comparable to that of copper(II)-L-Trpcomplexes. Since the copper(II)-D-Trp is less stable than copper(II)-L-Trp complex, it would be easier for amino groups on CS membraneto exchange with the amino group of one of the D-Trp moleculescomplexed with copper(II). Therefore more copper(II)-D-Trp com-plexes are adsorbed on the CS at equilibrium and the membraneselectivity shifts to D-selectivity.

As the initial copper(II) ion concentration continues to increase(higher than 0.2mg/ml), copper(II) amount is in excess in terms offorming complexes with D- and L-Trp. As a result the extra free cop-per(II) ions might compete with the copper(II)-D-Trp or copper(II)-L-Trp for the adsorption sites (amino groups) on CS membrane.Therefore the increased copper(II) ion concentration would result inthe increased amount of copper(II)-Trp in the solution. Accordingly,the overall Trp uptake is decreased.

Figs. 5 and 7(a) are actually cases of high copper(II) ion concentra-tion (fixed at 0.8mg/ml). When the initial Trp concentration is lowerthan 0.3mg/ml, the copper(II) ion is absolutely in excess in terms offorming complex with Trp and therefore copper(II)-Trp complexescould not compete with copper(II) ion for the adsorption sites (aminogroups) on the CS membrane. Correspondingly, the CS membranenearly shows no sorption capability and selectivity for Trp. With theincrease of Trp concentration (higher than 0.3mg/ml), the copper(II)-Trp complex could compete with copper(II) ion for the adsorptionsites on the CS membrane and meanwhile more D-Trp molecules areadsorbed onto the CS membrane (copper(II)-D-Trp complex is lessstable and therefore it is easier for amino group on the CS membraneto exchange with that of one of the D-Trp molecules complexed withcopper(II)). As a result the Trp uptake goes up significantly and sorp-tion selectivity of the CS membrane is given to play. Therefore thehigher the initial Trp concentration, the better the sorption capabilityand selectivity. However, the adsorption sites on the CS membraneare limited. Accordingly, as the initial Trp concentration continuesincreasing, the copper(II)-Trp complexes on the membrane tend tobe saturated and naturally the equilibrium Trp concentration in thebulk solution is increased. Clearly, in case of high copper(II) ion con-centration, only at high Trp concentrations could sorption capabilityand selectivity be given to full play.

From the above analysis, the selectivity reversion is attributable tothe stability difference between the copper(II)-D-Trp and copper(II)-L-Trp complexes, and the copper(II) ion concentration takes a vitalrole in the process of selectivity reversion. At low copper(II) ion con-centration (lower than 0.16mg/ml in this study), the CS membraneshows L-selectivity; but shifts to D-selectivity as copper(II) ion con-centration increased to higher level.

3.4. Performance comparison between CS-HT and CS-LT membranes

From the sorption resolution of Trp enantiomers, it is also foundthat the CS-HT membrane exhibits superior separation performanceover the CS-LT membrane (Figs. 6 and 7). Evidenced by the FT-IR

Figure 10. XRD diffractograms of CS-HT membrane (a) and CS-LT membrane (b).

spectra (Fig. 1), the chemical structures of the CS-HT and CS-LTmem-branes are almost identical. However, there is a significant differ-ence in the membrane resolution performance with respect to thetwo membranes. It is well-known that chitosan is semi-crystallinenatural polymer (Mucha et al., 2005), therefore it might be the crys-tallinity difference that could account for the difference inmembraneresolution performance. In order to reveal the difference in the crys-talline structures of CS-HT and CS-LT membranes, the XRD spectraof the two membranes are given in Fig. 10 . Both CS-HT and CS-LTmembranes show typical peaks at diffraction angles of around 10.8

and 20.1 (2), which is consistent with previous literature reports(Kumar et al., 2007). According to Eq. (3), the crystallinity indices(CrI, %) of CS-HT and CS-LT membranes are determined as 30% and37%, respectively. Clearly, the CS-LT membrane is more crystalline instructure and the CS-HT membrane is less crystalline, which meansthat the high temperature for membrane preparation favors amor-phous structure. The amino and hydroxyl groups residing within thecrystalline area, bonded by the strong interactions such as hydrogenbond, may be liable to be released to the amorphous region due tothe destruction of such interactions under the influence of high tem-perature for membrane preparation; while low temperature mighttend to retain or even strengthen the crystalline area. Therefore, theCS-LT membrane is more crystalline compared with the CS-HT. Inaddition, only the amino groups in the amorphous area could beeasily accessed (Wang et al., 2006), namely amorphous area actu-ally mainly account for the sorption resolution of Trp. Correspond-ingly, the membrane performance is directly correlated with theamount of amino groups within the amorphous area which could betermed as active amino groups in the present research; as a result,the more amorphous in structure, the better the membrane resolu-tion performance. Therefore, the differences in sorption selectivityand sorption capability with respect to CS-HT and CS-LT membranescan be explained. The CS-HT membrane evidently has more activeamino groups than the CS-LT due to the fact that the former is fab-ricated through solgel process at high temperature and the latterby a freeze-drying method. Furthermore, from the SEM images ofthe CS-LT and the CS-HT membranes in Figs. 2 and 3, smaller poresand more porous structures are observed in the case of the CS-HT

1472 H.-D. Wang et al. / Chemical Engineering Science 64 (2009) 1462 -- 1473

membrane. This indicates that the specific surface area per unit vol-ume is larger in the case of the CS-HT membrane. Thus, this largerspecific surface area may be also one of the reasons for the superior-ity of the CS-HT membrane, especially with respect to the sorptioncapability.

4. Conclusions

In the present research, the concept of CS chiral ligand exchangemembrane for sorption resolution of Trp enantiomers is proposedand proved feasible. To investigate the sorption resolution character-istics, CS porous membranes are prepared by freeze-drying methodand solgel process at high temperature, respectively.

It is found that the copper(II) ion concentration has considerableinfluence on the sorption selectivity of the CS membrane. At lowcopper(II) concentration (lower than 0.16mg/ml), the CS membraneshows L-selectivity while it reverses to D-selectivity as the copper(II)ion concentration rises up to high level. The selectivity reversion ofthe CS membrane is attributable to the stability difference betweenthe copper(II)-D-Trp and copper(II)-L-Trp complexes. At low cop-per(II) concentration, due to high stability of the copper(II)-L-Trpcomplex, the CSmembrane tends to bindmore copper(II)-L-Trp com-plex. While at high copper(II) concentration, since the amounts ofD- and L-Trp are comparable and the copper(II)-D-Trp is less stablethan copper(II)-L-Trp complex, it would be easier for amino groupson CS membrane to exchange with D-Trp molecules in copper(II)-D-Trp complexes. Therefore more copper(II)-D-Trp molecules are ad-sorbed on the CS at equilibrium.

In addition, the CS membrane prepared at high temperature(CS-HT) is found to be superior to that prepared at low temperature(CS-LT) with respect to the sorption selectivity and sorption capa-bility. The membrane preparation by the solgel process at hightemperature gives rise to relatively lower crystallinity which facil-itates the existence of active amino groups in larger content andaccordingly enhances the membrane performance. The superiorityof sorption capability of the CS-HT membrane is also attributableto the larger specific surface area of the CS-HT membrane thanthat of the CS-LT membrane. The results presented in this workare valuable for designing and fabricating chiral ligand exchangemembranes for enantioseparation in sorption mode.

Acknowledgements

The present work is financially supported by the National Natu-ral Science Foundation of China (20674054) the Key Project of theMinistry of Education of China (106131) National Basic ResearchProgram of China (2009CB623407) and Sichuan Youth Science andTechnology Foundation for Distinguished Young Scholars (08ZQ026-042). The authors gratefully acknowledge the kind help of Ms. DanYang at Sichuan University for her assistance in the HPLC analysis,Ms. Xin-Yuan Zhang of Analytical and Testing Center at Sichuan Uni-versity in the SEM observation as well as Prof. Lin-Fang Du (Schoolof Life Sciences) in the fluorescence titration experiment. Meanwhilemany thanks go to Mr. Chuan Xin at Chengdu Organic ChemicalsCompany Ltd. of Chinese Academy of Sciences for his useful advicein the XPS analysis.

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Chitosan chiral ligand exchange membranes for sorption resolution of amino acidsIntroductionExperimentalMaterialsPreparation of porous chitosan membranes by freeze-induced phase separationPreparation of porous chitosan membranes by dissolution of porogenChemical structure and morphological characterizationsSorption resolution of TrpHPLC analysisX-ray photoelectron spectroscopy (XPS) analysisFluorescence titrationX-ray diffraction (XRD) analysis

Results and discussionChemical structure and morphological characterizationsSorption resolution of TrpInvestigations on the mechanism of sorption resolutionPerformance comparison between CS-HT and CS-LT membranes

ConclusionsAcknowledgementsReferences