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Effect of triazine dye on electrical conductivity and extraction of bovine serum albumin in reversed micelles formed with cetyltrimethylammonium bromide
Colloids and Surfaces A: Physicochemical and Engineering Aspects 162 (1999) 259 – 264 www.elsevier.nl/locate/colsurfa
Effect of triazine dye on electrical conductivity and extraction of bovine serum albumin in reversed micelles formed with cetyltrimethylammonium bromide Tian-xi Zhang *, Hui-zhou Liu, Jia-yong Chen Laboratory of Separation Science and Engineering, Institute of Chemical Metallurgy, Chinese Academy of Sciences, P.O. Box 353, Beijing100080, People’s Republic of China Received 18 March 1999; accepted 27 May 1999
Abstract The effect of Cibacron Blue 3GA (CB) and bovine serum albumin (BSA) as guest molecules on the microstructure of reversed micelles has been investigated with electrical conductivity measurements. CB as an affinity ligand was directly introduced to reversed micelles formed with cationic surfactant, cetyltrimethylammonium bromide (CTAB). The anionic CB has electrostatic interactions with cationic surfactant and has affinity interaction with BSA. The conductivity of reversed micellar systems increases gradually with the increase of temperature either with or without the addition of CB. No electrical percolation appears with increase of temperature or water concentration. The conductivity of reversed micellar systems decreases with the addition of CB and decreases further with the addition of both CB and BSA. The conductivity of organic phase is three orders of magnitude lower than that of aqueous phase under the same CB concentration, which indicates that CB is probably confined to the closed microdomains of reversed micellar systems. The conductivity behavior of reversed micelles has not much difference with the addition of CB and BSA either by injection method or by phase transfer. For pH B pI, reversed micelles containing no CB solubilize no BSA due to electrostatic repulsion by phase transfer method. The enhancement of BSA transfer to the reversed micelles with addition of affinity CB indicates a strong interaction between the extracted BSA and the reversed micellar phase. The improvement of selectivity should be expected with the presence of biospecific CB because the affinity interaction is the unique driving force for BSA transfer. © 2000 Elsevier Science B.V. All rights reserved. Keywords: Conductivity; Affinity extraction; Reversed micelles; Cibacron Blue 3GA; Bovine serum albumin
1. Introduction
* Corresponding author. Tel.: + 86-10-62555005; fax: +8610-62561822. E-mail address: [email protected] (T.-x. Zhang)
Reversed micelles are nanometric aggregates stabilized by surfactants in organic solvents. Reversed micellar systems have been studied for the separation proteins in liquid–liquid extraction processes [1–3]. A significant improvement on the
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process selectivity can be achieved by introducing affinity ligands to the reversed micellar phase [4–6]. It seems that the effect of affinity ligands as guest molecules on the microstructure of reversed micelles has been paid little attention. The electrical conductivity of reversed micellar systems is very sensitive to the microstructure [7]. The conductivity of water-in-oil microemulsions usually shows variations over many orders of magnitudes. The conductivity is comparable to that of electrolyte solutions (0.1 – 1 S/m) in the presence of bicontinuous structures and drops sharply by 3 – 4 orders of magnitude due to percolative phenomenon [8,9]. The conductivity of reversed micelles in the presence of closed microdomains remains around 10 − 6 – 10 − 5 S/m, which are much higher than the typical conductivity of alkanes (10 − 16 – 10 − 12 S/m) [10,11]. In this work, Cibacron Blue 3GA (CB) as an affinity ligand was directly introduced to the reversed micelles formed with cationic surfactant, cetyltrimethylammonium bromide (CTAB). Anionic CB has electrostatic interaction with cationic surfactant and also has affinity interaction with bovine serum albumin (BSA). Various parameters that were used in the forward and backward transfer of BSA, such as concentrations of CB, BSA and hexanol, temperature, pH, on the effect of the conductivity of the reversed micellar systems were studied.
2. Experimental BSA (67 kDa, pI 4.7) was purchased from Beijing Hongxing Huaxue Factory, China. CB was obtained from Sigma. Beijing Chemical Reagents Company supplied CTAB, n-hexanol and n-hexane (analytical grade). Other chemicals were all commercially available reagents of analytical grade. Deionized and distilled water was used throughout the experiment. Conductivity determinations were carried out with a DDS-12A conductometer (Tianjin Scientific Instrument Factory, China) under thermostatically controlled conditions with a precision of 90.1°C. The original reversed micellar solutions used contained 50 mM CTAB and 15% (v/v) n-hexanol in n-hexane, denoted as CTAB (50 mM)/hexanol 15% (v/v)/hexane/H2O 0.8% (v/v). The organic phase for conductivity determination was made by injection method, except for those specially cited were made by phase transfer method (Table 1). Water concentration in organic phase was defined as the molar ratio of water to CTAB (W0). During the extraction of BSA, water concentration in the original organic phase was W0 = 8.89. The aqueous phase was 10 mM citrate or phosphate buffer solution. Protein solutions were made in buffer stocks in concentrations of 1.0 mg/ml. CB (typical 0.71 mmol/l) with the molar ratio of CB to CTAB (50 mM) of 0.0142 was dissolved in buffer solution before the forward
Table 1 The relationship of BSA transfer and the conductivity of organic phase using the phase transfer methoda BSA (mg/ml)b
PH
CB (mM)
BSA transferred to organic phase (%)c
K (mS/cm)
W0
0.0 1.0 0.0 1.0 0.0 1.0 0.0 1.0
4.50 4.50 4.50 4.50 6.29 6.29 6.29 6.29
0.0 0.0 0.71 0.71 0.0 0.0 0.71 0.71
– 0.0 – 28.90 – 100 – 100
0.119 0.118 0.115 0.111 0.111 0.106 0.110 0.105
24.68 24.60 25.75 25.91 25.08 26.42 25.66 26.30
a In the original aqueous phase: KBr= 45 mM, 10 mM buffer. In the original organic phase: CTAB (50 mM)/hexanol (15% v/v)/hexane, W0 = 8.89. b BSA concentration in the initial aqueous phase before the mixing of two phases. c BSA transfer into the organic phase after the mixing of two phases.
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extraction. Experiments were carried out in tightly stoppered 50 ml glass bottles. During the forward extraction, equal volumes (usually 3ml) of organic phase and aqueous phase were mixed with bottles placed in a water bath at 30.090.5°C with rate of agitation of 2.91 s − 1 for 10 min. The mixtures were then centrifuged at 50 s − 1 for 5 min to obtain a clear separation of two phases with no precipitation appeared at the interface. Blank experiments were performed simultaneously with the aqueous phase containing no protein. Unbound CB in the initial aqueous phase was transferred into the organic phase in all experiments of forward extraction. CB could not be detected in the aqueous phase after forward extraction, which indicated that the amount of CB remaining in the aqueous phase can be considered to be very small (as low as 3.8 nmol CB in the aqueous phase can be detected by visible light spectroscopy). The ionic strength of the aqueous phase was adjusted to the desired value with addition of KBr (typical 45 mM). BSA concentration in the aqueous phase was determined by UV spectroscopy, and in the reversed micellar phase by mass balance because the UV absorbance of organic phase could not be measured due to extremely high absorbance of CB. Water concentration (W0) in the organic phase after the phase transfer was determined using the Karl Fischer titration. The concentration of unbound CB in the aqueous phase was measured by visible light spectroscopy at 605 nm on a model 751G/Vis spectrophotometer. BSA concentrations in the aqueous solution were determined by measuring the absorbance at 278 nm, or by the Bradford method with the calibration curves prepared beforehand [12].
3. Results and discussion
3.1. Effect of temperature on electrical conducti6ity When the temperature or water concentration increases the conductivity of microemulsions increases gradually until a certain temperature or water concentration (W0) is reached of which a
261
Fig. 1. Variations of conductivity with temperature for reversed micelles. CTAB (50 mM)/hexanol (15.0% v/v)/hexane, W0 =20.0, injection method.
sudden increase of the conductivity is observed, which is known as electrical percolation. The values of the threshold of percolation can be modified by the addition of small quantities of additives [13,14]. Anionic CB has electrostatic interactions with cationic surfactant, in addition to, the affinity interaction with BSA. Fig. 1 shows the effect of temperature on the conductivity of reversed micellar systems with or without the addition of CB. As expected, the conductivity of systems increases gradually with the increase of temperature for cases in the presence of CB or not. However, there is no sudden increase of conductivity when temperature is below 48.1°C, which indicates no electrical percolation occurs. The curve for addition of CB is lower than that without addition of CB. The conductivity of reversed micellar systems decreases with the addition of CB. No electrical percolation occurs for the CTAB/chloroform/isooctane systems even at temperature as high as 58°C. On the contrary, the electrical percolation occurs for those systems with the addition of cetyl bromide and a percolation threshold temperature was observed to decrease as W0 increases [9]. There are not much differences of conductivity between CTAB/hexanol/hexane and CTAB/chloroform/isooctane systems without additives. Partial substitution of
262
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chloroform and isooctane by cetyl bromide is equivalent to the increase of the effective oil chain length resulted to the increase of surfactant aggregation number (N) and the rate constant ke for droplet collision with exchange of material [9]. In CTAB/hexanol/hexane system, the influence of conductivity with a small amount of CB addition can be considered as resulted from electrostatic interactions between CB and CTAB. The effect of microstructure of reversed micelles with different additives needs further investigation.
3.2. Effect of W0 on the conducti6ity of solutions with different hexanol concentrations The molar ratio of water to CTAB in reversed micelles is defined as W0. Fig. 2 shows the effect of water concentration on the conductivity of reversed micelles with different hexanol concentrations. The conductivity increases with W0 with the hexanol concentration of 24.1% (v/v), which leads to the turbid of system after W0 is above 17.8 mol/mol. The conductivity increases with W0 at first and decreases with W0 after reaching a maximum at hexanol concentration of 15.0% (v/ v). It also leads to the turbid of system after W0 is above 33.3 mol/mol. The reversed micelles can hold much more water and increase the radii of the droplets with decrease of hexanol concentration as it is generally accepted that the radius of
Fig. 3. Variations of conductivity with CB concentration in the aqueous or organic phase. CTAB (50 mM)/hexanol (15% v/v)/hexane, W0 =20.0, 30.0°C injection method.
the reversed micellar droplets increases with W0. No electrical percolation occurs for the reversed micellar systems with different hexanol concentrations, which probably indicate the reversed micelles composed of rather closed nanophases.
3.3. Effect of CB concentration on conducti6ity The anionic CB has electrostatic interactions with cationic surfactant and probably will influence the conductivity of reversed micellar systems. Fig. 3 shows the effect of CB concentration on conductivity with the addition of CB in the aqueous phase or organic phase. The conductivity of systems with addition of CB in aqueous phase is comparable with that of electrolyte solutions (102 –103 mS/cm), which indicates that CB presents electrolytic ions in the aqueous phase. However, the conductivity of organic phase is three orders of magnitude lower than that of aqueous phase at the same CB concentration, which indicates that CB is confined to the closed microdomains of reversed micellar systems.
3.4. Effect of BSA on conducti6ity
Fig. 2. Variations of conductivity of solution CTAB (50 mM)/hexanol/hexane by dilution with water. W0 = 8.89 for the original organic phase, 30.0°C, injection method.
BSA shows electrostatic interaction with cationic CTAB in the micellar phase. Fig. 4 indicates the effect of BSA concentrations on the conductivity with addition of BSA in the organic
T.-x. Zhang et al. / Colloids and Surfaces A: Physicochem. Eng. Aspects 162 (2000) 259–264
phase. The conductivity of systems decreases with increase of BSA concentration in the organic phase whether the aqueous solution with pH B pI or pH BpI, which indicates that the conductivity of systems has not been affected significantly by the electrostatic attraction or repulsion between BSA and CTAB. The conductivity of systems decreases further with addition of CB. The conductivity behavior can be considered as features characteristic of migration of charged droplets in the electrical field [10,11]. Charged droplets are formed by the exchange of material between droplets through collisions with temporary merging. The electrostatic interaction between BSA or CB with CTAB may be resulted to the decrease of the attractive interdroplet interactions and increase the stability of neutral droplets. The decrease of number of charged droplets would decrease the conductivity of systems studied.
3.5. BSA Transfer and conducti6ity in organic phase Anionic CB presents electrostatic interactions with cationic surfactant (CTAB) and also affinity interaction with BSA and can be transferred directly to the reversed micelles. Table 1 shows the relationship of BSA transfer and the conductivity of organic phase obtained by phase transfer method.
263
At pH B 4.50 (pHB pI), reversed micelles containing no CB solubilize no BSA due to repulsive electrostatic interaction. However, the BSA transfer to the reversed micelles increases with the addition of a small amount of unbound CB to the aqueous phase, which indicates a strong interaction between the extracted BSA and the reversed micellar phase in the presence of CB. The improvement of selectivity can be expected with the presence of biospecific CB because the affinity interaction is the unique driving force for BSA transfer. At pHB 6.29 (pHB pI), all BSA will be transferred into the reversed micelles either with or without the addition of CB, which indicates the electrostatic attraction between BSA and CTAB is the main driving force for BSA transfer. The conductivity values given in Table 1 show a very small decrease of conductivity accompanied by the BSA transfer into the reversed micelles. It is interesting that the conductivity of organic phase using phase transfer method is approximately equal to the maximum conductivity value obtained by using injection method, which might indicate that not much difference of microstructure exists for reversed micelles formed by using injection method or phase transfer method. The concentration of water (W0) in the organic phase increases with transfer of BSA or CB. The conductivity of reversed micelles is very sensitive to their structure. The rate constant ke for droplet collision with exchange of material
Fig. 4. Variations of conductivity with BSA concentration: (a) W0 =20.0, pH 7.04; (b) pH 4.51. CTAB (50 mM)/hexanol (15% v/v)/hexane, 30.0°C, injection method.
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must reach a value of at least (1 – 2)× 109 M − 1 s − 1 for percolative conduction to occur. The occurrence of electrical percolation corresponds to an increase of ke and also to the increase of attractive interactions between droplets [9,15]. Our data (Figures and Table 1) show no percolation phenomenon occurred with introduction of CB and BSA either with injection or phase transfer method, which might indicate that not much difference of reversed micellar structure exists between these two methods. CB and BSA could be confined in the closed microdomains of reversed micelles and exchanged between droplets with a slow rate.
celles containing no CB solubilize no BSA due to electrostatic repulsive interaction by using phase transfer method. The enhancement of BSA transfer to the reversed micelles with the addition of affinity CB indicates a strong interaction between the extracted BSA and the reversed micellar phase in the presence of CB. The improvement of selectivity can be expected with the presence of biospecific CB because the affinity interaction is the unique driving force for BSA transfer at pHB pI.
Acknowledgements We acknowledge the financial support of the National Natural Science Foundation of China (No. 29836130).
4. Conclusions CB as an affinity ligand is directly introduced to reversed micellar systems formed with cationic surfactant, CTAB. The anionic CB shows electrostatic interactions with cationic surfactant and affinity interactions with BSA. The electrical conductivity of reversed micellar systems is affected by the addition of CB and BSA. The conductivity of systems increases gradually with the increase of temperature either with or without the addition of CB. There is a maximum conductivity of reversed micelles with variation of water concentration (W0). However, there is no sudden increase of conductivity when temperature is below 48.1°C or with increase of W0 until turbid occurred, which indicates no electrical percolation occurs. The conductivity of reversed micellar systems decreases with the addition of CB and decreases further with the addition of CB and BSA. The conductivity of organic phase is three orders of magnitude lower than that of aqueous phase with the same CB concentration, which indicates that CB is probably confined to the closed microdomains of reversed micelles. The conductivity behavior of reversed micellar systems has not much difference with the addition of BSA and CB either by using injection method or by phase transfer. At pH B pI, reversed mi-
.
References [1] N.L. Abbott, T.A. Hatton, Chem. Eng. Prog. 84 (1988) 31. [2] M. Dekker, R. Hilhorst, C. Laane, Anal. Biochem. 178 (1989) 217. [3] P.L. Luisi, M. Giomini, P. Pileni, B.H. Robinson, Biochim. Biophys. Acta 947 (1988) 209. [4] B.D. Kelley, D.I.C. Wang, T.A. Hatton, Biotechnol. Bioeng. 42 (1993) 1199. [5] B.D. Kelley, D.I.C. Wang, T.A. Hatton, Biotechnol. Bioeng. 42 (1993) 1209. [6] Y. Sun, S. Ichikawa, S. Sugiura, S. Furusaki, Biotechnol. Bioeng. 58 (1998) 58. [7] D. Liu, J. Ma, H. Cheng, Z. Zhao, Colloids Surf. A 135 (1998) 157. [8] M.W. Kim, J.S. Huang, Phys. Rev. A 34 (1986) 719. [9] J. Lang, G. Mascolo, R. Zana, P.L. Luisi, J. Phys. Chem. 94 (1990) 3069. [10] H.F. Eicke, M. Borkovec, B. Das-Gupta, J. Phys Chem. 93 (1989) 314. [11] M. Giustini, G. Palazzo, G. Colafemmina, M.D. Monica, M. Giomini, A. Ceglie, J. Phys. Chem. 100 (1996) 3190. [12] M.M. Bradford, Anal. Biochem. 72 (1976) 248. [13] C. Mathew, P.K. Patanjali, A. Nabi, A. Maitra, Colloids Surf. 30 (1988) 253. [14] E. Alvarez, L. Garcia-Rio, J.C. Mejuto, J.M. Navaza, J. Chem. Eng. Data 43 (1998) 433. [15] A. Jada, J. Lang, R. Zana, J. Phys. Chem. 93 (1989) 10.
Effect of triazine dye on electrical conductivity and extraction of bovine serum albumin in reversed micelles formed with cetyltrimethylammonium bromide Tian-xi Zhang *, Hui-zhou Liu, Jia-yong Chen Laboratory of Separation Science and Engineering, Institute of Chemical Metallurgy, Chinese Academy of Sciences, P.O. Box 353, Beijing100080, People’s Republic of China Received 18 March 1999; accepted 27 May 1999
Abstract The effect of Cibacron Blue 3GA (CB) and bovine serum albumin (BSA) as guest molecules on the microstructure of reversed micelles has been investigated with electrical conductivity measurements. CB as an affinity ligand was directly introduced to reversed micelles formed with cationic surfactant, cetyltrimethylammonium bromide (CTAB). The anionic CB has electrostatic interactions with cationic surfactant and has affinity interaction with BSA. The conductivity of reversed micellar systems increases gradually with the increase of temperature either with or without the addition of CB. No electrical percolation appears with increase of temperature or water concentration. The conductivity of reversed micellar systems decreases with the addition of CB and decreases further with the addition of both CB and BSA. The conductivity of organic phase is three orders of magnitude lower than that of aqueous phase under the same CB concentration, which indicates that CB is probably confined to the closed microdomains of reversed micellar systems. The conductivity behavior of reversed micelles has not much difference with the addition of CB and BSA either by injection method or by phase transfer. For pH B pI, reversed micelles containing no CB solubilize no BSA due to electrostatic repulsion by phase transfer method. The enhancement of BSA transfer to the reversed micelles with addition of affinity CB indicates a strong interaction between the extracted BSA and the reversed micellar phase. The improvement of selectivity should be expected with the presence of biospecific CB because the affinity interaction is the unique driving force for BSA transfer. © 2000 Elsevier Science B.V. All rights reserved. Keywords: Conductivity; Affinity extraction; Reversed micelles; Cibacron Blue 3GA; Bovine serum albumin
1. Introduction
* Corresponding author. Tel.: + 86-10-62555005; fax: +8610-62561822. E-mail address: [email protected] (T.-x. Zhang)
Reversed micelles are nanometric aggregates stabilized by surfactants in organic solvents. Reversed micellar systems have been studied for the separation proteins in liquid–liquid extraction processes [1–3]. A significant improvement on the
0927-7757/00/$ - see front matter © 2000 Elsevier Science B.V. All rights reserved. PII: S 0 9 2 7 - 7 7 5 7 ( 9 9 ) 0 0 2 5 0 - 2
260
T.-x. Zhang et al. / Colloids and Surfaces A: Physicochem. Eng. Aspects 162 (2000) 259–264
process selectivity can be achieved by introducing affinity ligands to the reversed micellar phase [4–6]. It seems that the effect of affinity ligands as guest molecules on the microstructure of reversed micelles has been paid little attention. The electrical conductivity of reversed micellar systems is very sensitive to the microstructure [7]. The conductivity of water-in-oil microemulsions usually shows variations over many orders of magnitudes. The conductivity is comparable to that of electrolyte solutions (0.1 – 1 S/m) in the presence of bicontinuous structures and drops sharply by 3 – 4 orders of magnitude due to percolative phenomenon [8,9]. The conductivity of reversed micelles in the presence of closed microdomains remains around 10 − 6 – 10 − 5 S/m, which are much higher than the typical conductivity of alkanes (10 − 16 – 10 − 12 S/m) [10,11]. In this work, Cibacron Blue 3GA (CB) as an affinity ligand was directly introduced to the reversed micelles formed with cationic surfactant, cetyltrimethylammonium bromide (CTAB). Anionic CB has electrostatic interaction with cationic surfactant and also has affinity interaction with bovine serum albumin (BSA). Various parameters that were used in the forward and backward transfer of BSA, such as concentrations of CB, BSA and hexanol, temperature, pH, on the effect of the conductivity of the reversed micellar systems were studied.
2. Experimental BSA (67 kDa, pI 4.7) was purchased from Beijing Hongxing Huaxue Factory, China. CB was obtained from Sigma. Beijing Chemical Reagents Company supplied CTAB, n-hexanol and n-hexane (analytical grade). Other chemicals were all commercially available reagents of analytical grade. Deionized and distilled water was used throughout the experiment. Conductivity determinations were carried out with a DDS-12A conductometer (Tianjin Scientific Instrument Factory, China) under thermostatically controlled conditions with a precision of 90.1°C. The original reversed micellar solutions used contained 50 mM CTAB and 15% (v/v) n-hexanol in n-hexane, denoted as CTAB (50 mM)/hexanol 15% (v/v)/hexane/H2O 0.8% (v/v). The organic phase for conductivity determination was made by injection method, except for those specially cited were made by phase transfer method (Table 1). Water concentration in organic phase was defined as the molar ratio of water to CTAB (W0). During the extraction of BSA, water concentration in the original organic phase was W0 = 8.89. The aqueous phase was 10 mM citrate or phosphate buffer solution. Protein solutions were made in buffer stocks in concentrations of 1.0 mg/ml. CB (typical 0.71 mmol/l) with the molar ratio of CB to CTAB (50 mM) of 0.0142 was dissolved in buffer solution before the forward
Table 1 The relationship of BSA transfer and the conductivity of organic phase using the phase transfer methoda BSA (mg/ml)b
PH
CB (mM)
BSA transferred to organic phase (%)c
K (mS/cm)
W0
0.0 1.0 0.0 1.0 0.0 1.0 0.0 1.0
4.50 4.50 4.50 4.50 6.29 6.29 6.29 6.29
0.0 0.0 0.71 0.71 0.0 0.0 0.71 0.71
– 0.0 – 28.90 – 100 – 100
0.119 0.118 0.115 0.111 0.111 0.106 0.110 0.105
24.68 24.60 25.75 25.91 25.08 26.42 25.66 26.30
a In the original aqueous phase: KBr= 45 mM, 10 mM buffer. In the original organic phase: CTAB (50 mM)/hexanol (15% v/v)/hexane, W0 = 8.89. b BSA concentration in the initial aqueous phase before the mixing of two phases. c BSA transfer into the organic phase after the mixing of two phases.
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extraction. Experiments were carried out in tightly stoppered 50 ml glass bottles. During the forward extraction, equal volumes (usually 3ml) of organic phase and aqueous phase were mixed with bottles placed in a water bath at 30.090.5°C with rate of agitation of 2.91 s − 1 for 10 min. The mixtures were then centrifuged at 50 s − 1 for 5 min to obtain a clear separation of two phases with no precipitation appeared at the interface. Blank experiments were performed simultaneously with the aqueous phase containing no protein. Unbound CB in the initial aqueous phase was transferred into the organic phase in all experiments of forward extraction. CB could not be detected in the aqueous phase after forward extraction, which indicated that the amount of CB remaining in the aqueous phase can be considered to be very small (as low as 3.8 nmol CB in the aqueous phase can be detected by visible light spectroscopy). The ionic strength of the aqueous phase was adjusted to the desired value with addition of KBr (typical 45 mM). BSA concentration in the aqueous phase was determined by UV spectroscopy, and in the reversed micellar phase by mass balance because the UV absorbance of organic phase could not be measured due to extremely high absorbance of CB. Water concentration (W0) in the organic phase after the phase transfer was determined using the Karl Fischer titration. The concentration of unbound CB in the aqueous phase was measured by visible light spectroscopy at 605 nm on a model 751G/Vis spectrophotometer. BSA concentrations in the aqueous solution were determined by measuring the absorbance at 278 nm, or by the Bradford method with the calibration curves prepared beforehand [12].
3. Results and discussion
3.1. Effect of temperature on electrical conducti6ity When the temperature or water concentration increases the conductivity of microemulsions increases gradually until a certain temperature or water concentration (W0) is reached of which a
261
Fig. 1. Variations of conductivity with temperature for reversed micelles. CTAB (50 mM)/hexanol (15.0% v/v)/hexane, W0 =20.0, injection method.
sudden increase of the conductivity is observed, which is known as electrical percolation. The values of the threshold of percolation can be modified by the addition of small quantities of additives [13,14]. Anionic CB has electrostatic interactions with cationic surfactant, in addition to, the affinity interaction with BSA. Fig. 1 shows the effect of temperature on the conductivity of reversed micellar systems with or without the addition of CB. As expected, the conductivity of systems increases gradually with the increase of temperature for cases in the presence of CB or not. However, there is no sudden increase of conductivity when temperature is below 48.1°C, which indicates no electrical percolation occurs. The curve for addition of CB is lower than that without addition of CB. The conductivity of reversed micellar systems decreases with the addition of CB. No electrical percolation occurs for the CTAB/chloroform/isooctane systems even at temperature as high as 58°C. On the contrary, the electrical percolation occurs for those systems with the addition of cetyl bromide and a percolation threshold temperature was observed to decrease as W0 increases [9]. There are not much differences of conductivity between CTAB/hexanol/hexane and CTAB/chloroform/isooctane systems without additives. Partial substitution of
262
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chloroform and isooctane by cetyl bromide is equivalent to the increase of the effective oil chain length resulted to the increase of surfactant aggregation number (N) and the rate constant ke for droplet collision with exchange of material [9]. In CTAB/hexanol/hexane system, the influence of conductivity with a small amount of CB addition can be considered as resulted from electrostatic interactions between CB and CTAB. The effect of microstructure of reversed micelles with different additives needs further investigation.
3.2. Effect of W0 on the conducti6ity of solutions with different hexanol concentrations The molar ratio of water to CTAB in reversed micelles is defined as W0. Fig. 2 shows the effect of water concentration on the conductivity of reversed micelles with different hexanol concentrations. The conductivity increases with W0 with the hexanol concentration of 24.1% (v/v), which leads to the turbid of system after W0 is above 17.8 mol/mol. The conductivity increases with W0 at first and decreases with W0 after reaching a maximum at hexanol concentration of 15.0% (v/ v). It also leads to the turbid of system after W0 is above 33.3 mol/mol. The reversed micelles can hold much more water and increase the radii of the droplets with decrease of hexanol concentration as it is generally accepted that the radius of
Fig. 3. Variations of conductivity with CB concentration in the aqueous or organic phase. CTAB (50 mM)/hexanol (15% v/v)/hexane, W0 =20.0, 30.0°C injection method.
the reversed micellar droplets increases with W0. No electrical percolation occurs for the reversed micellar systems with different hexanol concentrations, which probably indicate the reversed micelles composed of rather closed nanophases.
3.3. Effect of CB concentration on conducti6ity The anionic CB has electrostatic interactions with cationic surfactant and probably will influence the conductivity of reversed micellar systems. Fig. 3 shows the effect of CB concentration on conductivity with the addition of CB in the aqueous phase or organic phase. The conductivity of systems with addition of CB in aqueous phase is comparable with that of electrolyte solutions (102 –103 mS/cm), which indicates that CB presents electrolytic ions in the aqueous phase. However, the conductivity of organic phase is three orders of magnitude lower than that of aqueous phase at the same CB concentration, which indicates that CB is confined to the closed microdomains of reversed micellar systems.
3.4. Effect of BSA on conducti6ity
Fig. 2. Variations of conductivity of solution CTAB (50 mM)/hexanol/hexane by dilution with water. W0 = 8.89 for the original organic phase, 30.0°C, injection method.
BSA shows electrostatic interaction with cationic CTAB in the micellar phase. Fig. 4 indicates the effect of BSA concentrations on the conductivity with addition of BSA in the organic
T.-x. Zhang et al. / Colloids and Surfaces A: Physicochem. Eng. Aspects 162 (2000) 259–264
phase. The conductivity of systems decreases with increase of BSA concentration in the organic phase whether the aqueous solution with pH B pI or pH BpI, which indicates that the conductivity of systems has not been affected significantly by the electrostatic attraction or repulsion between BSA and CTAB. The conductivity of systems decreases further with addition of CB. The conductivity behavior can be considered as features characteristic of migration of charged droplets in the electrical field [10,11]. Charged droplets are formed by the exchange of material between droplets through collisions with temporary merging. The electrostatic interaction between BSA or CB with CTAB may be resulted to the decrease of the attractive interdroplet interactions and increase the stability of neutral droplets. The decrease of number of charged droplets would decrease the conductivity of systems studied.
3.5. BSA Transfer and conducti6ity in organic phase Anionic CB presents electrostatic interactions with cationic surfactant (CTAB) and also affinity interaction with BSA and can be transferred directly to the reversed micelles. Table 1 shows the relationship of BSA transfer and the conductivity of organic phase obtained by phase transfer method.
263
At pH B 4.50 (pHB pI), reversed micelles containing no CB solubilize no BSA due to repulsive electrostatic interaction. However, the BSA transfer to the reversed micelles increases with the addition of a small amount of unbound CB to the aqueous phase, which indicates a strong interaction between the extracted BSA and the reversed micellar phase in the presence of CB. The improvement of selectivity can be expected with the presence of biospecific CB because the affinity interaction is the unique driving force for BSA transfer. At pHB 6.29 (pHB pI), all BSA will be transferred into the reversed micelles either with or without the addition of CB, which indicates the electrostatic attraction between BSA and CTAB is the main driving force for BSA transfer. The conductivity values given in Table 1 show a very small decrease of conductivity accompanied by the BSA transfer into the reversed micelles. It is interesting that the conductivity of organic phase using phase transfer method is approximately equal to the maximum conductivity value obtained by using injection method, which might indicate that not much difference of microstructure exists for reversed micelles formed by using injection method or phase transfer method. The concentration of water (W0) in the organic phase increases with transfer of BSA or CB. The conductivity of reversed micelles is very sensitive to their structure. The rate constant ke for droplet collision with exchange of material
Fig. 4. Variations of conductivity with BSA concentration: (a) W0 =20.0, pH 7.04; (b) pH 4.51. CTAB (50 mM)/hexanol (15% v/v)/hexane, 30.0°C, injection method.
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must reach a value of at least (1 – 2)× 109 M − 1 s − 1 for percolative conduction to occur. The occurrence of electrical percolation corresponds to an increase of ke and also to the increase of attractive interactions between droplets [9,15]. Our data (Figures and Table 1) show no percolation phenomenon occurred with introduction of CB and BSA either with injection or phase transfer method, which might indicate that not much difference of reversed micellar structure exists between these two methods. CB and BSA could be confined in the closed microdomains of reversed micelles and exchanged between droplets with a slow rate.
celles containing no CB solubilize no BSA due to electrostatic repulsive interaction by using phase transfer method. The enhancement of BSA transfer to the reversed micelles with the addition of affinity CB indicates a strong interaction between the extracted BSA and the reversed micellar phase in the presence of CB. The improvement of selectivity can be expected with the presence of biospecific CB because the affinity interaction is the unique driving force for BSA transfer at pHB pI.
Acknowledgements We acknowledge the financial support of the National Natural Science Foundation of China (No. 29836130).
4. Conclusions CB as an affinity ligand is directly introduced to reversed micellar systems formed with cationic surfactant, CTAB. The anionic CB shows electrostatic interactions with cationic surfactant and affinity interactions with BSA. The electrical conductivity of reversed micellar systems is affected by the addition of CB and BSA. The conductivity of systems increases gradually with the increase of temperature either with or without the addition of CB. There is a maximum conductivity of reversed micelles with variation of water concentration (W0). However, there is no sudden increase of conductivity when temperature is below 48.1°C or with increase of W0 until turbid occurred, which indicates no electrical percolation occurs. The conductivity of reversed micellar systems decreases with the addition of CB and decreases further with the addition of CB and BSA. The conductivity of organic phase is three orders of magnitude lower than that of aqueous phase with the same CB concentration, which indicates that CB is probably confined to the closed microdomains of reversed micelles. The conductivity behavior of reversed micellar systems has not much difference with the addition of BSA and CB either by using injection method or by phase transfer. At pH B pI, reversed mi-
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