# Extraction and Transport of Amino Acids Using Kryptofix 5 as Carrier through Liquid Membrane

Authors: Pankaj Raizada; Uma Sharma
Journal: Journal of Chemistry
 (2013)

---

## Abstract

The present work explores  membrane-mediated extraction and transport studies of  amino acids through artificial bulk liquid membrane system with  kryptofix 5  as a carrier. The various reaction parameters  such as  amino acid concentration, carrier concentration, time, pH, and stirring effect  were studied to optimize reaction conditions. The  stirring of source and receiving phases increased the efficiency of extraction process. Noncyclic receptor  kryptofix 5 with five oxyethylene units and terminal aromatic donor end groups governs its transport and extraction efficiency. The  extraction and transport efficiency  followed  the following trend:  valine > alanine > glycine > threonine. Supported liquid membrane (SLM) studies were performed using cellulose nitrate, PTFE, eggshell, and onion membranes. The egg shell membrane support proved to be  most efficient due to  intricate network of water insoluble proteins fibers with very high surface area and homogeneity.


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## Body

## 1. Introduction

The carrier-mediated transport of amino acids and metal ions through liquid membrane plays an important role in simulating biological membrane functions and separation technologies because of high transport efficiency, excellent selectivity, and economic advantages of liquid membrane over other separation techniques [1, 2].Recently, artificial liquid membrane has emerged as a novel and effective separation technique in the field of analytical chemistry. These membranes display high selectivity due to specific complexation of guest molecule by the carrier [3, 4].Macrocyclic receptors, lipophilic ionic carriers, and podands have been employed as selective carriers in these membranes. The binding efficiency and selectivity of podands have been reported by Katrizky et al. [5].Amino acids are important bioactive molecules, constituents of proteins and can take part in various physiological processes and perform structural, hormonal, and catalytic functions [6]. Glycine is an important source of energy for muscle tissue and central nervous system and strengthens the immune system by producing antibodies and helps to trigger the release of oxygen to the energy requiring cell-making processes. Threonine is an important constituent of collagen, elastin and enamel protein and helps in fat buildup in the liver. Valine promotes mental vigor, muscle coordination and calm emotions [7].The amino acids are the important precursors of metabolic processes and the most important biochemicals to be treated by bulk liquid membrane (BLM) and supported liquid membrane (SLMs) techniques [8]. Bulk liquid membrane systems are easy to construct and have determined mass transfer area and membrane thickness. However, due to low contact area, BLM techniques have limited analytical applications. Supported liquid membranes (SLM) are the most extensively used liquid membranes (LMs) due to their high enrichment factor, low reagent consumption, and wide variety of configurations. Supported liquid membrane studies involve simultaneously three occurring processes: molecule extraction from an aqueous source phase, diffusion through receptor immobilized solid membrane support, and release to an aqueous receiving phase [9, 10]. Our previous studies investigated extraction and transport studies of alkali and alkaline earth metal cations using noncyclic receptors containing naphthyl end groups [11].Present work explores the carrier ability (bulk liquid membrane and supported liquid membrane transport studies) and extractability of kryptofix 5 as a synthetic receptor for amino acids (glycine, alanine, threonine, and valine) in organic membrane under different reaction conditions.


## 2. Experimental

Analytical grade L-amino acids (glycine, valine, alanine, and threonine) were purchased from S.D Fine chemicals (India). Analytical grade kryptofix 5 was obtained from Merck and used without further purification. The solvent CHCl3 was obtained from Qualigens (India) and used without further purification. Systronics-167 Visible spectrophotometer was employed for the estimation of amino acids [12]. The pH was measured by a digital Systronics pH-meter 335 with a combined electrode.


### 2.1. Estimation of Amino Acid

The 0.5 mL aqueous amino acid (0.1 M) was taken in 10 mL standard flask, and the 0.2 mL ninhydrin solution (0.5%) was added and kept in the boiling water for 10 min. Thereafter, 6 mL of 60% ethanol was added with double distilled water until the total volume was 10 mL. In this way, calibration curve was plotted ranging from1.0×10-5 M 1.0×10-1 M at λmax 570 nm [12].


### 2.2. Extraction Studies

10 mL of aqueous amino acid solution and 10 mL of receptor solution in chloroform were taken in a 50 mL beaker and stirred on a magnetic stirrer for 4 h at 25°C [3]. After stirring, the mixture was allowed to stand for 5 min for the separation of two phases, and the aqueous phase was analyzed for extracted amino acid by determining the difference in the concentration of amino acid in aqueous phase before and after extraction. The efficiency of extraction and transport was calculated by using (1), and distribution ratio was calculated by (2) [3, 13]
(1)Efficiency(%)=A0-AA0×100,
where A0 and A are the concentrations of aqueous phases before and after extraction
(2)Dm=CreceivingphaseCsourcephase,
where Csourcephase and Creceivingphase are the concentrations of amino acids in source and receiving phase during extraction process.


### 2.3. Bulk Liquid Membrane Transport Studies

Transport studies were performed in a “U” tube glass cell at 25°C. 15 mL of chloroform containing appropriate concentration of receptor was taken as membrane phase [3, 11]. The source phase was composed of 10 mL of different concentration of amino acid in one limb of the “U” tube and 10 mL of deionised water served as the receiving phase in the other limb. The membrane phase was constantly stirred for 24 h. Flux for transport studies was calculated by using (3) [3, 13]
(3)Jm=C(rec.)VAt,
where C is concentration of amino acid in receiving phase (mol/dm3), V is volume of receiving phase (mol/dm3), A is effective area of membrane (m2), and t is time in (s).


### 2.4. Supported Liquid Membrane (SLM) Transport

Supported liquid membrane transport studies of amino acids were carried out by using various membrane supports, eggshell membrane, onion membrane, cellulose nitrate, and PTFE in order to find out the best membrane support with its applicability for the separation of amino acids (alanine, glycine, valine, and threonine). The supported liquid membrane experiments were carried out in a permeation cell consisting of two identical cylindrical compartments (half-cells). 50 mL aqueous solution of amino acid served as source phase, and 50 mL of deionised water served as receiving phase. SLM consisted of a natural or artificial membrane immobilized in chloroform solution containing receptor. The transported amount of amino acid was determined by using Systronics UV-visible spectrophotometer.


## 2.1. Estimation of Amino Acid

The 0.5 mL aqueous amino acid (0.1 M) was taken in 10 mL standard flask, and the 0.2 mL ninhydrin solution (0.5%) was added and kept in the boiling water for 10 min. Thereafter, 6 mL of 60% ethanol was added with double distilled water until the total volume was 10 mL. In this way, calibration curve was plotted ranging from1.0×10-5 M 1.0×10-1 M at λmax 570 nm [12].


## 2.2. Extraction Studies

10 mL of aqueous amino acid solution and 10 mL of receptor solution in chloroform were taken in a 50 mL beaker and stirred on a magnetic stirrer for 4 h at 25°C [3]. After stirring, the mixture was allowed to stand for 5 min for the separation of two phases, and the aqueous phase was analyzed for extracted amino acid by determining the difference in the concentration of amino acid in aqueous phase before and after extraction. The efficiency of extraction and transport was calculated by using (1), and distribution ratio was calculated by (2) [3, 13]
(1)Efficiency(%)=A0-AA0×100,
where A0 and A are the concentrations of aqueous phases before and after extraction
(2)Dm=CreceivingphaseCsourcephase,
where Csourcephase and Creceivingphase are the concentrations of amino acids in source and receiving phase during extraction process.


## 2.3. Bulk Liquid Membrane Transport Studies

Transport studies were performed in a “U” tube glass cell at 25°C. 15 mL of chloroform containing appropriate concentration of receptor was taken as membrane phase [3, 11]. The source phase was composed of 10 mL of different concentration of amino acid in one limb of the “U” tube and 10 mL of deionised water served as the receiving phase in the other limb. The membrane phase was constantly stirred for 24 h. Flux for transport studies was calculated by using (3) [3, 13]
(3)Jm=C(rec.)VAt,
where C is concentration of amino acid in receiving phase (mol/dm3), V is volume of receiving phase (mol/dm3), A is effective area of membrane (m2), and t is time in (s).


## 2.4. Supported Liquid Membrane (SLM) Transport

Supported liquid membrane transport studies of amino acids were carried out by using various membrane supports, eggshell membrane, onion membrane, cellulose nitrate, and PTFE in order to find out the best membrane support with its applicability for the separation of amino acids (alanine, glycine, valine, and threonine). The supported liquid membrane experiments were carried out in a permeation cell consisting of two identical cylindrical compartments (half-cells). 50 mL aqueous solution of amino acid served as source phase, and 50 mL of deionised water served as receiving phase. SLM consisted of a natural or artificial membrane immobilized in chloroform solution containing receptor. The transported amount of amino acid was determined by using Systronics UV-visible spectrophotometer.


## 3. Results and Discussion

The blank experiments for the extraction and transport studies of each amino acid in which membrane was devoid of carrier, no leakage of amino acid from the source phase into the receiving phase was observed. All measurements were performed in triplicate to check reproducibility.


### 3.1. Effect of Receptor and Amino Acid Concentration

For optimization of receptor, the concentration was varied from1.0×10-6 to 1.0×10-2 M at constant amino acid concentration (5.0×10-3 M). At optimal receptor concentration (1.0×10-4 M), amino acid reacted with the receptor and resulted in the formation of amino acid receptor complex (Table 1). Above optimal concentration extraction rate decreased with further increase in the receptor concentration. The flux Jm was controlled by the complex formation at the source receiving-membrane interface which was governed by the complexation and decomplexation at the optimal concentration. The amino acid concentration was varied from 2.0×10-3 to 7.0×10-3 M to study the influence on transport and extraction processes. Both transport and extraction rates increased with increase in the concentration of amino acid with optimal concentration of 5.0×10-3 M (Figures 1 and 2).Table 1
Amount of amino acids transported into receiving phase through SLM using various  membrane supports after 24 h with kryptofix 5 in chloroform.
S. number
Amino acids
Egg shell membrane
PTFE membrane
Onion membrane
Cellulose nitrate membrane
Transported amino acid[×10-3M]
J
m
×
10
-
7 [mol/cm2s]
Transported amino acid[×10-3M]
J
m
×
10
-
7 [mol/cm2s]
Transported amino acid[×10-3M]
J
m
×
10
-
7 [mol/cm2s]
Transported amino acid[×10-3M]
J
m
×
10
-
7 [mol/cm2s]
(
1
)
Alanine
2.05
2.90
1.23
3.50
—
—
1.15
2.19
(
2
)
Glycine
3.15
5.30
1.50
3.75
—
—
2.50
4.38
(
3
)
Threonine
2.75
3.89
1.20
1.69
—
—
1.30
1.84
(
4
)
Valine
2.95
4.17
1.30
1.89
—
—
1.65
1.90
Concentration of amino acid in source phase:  5.0× 10−3 M.
Concentration of kryptofix 5: 1.0× 10−4 M in 50 mL chloroform.
Stirring speed: 200 rpm, pH: 6, and temperature: 30°C.Figure 1
Amount of amino acid transported after 24 h with kryptofix 5. Concentration of kryptofix 5 = 1.0×10-5 M in 15 mL chloroform. Stirring speed = 200 rpm, pH = 6, and temperature =30°C.Figure 2
Amount of amino acid extracted after 4 h with kryptofix 5. Concentration of kryptofix 5 =1.0×10-4 M in 10 mL chloroform. Stirring speed = 200 rpm, pH = 6, and temperature = 30°C.


### 3.2. Effect of Stirring

The extraction efficiency decreased without stirring in comparison to the experiments with stirring speed of 200 rpm. For valine, 76% and 26% of extraction efficiencies were observed with and without stirring, respectively (Figure4). This signifies that the stirring of aqueous phase considerably enhanced extraction and transport rate of amino acids. Similar trend was found for other investigated amino acids (Figure 3).Figure 3
Efficiency of amino acids extracted with stirring. Concentration of amino acid in source phase =5.0×10-3 M. Concentration of kryptofix 5 = 1.0×10-4 M in 10 mL chloroform. Stirring speed = 200 rpm, pH = 6, and temperature = 30°C.Figure 4
Efficiency of amino acids extracted without stirring. Concentration of amino acid in source phase =5.0×10-3 M. Concentration of kryptofix 5 = 1.0×10-4 M in 10 mL chloroform, pH = 6, and temperature = 30°C.


### 3.3. Effect of Time and Back Extraction

Figure5 shows the time dependence of amino acids transport and extraction through liquid membrane designed under optimal experimental conditions. Maximal extraction of amino acids took place within the first 2 h indicating that the time for attaining equilibrium is 2 h. For back extraction studies, aliquots from the loaded organic phase were withdrawn and subsequently back extracted for about 4 h with the same volume of the strippent which is deionised water in the present study. Glycine and threonine were found to be efficiently recovered due to easy decomplexation at the membrane receiving interface (Figure 6).Figure 5
Effect of time on extraction of amino acid with kryptofix 5. Concentration of amino acid in source phase =5.0×10-3 M. Concentration of kryptofix 5 = 1.0×10-4 M in 10 mL chloroform. Stirring speed = 200 rpm, pH = 6, and temperature =30°C.Figure 6
Efficiency of amino acids back extracted after 4 h with kryptofix 5. Concentration of amino acid in source phase =5.0×10-3 M. Concentration of kryptofix 5 = 1.0×10-4 M in 10 mL chloroform. Stirring speed = 200 rpm, pH = 6, and temperature =30°C.


### 3.4. Effect of pH

pH plays an important role in the extraction of amino acids. Maximum extraction of amino acids was observed at pH 6. At optimal pH, the carrier diffusion process seems to govern the transport phenomenon. The amino acid was extracted by the complexation of its H4N+ moiety by the flexible donor oxygen atoms. This can be explained by nature and hydrophobicity of amino acids. Therefore, valine and alanine were extracted more efficiently (Figure 7).Figure 7
Effect of pH. Concentration of amino acid in source phase =5.0×10-3 M. Concentration of kryptofix 5 = 1.0×10-4 M in 10 mL chloroform. Stirring speed = 200 rpm, pH = 6, and temperature = 30°C.Figures1 and 2 display the results of transport and extraction studies of amino acids using kryptofix 5 as a carrier at optimal concentration. The trend of extraction and transport efficiency of kryptofix was observed to be valine > alanine > glycine > threonine. The receptor proved to be a selective extractant for valine and alanine because it interacted with the amino acids by hydrogen bonding interactions between H4N+ moiety of amino acids and donor oxygen atoms of the receptor. The ability of amino acid receptor complex formation enhanced due to the presence of Π electrons of the quinoyl end groups of the receptor [14]. Besides, the extraction behaviour of receptor with amino acids strongly depends on the hydrophobicity of amino acids [3, 15]. The high hydrophobicity of valine resulted in more distribution at the aqueous organic interface thus accounting for its more tendency to pass from aqueous phase to organic phase giving better extraction of valine with receptor. Higher distribution ratio values were observed for highly hydrophobic amino acids (Figure 8).Figure 8
Hydrophobicity scale and flux values for amino acids.The order of transport ability of kryptofix 5 for amino acids was observed similar to the trend of extractability. The flexible ether chain of receptor adopted itself to H4N+, moiety of amino acids, whereas the insertion of quinoyl end group of receptor reduced its flexibility and permitted effective conformational changes in the binding and releasing processes [15]. The hydrophobic exterior and flexible donor sites with most suited conformation facilitated the passage of amino acid receptor complex from the organic phase to the aqueous receiving phase. The accumulation of amino acid in the membrane phase decreased the overall efficiency of transport process. Figure 9 describes the schematic representation of liquid membrane studies and possible sites of interaction of kryptofix 5 with guest amino acids.Figure 9
Schematic representation of liquid membrane studies of amino acids using kryptofix 5 as carrier in chloroform liquid membrane system. AA means amino acid. (a) represents interaction between –NH4+ and active –OH sites of kryptofix 5. (b) CH-π interactions between alkyl part of amino acid and Π electrons of kryptofix5.


### 3.5. Supported Liquid Membrane Transport Studies

The results of supported liquid membrane transport studies (SLM) of amino acids with kryptofix 5 are presented in (Table1). The transport of amino acids by receptor across eggshell and cellulose nitrate membrane support followed the following trend: glycine > valine > threonine > alanine. The sequence for PTFE membrane was observed as glycine > valine > alanine > threonine. The efficacy of membrane supports followed the following trend: eggshell membrane > cellulose nitrate membrane > PTFE. The eggshell membrane is composed of intricate network of water insoluble proteins fibres as major constituents with small amount of carbohydrates and lipids with very high surface area and homogeneity (Figure 10). It possessed collagen-like proteins with high content of amide, carboxylate, and amine surface functional groups. The eggshell membrane (ESM) had active sites to interact with the guest amino acids because of its hydrophobic nature and exhibits electrostatic interactions between positively charged –H4N+ group of amino acid and –COOH group of eggshell membrane. More number of receptor molecules adhered due to hydrophobic nature resulting in good transportability. The structure of the hydrophobic receptor with appropriate complementary binding sites was responsible for transport of amino acids across eggshell membrane support. The transport of amino acids through supported liquid membrane mainly depends upon the structure of the receptor, nature, and structure of membrane support [10, 11].Figure 10
SEM micrograph receptor loaded eggshell membrane.The amount of amino acids transported by SLM was greater as compared to BLM. Figure10 shows the SEM image of receptor loaded eggshell membrane with high cross linking and homogeneity.


## 3.1. Effect of Receptor and Amino Acid Concentration

For optimization of receptor, the concentration was varied from1.0×10-6 to 1.0×10-2 M at constant amino acid concentration (5.0×10-3 M). At optimal receptor concentration (1.0×10-4 M), amino acid reacted with the receptor and resulted in the formation of amino acid receptor complex (Table 1). Above optimal concentration extraction rate decreased with further increase in the receptor concentration. The flux Jm was controlled by the complex formation at the source receiving-membrane interface which was governed by the complexation and decomplexation at the optimal concentration. The amino acid concentration was varied from 2.0×10-3 to 7.0×10-3 M to study the influence on transport and extraction processes. Both transport and extraction rates increased with increase in the concentration of amino acid with optimal concentration of 5.0×10-3 M (Figures 1 and 2).Table 1
Amount of amino acids transported into receiving phase through SLM using various  membrane supports after 24 h with kryptofix 5 in chloroform.
S. number
Amino acids
Egg shell membrane
PTFE membrane
Onion membrane
Cellulose nitrate membrane
Transported amino acid[×10-3M]
J
m
×
10
-
7 [mol/cm2s]
Transported amino acid[×10-3M]
J
m
×
10
-
7 [mol/cm2s]
Transported amino acid[×10-3M]
J
m
×
10
-
7 [mol/cm2s]
Transported amino acid[×10-3M]
J
m
×
10
-
7 [mol/cm2s]
(
1
)
Alanine
2.05
2.90
1.23
3.50
—
—
1.15
2.19
(
2
)
Glycine
3.15
5.30
1.50
3.75
—
—
2.50
4.38
(
3
)
Threonine
2.75
3.89
1.20
1.69
—
—
1.30
1.84
(
4
)
Valine
2.95
4.17
1.30
1.89
—
—
1.65
1.90
Concentration of amino acid in source phase:  5.0× 10−3 M.
Concentration of kryptofix 5: 1.0× 10−4 M in 50 mL chloroform.
Stirring speed: 200 rpm, pH: 6, and temperature: 30°C.Figure 1
Amount of amino acid transported after 24 h with kryptofix 5. Concentration of kryptofix 5 = 1.0×10-5 M in 15 mL chloroform. Stirring speed = 200 rpm, pH = 6, and temperature =30°C.Figure 2
Amount of amino acid extracted after 4 h with kryptofix 5. Concentration of kryptofix 5 =1.0×10-4 M in 10 mL chloroform. Stirring speed = 200 rpm, pH = 6, and temperature = 30°C.


## 3.2. Effect of Stirring

The extraction efficiency decreased without stirring in comparison to the experiments with stirring speed of 200 rpm. For valine, 76% and 26% of extraction efficiencies were observed with and without stirring, respectively (Figure4). This signifies that the stirring of aqueous phase considerably enhanced extraction and transport rate of amino acids. Similar trend was found for other investigated amino acids (Figure 3).Figure 3
Efficiency of amino acids extracted with stirring. Concentration of amino acid in source phase =5.0×10-3 M. Concentration of kryptofix 5 = 1.0×10-4 M in 10 mL chloroform. Stirring speed = 200 rpm, pH = 6, and temperature = 30°C.Figure 4
Efficiency of amino acids extracted without stirring. Concentration of amino acid in source phase =5.0×10-3 M. Concentration of kryptofix 5 = 1.0×10-4 M in 10 mL chloroform, pH = 6, and temperature = 30°C.


## 3.3. Effect of Time and Back Extraction

Figure5 shows the time dependence of amino acids transport and extraction through liquid membrane designed under optimal experimental conditions. Maximal extraction of amino acids took place within the first 2 h indicating that the time for attaining equilibrium is 2 h. For back extraction studies, aliquots from the loaded organic phase were withdrawn and subsequently back extracted for about 4 h with the same volume of the strippent which is deionised water in the present study. Glycine and threonine were found to be efficiently recovered due to easy decomplexation at the membrane receiving interface (Figure 6).Figure 5
Effect of time on extraction of amino acid with kryptofix 5. Concentration of amino acid in source phase =5.0×10-3 M. Concentration of kryptofix 5 = 1.0×10-4 M in 10 mL chloroform. Stirring speed = 200 rpm, pH = 6, and temperature =30°C.Figure 6
Efficiency of amino acids back extracted after 4 h with kryptofix 5. Concentration of amino acid in source phase =5.0×10-3 M. Concentration of kryptofix 5 = 1.0×10-4 M in 10 mL chloroform. Stirring speed = 200 rpm, pH = 6, and temperature =30°C.


## 3.4. Effect of pH

pH plays an important role in the extraction of amino acids. Maximum extraction of amino acids was observed at pH 6. At optimal pH, the carrier diffusion process seems to govern the transport phenomenon. The amino acid was extracted by the complexation of its H4N+ moiety by the flexible donor oxygen atoms. This can be explained by nature and hydrophobicity of amino acids. Therefore, valine and alanine were extracted more efficiently (Figure 7).Figure 7
Effect of pH. Concentration of amino acid in source phase =5.0×10-3 M. Concentration of kryptofix 5 = 1.0×10-4 M in 10 mL chloroform. Stirring speed = 200 rpm, pH = 6, and temperature = 30°C.Figures1 and 2 display the results of transport and extraction studies of amino acids using kryptofix 5 as a carrier at optimal concentration. The trend of extraction and transport efficiency of kryptofix was observed to be valine > alanine > glycine > threonine. The receptor proved to be a selective extractant for valine and alanine because it interacted with the amino acids by hydrogen bonding interactions between H4N+ moiety of amino acids and donor oxygen atoms of the receptor. The ability of amino acid receptor complex formation enhanced due to the presence of Π electrons of the quinoyl end groups of the receptor [14]. Besides, the extraction behaviour of receptor with amino acids strongly depends on the hydrophobicity of amino acids [3, 15]. The high hydrophobicity of valine resulted in more distribution at the aqueous organic interface thus accounting for its more tendency to pass from aqueous phase to organic phase giving better extraction of valine with receptor. Higher distribution ratio values were observed for highly hydrophobic amino acids (Figure 8).Figure 8
Hydrophobicity scale and flux values for amino acids.The order of transport ability of kryptofix 5 for amino acids was observed similar to the trend of extractability. The flexible ether chain of receptor adopted itself to H4N+, moiety of amino acids, whereas the insertion of quinoyl end group of receptor reduced its flexibility and permitted effective conformational changes in the binding and releasing processes [15]. The hydrophobic exterior and flexible donor sites with most suited conformation facilitated the passage of amino acid receptor complex from the organic phase to the aqueous receiving phase. The accumulation of amino acid in the membrane phase decreased the overall efficiency of transport process. Figure 9 describes the schematic representation of liquid membrane studies and possible sites of interaction of kryptofix 5 with guest amino acids.Figure 9
Schematic representation of liquid membrane studies of amino acids using kryptofix 5 as carrier in chloroform liquid membrane system. AA means amino acid. (a) represents interaction between –NH4+ and active –OH sites of kryptofix 5. (b) CH-π interactions between alkyl part of amino acid and Π electrons of kryptofix5.


## 3.5. Supported Liquid Membrane Transport Studies

The results of supported liquid membrane transport studies (SLM) of amino acids with kryptofix 5 are presented in (Table1). The transport of amino acids by receptor across eggshell and cellulose nitrate membrane support followed the following trend: glycine > valine > threonine > alanine. The sequence for PTFE membrane was observed as glycine > valine > alanine > threonine. The efficacy of membrane supports followed the following trend: eggshell membrane > cellulose nitrate membrane > PTFE. The eggshell membrane is composed of intricate network of water insoluble proteins fibres as major constituents with small amount of carbohydrates and lipids with very high surface area and homogeneity (Figure 10). It possessed collagen-like proteins with high content of amide, carboxylate, and amine surface functional groups. The eggshell membrane (ESM) had active sites to interact with the guest amino acids because of its hydrophobic nature and exhibits electrostatic interactions between positively charged –H4N+ group of amino acid and –COOH group of eggshell membrane. More number of receptor molecules adhered due to hydrophobic nature resulting in good transportability. The structure of the hydrophobic receptor with appropriate complementary binding sites was responsible for transport of amino acids across eggshell membrane support. The transport of amino acids through supported liquid membrane mainly depends upon the structure of the receptor, nature, and structure of membrane support [10, 11].Figure 10
SEM micrograph receptor loaded eggshell membrane.The amount of amino acids transported by SLM was greater as compared to BLM. Figure10 shows the SEM image of receptor loaded eggshell membrane with high cross linking and homogeneity.


## 4. Conclusion

The liquid-liquid extraction and transport studies with kryptofix 5 have been carried out in order to investigate the molecular recognition properties of kryptofix 5 for various amino acids. The high extractability of kryptofix 5 for amino acids was created by the binding of H4N+moiety of amino acids with flexible donor sites of receptor. Kryptofix 5 was found as selective extractant for valine and alanine. The objective of this precedent research work is to find out the applicability of synthetic receptor for the separation of metal cations and biomolecules by affinity chromatography. Kryptofix 5 is expected to be a novel recognition tool for biologically important amino acids, and, in future, its derivatives can be synthesized and used for the liquid membrane studies of biomolecules-like proteins, peptides, and nucleotides.



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*Source: 701570-2012-10-17.xml*