Analytica Chimica Acta 431 (2001) 5967

Facilitated transport and separation of aromatic amino acids through activated composite membranes
Juan Antonio Calzado, Cristina Palet, Manuel Valiente
Qumica Analtica, Universitat Autnoma de Barcelona, E-08193, Bellaterra, Catalunya, Spain Received 25 July 2000; received in revised form 18 October 2000; accepted 9 November 2000

Abstract Activated composite membranes (ACM) containing bis-(2-ethyl-hexyl) phosphoric acid (DEHPA) have been found to facilitate the transport and separation of aromatic amino acids. The ACM, prepared according to a previously established procedure, were used to experimentally determine their behavior for the transport of phenylalanine (Phe), that was used as target amino acid. A general characterization of the Phe transport through DEHPA-ACM membranes including: the existence of facilitated counter transport, reproducibility and the effect of buffering the feed solution has been performed prior to a systematic study of the inuence of some chemical transport parameters. In this concern, the initial Phe concentration, the nature and concentration of the stripping agent and the pH of the feed solution have been optimized (the feed phase was 0.5 mM of Phe in a 2 M NaCl solution at pH 3 and a 2 M HCl solution was used as stripping phase). Also, the inuence of the DEHPA concentration in the casting solution used to prepare the membrane is established. It is found that the membrane phase controls the rate transport up to a DEHPA concentration of 1200 mM. Finally, although the afnity of the aromatic amino acids phenylalanine, tryptophan (Trp) and tyrosine (Tyr) for DEHPA is in the order Phe > Trp > Tyr, the selectivity of the DEHPA-ACM was found to be Trp > Phe > Tyr. 2001 Elsevier Science B.V. All rights reserved.
Keywords: Amino acid; Phenylalanine; Bis-(2-ethyl-hexyl) phosphoric acid (DEHPA); Facilitated transport; Activated composite membrane

1. Introduction In the last decade, the production of amino acids has increased enormously. The introduction of sweeteners for hypocaloric beverages, the enrichment of the animal and human diet with these metabolites, and the use of some of them as precursors of some synthetic penicillin has produced this enhancement. At the moment, the production of amino acids is carried out by chemical or biochemical processes [1]. In the case of the chemical process, a racemic mixture
Corresponding author. Tel.: +34-93-5812903; fax: +34-93-5811985. E-mail address: manuel.valiente@uab.es (M. Valiente).

of corresponding amino acid is obtained, whereas the l separated form is obtained by the natural-biochemical process, which is carried out by means of either an enzymatic or a microbial system. In the fermentation broth media, the concentration of amino acids is very low and they are present in a mixture of different compounds. So, separation and purication steps are necessary during the down stream production of amino acids. Some of the separation techniques actually used include ion exchange, chromatography, ltration, evaporation, reverse osmosis, and electrodialysis. Among the problems that those techniques can introduce, the big waste liquor produced during the regeneration of the ion exchange resins or the high energy consumption for the

0003-2670/01/$ see front matter 2001 Elsevier Science B.V. All rights reserved. PII: S 0 0 0 3 - 2 6 7 0 ( 0 0 ) 0 1 2 9 3 - 9

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evaporation process are some of the most important drawbacks [2]. The use of liquid membranes is an alternative to those mentioned separation techniques [1,3]. The study of liquid membrane systems for the separation and preconcentration of amino acids has received an important attention during the last years. Different congurations, i.e. supported liquid membranes (SLM), emulsion liquid membranes (ELM), and carriers (neutral, anionic, cationic and metallic) have been employed to accomplish for this purpose. Some neutral [46], anionic [79], cationic [1013], and some metal bearing organic compounds [1417] have been used as carriers and have been assayed for different purposes. Different problems arise from the use of these liquid membrane systems. When using an ELM conguration, the main disadvantage is the swelling of the membrane that produces a dilution of the separated product in the inner aqueous phase (in the water/oil/water emulsions), or also the breakage of the membrane itself [1,2]. The SLM conguration, instead, have the advantage of the low operation cost, the single step operation and the exibility and the selectivity of those systems by just making the correct choice of the carrier. However, they also present some disadvantages, being the most important, the low stability of the liquid membrane itself. Usually, the carrier, the solvent or both suffer from continuos leaching out from the membrane support to the aqueous phases, so, the short lifetime of the liquid membrane reduces the industrial exploitation of these membranes [18]. The leaching of organic compounds is also an inconvenient because the nal product, present in the receiving aqueous phase, will be contaminated with such compounds. Different approaches have been assayed to improve the stability of these membranes; the use of ion exchange membranes with cationic or anionic groups covalently bonded to the structure of the polymeric support [19,20], the formation of a layer on the surface of the impregnated support [2123] or the incorporation of the carrier and solvent into a polymer by different ways [24,25]. Recently, the activated composite membranes (ACM) have been presented as a good alternative to have stable liquid membranes, immobilized onto polymeric supports [26]. These membranes have been demonstrated to enhance the lifetime of the liquid membrane systems. In this case, the carrier

used was mainly bis-(2-ethyl-hexyl) phosphoric acid (DEHPA). On the other hand, this carrier has also been extensively used for different congurations of liquid membranes with good success in the transport of amino acids [2,10,11,27]. In the view of these properties, DEHPA was selected as membrane carrier to accomplish for the transport of amino acids through activated composite membranes in the present work. The aim of this paper is to demonstrate the possibilities of such a kind of membranes for the transport of amino acids, being the phenylalanine (Phe) used as a target molecule. The involved chemical transport parameters have been evaluated by including the use of different stripping agents, the inuence of the carrier concentration and pH of the feed solution. A selectivity study has been also carried out by using mixtures of some aromatic amino acids. 2. Experimental section 2.1. Reagents d,l-Phenylalanine, tyrosine and tryptophan were provided by Sigma (St. Louis, MO, USA) and used without further purication. Bis-(2-ethyl-hexyl) phosphoric acid was obtained from Lancaster (England, UK). Polysulfone (PS), 1,3,5-benzenetricarbonyl trichloride and sodium hydroxide were purchased from Aldrich (Steinheim, Germany). Fluka (Swiss or Germany) supplied N,N-dimethylformamide (DMF) reagent grade, sodium dodecyl sulfate (SDS) and sodium formate. Sodium chloride was from Carlo Erba (Milano, Italy). Sodium di-hydrogenphosphate and 1,3-phenylenediamine (PDA) were purchased from Merck (Darmstadt, Germany). The rest of the products used were purchased from Panreac (Barcelona, Spain) and were PA and ACS-ISO grade. All the Milli-Q water used proceeds from a millipore water purication system (Molsheim, France). The non-woven fabric 3329 was supplied by Holytex (France). 2.2. Apparatus A rotary mixer (Heidolph, model RZR1, Germany) was used for the dilution of the polysulfone. The cell used for the amino acid transport experiments,

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consisting of two separate compartments for the aqueous feed and stripping solutions connected by a circular window where the membrane was placed, was home made [28]. The d.c. stirring motors (JVC and Maxon A-max, Switzerland) were used to stir the contents of the two compartments. A tachometer (Ika-Tron, model DZM 1, Germany) was employed to monitor the stirring rate. A capillary electrophoresis system (System MDQ Beckman, Palo Alto, CA) and an UVVIS spectrometer (UV-2, ATI-Unicam, UK) were alternatively used to determine the concentration of the studied amino acids in the feed and the stripping aqueous solutions, depending on the media and on the presence of other amino acids, specially for the selectivity studies. 3. Methodology 3.1. Membrane casting preparation The membranes were prepared in the laboratory according to an established protocol [29,30]. The casting solution of polysulfone (15% in mass) was prepared by dilution overnight of the commercial polysulfone in N,N-dimethylformamide. This casting solution was used for making the PS membranes over the non-woven fabric by phase inversion technique, as described elsewhere [31]. The preparation of the thin dense layer of polyamide containing different amounts of DEHPA (2501200 mM) was carried out by interfacial polymerization technique [31]. This dense layer was prepared from an aqueous amine solution (PDA) that was used to impregnate the hydrophilic PS membrane and a hexane solution containing DEHPA and 1,3,5-benzenetricarbonyl trichloride. The excess of solution was removed with distilled water and nally all the membrane was dried out in an oven at 60 C during 15 min and then stored for its later use. The membrane prepared is made of two layers, the macroporous polysulfone and the denser polyamide. The DEHPA is retained mainly in the dense layer but also is present in the macroporous one. 3.2. Determination of amino acids Direct UV spectrophotometric analysis of the single aqueous solutions of Phe, Trp and Tyr was used to

follow up their transport process through the activated composite membrane. In the case of mixtures, of these amino acids or in presence of media with a high absortivity in the range of the wavelength used, capillary electrophoresis (CE) was used to monitor the transport of the different amino acids through the membrane. The direct analysis was performed at 218224 nm. Either the aqueous feed or stripping solutions in absence of the amino acid was placed in the reference beam. Corresponding calibration curves were obtained prior to the measurement of the samples to determine the concentration of the given amino acid. To ensure both, the correct way of analysis by using the direct method and to check the absence of looses of organic compounds from the membrane, the UV spectra of the single amino acid samples and of some of the samples of amino acids mixture were registered. The CE analysis was performed in a 75 m internal diameter, fused-silica capillary of 50 cm to the photometric detector (60 cm total length). The optimized running buffer was a 50 mM solution of NaH2 PO4 with the pH adjusted to 2.0 with drops of HCl 1.0 M. The applied voltage was 20 kV (it means a eld strength of 333 V cm1 ) and the capillary temperature was set-up at 25 C. Samples were introduced by hydrodynamic injection for 5 s at 0.5 psi. The electropherograms were recorded at 214 and 254 nm with the PACE-MDQ apparatus. In this system, the samples were stored at 10 C, in the same instrument during analysis. The capillary was conditioned daily by washing it with water (3 min) followed by freshly prepared 0.1N NaOH (5 min) and water (5 min). All the rinsing processes were made at 20 psi. In order to optimize the reproducibility of the migration time, the capillary was ushed before each sample analysis with water (1 min), 0.1N H3 PO4 (5 min) and also with buffer (10 min). After each sample analysis, the capillary was ushed with water (1 min). At the end of the day, the capillary was ushed with water (2 min), 0.1N NaOH (3 min), water (1 min) and methanol (1 min) in order to remove any rest of organic material and to facilitate the cleaning and drying of the capillary for next work. 3.3. Transport experiments The membrane support, prepared as described above, was placed in the circular window separating two compartments of the transport cell with the

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polyamide layer facing the feed solution [28]. The feed and the stripping aqueous phases were placed in the corresponding compartments and the experiment started by switching on the stirring motors used for the agitation of the aqueous solutions. In each experiment, the corresponding stirring rates were kept constant during the experiment, during 6 h. All the experiments were performed at 24 1 C in a thermostated room. Different chemical parameters, including membrane and aqueous solution compositions, were varied systematically to determine their inuence on the mass transfer of the phenylalanine (Phe) through the activated composite membrane (ACM) containing DEHPA as carrier. After a previous characterization of the DEHPA-ACM system for Phe transport, its initial concentration in the feed solution was varied in the range of 0.051 mM, in order to evaluate its inuence on the Phe transport rate. Different acid stripping solutions were investigated, so different mineral acids were tested. Among them, HCl solution was selected and studied at concentrations ranging between 0.001 and 2.0 M. The inuence of the amount of carrier immobilized in the membrane was evaluated, in separate experiments, by varying the DEHPA concentration in the casting solution [32], ranging from 0 to 1200 mM. The inuence of the initial pH of the feed solution onto the Phe transport was also studied by varying the pH from 0.5 to 4. Unless otherwise noted, the conditions for the membrane experiments were: a 0.5 mM Phe solution in 2.0 M NaCl at pH 3 (adjusted with HCl) as feed solution, 2.0 M of HCl as stripping solution and an activated composite membrane prepared with a solution of 1000 mM of DEHPA as casting solution. To determine the selectivity of the DEHPA-ACM over other aromatic amino acids than Phe, tryptophan (Trp) and tyrosine (Tyr) were assayed. Three sets of experiments were performed. Each set involves experiments using a single amino acid solution as feed phase, and additional experiments with corresponding binary mixtures of the mentioned amino acids. In order to calculate the amino acid transport rate, samples were periodically withdrawn during 6 h from both feed and stripping aqueous solutions to determine the amino acid concentration. For this, UV direct analysis was performed, except for the selectivity studies or in presence of a highly absorbent media where the CE technique was employed, as mentioned

before. Also, the pH of the feed solution was monitored during the experiment in order to control a possible ux of protons. Each data value reported is the result of at least two replicates.

4. Results and discussion The transport data obtained as described in the experimental section were transformed into permeability coefcient values dened by the following equation [28]. P = Vf dC CQ dt (1)

where P is the permeability coefcient of the membrane (cm min1 ), Vf the volume of the feed solution (ml), and Q the area of the liquid membrane phase in contact with the aqueous phases (cm2 ). After integration of this equation we obtain ln Q Cf = Pt Cf0 Vf (2)

where Cf0 and Cf (M) are the concentrations of the corresponding amino acid in feed solution at time zero and t, respectively. The permeability is a measure of the transport rate or ux and can be calculated by plotting the rst term of Eq. (2) against time. The concentration of the amino acid at the feed solution at different times can be used directly as Cf in Eq. (2). As reported previously [33], when the stripping process is slower than the extraction of the analyte by the liquid membrane from the feed solution, the stripping permeability (Ps ) can be used, as it is in the present case. This is based on the periodical measurement of the amino acid concentration in the stripping solution. The permeability coefcient, Ps , was used to assess the inuence of the chemical variables on the ACM performance. 4.1. Characterization of the DEHPA-ACM To establish the inuence of the mentioned chemical parameters on the amino acid transport through a DEHPA-ACM, a systematic study was carried out including basic aspects of the membrane transport, i.e.

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the verication of the facilitated transport, the existence of a counter transport mechanism, the reproducibility of the transport system and the effect of buffering the aqueous feed solution. Thus, a blank experiment was performed by using a ACM containing no DEHPA (blank membrane) and a negligible transport was observed (0.12 103 5109 cm min1 ). Also, the counter transport of Phe against its concentration gradient was conrmed by using an acidic stripping solution (as indicated previously) containing the same initial amount of Phe that on the feed side. The transport rate obtained in this case was the same than when no Phe is present in the receiving phase from the beginning. The reproducibility of the ACM transport system was determined by specic experiments using two membranes prepared separately with the same DEHPA composition. Five consecutive transport experiments were performed with each ACM. The membranes were rinsed with an acidic solution between every experiment, to remove any residue of amino acid from the previous experiment. Each transport experiment was performed during 6 h (membrane 1: 5.05 103 9 107 cm min1 ; membrane 2: 4.30 103 6 107 cm min1 ). A statistical evaluation of the ob95% tained results for Phe transport (t exp = 1.392; tteo = 2.306) revealed no signicant differences between the performance of both membranes and the relative standard deviation was of an 18%. So, the procedure, based in the interfacial polymerization technique, to prepare the DEHPA activated membranes is quite reproducible. Further more, the results obtained from these ve consecutive experiments demonstrates a good stability of the membrane. Also the effect of buffering the aqueous feed solution was evaluated by using 0.5 M of formic acid/formate buffer at pH 3.5. No difference on Phe transport was appreciated for the rst 6 h of experiment comparing to its transport in absence of buffer. Thus, no buffer was used on the feed solution later on. 4.2. Inuence of the amino acid initial concentration on the feed solution The initial concentration of Phe in the feed solution was varied between 0.05 and 1.0 mM in order to evaluate its inuence on the Phe transport rate. Results

Table 1 Effect on the ACM transport of the initial concentration of amino acid in the aqueous feed phasea [Phe]initial (mM) 0.05 0.10 0.50 1.00 Ps (103 cm min1 ) 11 7 3 3 3 4 1 1

a The rest of parameters was kept as the standard conditions reported in the methodology section.

collected in Table 1 shows two trends of Phe transport in this amino acid concentration range. It is observed that at high initial Phe concentrations, the amount of amino acid that cross the membrane is relatively high, so it is easy to monitor the process by following the Phe concentration in the stripping (the uncertainty is low), although the relative concentration transported is small. Thus, bigger amounts of amino acid found in the receiving phase, do not represent bigger permeabilities or faster transport. On the other hand, lower initial concentration results in a lower amount of amino acid transported and the Phe determination is less accurate. According, to these results, an initial concentration of 0.5 mM was selected as a compromise of both feasibility and sensibility for the characterization of the transport. 4.3. Effect of the composition and concentration of the stripping agent As it is reported in the literature [11], the facilitated transport of amino acids by DEHPA-SLM systems is accomplished from feed solutions at pH near to the pKa of DEHPA (1.27) to an acidic stripping solution. It is also known that in these SLM systems, a HCl stripping solution produces the highest amino acid transport against other mineral acids, such as H2 SO4 and HNO3 [11]. To compare the behavior with our DEHPA-ACM system, corresponding experiments were carried out under similar conditions of those previously reported for SLM [11], so these three mineral acids were used as stripping agents. Relative to the HCl transport, a 20 and an 80% reduction of the permeability coefcient was found for H2 SO4 and HNO3 , respectively. This behavior of DEHPA-ACM follows the same

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Fig. 1. Inuence of the acidity of the stripping solution on the permeability coefcients for Phe transport through DEHPA-ACM. Where () stands for the transport experiments with equal ionic strength in both the feed and the stripping aqueous solutions, by a proper addition of NaCl salt. In ( ) the ionic strength was not balanced. In all these experiments, the rest of parameters were keep at the reported default values (see methodology). Bars indicate the standard deviation of the experiments. The dotted line does not represent a model.

pattern of the corresponding DEHPA-SLM [11]. As the results using HCl correspond to the highest Phe transport rate, this acid was chosen as striping agent. The effect of HCl concentration was studied. To this purpose, two sets of experiments were carried out. In the rst one the ionic strength of both aqueous phases was adjusted to be the same, on the contrary, for the experiments in the second set, this condition was not established. NaCl was the added salt to the feed phase in the rst set to avoid the introduction of another different anion on the system. As seen in Fig. 1, the stripping permeability coefcient, Ps , increases with proton concentration in the receiving phase. This behavior is a consequence of the different speciation of both, the DEHPA used as carrier (immobilized in the ACM) and the analyte Phe, with the pH. So, at higher acidity of the stripping solution, the respective protonated forms of DEHPA and Phe predominate and the corresponding DEHPAPhe complex breaks down to accomplish for the Phe transport to this acidic receiving phase. Also, no signicant inuence of the aqueous ionic strength was observed, i.e. the

results from both sets of experiments are practically the same. 4.4. Inuence of the carrier concentration It is well-known that the rate of facilitated transport of the analytes is directly affected by the amount of carrier immobilized in the corresponding liquid membrane. A linear relationship between the concentration of DEHPA present in the casting solution and the amount of DEHPA immobilized in the ACM has been reported elsewhere [32]. So, based on these results, ACM prepared with different casting solutions at different concentrations of DEHPA were used to characterize the Phe transport. The results collected in Fig. 2 show the same prole that those typically obtained with other transport systems based on liquid membrane congurations (i.e. SLM). From these results, a diffusion limited transport can also be suspected for our ACM system. A 1000 mM DEHPA casting solution was selected to prepare the DEHPA-ACM to get the maximum Phe transport as well as the best DEHPA immobilization.

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Fig. 2. Inuence of the amount of DEHPA immobilized onto the ACM on the Phe transport by varying its concentration in the corresponding casting solution.

4.5. Inuence of the initial pH of the feed solution It is observed that the small change in the pH of the feed solution occurring during 6 h of the transport experiments produces no effect on the transport of Phe. On the other hand, the variation of the feed initial pH

results in a variation of the ACM permeability (see Fig. 3). As seen, the permeability of ACM decreases with pH lower than 3, specially when pH is lower than the amino acid pKa 1. Thus, the mechanism of transport seems to be the same observed in other SLM [2,10], and it is limited by the availability of the

Fig. 3. Inuence of the initial pH of the feed phase in the Phe transport through a DEHPA-ACM system.

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dimmeric or the anionic form of the carrier to perform the corresponding cation exchange reaction with the positively charged Phe. Thus, the corresponding complex formation may follow the Eq. (3) as Phe+ + 2(HL)2 PheL(HL)3 + H+ Kex (3)

where bars design species in the organic phase. This equation supposes the partially loss of the proton of DEHPA and its interaction with the positively charged amino acid. The plateau observed at pH > 3 is compatible with this reaction and can be explained by the diffusion resistance of the membrane rather than the chemical properties. 4.6. Selectivity of the DEHPA-ACM Once the conditions of ACM transport for Phe were characterized, other aromatic amino acids, such as tryptophan (Trp) and tyrosine (Tyr) were assayed in order to ascertain a possible discrimination by the membrane performance. Experiments were done both using single amino acid solutions and binary mixtures, as described in the experimental part. The experimental conditions were kept as stated before. The obtained results are collected in Fig. 4. The results are displayed

as follows: data on Fig. 4A represents permeability coefcients of the Phe both in absence and in presence of another target amino acid (either Tyr or Trp). Data on Fig. 4B concerns to Trp transport and Fig. 4C are the related data for Tyr. With reference to the transport of single amino acids, it is observed that Phe and Trp have very similar transport rate while Tyr is almost three times slower. Such differences can be attributed to the respective afnity of these amino acids for the extractant DEHPA, being in the order of Phe > Trp > Tyr. The respective lipophilic characteristics of the target amino acids contribute to maintain this relationship [34]. On the other hand, binary mixtures of these amino acids respond to membrane transport in a very different form. Thus, while Phe transport decreases sharply in presence of the other amino acids (specially Trp), Trp does not accuse so big difference when it is carried by membrane from their respective binary mixtures. Such a difference, interpreted in terms of competitive transport, can be attributed to the known capability of the aromatic rings to establish hydrogen bonds with other molecules bearing acidic hydrogens, as it has been previously reported for Phe and, specially, for Trp because of its indol group [35].

Fig. 4. Inuence of binary equimolar mixtures of aromatic amino acids in the transport of the rst one. Plot A represents the permeability coefcient of Phe alone or in presence of Trp or Tyr (Phe, PheTrp and PheTyr in x-axis, respectively). Plot B stands for Trp and C for Tyr. The rest of conditions are as the reported default values.

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In the case of Trp, its higher molecular surface and the presence of the indol group seem to be the principal reason to have a stronger interaction with the corresponding ACM containing DEHPA as carrier. Furthermore, the stability constant of the extraction reaction in n-dodecane reported in the literature for Trp (0.0554 l mol1 ) is lower than the stability constant for Phe (0.117 l mol1 ) [10]. This thermodynamic difference may explain, together with the solubility differences, the easier mobility of Trp against Phe inside of the ACM, which is known to be the higher resistance path for the facilitated transport an thus the step controlling the transport rate. Acknowledgements The present work has been carried out under the nancial support of CICYT (The Spanish Commission for Research and Development), project (QUI99-0749-C03-01). J.A. Calzado wants to acknowledge the CIRIT (Commission for Research and Development of Catalunya) the personal support received by means of a pre-doctoral scholarship. References
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