Roumanian Biotechnological Letters
Copyright © 2006 Bucharest University
Roumanian Society of Biological Sciences
Vol. 11, No. 5, 2006, pp. 2915-2921
Printed in Romania. All rights reserved
ORIGINAL PAPER
Extraction and transport of neutral amino acids through liquid
membranes
Received for publication, July 15, 2006
Accepted, September 15, 2006
ALEXANDRA CRISTINA BLAGA1), ANCA-IRINA GALACTION2), DAN CAŞCAVAL1)*,
ELENA FOLESCU1)
1)
Technical University "Gh. Asachi" of Iasi, Faculty of Chemical Engineering, Dept. of
Biochemical Engineering, 71 D. Mangeron Avenue, 700050 Iasi, Romania, email:
dancasca@ch.tuiasi.ro
2)
University of Medicine and Pharmacy, Faculty of Medical Bioengineering, Dept. of
Biotechnology, 16 University Street, 700115 Iasi, Romania, email: galact@from.ro
* the corresponding author
Abstract
This study is focused on facilitated pertraction of some neutral amino acids (L-cysteine, Ltryptophan, L-glycine, L-alanine) with D2EHPA dissolved in dichloromethane. The results indicated
the significant influences of the pH-gradient between the aqueous phases, carrier concentration and
mixing intensity. The maximum mass flows of the amino acids through liquid membrane can be
reached at the pH-value of the feed phase of 3, pH-value of the stripping solution of 1, D2EHPA
concentration in solvent layer of 60 g l-1, using an intense mixing of the two aqueous phases.
In function of the pH-gradient between the aqueous phases, it is possible to separate
selectively the considered amino acids.
Keywords: amino acids, L-cysteine, L-tryptophan, L-glycine, L-alanine, D2EHPA,
pertraction, liquid membrane, carrier, mass flow, permeability factor.
Introduction
Biotechnology has much progressed and represents the principal way for obtaining of
some very important products for human activity. In this context, the production of amino
acids, which are the main structural components of proteins and enzymes, has been
significantly increased. The amino acids can be obtained by biosynthesis or from protein
hydrolysis, but their separation from fermentation broths or protein hydrolysates is rather
difficult.
Generally, the techniques used for separation or purification of amino acids include
chromatography, adsorption, ion-exchange, and crystallization at the isoelectric point [1]. But
these techniques are difficult to scale-up, consequently the production of amino acid is limited
and the technology is rather expensive.
Recently the separation of amino acids by liquid-liquid extraction has been analyzed.
Because amino acids dissociate in aqueous solutions, forming characteristic ionic species as a
function of the solution pH value, their solubility in organic solvents, especially in low-polar
ones, is very low. The extraction of amino acids is only possible by adding into the organic
phase extractants such as phosphoric acid derivatives [1-5], high molecular weight quaternary
aliphatic amines [6,7] or crown-ethers [1,8,9].
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ALEXANDRA CRISTINA BLAGA, ANCA-IRINA GALACTION, DAN CAŞCAVAL,
ELENA FOLESCU
Extraction and transport through liquid membrane, also called pertraction, represents a
development of the extraction processes. This technique can be applied for amino acids
separation and mainly uses emulsion liquid membranes [10-12] and supported liquid
membranes [13-15]. Because the main limitations of these processes are the swelling of
emulsion liquid membrane and the low stability of supported liquid membrane, the extraction
and transport of amino acids can be carried out through the free liquid membranes, which do
not need to stabilize the membrane by adding surfactants or by including into porous
membranes or hollow fibers [1,16].
The previous studies have been focused on the separation the acidic amino acids (Laspartic and L-glutamic acids) by facilitated pertraction with di-(2-ethylhexyl) phosphoric
acid (D2EHPA) [16]. In this paper, the studies are continued by analyzing the possibility to
separate some neutral amino acids (L-cysteine, L-tryptophan, L-glycine, L-alanine) using the
same technique, in the purpose to establish the optimum conditions required for selective
separation of amino acids from fermentation broths or protein hydrolysates.
Materials and methods
The experiments have been carried out using a patented pertraction equipment of U-type
cell, which allows obtaining and easily maintaining the free liquid membrane and was
described in the previous paper [16,17].
The liquid membrane phase consists of a solution of 20 - 100 g/l D2EHPA as carrier
dissolved in dichloromethane. The feed phase contains initial solutions of 3 g l-1 L-cysteine/
L-tryptophan/L-glycine/L-alanine.
The pH of the feed phase varied from 2 to 6, being adjusted at the prescribed values with
solutions of 4% H2SO4 or 4% NaOH. The pH of stripping solution has been adjusted with 4%
HCl solution in the pH-domain of 1 to 5. The pH values were determined using a digital pHmeter of Consort C831 type.
The aqueous solutions are independently mixed by means of double blades stirrers with
6 mm diameter and 3 mm height, having a rotation speed between 0 and 600 rpm. In order to
reach high diffusion rates through the solvent layer, the organic phase has been mixed with
one stirrer of the same design, at a constant rotation speed of 500 rpm. The area of mass
transfer surface, both for extraction and for re-extraction, was of 1.59x10-3 m2. At this mixing
intensity level, the interfaces between the phases remained flat, and, consequently, the
interfacial area constant.
The experiments have been carried out in a continuous system, at the steady state
conditions for aqueous solutions, these solutions being separately fed with a volumetric flow
of 1.92 l h-1.
The evolution of pertraction was followed by means of the amino acid mass flows and
permeability factors through liquid membrane. The initial mass flows, ni, represents the flux
of amino acid transferred from the feed phase to the solver layer, and the final mass flow, nf,
also called overall mass flow, represents the flux of amino acid transferred from the organic
solvent to the striping phase. The permeability factor, P, is a measure of the capacity to
transport the solute through the liquid membrane, being defined as the ratio between the final
mass flow and the initial mass flow of amino acid.
For calculating these parameters, the amino acids concentration in the feed and
stripping solutions have been measured by high performance liquid chromatography
technique (HPLC) with a HP 1090 liquid chromatography, then the mass balance being used.
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Roum. Biotechnol. Lett., Vol. 11, No. 5, 2915-2921 (2006)
Extraction and transport of neutral amino acids through liquid membranes
Results and discussion
Extraction and transport through liquid membranes is strongly influenced by the pHgradient between the feed and stripping phases, carrier concentration in liquid membrane, and
mixing intensity of the three phases. In the case of amino acids pertraction, the influence of
the pH-gradient between is enhanced by the formation of the ionized forms of amino acids in
the aqueous phases and controls the efficiency both of extraction/re-extraction and the
transport rate through the solvent layer.
Thus, from Figure 1 it can be observed that for all studied neutral amino acids the initial
mass flows increase with the increase of feed phase pH, reach a maximum value at pH = 3,
followed by their strong decrease.
L-Cysteine
L-Tryptophan
1.0
-1
1.0
-2
n, moles m h
P
-2
n, moles m h
-1
1.0
0.8
0.8
0.6
0.6
0.4
P
0.8
0.8
0.6
0.6
0.4
ni
nf
P
0.2
0.4
0.4
ni
nf
P
0.2
0.2
0.0
0.0
0.2
2
3
4
5
2
6
3
4
pH of feed phase
L-Glycine
L-Alanine
1.0
1.0
1.0
-2
0.8
0.6
0.6
0.4
-2
-1
P
n, moles m h
-1
0.8
n, moles m h
5
6
pH of feed phase
P
0.8
0.8
0.6
0.6
0.4
ni
nf
P
0.2
0.4
0.2
0.0
2
3
4
5
6
0.4
ni
nf
P
0.2
0.2
0.0
2
pH of feed phase
3
4
5
6
pH of feed phase
Figure 1. Variation of the amino acids mass flows and permeability factor in function of the pH-value of the
feed phase (pH of stripping phase = 2, D2EHPA concentration = 40 g l -1, rotation speed = 500 rpm).
This evolution is the result of the reactive extraction mechanism of amino acids with
D2EHPA, which occurs by means of an interfacial chemical reaction of ionic exchange type
controlled by the pH of aqueous phase. The carrier, D2EHPA, reacts only if the amino acids
exist in aqueous solution in their cationic form (pH of aqueous phase has to be below pKi), the
general expression of the chemical reaction being as follows:
R-CH(NH3+)-COOH(aq) + HP(o)
R-CH(NH3+)-COOH.P-(o) + H+(aq)
where HP is the carrier [4].
The maximum of mass flows is the result of two opposite phenomena: the increase of
the concentration of extractant active form, which is able to react with the amino acid, and the
Roum. Biotechnol. Lett., Vol. 11, No. 5, 2915-2921 (2006)
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ALEXANDRA CRISTINA BLAGA, ANCA-IRINA GALACTION, DAN CAŞCAVAL,
ELENA FOLESCU
decrease of the total amount of amino acid existing in cationic form. The supplementary
increase of pH-value of feed phase to the isoelectric point determines the increase of
zwitterions concentration, that significantly reducing the initial mass flows of the amino acids
(at the isoelectric point the reactive extraction of amino acids becomes impossible [4]). Due to
its lower isoelectric point, this effect is more pronounced for L-cysteine (pKi = 5.1)
comparatively with the other considered amino acids (at pH = 6 the pertraction efficiency for
L-tryptophan, L-glycine, L-alanine is very low, but becomes 0 for L-cysteine).
The highest values of initial mass flows were recorded for L-tryptophan, owing to its
aromatic radical R which induces superior hydrophobicity.
Similar to the acidic amino acids, the final mass flows of neutral amino acids initially
increase with pH of feed solution, due to their accumulation in the liquid membrane. But,
unlike the aspartic and glutamic acids [16], the final mass flows of all neutral amino acids
reach a maximum value at pH = 4. The further increase of pH leads to the decrease of the
final mass flows, owing to the change of the sense of pH-gradient which controls the direction
of solute transfer through liquid membrane.
The permeability factor strongly increases with the pH increase, becoming higher than
1 for pH  3-3.5. This variation indicates that the final mass flows become superior to the
initial ones, phenomena that are possibly due to the re-extraction of the supplementary
amount of amino acids accumulated into the organic layer.
The increase of the pH-value of the stripping phase determines the reducing of both
initial and final mass flows, the same evolution being registered for the permeability factor, as
it can be seen from Figure 2. For all studied neutral amino acids, the maximum values of the
permeability factors are over 0.9, being reached for the pH-value of stripping phase of 1 and
pH for the feed phase of 3.
L-Cysteine
L-Tryptophan
-2
0.8
0.8
0.6
0.6
1.0
-1
1.2
P
-2
n, moles m h
P
n, moles m h
1.0
-1
1.0
1.0
0.8
0.8
0.6
0.6
0.4
0.4
0.4
0.4
ni
nf
P
0.2
0.2
0.0
0.2
2
3
4
0.2
0.0
0.0
1
ni
nf
P
0.0
1
5
2
3
4
5
pH of stripping phase
pH of stripping phase
L-Glycine
L-Alanine
1.2
1.0
0.8
0.8
0.6
0.6
-2
n, moles m h
P
-2
n, moles m h
-1
-1
1.0
1.0
P
1.0
0.8
0.8
0.6
0.6
0.4
0.4
0.4
0.4
ni
nf
P
0.2
0.0
1
2
3
4
5
pH of stripping phase
0.2
0.2
0.0
0.0
ni
nf
P
0.2
0.0
1
2
3
4
5
pH of stripping phase
Figure 2. Variation of the amino acids mass flows and permeability factor in function of the pH-value of the
stripping phase (pH of feed phase = 3, D2EHPA concentration = 40 g l-1, rotation speed = 500 rpm).
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Roum. Biotechnol. Lett., Vol. 11, No. 5, 2915-2921 (2006)
Extraction and transport of neutral amino acids through liquid membranes
The carrier concentration inside the liquid membrane is another important factor that
controls the amino acids pertraction efficiency. From Figure 3 it can be observed that the
initial and final mass flows of amino acids are continuously intensified with the increase of
D2EHPA concentration to 60 g l-1, due to the increase of the concentration of one of the
reactants and, consequently, to the accumulation of the interfacial compound into the organic
layer. Over this level, the increase of mass flows becomes slower, this variation being more
accentuated for the final mass flows.
Contrary to the above mentioned variations, the permeability factors initially increase
with D2EHPA concentration to a maximum value, followed by its decreasing. For all neutral
amino acids, the maximum level of permeability factor corresponds to a carrier concentration
of 60 g l-1. Therefore, similar to the acidic amino acids, the pertraction system reaches its
maximum capacity of solute transfer at 60 g l-1 carrier concentration, for the given
experimental conditions.
L-Cysteine
L-Tryptophan
2.0
-2
n, moles m h
P
1.4
1.2
0.9
1.0
0.8
P
1.6
0.9
1.2
0.8
ni
nf
P
0.6
1.0
-1
1.0
-2
n, moles m h
-1
1.6
ni
nf
P
0.8
0.4
0.7
20
40
60
80
0.8
0.4
100
20
40
-1
Carrier concentration, gl
L-Glycine
60
80
100
Carrier concentration, g/l
L-Alanine
1.6
1.0
1.0
-1
P
-2
n, moles m h
P
1.4
-2
n, moles m h
-1
1.6
1.2
0.9
1.0
1.2
0.9
0.8
0.8
0.8
ni
nf
P
0.6
ni
nf
P
0.4
0.4
0.8
0.7
20
40
60
80
100
20
-1
Carrier concentration, gl
40
60
80
100
Carrier concentration, g/l
Figure 3. Variation of the amino acids mass flows and permeability factor in function of the carrier
concentration (pH of feed phase = 3, pH of stripping phase = 2, rotation speed = 500 rpm).
The dependences of amino acids mass flows on rotation speed, plotted in Figure 4,
suggest that the overall separation process could be controlled by the diffusional processes or
interfacial chemical reactions. The mixing intensification of the two aqueous phases induces
the enhancement both of the initial and final mass flows for all neutral amino acids, owing to
the diminution of resistance to the diffusion through the interfacial boundary layers, evolution
Roum. Biotechnol. Lett., Vol. 11, No. 5, 2915-2921 (2006)
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ALEXANDRA CRISTINA BLAGA, ANCA-IRINA GALACTION, DAN CAŞCAVAL,
ELENA FOLESCU
that is recorded also for the permeability factor. The recorded influence is more important for
rotation speed values below 500 rpm, over this level the kinetic resistance becoming the
limiting step.
The increase of permeability factors with the rotation speed intensification indicates a
stronger influence of mixing on final mass flows, due to the more accentuated resistance to
the diffusion through the boundary layer on the stripping phase side.
L-Cysteine
L-Tryptophan
0.8
0.6
-1
1.0
P
-2
P
1.0
n, moles m h
0.8
-2
n, moles m h
-1
1.0
0.8
0.8
0.6
0.6
0.6
0.4
0.4
ni
nf
P
0.2
200
300
0.4
ni
nf
P
200
400
500
600
Rotation speed, rpm
300
400
500
600
Rotation speed, rpm
L-Glycine
L-Alanine
-1
P
-2
0.8
0.6
-1
1.0
P
-2
0.8
1.0
n, moles m h
1.0
n, moles m h
0.4
0.2
0.8
0.8
0.6
0.6
0.6
0.4
0.4
ni
nf
P
0.2
200
300
0.4
400
500
600
Rotation speed, rpm
ni
nf
P
0.4
0.2
200
300
400
500
600
Rotation speed, rpm
Figure 4. Variation of the amino acids mass flows and permeability factor in function of the
rotation speed (pH of feed phase = 3, pH of stripping phase = 2, D2EHPA concentration = 40
g l-1).
Conclusions
The studies on facilitated pertraction of some neutral amino acids (L-cysteine, Ltryptophan, L-glycine, L-alanine) through a liquid membrane of dichloromethane and
D2EHPA as a carrier underlined the major influence of pH-gradient between the feed and
striping phases, carrier concentration in organic layer and mixing intensity of aqueous phases.
In the purpose to reach the maximum values of amino acids mass flows, the facilitated
pertraction has to be carried out at the pH of feed phase of 3, pH of stripping phase of 1,
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Roum. Biotechnol. Lett., Vol. 11, No. 5, 2915-2921 (2006)
Extraction and transport of neutral amino acids through liquid membranes
D2EHPA concentrations in the liquid membrane of 60 g l-1, and the aqueous phases must be
intensively mixed.
By modifying the pH of the feed phase it is possible to separate selectively the studied
amino acids. For example, at pH of 5 only L-tryptophan, L-glycine and L-alanine are
extracted and transported through liquid membrane, L-cysteine remaining in the feed phase.
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