liquid membranes in separation of relevant biological

Systems Membranes-complex roadmaps towards functional devices and coupled processes SYSMEM

LIQUID MEMBRANES IN SEPARATION OF RELEVANT BIOLOGICAL SPECIES Lucia MUTIHAC

University of Bucharest, Department of Analytical Chemistry, 4-12, Regina Elisabeta Blvd., Bucharest, 030018, Romania Abstract The selectivity of macrocyclic hosts such as crown ethers, modified cyclodextrins, and functionalized calixarenes to various biorelevant guests and their ability to be used as carriers through membrane processes is presented. Moreover the liquid membrane separation of amino acids, peptides, proteins, and saccharides bu using these receptors is summarize hereafter. Likewise, the mechanisms of transport through liquid membranes and factors involved in separation of biological compounds are discussed. Aspects of membrane ultrafiltration of proteins, blood plasma processing by membrane separation, permeation of amino acid through ultrafiltration membranes, and separation of some amino acids through liquid membranes from acid hydrolisates originanting in proteins, constitute some topics overviewed in the present work.

1. INTRODUCTION The recognition and transportation of biological substrates such as amino acids, peptides, proteins, saccharides, and nucleic acids, are of great importance in chemistry, biochemistry, and separation science. The analysis of natural biological compounds for understanding the mechanistic and structural aspects of their behavior has constituted the main topic of a large number of studies [1-5]. The liquid membrane processes are efficient techniques for selective separation, concentration, and purification of chemical and biological compounds. Liquid membranes in separation of various compounds have the advantage of enhanced transport by using a selective carrier of anionic or cationic form dissolved in an organic solvent. Moreover the separation of enantiomer compounds may be carried out by using liquid membranes containing chiral transportors. It is well known that the membrane processes have already been successfully applied in advanced components of analytical instruments (i.e., membrane sensors), biomedical and

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biotechnological applications (i.e., artificial organs), textile and pharmaceutical industry [6-9]. The main potential applications of membranes and membrane processes are indicated by the growing interest in (i) the use of membrane processes for recovery of valuable raw materials, (ii) removing bacteria and suspended solids to reach drinking water quality, (iii) producing demineralized water from natural sources. Membrane technologies are able to produce high purity chemicals using biotechnological processes. Pigments and dyes can be easily removed and concentrated by membranes.One of the most important fields in membrane operation stems from combining selective mass transfer through membrane with a specific chemical reaction in a biological process. This combination is possible using the membrane itself as a reactor. In these systems, the chemical transformation and physical separation of species from the products may take place in the same time while minimizing product inhibition phenomena. This kind of biological membrane reactor has already been used in the wastewater treatment [10]. Membrane separation in bioreactors is one of the most attractive operations applied in biochemical processes. Enzyme membranes increase the potentiality of membrane separation in biochemical processes as well. Drioli et al. [8] investigated the possibility of catalytic membrane systems. Thus, membrane reactor configuration, the applications of dense and porous membranes to various reactions, their ability to work in gas or liquid phase using various driving forces (i.e., transmembrane concentration gradient, transmembrane gradient pressure, and transmembrane gradient temperature) have been employed in recent applications: decomposition reactions (e.g., H2S, etc.), dehydrogenation reactions (e.g., cyclohexane, ethylbenzene, etc.), and hydrogenation reactions (e.g., butadiene, acetylene, etc.). The processes in a membrane bioreactor can be considered as the combination of two basic processes: (i) biological reaction and (ii) membrane separation into a single process. Membrane processes are becoming new techniques in drinking water treatment, industrial water, and industrial effluents and domestic wastewater due to the growing concern regarding the rapid increase in level of environmental pollution over recent decades.2-5 The cleaning of contaminants that are released in the environment in appreciable amount causing pollution problems in all part of the world is one of the most important problems today. The application of molecular biology (DNA microchips, genetic probes) for toxicological tests and epidemiological studies permit , at present time the evaluation of the microbial health risks. Now, the evaluation of the microbiological safety of water is mainly based on the ability of

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bacteria to grow up on a special nutritional media. So, with new microbial methods can be differentiate the pathogenic species, their viability and virulence, viruses and parasites such as Giardia and Cryptosporidium or bacteria such as Escherichia Coli and Salmonella. Macrocycles pre-designed to selectively interact with target inorganic or organic guests had an important impact in separations chemistry also. In this respect, crown ethers, cyclodextrins, and calixarenes were widely used as extractants or carriers through liquid membranes concerning the separation of different chemical and biological compounds. It is well known that biogenic amines, amino acids, hormones, sugars, peptides, nucleic acids, and proteins constitute the most fundamental substrates in biological and artificial processes. The understanding of specific biomolecular interactions plays a key role in modern supramolecular chemistry. The present work focuses on recent studies on the separation through liquid membranes of some biological compounds by using crown ethers, modified cyclodextrins, and functionalized calix[n]arenes as carriers, and meaningful aspects concerning the membrane ultrafiltration of proteins, blood plasma processing by membrane separation, and the permeation of amino acid through ultrafiltration membranes. In this respect, the transport mechanisms and the factors involved in separation will be briefly presented. 2. MEMBRANE ULTRAFILTRATION OF PROTEINS Proteins are generally purified and fractionated using chromatographical methods like affinity chromatography. Membrane technology can be involved in many other cases using the same types of separation principles as in chromatography in addition to the natural separation depending on size. The physico-chemical properties of the membranes and proteins influence the fractionation process of proteins [11]. It was shown that the best pH values for fractionation of globular proteins were such that one protein had its izoelectric points at this pH and, consequently, permeated the membrane, while the other one was held back in the retentate because of charge repulsion with the membrane. There were employed only small concentrations of proteins and the kind of membranes used were hydrophilic or hydrophobic ultrafiltration membranes of different types of materials. The membrane modules were cross-flow modules. Cusserand and Aimar [12] reported a theoretical model in which exclusion of proteins by electrostatic repulsion is presented as a function of pore size, molecular size, and charge density in the electrolyte. A partition coefficient of a charged protein between the

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bulk and the porous medium can be computed using Boltzmann distribution. The results were in good agreement with the literature data. To conclude with, the electrostatic interaction between the membrane material and the charged molecules may significantly modify the selectivity of the membrane. Based on Boltzmann statistics, Cusserand and Aimar [12] estimated the following equation for the probability distribution of the proteins between bulk and porous medium

 ∆F  2 (1) = (1 − λ ) exp −  cb  kT  where ∆F is the free energy, k is the characteristic Debye length, and λ is the pore radius ( λ < 0.2 ). The partition coefficient Φ was cp

deducted from the fusion flux measurements using the following diffusion flux of bovine serum albumin and α-lactalbumin through porous ultrafiltration membranes (sulfonated polysulfone IRIS membranes, 100 kD)

J d = D 0 ∆C where

ρ l

ρ l

(2)

Φ

denotes the porosity to membrane thin layer thickness

ratio. This ratio was determined by diffusion measurements of molecules of different sizes at high ionic strengths and resulted a value of 4×10 3 m-1. The model presented could be useful for membrane characterization. Le Berre and Daufin [13] used the cross-flow microfiltration for separating β-casein from sodium caseinate. The separation was possible owing to the selective solubilization of β-casein monomers from whole casein submicelles at low temperature. Separation of βcasein using membranes was mainly controlled by a particle cake build up. A special interest in membrane processes is the membrane ultrafiltration of protein solutions. The most important factors involved are: (i) the role of concentration and/or gel polarization, (ii) the increase of solvent flux, and (iii) the solute retention behavior of some particular membranes. The last two issues particularly stimulated the attention paid to the development of sophisticated fluid management techniques to reduce polarization and to increase solvent flux,20,21 while simultaneous efforts were mounted to understand polarization in protein ultrafiltration in terms of a gel layer as well as osmotic limitations.22,23 In the same time, industry is acting towards membrane

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fabrication with sharp cutoff behavior aiming to efficient utilization of existing membranes with diffuse cutoff behavior. Further, some other important factors in protein ultafiltration may be considered, such as (i) the role of protein adsorption on the membrane, (ii) the effect of pH and protein aggregation, (iii) the unsteady initial behavior of membranes having a limited solute permeability, (iv) the ionic strength, (v) some phases stirring, and (vi) the membrane hysteresis. An additional issue of interest is reproducibility as related to membrane cleaning. The experimental results suggest that the protein adsorption on and within the ultrafiltration membrane may influence the membrane performances. The aggregations of protein molecules determined by the pH, ionic strength, and so on, affect significantly the membrane transport behavior [24]. 3. BLOOD PLASMA PROCESSING BY MEMBRANES The study of the function of an isolated protein is a main topic in modern protein chemistry. The blood plasma proteins involved in hemostasis, inflammations, and host defense mechanisms are of special interest. These proteins are trace components in blood and circulate in zymogen or preactivated forms which convert to biologically active states in vivo following appropriate stimuli. Experiments are focused on plasma protein fractionation that allows separation of trace plasma protein components, yet keeping unaltered their functional and physico-chemical properties. Bing et al. [14] carried out separation of trace protein components from large volumes of human plasma by electrodyalisis across ion-selective semipermeable membranes. The resulting plasma fractions were then used, or proposed to be used, for further fractionation by other chemical methodologies. In aqueous solutions proteins are polyionic and bind water as well as ions. The fractional precipitation of plasma proteins by controlling ionic strength and pH is based on the protein solubility properties and reflects the balance between hydrophilic and hydrophobic group on the molecule. The relationship between the solubility (S) of a protein and ionic strength (I) is expressed by log S = − K S I (3) where K S is the salting-out coefficient which is dependent on the pH and temperature. The schematic representation of the electrodialysis process used in plasma deionization is presented in Fig. 1 [14].

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4. PERMEATION OF AMINO ACIDS THROUGH ULTRAFILTRATION MEMBRANES Membrane systems are also used to produce and/or separate amino acids, which are basically pharmaceutical and food additives. Kimura et al. [15] used charged ultrafiltration membrane reactors to separate amino acids. Along the same topics, Tone et al. [16] optically resolved racemic tryptophan, phenylalanine, and tyrosine using an ultrafiltration plasma polimerized membrane. Drioli et al. [28] employed the bacteria Radiobacter containing hydantoinase to produce intermediates of D-amino acids in a membrane segregated enzyme reactor with tube-and-shell configuration. The advantage of the latter system consists in the D-amino acid intermediate (in this case N-carbamil-p-OH-phenyl hydantoine) which is continuously produced and separated. Further, the acid may be converted in a subsequent reaction, i.e., by using carbamilase enzyme, to the corresponding D-amino acid. ( = Plasm a Stream, Electrode Stream

_ X

X

A

M

_ X

M

C

X

+

Electrode Stream

_ +

M

M +

+

M

M

X

A

C

A

_ X

+

M +

C _ X

_ +

+

M

(Anode)

C

A

C

A

= Transfer Stream )

_

_ X D*

D* C* D* C* D*

(Cathode)

C* D*

H2, MOH

X2, O2 HX

( = Desalted Pla sm a Stream, = Concentrated Transfer Stream )

Fig.1. Schematic representation of the electrodialysis process used in plasma deionization; A: anion transfer membrane, C: cation transfer : + membrane, X anion; M : cation, D*: desalted plasma cell, C*: concentrating cell [14].

Creagh [17] investigated micellar-enhanced ultrafiltration as a large scale technique for separating D-phenylalanine. L-5-cholesterol glutamate, a chiral ligand-exchange cosurfactant, was used together

246


with a non-ionic surfactant to form mixed micelles that preferentially bind D-phenylalanine over L-phenylalanine in the presence of copper (II). Micelles containing the chiral-ligand cosurfactant were compartmentalised in a continuous stirred cell by a cellulose ultrafiltration membrane. Amino acids having an amino group and a carboxyl group are positively charged by protonation in low pH solutions, whereas they dissociate into anions in high pH solutions. As a consequence, they show specific permeation characteristics when they permeate through the charged membranes at various pH values. Masawaki et al. [18] reported the mechanism of aqueous amino acid solutions at various pH values through positively and negatively charged ultrafiltration membranes. The friction parameters are evaluated according to the transport equation using experimental data. In permeating through the membrane, the zwitterion of the amino acid has no net charge and these fluxes are not affected by the fixed charge of the membrane. From the experimental data, the pure water permeability of the negatively charged membrane was smaller than that of the positively charged membrane. It means that the size of the membrane pore in negatively charged membrane may be smaller than that of the positively charged membranes. Taking by example the glycine and histidine separation, although the molecular size of glycine is smaller than that of histidine, the friction parameter between glycine zwitterion and the membrane pore wall in negatively charged membranes becomes larger than that between histidine and the membrane pore wall in positively charged membranes. 5. SEPARATION OF AMINO ACIDS AND DIPEPTIDES THROUGH BULK LIQUID MEMBRANES FROM PROTEINS Crown ethers as carriers through liquid membrane Since Pedersen in 1967 [19] reported the first example of synthetic molecular recognition of polyethers, many other examples of synthetic models have been developed and reported. In this respect, the design and synthesis of efficient artificial receptors for selective binding of biologically important species has been a dynamic field of intense interest over the past decade and has come out with many applications [1, 20, 21]. The relatively new class of chemical compounds called macrocyclic ligands [1], which are able to specifically recognize through controlled interactions according to size, shape, and structure of various cations, and then by means of these, various anions, has

247


allowed the study the amino acids whose amphion type molecule may appear either as cation in acid medium or as anion in basic medium (Fig. 2). The amino acids may be considered the fundamental constituents of a wide variety of biological macromolecules. In this respect the separation of these compounds is important for both analytical point of view and biochemical as well. Moreover, the understanding of selective recognition and transport of amino acids is of fundamental interest, in part from the point of view of mimicking natural biological systems. Due to their applications in separation, diagnostic or biological areas, short peptides are important targets for molecular recognition. Short peptides exhibit the binding ability because they are usually conformational flexible. Using an ion-pairing mechanism some protonated peptides (Glycyl-glycine, Glycyl-L-α-alanine, Glycyl-Lvaline, Glycyl-L-leucine, Glycyl-L-phenylalanine, Glycyl-glycyl-glycine, Glycyl-L-aspartic acid, and L-leucyl-glycyl-glycine) were transported through chloroform liquid membrane by using crown ethers as carriers in the presence of counterions [22]. Mixtures of α-amino acids may be separated in cationic form through a liquid membrane of 18-crown-6 (18C6), benzo-18-crown-6 (B18C6), dibenzo-18-crown-6 (DB18C6) in 1,2-dichloroethane in presence of picrate anion. The separation process is performed according to character (more hydrophobic or hydrophilic) of the Rchain from the amino acid molecule (R − CH(NH 2 )COOH) . Therefore, a classification of amino acids is eligible: (i) extractibles (i.e., L-Met, L-Ile, L-Phe, L-Leu, L-α-Ala, L-Cys), and (ii) nonextractibles (i.e., L-Asp Acid, L-Glu acid, L-Ser, L-Thr, L-Pro, L-Lys, LTyr, L-Arg, L-Hys) from mixtures [23].

Fig.2. Chemical structures of some crown ethers used as carriers.

Models which describe the macrocyclic mediated transport of cations across bulk liquid membranes indicate that the membrane solvent plays an important role in global membrane performance. There are four of the parameters mainly affecting the mass transfer

248


which are solvent-dependent: (i) the equilibrium constant for cationmacrocyclic interaction in the membrane, (ii) the thickness of the boundary layers of the membrane, (iii) the partition coefficients, and (iv) the diffusivities of the species involved in transport. As a general rule, any solvent employed as an organic phase should be one that retains the mediating carrier to a very large extent and yet can accommodate relatively high concentrations of water to aid in the transfer of hydrated ionic species without loss of carrier within the aqueous phases. Both requirements are influenced by the polarity of the solvent, which appears to be the most important factor in determining its effectiveness as membrane medium [24]. The overall rate of substrate transport through bulk liquid membranes can be controlled by one of a series of resistances to mass transfer in the membrane system. Specifically, in carrier mediated transport, the rate may be limited by resistances associated with mass transfer of substrate within the source or receiving phases, diffusion across the organic phase, or interfacial complexation/decomplexation reactions. Estimation of the ratelimiting process provides the possibility to optimize both the substrate transport in separation techniques (i.e., mass transfer and selectivity), as well as the design of new synthetic carrier molecules.

Fig.3. Mechanism of carrier (L) mediated active transport of ion pair +

−

( [L - AA] A ) through bulk liquid membranes assisted by pH gradient; AA+ - amino acid in protonated form; A- - counter ion; L - ligand macrocyclic

Generally, the bulk liquid membranes do not permit a good control of the hydrodynamic behavior of the systems involved. An important issue in their study concerns the diffusion processes, which depend on stirring rate variations, whereas interfacial processes do not.

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Several mathematical models have been proposed in the literature to characterize the active transport processes in bulk liquid membranes. The conceptual model of amino acid mediated transport by macrocyclic ligands through bulk liquid membranes assisted by pH gradient is presented in Fig. 3. Modelling ion pair transport of amino acid or dipeptide by macrocyclic receptors through liquid membrane The transport of amino acid or dipeptide in protonated form from the source aqueous phase to the organic phase as ion pair can be represented by the following equation:

(R − NH ) + (L) + (A ) + 3 w

−

o

w

⇔ (R − NH3LA )o

extraction

(4)

characterized by the equilibrium constant:

[

K ex = [R − NH3LA ]o R − NH3+

] [A ] [L] −

w

w

(5)

o

where the subscripts w and o define the aqueous phase and the organic phase respectively. Further on, the transport of amino acid or dipeptide as ion pair through the organic phase may be written:

(R − NH3LA )os ⇔ (R − NH3LA )or

membrane transport

(6)

where the subscripts os and or denote membrane/source interface and membrane/receiving interface, respectively,

(R − NH3LA )or

( ) + (L)

⇔ (R − NH2 )w + A −

w

o

receiving

(7)

The active transport, assisted by pH gradient of amino acid or dipeptide in protonated form as ion pairs in the presence of picric acid or tropaeolin 00 was performed. The experimental results of studies on amino acid active transport through liquid membranes were applied to acid hydrolisated samples originating in proteins from milk Casein (Table 1) and Albumina Bovis (Table2). According to the experimental data from Table 1 and 2, the extraction yields within 50% and 81% (depending on the amino acid) for β-Casein and within 55% and 87% for Albumina Bovis are fairly high. For some amino acids like lysine, the extraction yields (6% for β-Casein and 7% for Albumina Bovis) were evidently much lower as compared with the model mixtures in the same conditions [25].

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Table 1. Experimental results obtained in separation through liquid membrane of the amino acids from proteic hydrolysate of Casein [25] Initial amount µg / 5 mL 91.50

Source

13.72

Casein phase (5 mL) % 15

Leucine

112.50

11.97

11

88.30

79

Phenylalanine

66.00

20.79

32

39.64

60

Valine

90.00

34.65

39

47.07

52

Methionine

53.50

21.50

42

31.69

59

Alanine

41.00

18.04

44

20.83

51

Cystine

14.00

6.30

45

7.03

50

Lysine

131.50

118.61

90

8.05

6

Histidine

42.00

42.19

100

_

_

Arginine

62.50

60.75

97

_

_

Aspartic acid

121.50

115.47

95

_

_

Treonine

70.00

72.93

104

_

_

Serine

90.00

89.86

100

_

_

Glutamic acid

321.50

308.80

96

_

_

Proline

116.50

110.67

95

_

_

Glycine

36.50

35.77

98

_

_

Tyrosine

116.50

113.58

98

_

_

Amino Acid

Isoleucine

µg

Receivin g

µg

73.67

phase (10 mL) % 81

Source phase: amino acid; [picric acid] = 1.6 × 10 −3 M; HCl, 0.05 N (pH=2.02); Receiving phase: LiOH, 0.1 N (pH=13.01); Membrane: [18crown-6] =

10 −2 M/1,2-dichloroethane; Phase ratio: 5:50:10 (v/v/v).

The interfaces thermodynamic equilibria by complexation and decomplexation influence the rate of transport through membrane when the diffusion process of the complex through membrane phase is rate-limiting. Several mathematical models were proposed in the literature to characterize the active transport processes in bulk liquid membranes .

251


Table 2. Experimental results obtained in separation through liquid membrane of the amino acids from proteic hydrolysate of Albumina Bovis [25]

µg

Albumina phase (5 mL) %

Isoleucine

43.50

5.22

12

37.63

87

Leucine

135.00

14.17

11

114.75

85

Phenylalanine

84.50

16.97

20

64.32

76

Valine

81.00

25.11

31

52.93

65

Methionine

100.50

35.24

35

63.06

63

Alanine

26.00

8.29

32

14.70

56

Cystine

182.50

73.36

40

100.37

55

Lysine

179.00

161.99

91

12.17

7

Histidine

57.00

54.76

96

_

_

Arginine

93.00

91.97

99

_

_

Aspartic acid

125.00

123.75

99

_

_

Treonine

91.50

86.19

94

_

_

Serine

66.50

63.51

96

_

_

Glutamic acid

234.50

231.45

99

_

_

Proline

66.50

64.30

97

_

_

Glycine

10.00

9.50

95

_

_

Tyrosine

86.00

84.62

98

_

_

Amino Acid

Initial amount

µg

/ 5 mL

Source

Bovis Receiving

µg

phase (10 mL) %

Source phase: amino acid; [picric acid] = 1.6 × 10 −3 M; HCl, 0.05 N (pH=2.02); Receiving phase: LiOH, 0.1 N (pH=13.01); Membrane: [18-crown-6] = 10-2 M/1,2-dichloroethane; Phase ratio: 5:50:10 (v/v/v).

It is well known that the partition of crown ether and compounds under study, complexation, and the interaction of carrier molecules at the interfaces between the membrane and aqueous

252


phases are physicochemical parameters that characterize the transport through liquid membrane. The structure of the anion used as the counter ion for cationligand complexes exhibits influence upon the rate of cation transport through liquid membrane by modifying the phase distribution of the protonated peptides [22]. The experimental data of the protonated peptides transport though chloroform liquid membrane using 18C6, B18C6 and DB18C6 as carriers are given in Fig. 4 [22].

Fig.4. Experimental data of the transport of some peptides through a chloroform liquid membrane by 18C6, B18C6, and DB18C6 at 298.15 K. Source phase: [peptide]=0.5 mM; [Tropaeolin 00] = 0.5 mM; HCl 0.05 N (pH≈2.01); 5 mL; Membrane: chloroform; [carrier] = 10 mM; 30 mL.; Receiving phase: LiOH 0.01 N (pH ≈ 13.02); 5 mL. % Percentage of peptide found in the receiving phase after 10 hours of stirring

The active transport, assisted by pH gradient of peptides in protonated form as ion pairs in the presence of Tropaeolin 00 using 18C6, B18C6 and DB18C6 as carriers through chloroform liquid membrane was performed. The stability of membrane is influenced by the nature of solvent. In our experiments we used chloroform as solvent ( ε r = 4.81 ). The solvent dielectric constant is an important feature of the electrostatic interaction in forming of ion pairs [26]. The ion pairing increases as the solvent dielectric decreases. Wipff et al. [27]

253


reported interesting studies about the effect of solvent on the conformational state and recognition properties of macrocyclic ligands by molecular dynamic investigations. The differences in the transport yields of peptides with crown ethers used in our experiments can be attributed to the different structural characteristic of these ligands and to the hydrophobicity of peptides as well. Although no significant variation is observed for the values of extraction constants of the peptides under study with above mentioned ligands, the transport yields differ significantly for the same peptides like Gly-L-α-Ala, Gly-L-Val, Gly-L-Asp, Gly-L-Phe. For GlyGly-Gly, the yield of the transport was 77% using DB18C6 as carrier, 71% using 18C6 as carrier and 57% using B18C6 as carrier. Comparing with the others peptides under study, Gly-Gly is transported with the lower yield of the transport using 18C6 and DB18C6 as carriers. Thus, there is a relationship between the stability constants and the rates of cation transport through liquid membrane for metal cations that has been demonstrated by studies of Lehn et. al [28] and Lamb et. al. [29]. It is of interest, therefore to examine the relationship between the stability constants of peptide complexes and their transport through liquid membrane.

80

G ly -L -P h e *

G ly -G ly-G ly Transport Yield (%)

70

G ly-L -L e u *

G ly -L -A s p

60

G ly -L -V a l* 50

G ly-L -A la *

40

30

G ly -G ly* 3 .0

3 .2

3 .4

3 .6

3 .8

4 .0

4 .2

4 .4

4 .6

4 .8

5 .0

S ta b ility C o n s ta n t (lo g K )

Fig.5. Correlation between stability constants (log K) of peptide complexes with 18C6 and their transport yield (%) through chloroform liquid membrane.

In Fig. 5 is displayed the correlation between the values of stability constants (log K) for some peptides with 18C6 and their transport through chloroform membrane.

254


As illustrated in Fig. 5, the following peptides Gly-L-Leu, GlyGly-Gly, Gly-L-Asp and Gly-L-Phe showed better transport through membrane than Gly-L-α-Ala, Gly-L-Val and Gly-Gly. Even though the log K of Gly-Gly is higher than that of Gly-L-Leu, the yield of transport of Gly-L-Leu is higher than that of Gly-Gly. For experimental reasons the log K values were determined in conditions different from those of the transport measurements. So, there is not the proportionality between the transport yield and the complex stability for the peptides under study. Liquid membranes containing modified cyclodextrin as carriers Cyclodextrins are cyclic oligosaccharides composed of six (αcyclodextrin), seven (β- cyclodextrin), eight (γ- cyclodextrin) or more glucose units per macrocycle, respectively (Fig. 6). The size of the cyclodextrin cavities, which is controlled by the number of glucose residues in the cyclodextrin ring, is characterized by an internal diameter 4.5, 7, and 8.5 Å for α-, β- and γ-cyclodextrin, respectively. As a result of different cyclodextrin ring sizes for any given guest complexation, complexes are produced with different stabilities.60-66 The cyclodextrins have a hydrophilic exterior and a hydrophobic cavity able to extract a large variety of compound guests as a function of size, shape, and hydrophobicity of both the cyclodextrin and the guest compound (Fig. 6) [30].

α-cyclodextrin

β-cyclodextrin γ-cyclodextrin Fig. 6. The structure of cyclodextrins

Biologically derived cyclodextrins and their synthetically derivatized analogs are used in host-guest chemistry [30]. Cyclodextrins and their derivatives have also been used in membrane

255


separation processes. They are good candidates for employing in different separation techniques such as chromatographic separations, capillary electrophoresis, and liquid membranes. As compared with their use in some other applications, relatively few reports have been published on the use of cyclodextrins in membrane separation. By means of the transport of the aromatic hydrocarbon (o-, m-, and p-xylene, naphthalene, anthracene, and pyrene) from one hexane phase to another through aqueous phase with α-cyclodextrin and β-cyclodextrin, Poh et al. [31] determined the association constants of the cyclodextrin-aromatic hydrocarbon complexes. The values of association constants of 1:1 complexes formed are in good agreement with those determined by other methods. By using a cyclodextrin dimer 1 Ikeda et al. [32] reported a selective transport of saccharides (Chart 1) through liquid membrane. The separation of a mixture of saccharides is difficult because most saccharides are isomers that only differ in the configuration of specific hydroxyl groups.

1

Chart 1

The system of liquid membrane is useful not only for separation of a mixture of saccharides, but for clarifying the mechanism of action of saccharide transport through a biomembrane as well.

256


The reported system is more successful for the transport of the saccharide through the liquid membrane than the system using the cyclodextrin monomer (Fig. 7)

Fig.7. Transport rates of monosaccharides through liquid membrane mediated by cyclodextrin derivatives [32].

Cyclodextrins were also used for selective extraction of lipophilic guest compounds from organic phase into aqueous phase [30]. Selective transport through liquid membrane by using calix[n]arenes as carriers The family of calix[n]arenes is deeply involved in molecular recognition of chemical and biological compounds (Fig. 8). The calix[n]arenes have an important role in chemical separations, apart from other important applications like chromogenic and fluorogenic sensors and field effect transistors, ion selective electrodes, calixarene molecules for nonlinear optical devices, as liquid crystals or as catalysts in synthetic reactions [33, 34]. By using calix[n]arene derivatives with functional groups such as ether, amide, ketonic, ester, and crown ether increases further their potential applications [35].

257


Fig.8. Structure of p-tert-butylcalix[n]arenes, n = 4, 6, 8.

The molecular dynamic simulations on the structural properties of the complexes of calix[6]arene-crown-6 with ammonium ions ( NM +4 and NH +4 ) in the presence of acetate as counterion were performed in chloroform solution establishing the location of the guest inside the host. The transport of N-benzoyl amino acids through a chloroform liquid membrane by using a calix[6]arene ethyl ester derivative as selective carrier at was reported by Chang et al. [36] The transport rate was function of hydrophobicity of the guest anions and the size of alkaline metal cations, which coexisted in the source phase. The transport rate for a given cation increased with increasing hydrophobicity of amino acids as follows: Bz-Gly < Bz-Ala < Bz-Val ≅ Bz-Trp < Bz-Phe. In these experiments, the separation of amino acids was carried out in carboxylate form of amino acids, a common form of amino acids and proteins in physiological fluids. The experimental results obtained by the same author suggested that the ethoxycarbonylmethyl derivative of p-tert-butylcalix[6]arene can be used as a carrier for selective recognition and separation of phenylalanine and tryptophan over glycine, alanine and 4aminobutyric acid (GABA). A schematic mechanism concerning the interaction between the phenylalanine and tryptophan ester on one side, and the calix[6]arene receptor on the other side was drawn in the same time. By designing a new calix[4]arene having chiral pendant groups, Okada et al. [37] performed the transport through liquid membrane of some amino acid ethyl and methyl esters and Z-amino acid carboxylates into CH2Cl2. The transport rates of amino acids esters, however, were lower than those of Z- carboxylates due to deep inclusion into cavity. The amino acid esters have higher enantioselectivity than Z-carboxylates in order to interact directly with the binding sites. These results could be explained by the pendant group readily fixed the substrate amino acid of the same chirality to form hydrogen-bonded aggregates in lipophilic media. Hu et al.[38]

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synthetized (R)-cysteine-containing calix[4]arenes which might serve as good chiral macrocyclic ligands in the studies of chiral recognition and chiral catalysis. The studies concerning the ability of calix[6]arene hexacarboxylic acid derivatives to act as carriers through liquid membrane for transporting aromatic amino acids were reported by Oshima et al. [39]. The calix[6]arene hexacarboxylic acid has a cyclic structure capable to include an amino acid ester and bears six ionizable carboxylic acids contributing to electrostatic interaction, as a carrier through liquid membrane. It was also demonstrated that a calix[6]arene acid derivative exhibits high extractability for amino acids compared to ordinary commercial extractants and their structural analogues. The hydrophobic amino acid esters (L,D-tryptophan methyl ester hydrochloride, L-phenylalanine methyl ester hydrochloride, and L-tyrosine methyl ester hydrochloride) and L-tryptophan were transported by the carrier above mentioned. Based on complexation characterized by a proton-exchange mechanism, the transport through membrane was controlled by changing the pH gradient between the source and the receiving aqueous phases. The calix[6]arene hexacarboxylic acid exhibited a high transport ability compared to the other calix[n]arene derivatives (n = 4, 8). By combining an enzyme reaction and a liquid membrane transport with the calix[6]arene, an optical resolution system for a racemate of tryptophan methyl ester was developed. Thus, it was realized a novel liquid membrane system for the chiral separation. Shinkai et al.[40] showed spectroscopically that the pseudo-C2-symmetrical homooxacalix[3]arene exibit enantiomeric recognition properties toward alanine ethyl ester and phenylalanine ethyl ester. Using a supported liquid membrane composed of a porous polymeric support, Antipin et al. [41] studied the separation of zwitterionic form of aromatic amino acids by calix[4]arene based αaminophosphonates. Thus, calix[4]arene functionalized with αaminophosphonates in lower rim 1 or upper rim 2 exhibited high selectivity as carriers of the zwitterionic forms of aromatic amino acids transport through a supported liquid membranes in NitroPhenylOctylEther (NPOE). It is worth noting that employing receptor 2 the phenylalanine is transported 40 times faster than tryptophan.

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1

2

The ability of p-tert-butylcalix[n]arene (n = 4, 6, 8) (Fig.8) to act as useful carriers in the transport through liquid membrane and even as extractants in liquid-liquid extraction of amino acid methylesters as ion pairs in the presence of picrate [42] or tropaeolin 00 ([4-4’-(anilinophenylazo)benzenesulphonic acid]) [43] as counterions was also investigated. Amino acid methylesters (Ltryptophan, L-phenylalanine, L-tyrosine, L-leucine, L-valine, Lcysteine, L-isoleucine, and L-serine) were extracted from aqueous phase (pH = 5.0) into organic phase and transported through liquid membrane by calixarenic receptors in the presence of tropaeolin 00 as ion pairs [43]. The device used for the transport experiments is presented in Fig.9. As in extraction experiments, p-tert-butylcalix[6]arene exhibited a high transport ability towards L-tryptophane in comparison with p-tert-butylcalix[n]arenes ( n = 4,8 ). The transport of amino acid methylesters through liquid membrane by the p-tert-butylcalix[4]arene as carrier is smaller than that of p-tert-butylcalix[n]arenes ( n = 6 ,8 ) as carriers. The sequence of decreasing transport yields of amino acids using p-tertbutylcalix[4]arene as carrier was the following: L-TrpOMe > LPheOMe > L-LeuOMe > L-TyrOMe. In the membrane system, the p-tert-butylcalix[8]arene exhibited better transport ability than both p-tert-butylcalix[n]arenes ( n = 4,6 ) for the amino acids methylesters through chloroform liquid membrane, except L-tryptophane with p-tert-butylcalix[6]arene.

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Fig.9. The device employed in amino acids transport through chloroform liquid membrane.

In Fig. 10, the transport yields of amino acid methylesters through chloroform liquid membrane with p-tert-butylcalix[n]arene ( n = 4,6 ,8 ) is displayed. The sequence of the transport yields of amino acids using ptert-butylcalix[6]arene as carrier was the following: L-TrpOMe > LPheOMe > L-LeuOMe > L-IleOMe ≅ L-ValOMe > L-TyrOMe and with the p-tert-butylcalix[8]arene as carrier the sequence of amino acid yields is the following: L-LeuOMe > L-TrpOMe > L-PheOMe > LValOMe > L-IleOMe ≅ L-SerOMe > L-TyrOMe. The results pointed out that the structure of calixarene is one of the most important parameter for recognition of amino acids. As in the case of p-tert-butylcalix[n]arenes ( n = 6 ,8 ), p-tertbutylcalix[4]arene doesn’t show any transport ability towards LCysOMe through chloroform liquid membrane. The structure of Lcysteine could be responsible of this behavior. The same situation subsists for L-Ser and L-Val. The hydrophobicity of the amino acid is an important parameter, which has to be considered in both the extraction and transport experiments. The transport was proved to be essentially controlled by the structure of calix[n]arene and the nature of the amino acid. The physicochemical parameters, such as the pH, the nature of solvent, and the nature of the anion used as counterion influence also the separation process. Moreover, the influence of the composition and structure of the compounds under study upon the partition processes occurring in biphasic and triphasic systems is an important issue.

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Fig.10. The transport yield of some amino acid methylester hydrochlorides through chloroform liquid membrane by p-tert- butylcalix[n]arenes ( n = 4,6 ,8 ) as carriers in the presence of tropaeolin 00 after 24 hours of stirring 200rpm. Source phase: [amino acid methylester hydrochloride] = 3 × 10 −3 M; pH = 5.0 ; [tropaeolin 00] = 1 × 10 −3 M; 10 mL; Membrane: Chloroform, [calixarene] = 5 × 10 −3 M, 35 mL; Receiving phase: LiOH 0,01 N ( pH = 13.0 ), 10 mL.

The results suggested further possibilities for optimal separation of amino acids derivatives and other biological species by means of derivative calixarenes. 6. CONCLUSIONS The use of membranes in separation and purification of biological compounds was unanimously recognized as one of the most successful novel applications developed during the last years. Future developments of membrane processes in biotechnology and artificial organs should proceed together. Mixtures of amino acids can be separated in cationic form through a liquid membrane using macrocyclic ligands as carriers. Separation is carried out accordingly to the hydrophobic or hydrophilic character of the R-chain from the amino acid structure. In the case of membrane ultrafiltration of protein solutions, there are involved many factors, such as the protein adsorbtion, the pH effect, ionic strength, stirring, and so on. These

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factors affect membrane transport behavior and implicitly the membrane performance. The wide interest in the use of cyclodextrins and their derivatives in the separation field arises from the fact that cyclodextrins can improve the selectivity of separation because of their ability to form inclusion complexes with a large number of organic and inorganic compounds. Calix[n]arenes and their derivatives have been attracting much attention recently as interesting novel type host compounds with applications in separation science especially in liquid membrane. The main challenges are likely to be in the achievement of greater selectivity and higher efficiency. Therefore, it will be essentially an adequate blending of new theories and practical considerations. REFERENCES [1] J.-M. Lehn, Supramolecular Chemistry, Concepts an Perspectives, VCH, Weinheim, 1995. [2] M. W. Peczuh, A. D. Hamilton, Peptide and protein recognition by designed molecules, Chem.Rev., 100 (2000) 2479-2494. [3] J.-M. Lehn, R. Meric, J.-P. Vigneron, M. Cesario, J.Gujihem, C. Pascard, Z. Asfari, J. Vicens, Binding of acethylcholine and other quaternary ammonium cations by sulfonated calixarenes. Crystal structure of a [cholinetetrasulfonated calix[4]arene] complex, Supramol. Chem. 5 (1995) 97-103. [4] L. Mutihac, H.-J. Buschmann, R.-C. Mutihac, E. Schollmeyer, Complexation and separation of amines, amino acids, and peptides by functionalized calix[n]arenes, J. Incl. Phenom. Macrocyclic Chem. 51 (2005) 1-10. [5] R. M. Izatt, J. D. Lamb, R. T. Hawkins, P. R. Brown, S. R. Izatt, J. J. + + Christensen, Selective M /H coupled transport of cations through a liquid membrane by macrocyclic calixarene ligands, J. Am. Chem. Soc. 105 (1983) 1782-1785. [6] E. Drioli, G. Iorio and G. Catapano, Handbook of Industrial Membrane Technology (M. C. Porter, ed.), Park Ridge, New Jersey, 1989, 401-81. [7] R. Rautenbach, R. Albert, Membrane Processes, John Wiley and Sons,

New York, 1989, 29-30. [8] E. Drioli, L. Giorno, L. Donato, R. Molinari, A. Basile, Properties and Chemistry of Biomolecular Systems, N. Russo et al. (eds.), Kluwer Academic Publishers, Amsterdam, 1994, 193-204. [9] M. Mulder, Basic Principle of Membrane Technology, Kluwer Academic Publishers, Dordrecht, 1991. [10] P. Aptel, J.M. Rovel, Water Treatment Handbook, Edited by Degremont, Paris, Lavoisier, VI edition, 1991. [11] Gh. Nechifor, E.Totu, O. Popescu, S. Picioreanu, Analytical separation methods, Ars Docendi, 1999. [12] C. Causserand, P. Aimar, Récents Progrès en Génie des Procédés, 6 (1992) 245-250.

263


[13] O. Le Berre, G. Daufin, Récents Progrès en Génie des Procédés, 6 (1992) 297-298. [14] D. H. Bing, A. C. DiDonno, M. Regan, C. J. Strang, Membrane Separations in Biotechnology, W. Courtney McGregor (ed.), Marcel Dekker Inc., New York, Basel, 1986, 135-160. [15] S. Kimura, A. Tamano, Membranes and Membrane Processes, E. Drioli, M. Nakagaki (eds.), New York, Plenum Press, 1986. [16] S. Tone, T. Masawaki, T. Hamada, The optical resolution of amino acids by ultrafiltration membranes fixed with plasma polymerized-menthol, J. Membrane Sci., 103 (1995) 57-63. [17] A. L. Creagh, B. B. E. Hasenack, A. Van der Padt, E. J. R. Sudholter, K. Van’t Riet, Separation of amino acid enantiomers using micellar-enhanced ultrafiltration, Biotechnol. Bioeng., 44 (1994) 690-698. th [18]. T. Masawaki, S. Tone, Preprints, 7 International Symposium on Synthetic Membranes in Science and Industry, Tubingen, Germany, Aug. 29 –Sep.1 (1994) 262-265. [19] C. J. Pedersen, Cyclic polyethers and their complexes with metal salts, J. Am. Chem. Soc., 89 (1967) 2495-2496. [20] C. D. Gutsche, Calixarenes Revisited, The Royal Society of Chemistry, Cambridge, UK,1998. [21] C. Luca, L. Mutihac, R. Mutihac, A.-M. Josceanu, Mecanisme de transport prin membrane lichide, Editura Academiei Române, Bucureşti, 1998. [22] H.-J. Buschmann, L. Mutihac, Complexation, liquid-liquid extraction, and transport through liquid membrane of protonated peptides using crown ethers, Anal. Chim. Acta, 466 (2002) 101-108. [23] C. Luca, L. Mutihac, T. Constantinescu, Separation of amino acids by liquid membranes with macrocyclic carriers, Rev. Roum. Chim. 35 (1990) 467-473. [24] L. Mutihac, R. Mutihac, Modelling and simulation of carrier mediated transport of ion pairs through liquid membranes, Acta Chim. HungaricaModels in Chemistry, 129 (1992) 573-577. [25] L. Mutihac, C. Baltariu, A. Hotaranu, R. Mutihac, Liquid membranes involved in some biological compounds separation, Rev. Roum.Chem. 51 (2006) 1041-1051. [26] H.-J. Buschmann, R.-C. Mutihac, E. Schollmeyer, Interactions between crown ethers and water, methanol, acetone, and acetonitrile in halogenated solvents, J. Solution Chem. 39 (2010) 291-299. [27] N. Sieffert, A. Chaumont, W. Wipff, Importance of the liquid-liquid interface in assisted ion extraction: new molecular dynamics studies of cesium picrate extraction by a calix[4]arene. J. Phys. Chem. C, 113 (2009) 10610-10622. [28] M. Kirch, J.-M. Lehn, Selective Transport of Alkali Metal Cations through a Liquid Membrane by Macrobicyclic Carriers Angew. Chem., Int.

Ed. Engl. 14 (1975) 555.

264


[29] J. D. Lamb, J. J. Christensen, J. L. Oscarson, B. L. Nielsen, B. W. Assay, R. M. Izatt, The relationship between complex stability constants and rates of cation transport through liquid membranes by macrocyclic carriers, J. Am. Chem. Soc. 102 (1980) 6820-6824. [30] Szejtli, J.; Osa, T. Comprehensive Supramolecular Chemistry; Elsevier, Oxford, Vol. 3, Cyclodextrins, 1996. [31] B.-L.Poh, Y. Mooi Chow, Transport method for determining the association constants of complexes formed between aromatic hydrocarbons and α- and β-cyclodextrin in water, J. Incl. Phenom. 14 (1992) 85-90. [32] H. Ikeda, A. Matsuhisa, A. Ueno, Selective transport of saccharides through a liquid membrane using cyclodextrin dimer, J. Incl. Phenom. Macrocyclic Chem. 44 (2002) 347-350. [33] G. L. Lumetta, R. D. Rogers, A. S. Gopalan (Eds.) Calixarenes for Separations (ACS Symposium Series 757), Washington, 2000. [34] J. Vicens and J. Harrowfield (eds): Calixarene in the Nanoworld, Springer, Dordrecht 2007. [35] L. Mutihac, Functionalized calix[n]arenes as membrane transporters for biological compounds. A minireview, Curr. Drug Discovery Technol. 5 (2008) 98-104. [36] S.-K. Chang, H.-S Hwang, H. Son, J. Youk, Y. S. Kang, Selective transport of amino acid esters through in chloroform liquid membrane by a calix[6]arene-based ester carrier. J. Chem. Soc., Chem. Commun. (1991) 217-218. [37] Y. Okada, Y. Kasai, J. Nishimura, The selective extraction and transport of amino acids by calix[4]arene-derived esters. Tetrahedron Lett. 36 (1995) 555-558. [38] H. Hu, J. He, A. S. C. Chan, X. Han, J.-P. Cheng, New chiral macrocyclic ligands. Design and synthesis of (R)-cysteine-containing calix[4]arenes. Tetrahedron: Asymmetry 10 (1999) 2685-2689. [39] T. Oshima, K. Inoue, S. Furusaki, M. Goto, Liquid membrane transport of amino acids by a calix[6]arene carboxylic acid derivative. J. Membr. Sci. 217 (2003) 87-97. [40] Araki, K., Inada, K., Shinkai, S.: Chiral recognition of α-amino acid derivatives with a homooxacalix3arene: Construction of a pseudo-C2symmetrical compound from a C3 symmetrical macrocycle. Angew. Chem. Int. Ed. Engl. 35 (1996) 72-74. [41] I. S. Antipin, I. I. Stoikov, E. M. Pinkhassik, N. Fitseva, I. Stibor, A. I. Konovalov, Calix[4]arene based -aminophosphonates: Novel carriers for zwitterionic amino acids transport. Tetrahedron Lett. 38, (1997) 58655868. [42] L. Mutihac, H.-J. Buschmann, E. Diacu, Calixarene derivatives as carriers in liquid membrane separation. Desalination 148 (2002) 253- 256. [43] L. Mutihac, R. Mutihac, Liquid-liquid extraction and transport through membrane of amino acid methylesters by calix[n]arene derivatives. J. Incl. Phenom. Macrocyclic Chem. 59 (2007) 177-181.

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