- Email: [email protected]
Adsorption of single component and binary mixtures of protein and surfactants at the oil–water interface
Colloids and Surfaces B: Biointerfaces 13 (1999) 195 – 202
Adsorption of single component and binary mixtures of protein and surfactants at the oil–water interface O.S. Sudah a, G. Chen a, Y.C. Chiew a,b,* b
a Department of Chemical and Biochemical Engineering, Rutgers Uni6ersity, Piscataway, NJ 08854, USA Department of Chemical and En6ironmental Engineering, National Uni6ersity of Singapore, Kent Ridge Crescent, Singapore 119260, Singapore
Received 14 December 1998; accepted 26 January 1999
Abstract The dynamic interfacial tensions of non-ionic Triton X-100 surfactants, chicken-egg-white lysozyme proteins, and binary mixtures of Triton X-100–lysozyme at the dodecane – water interface were measured using a capillary wave technique. Consistent with results reported in the literature, the adsorption of Triton X-100 was found to be diffusion-controlled. The adsorption of lysozyme is well represented by a series of two first-order relaxation processes, which, respectively, had been identified in the literature as related to the adsorption/penetration of protein onto the interface and rearrangement/unfolding to its equilibrium in the interface. Our data show that the second process is the slower of the two. For Triton X-100–lysozyme binary mixtures, we found that the presence of nonionic Triton surfactants has little or no effect on the first process. In contrast, the Triton surfactants play a significant role in impeding the rearrangement/unfolding of the protein molecules in the interface. © 1999 Elsevier Science B.V. All rights reserved. Keywords: Kinetics of adsorption; Proteins; Surfactants; Dynamic interfacial tensions; Surface waves diffraction
1. Introduction The study of adsorption of surfactants, emulsifiers and biological molecules at the fluid interface is a problem that has received considerable theoretical as well as experimental attention. An understanding of the adsorption behavior of surface active materials is relevant to a variety of * Corresponding author. Tel.: +65-779-2196; fax: + 65779-1936. E-mail address: [email protected] (Y.C. Chiew)
practical problems involving agriculture, paint and food products. Measurements of static and dynamic interfacial tensions at fluid interfaces have provided information and insights on the surface activities of surfactants and proteins. A great deal of effort has been directed to investigating the adsorption kinetics of surfactants and proteins at fluid interfaces. Much of the work centers on adsorption of surfactants onto the air–liquid [1–4] and liquid–liquid interfaces [1,5– 7]. Recently, studies of protein adsorption have begun to appear in the literature [8–16]. Much of
0927-7765/99/$ - see front matter © 1999 Elsevier Science B.V. All rights reserved. PII: S 0 9 2 7 - 7 7 6 5 ( 9 9 ) 0 0 0 1 1 - 9
196
O.S. Sudah et al. / Colloids and Surfaces B: Biointerfaces 13 (1999) 195–202
this work, however, was concerned with adsorption of a single component protein at the air–water interface. Relatively little attention has been directed at examining adsorption of a binary mixture of surfactant – protein at the oil–water interface. In this work we studied the adsorption kinetics of a binary mixture of lyophilized chicken eggwhite protein lysozyme and nonionic surfactant TritonX-100 at the dodecane – water interface. The electrocapillary wave technique [3,4] was employed to obtain static and dynamic interfacial tension measurements at the dodecane– water interface. This is a noninvasive technique that has been previously used in our laboratory to measure the surface tension of air–water interface. This paper is organized as follows. The experimental technique and procedure employed in this work is presented in Section 2. Section 3 reports on the static and dynamic interfacial tension of nonionic surfactant TritonX-100, lysozyme and binary mixtures of TritonX-100 – lysozyme. Concluding remarks are made in Section 4.
2.2. Capillary wa6e apparatus and methods Interfacial tensions of the adsorbed dodecane– water interface were measured by means of a capillary wave technique. The method used here is a modification of the technique developed in our laboratory for studying air–water interfaces [3,4,17]. A schematic of the experimental set-up is shown in Fig. 1. The apparatus consists of a Helwett-Packard (model 3325B) function generator, an EG&G Princeton Applied Research twophase lock-in amplifier (model 5208), a Kepco power supply unit, a Tektronix oscilloscope, and a Uniphase He–Ne laser source. The dodecane and water are contained in a rectangular aluminum trough with dimensions 39×23× 9 cm. The detection principle is based on the Kelvin equation for a liquid–liquid interface: v2 3 s= (r+r%) l (2p)3
(1)
which relates the interfacial tension s to liquid densities r and r% , and the capillary wave number k at a given capillary wave frequency v. The
2. Experimental methods and procedure
2.1. Materials Nonionic surfactants Triton X-100 (molecular weight 628 g/mol) and n-dodecane (+99% purity) were purchased from Fisher Scientific. Crystallized and lyophilized chicken-egg-white lysozyme proteins (molecular weight 14300 g/mol) were purchased from Sigma. The buffers used were 0.1 M boric acid – potassium chloride– sodium hydroxide (pH 9), 0.1 M potassium phosphate–monobasic sodium hydroxide (pH 7), and 0.05 M potassium biphthalate – sodium hydroxide (pH 5), all purchased from Fisher Scientific. Earlier studies in our laboratory showed that these buffers are surfactant free. Purified water from a Milli-Q ultrapure water system with a resistivity of 18.2 mV cm was used in preparing protein solutions. All measurements were performed at 2391°C.
Fig. 1. Schematic diagram representing the electrocapillary wave generation and detection system.
O.S. Sudah et al. / Colloids and Surfaces B: Biointerfaces 13 (1999) 195–202
capillary wave is generated at the dodecane–water interface by applying an AC voltage coupled with a DC offset to a metal blade that is located in the dodecane (note that the oil phase is nonconductive) and just above the dodecane – water interface. In this experimental set-up, there are two phase interfaces: the air – dodecane interface and the dodecane – water interface. Both interfaces reflect the incident laser beam. Because the beam reflected from the air – dodecane interface is very close to that reflected from the dodecane–water interface, a small glass plate tilted at a small angle is placed at the air – dodecane interface to separate these two reflected beams to ensure that only the laser beam reflected from the dodecane–water interface is directed to a position sensitive photodiode (PSD) for data collection. The PSD measures the phase angle of the wave (at wave frequency v) propagating at the dodecane–water interface through the use of a lock-in amplifier. The wavelength is related to the phase angle f by: f=
360l l
(2)
where l is the distance between the blade (wave generator) and the incident laser beam (detection point). The phase angle f given by Eq. (2) is related to the experimentally measured phase angle fm by f =fm −f0 where the reference angle f0 is set by the amplifier. During the course of an adsorption experiment, as surface active materials adsorbed onto the dodecane–water interface, the interfacial properties change with time. Consequently, measured values of phase angle fm(t) varies with time. The dynamic interfacial tension s(t) at time t can be obtained from: v2 s(t)= (r +r%) l(t)3 (2p)3 where l(t)=
1 f (t) − fm( ) − m l( ) 360l
(3)
n
−1
.
(4)
Here, fm( ) and l( ) correspond to the measured phase angle and wavelength at ‘infinite’ time (t= ) after the interface has attained equilibrium.
197
To measure the static interfacial tension s(t= ), the distance l between the wave generator and detector is varied by moving the laser beam away from the blade at a known velocity through the use of a computer controlled DC motor; this yields the slope dfm/dl and hence the wavelength l(t= ) through Eq. (2).
2.3. Experimental procedure and preparation In a typical experiment, 5000 ml water (buffered to pH 6.8) was poured into an aluminum trough. The metal blade (wave generator) was then placed at the center of the trough above the air–water interface. The laser beam (detection spot) was then moved to a distance of approximately 0.5 cm from the blade. The blade was then lowered to the air–water interface until a steady signal was observed on the oscilloscope. It should be mentioned that measurements of the phase angle fm and dfm/dl give an air–water surface tension of 72.5 dyn/cm. Once this was observed, 1000 ml dodecane was slowly and carefully poured onto the top of the water phase. As mentioned earlier, a microscope glass slide was placed on top of the air–dodecane interface to separate the laser beam reflected from the dodecane–water interface from that of the air–dodecane interface. The static interfacial tension of the dodecane– water interface was measured using the capillary wave method and calculated using Eqs. (3) and (4). Tension measurement was performed at a frequency v= 50 Hz, the dodecane–water interfacial tension agreed with the theoretical one of 51.5 dyn/cm [18–20] to 91 dyn/cm accuracy. Appropriate amounts of lysozyme solution (pH 6.8) at 1 × 10 − 4 g/ml were injected into the aqueous phase to make protein solutions of the desired concentration. This was achieved through three 22 gauge plastic tubes attached to the bottom of the trough. The injection points were placed at an equal distance from each other to maximize the distribution of injected solutes. It was found that this simple method produces reproducible results when measurements were repeated. A similar procedure was used for the TritonX-100 solution. For binary solutions of Triton X-100–lysozyme, solutions of the desired con-
198
O.S. Sudah et al. / Colloids and Surfaces B: Biointerfaces 13 (1999) 195–202
local equilibrium at the interface, Eq. (5) is combined with the Gibbs adsorption isotherm to yield the following equation for dynamic interfacial tension s: s0 − s(t)=
Fig. 2. Equilibrium interfacial tension s of nonionic surfactants at the dodecane–water interface plotted as a function of bulk surfactant concentration.
centration and composition were made by injecting appropriate quantities of Triton X-100– lysozyme into the water phase. This proved to be effective in preparing solutions for our studies.
3. Results and discussion
The static tensions of Triton X-100 adsorbed onto the dodecane – water interface were measured at different bulk Triton X-100 concentrations and plotted in Fig. 2. It can seen that the interfacial tension decreases with surfactant concentration and becomes constant when the Triton X-100 concentration reaches the critical micelle concentration at approximately 3× 10 − 5 wt.%. Dynamic interfacial tensions of Triton X-100 at the dodecane – water interfaces were measured at 2×10 − 5 wt.% surfactant bulk concentration. Earlier work by Liggieri et al. [5] showed that the Freundlich isotherm: G= Kc m
(5)
fits the experimental adsorption data of Triton very well. In Eq. (5), G represents the equilibrium adsorption, c is the surfactant concentration, and K and m are empirical parameters. If we assume
RTKc m m
(6)
where s0 refers to the initial interfacial tension, R is the gas constant, and T represents absolute temperature. When the Freunlich isotherm is applied to the static interfacial tension data we found that K=2.459× 10 − 9 and m= 0.144; these parameter values are in the same order of magnitude as those found for the hexane–aqueous Triton X-100 interface [5]. For diffusion-controlled adsorption, the model of Ward and Tordai [21] yields the following equation for the surface concentration G: G(t)= 2c
& Dat p
1/2
−
1/2
Da p
t
0
cs(t) dt (t− t)1/2
(7)
where c is the bulk concentration, cs is the subsurface concentration, Da is the apparent diffusion coefficient, and t is time. When t is small (short time), Eq. (7) simplifies to: G(t)= 2c
3.1. Triton X-100
Dat p
1/2
.
(8)
By combining Eqs. (5), (6) and (8), we have: s0 − s(t)=
2RTc Dat m p
1/2
.
(9)
For a long t, Joos [3] suggested that cs is almost at equilibrium and may be assumed to no longer change with time, hence allowing the integral in Eq. (7) to be evaluated to give: s(t)= se +
2RTG2 p Dat m
1/2
(10)
where se represents the equilibrium interfacial tension. Dynamic interfacial tensions s(t) of a 2 ×10 − 5 wt.% Triton X-100 adsorbed at the dodecane–water interface were fitted to the short-time equation, Eq. (9), and the apparent diffusion coefficient Da was found to be 9.36 ×10 − 6 cm2/s. In the long-time limit, the dynamic interfacial tension s(t) is plotted against t − 1/2 according to
O.S. Sudah et al. / Colloids and Surfaces B: Biointerfaces 13 (1999) 195–202
199
Eq. (10) as shown in Fig. 3. The fitting procedure gives Da = 8.23 × 10 − 6 cm2/s. The value of Da obtained here is in agreement with that reported by Liggieri et al. [5].
3.2. Lysozyme The equilibrium static interfacial tensions of lysozyme adsorbed at the dodecane – water interface at varying protein concentrations were also measured and are shown in Fig. 4. As expected the tension s is observed to decrease with increasing protein concentration; however, the tension becomes constant when the bulk lysozyme concentration reaches approximately 2×10 − 4 wt.%. This leveling off of the tension s was also observed for proteins adsorbed at air – water and oil–water interfaces [8 – 10]. The kinetics of protein adsorption is more complex than that of surfactants and has been shown to be affected by the following processes: (1) solute transport by convection or diffusion or both from the bulk aqueous solution to the subsurface just below the interface, (2) adsorption and penetration from the sub-surface onto the interface, and (3) molecular conformational changes that are accompanied by unfolding and stretching of adsorbed molecules. Dynamic interfacial tensions s(t) of lysozyme adsorbing onto the dodecane/water interface were
Fig. 3. Dynamic interfacial tension s(t) of Triton X-100 surfactant plotted as a function of t − 1/2 at 2 × 10-5 wt.% bulk concentration.
Fig. 4. Equilibrium interfacial tension s of lysozyme at the dodecane – water interface plotted as a function of bulk protein concentration.
measured at different bulk lysozyme concentrations. The s(t) data of adsorbed protein layers have been successfully modeled as first-order relaxation processes [8–10], i.e., by plotting the dynamic interfacial tension s(t) in the following form: ln
s(t)−se t =− . s0 − se t
(11)
Here, se refers to the equilibrium interfacial tension, s0 denotes the interfacial tension at t=0, and t represents a characteristic time for the relaxation process. Fig. 5 shows a plot of ln ([s(t)− se]/[s0 −se]) versus time t for a 2× 10 − 4 wt.% lysozyme solution. It is observed that the data fall into two linear regimes with distinct slopes with time constants t1 = 2.3 and t2 = 5.3 h indicating that the adsorption of lysozyme is characterized by two different relaxation processes. This behavior was also observed by Grahams and Philips [8–10] for the adsorption of proteins onto the air–water interface. Grahams and Philips measured both the dynamic interfacial pressure P(t) (defined as s(t)− s0, where s0 refers to the interfacial tension of the dodecane–water interface with no protein) and the interfacial protein concentration G(t). They found that both P(t) and G(t) increase with time in regime 1; however, only P(t) increases while the surface concentration G(t) remains con-
200
O.S. Sudah et al. / Colloids and Surfaces B: Biointerfaces 13 (1999) 195–202
teristic time for the first relaxation process is seen to decrease with increasing bulk lysozyme concentration. The time decreases from 4.4 to 2.3 h as the protein bulk concentration increases from 8× 10 − 5 to 2× 10 − 4 wt.%; however, it remains practically unchanged when the bulk concentration increases from 2×10 − 4 to 1.8× 10 − 3 wt.%. Similar behavior is observed for the time constant t2; it decreases from 15.9 to 5.3 h when the lysozyme bulk concentration increases from 8×10 − 5 to 2× 10 − 4 wt.% and remains fairly constant at the two higher concentrations.
Fig. 5. A plot of ln ([s(t)−se]/[s0 − se]) versus time t for a 21 ×0 − 4 wt.% lysozyme solution. The data are characterized by a series of two first-order processes with time constants t1 =2.3 and t2 = 5.3 h.
stant in regime 2. They suggest that the first relaxation process is related to the transport and adsorption/penetration of protein molecules into the interface, while the second is related to the rearrangement and unfolding of adsorbed proteins to its equilibrium state at the interface. For a 2×10 − 4 wt.% lysozyme solution, our results show that the second process (protein rearrangement/unfolding) is at least twice as slow as the first (adsorption/ penetration). The dynamic interfacial tension s(t) and the characteristic times t1 and t2 for lysozyme adsorbing onto the dodecane – water interface at three different bulk protein concentrations were obtained and are displayed in Table 1. An examination of the data shows that t2 is consistently larger than t1 indicating that the second relaxation process is slower than the first. The characTable 1 Relaxation times t1 and t2 for lysozyme at different bulk concentrations Lysozyme concentration (wt.%)
8×10−5 2×10−4 1.8×10−3
Relaxation times (h) t1
t2
4.4 2.3 1.9
15.9 5.3 5.9
3.3. Binary lysozyme–Triton X-100 In this section, we consider the effect of nonionic surfactant Triton X-100 on the adsorption kinetics of lysozyme at the dodecane–water interface. The dynamic interfacial tensions of a lysozyme–Triton X-100 mixture with compositions 2×10-4/2×10 − 5 wt.% were measured and plotted according to Eq. (11) to determine the effect of nonionic Triton surfactant on the adsorption of lysozyme. Fig. 6 shows ln ([s(t)−se]/ [s0 − se]) versus time t for the lysozyme–Triton X-100 mixture. Similar to the adsorption behavior of the one-component lysozyme system (shown in Fig. 5), two relaxation processes are observed. The corresponding characteristic times are t1 = 2.4 and t2 = 15.2 h compared with 2.3 and 5.3 h in the absence of surfactants. We see that t1 remains unchanged at approximately 2.4 h and is unaffected by the presence of nonionic Triton surfactants. This result suggests that, in this protein–surfactant mixture, the rate of the first relaxation process is dominated by the adsorption/penetration of lysozyme into the interface. The presence of Triton surfactants has little effect on t1 since the rate of adsorption of Triton is much faster than that of lysozyme. Fig. 6 further shows that, for 1.5 BtB 4 h, there exists a period during which the interfacial tension s(t) increases with time before the second relaxation process commences. This ‘transition’ may be attributed to competitive adsorption between the protein and surfactant molecules leading to molecular displacement at the interface.
O.S. Sudah et al. / Colloids and Surfaces B: Biointerfaces 13 (1999) 195–202
201
Fig. 6. Variation of interfacial tension s(t) with time for a lysozyme – Triton 2× 10 − 4/2 ×10 − 5 wt.% binary mixture. The quantity ln ([s(t) −se]/[s0 − se]) is plotted as a function of time. The time constants are found to be 2.4 and 15.2 h, respectively.
Fig. 7. Variation of interfacial tension s(t) with time for a lysozyme – Triton 1.8× 10 − 3/2 ×10 − 5 wt.% binary mixture. The quantity ln ([s(t) −se]/[s0 − se]) is plotted as a function of time. The time constants t1 and t2 are found to be 2.1 and 9.7 h, respectively.
In contrast, the second relaxation process, i.e., the unfolding/rearrangement of protein molecules at the interface, is significantly affected by the Triton surfactant as indicated by the 3-fold increase in the characteristic time t2 from 5.3 to 15.2 h. It implies that the unfolding/rearrangement process is an extremely slow one before the protein molecules reach an equilibrium state.
Fig. 7 shows the variation of ln ([s(t)−se]/ [s0 − se]) versus time t for at a higher protein concentration with lysozyme–Triton X-100 composition 1.8×10 − 3/21.8× 10 − 3/2×10 − 5 wt.%. We found that Triton surfactants have little influence on the time constant t1 but significantly affected t2. Values of these time constants are shown in Table 2.
O.S. Sudah et al. / Colloids and Surfaces B: Biointerfaces 13 (1999) 195–202
202
Table 2 Relaxation times t1 and t2 at different concentrationsa Lysozyme concentration (wt.%)
Lys Lys Lys Lys a
2×10−4 2×10−4–Triton 2×10−5 1.8×10−3 1.8×10−3–Triton 2×10−5
Relaxation times (h) t1
t2
2.3 2.4 1.9 2.1
5.3 15.2 5.9 9.7
Triton X-100 concentration is fixed at 2×10−5 wt.%.
4. Conclusion In this work we studied the kinetics of adsorption of nonionic surfactant Triton X-100 and a model protein lysozyme onto the dodecane–water interface. It was found that, consistent with earlier studies, the adsorption of Triton is diffusion-limited. In contrast, the adsorption of lysozyme is not diffusion-limited. The adsorption kinetics can be described by a series of two first-order relaxation processes consistent with the results of Graham and Philips [8 – 10]. The first relaxation may be attributed to the adsorption/penetration of protein molecules into the interface while the second relaxation is related to the relaxation or unfolding of adsorbed protein molecules to their equilibrium state. The second relaxation is slower than the first as indicated by the respective characteristic time constants. In binary Triton X-100– lysozyme mixtures, the presence of surfactants has little or no effect on the time constant of the first relaxation but has a significant influence in impeding the second relaxation process.
.
Acknowledgements This research was partially supported by the Center for Advanced Food Technology, Rutgers University.
References [1] G. Kretzschmar, R. Miller, Adv. Colloid Interface Sci. 37 (1991) 97. [2] P. Joos, J. P. Fang, G. Serrien, J. Colloid Interface Sci. 151 (1992) 144. [3] Y.C. Chiew, Q. Jiang, J.E. Valentini, J. Colloid Interface Sci. 155 (1993) 8. [4] Y.C. Chiew, Q. Jiang, Langmuir 9 (1993) 273. [5] L. Liggieri, F. Ravera, A. Passerone, J. Colloid Interface Sci. 169 (1995) 226. [6] L. Liggieri, F. Ravera, E. Ricci, A. Passerone, J. Colloid Interface Sci. 169 (1995) 228. [7] J. Van Hunsel, G. Bleys, P. Joss, J. Colloid Interface Sci. 114 (1986) 432. [8] D.E. Graham, M.C. Phillips, J. Colloid Interface Sci. 70 (1979) 403. [9] D.E. Graham, M.C. Phillips, J. Colloid Interface Sci. 70 (1979) 415. [10] D.E. Graham, M.C. Phillips, J. Colloid Interface Sci. 70 (1979) 427. [11] S. Damodaran, S. Xu, Langmuir 8 (1992) 2021. [12] S. Damodaran, S. Xu, Langmuir 10 (1994) 472. [13] F. Uraizee, G. Narasimhan, Biotechnol. Prog. 8 (1992) 187. [14] G. Narasimhan, F. Uraizee, J. Colloid Interface Sci. 146 (1991) 169. [15] J.D. Andrade, J.J. Magda, B.C. Tripp, J. Colloid Interface Sci. 173 (1995) 16. [16] A.L. De Roos, P. Walstra, Food Rev. Int. 9 (1993) 503. [17] Y.C. Chiew, Q. Jiang, Am. Chem. Soc. 14 (1995) 183. [18] C.J. Radke, C.A. MacLeod, J. Colloid Interface Sci. 160 (1993) 435. [19] M.F. Fowkes, J. Phys. Chem. 67 (1963) 2538. [20] H.K. Livingston, C.S. Swingley, M. Nakonecznyj, J.J. Jasper, J. Phys. Chem. 74 (1970) 1535. [21] A. Ward, L. Tordai, J. Chem. Phys. 14 (1946) 453.
Adsorption of single component and binary mixtures of protein and surfactants at the oil–water interface O.S. Sudah a, G. Chen a, Y.C. Chiew a,b,* b
a Department of Chemical and Biochemical Engineering, Rutgers Uni6ersity, Piscataway, NJ 08854, USA Department of Chemical and En6ironmental Engineering, National Uni6ersity of Singapore, Kent Ridge Crescent, Singapore 119260, Singapore
Received 14 December 1998; accepted 26 January 1999
Abstract The dynamic interfacial tensions of non-ionic Triton X-100 surfactants, chicken-egg-white lysozyme proteins, and binary mixtures of Triton X-100–lysozyme at the dodecane – water interface were measured using a capillary wave technique. Consistent with results reported in the literature, the adsorption of Triton X-100 was found to be diffusion-controlled. The adsorption of lysozyme is well represented by a series of two first-order relaxation processes, which, respectively, had been identified in the literature as related to the adsorption/penetration of protein onto the interface and rearrangement/unfolding to its equilibrium in the interface. Our data show that the second process is the slower of the two. For Triton X-100–lysozyme binary mixtures, we found that the presence of nonionic Triton surfactants has little or no effect on the first process. In contrast, the Triton surfactants play a significant role in impeding the rearrangement/unfolding of the protein molecules in the interface. © 1999 Elsevier Science B.V. All rights reserved. Keywords: Kinetics of adsorption; Proteins; Surfactants; Dynamic interfacial tensions; Surface waves diffraction
1. Introduction The study of adsorption of surfactants, emulsifiers and biological molecules at the fluid interface is a problem that has received considerable theoretical as well as experimental attention. An understanding of the adsorption behavior of surface active materials is relevant to a variety of * Corresponding author. Tel.: +65-779-2196; fax: + 65779-1936. E-mail address: [email protected] (Y.C. Chiew)
practical problems involving agriculture, paint and food products. Measurements of static and dynamic interfacial tensions at fluid interfaces have provided information and insights on the surface activities of surfactants and proteins. A great deal of effort has been directed to investigating the adsorption kinetics of surfactants and proteins at fluid interfaces. Much of the work centers on adsorption of surfactants onto the air–liquid [1–4] and liquid–liquid interfaces [1,5– 7]. Recently, studies of protein adsorption have begun to appear in the literature [8–16]. Much of
0927-7765/99/$ - see front matter © 1999 Elsevier Science B.V. All rights reserved. PII: S 0 9 2 7 - 7 7 6 5 ( 9 9 ) 0 0 0 1 1 - 9
196
O.S. Sudah et al. / Colloids and Surfaces B: Biointerfaces 13 (1999) 195–202
this work, however, was concerned with adsorption of a single component protein at the air–water interface. Relatively little attention has been directed at examining adsorption of a binary mixture of surfactant – protein at the oil–water interface. In this work we studied the adsorption kinetics of a binary mixture of lyophilized chicken eggwhite protein lysozyme and nonionic surfactant TritonX-100 at the dodecane – water interface. The electrocapillary wave technique [3,4] was employed to obtain static and dynamic interfacial tension measurements at the dodecane– water interface. This is a noninvasive technique that has been previously used in our laboratory to measure the surface tension of air–water interface. This paper is organized as follows. The experimental technique and procedure employed in this work is presented in Section 2. Section 3 reports on the static and dynamic interfacial tension of nonionic surfactant TritonX-100, lysozyme and binary mixtures of TritonX-100 – lysozyme. Concluding remarks are made in Section 4.
2.2. Capillary wa6e apparatus and methods Interfacial tensions of the adsorbed dodecane– water interface were measured by means of a capillary wave technique. The method used here is a modification of the technique developed in our laboratory for studying air–water interfaces [3,4,17]. A schematic of the experimental set-up is shown in Fig. 1. The apparatus consists of a Helwett-Packard (model 3325B) function generator, an EG&G Princeton Applied Research twophase lock-in amplifier (model 5208), a Kepco power supply unit, a Tektronix oscilloscope, and a Uniphase He–Ne laser source. The dodecane and water are contained in a rectangular aluminum trough with dimensions 39×23× 9 cm. The detection principle is based on the Kelvin equation for a liquid–liquid interface: v2 3 s= (r+r%) l (2p)3
(1)
which relates the interfacial tension s to liquid densities r and r% , and the capillary wave number k at a given capillary wave frequency v. The
2. Experimental methods and procedure
2.1. Materials Nonionic surfactants Triton X-100 (molecular weight 628 g/mol) and n-dodecane (+99% purity) were purchased from Fisher Scientific. Crystallized and lyophilized chicken-egg-white lysozyme proteins (molecular weight 14300 g/mol) were purchased from Sigma. The buffers used were 0.1 M boric acid – potassium chloride– sodium hydroxide (pH 9), 0.1 M potassium phosphate–monobasic sodium hydroxide (pH 7), and 0.05 M potassium biphthalate – sodium hydroxide (pH 5), all purchased from Fisher Scientific. Earlier studies in our laboratory showed that these buffers are surfactant free. Purified water from a Milli-Q ultrapure water system with a resistivity of 18.2 mV cm was used in preparing protein solutions. All measurements were performed at 2391°C.
Fig. 1. Schematic diagram representing the electrocapillary wave generation and detection system.
O.S. Sudah et al. / Colloids and Surfaces B: Biointerfaces 13 (1999) 195–202
capillary wave is generated at the dodecane–water interface by applying an AC voltage coupled with a DC offset to a metal blade that is located in the dodecane (note that the oil phase is nonconductive) and just above the dodecane – water interface. In this experimental set-up, there are two phase interfaces: the air – dodecane interface and the dodecane – water interface. Both interfaces reflect the incident laser beam. Because the beam reflected from the air – dodecane interface is very close to that reflected from the dodecane–water interface, a small glass plate tilted at a small angle is placed at the air – dodecane interface to separate these two reflected beams to ensure that only the laser beam reflected from the dodecane–water interface is directed to a position sensitive photodiode (PSD) for data collection. The PSD measures the phase angle of the wave (at wave frequency v) propagating at the dodecane–water interface through the use of a lock-in amplifier. The wavelength is related to the phase angle f by: f=
360l l
(2)
where l is the distance between the blade (wave generator) and the incident laser beam (detection point). The phase angle f given by Eq. (2) is related to the experimentally measured phase angle fm by f =fm −f0 where the reference angle f0 is set by the amplifier. During the course of an adsorption experiment, as surface active materials adsorbed onto the dodecane–water interface, the interfacial properties change with time. Consequently, measured values of phase angle fm(t) varies with time. The dynamic interfacial tension s(t) at time t can be obtained from: v2 s(t)= (r +r%) l(t)3 (2p)3 where l(t)=
1 f (t) − fm( ) − m l( ) 360l
(3)
n
−1
.
(4)
Here, fm( ) and l( ) correspond to the measured phase angle and wavelength at ‘infinite’ time (t= ) after the interface has attained equilibrium.
197
To measure the static interfacial tension s(t= ), the distance l between the wave generator and detector is varied by moving the laser beam away from the blade at a known velocity through the use of a computer controlled DC motor; this yields the slope dfm/dl and hence the wavelength l(t= ) through Eq. (2).
2.3. Experimental procedure and preparation In a typical experiment, 5000 ml water (buffered to pH 6.8) was poured into an aluminum trough. The metal blade (wave generator) was then placed at the center of the trough above the air–water interface. The laser beam (detection spot) was then moved to a distance of approximately 0.5 cm from the blade. The blade was then lowered to the air–water interface until a steady signal was observed on the oscilloscope. It should be mentioned that measurements of the phase angle fm and dfm/dl give an air–water surface tension of 72.5 dyn/cm. Once this was observed, 1000 ml dodecane was slowly and carefully poured onto the top of the water phase. As mentioned earlier, a microscope glass slide was placed on top of the air–dodecane interface to separate the laser beam reflected from the dodecane–water interface from that of the air–dodecane interface. The static interfacial tension of the dodecane– water interface was measured using the capillary wave method and calculated using Eqs. (3) and (4). Tension measurement was performed at a frequency v= 50 Hz, the dodecane–water interfacial tension agreed with the theoretical one of 51.5 dyn/cm [18–20] to 91 dyn/cm accuracy. Appropriate amounts of lysozyme solution (pH 6.8) at 1 × 10 − 4 g/ml were injected into the aqueous phase to make protein solutions of the desired concentration. This was achieved through three 22 gauge plastic tubes attached to the bottom of the trough. The injection points were placed at an equal distance from each other to maximize the distribution of injected solutes. It was found that this simple method produces reproducible results when measurements were repeated. A similar procedure was used for the TritonX-100 solution. For binary solutions of Triton X-100–lysozyme, solutions of the desired con-
198
O.S. Sudah et al. / Colloids and Surfaces B: Biointerfaces 13 (1999) 195–202
local equilibrium at the interface, Eq. (5) is combined with the Gibbs adsorption isotherm to yield the following equation for dynamic interfacial tension s: s0 − s(t)=
Fig. 2. Equilibrium interfacial tension s of nonionic surfactants at the dodecane–water interface plotted as a function of bulk surfactant concentration.
centration and composition were made by injecting appropriate quantities of Triton X-100– lysozyme into the water phase. This proved to be effective in preparing solutions for our studies.
3. Results and discussion
The static tensions of Triton X-100 adsorbed onto the dodecane – water interface were measured at different bulk Triton X-100 concentrations and plotted in Fig. 2. It can seen that the interfacial tension decreases with surfactant concentration and becomes constant when the Triton X-100 concentration reaches the critical micelle concentration at approximately 3× 10 − 5 wt.%. Dynamic interfacial tensions of Triton X-100 at the dodecane – water interfaces were measured at 2×10 − 5 wt.% surfactant bulk concentration. Earlier work by Liggieri et al. [5] showed that the Freundlich isotherm: G= Kc m
(5)
fits the experimental adsorption data of Triton very well. In Eq. (5), G represents the equilibrium adsorption, c is the surfactant concentration, and K and m are empirical parameters. If we assume
RTKc m m
(6)
where s0 refers to the initial interfacial tension, R is the gas constant, and T represents absolute temperature. When the Freunlich isotherm is applied to the static interfacial tension data we found that K=2.459× 10 − 9 and m= 0.144; these parameter values are in the same order of magnitude as those found for the hexane–aqueous Triton X-100 interface [5]. For diffusion-controlled adsorption, the model of Ward and Tordai [21] yields the following equation for the surface concentration G: G(t)= 2c
& Dat p
1/2
−
1/2
Da p
t
0
cs(t) dt (t− t)1/2
(7)
where c is the bulk concentration, cs is the subsurface concentration, Da is the apparent diffusion coefficient, and t is time. When t is small (short time), Eq. (7) simplifies to: G(t)= 2c
3.1. Triton X-100
Dat p
1/2
.
(8)
By combining Eqs. (5), (6) and (8), we have: s0 − s(t)=
2RTc Dat m p
1/2
.
(9)
For a long t, Joos [3] suggested that cs is almost at equilibrium and may be assumed to no longer change with time, hence allowing the integral in Eq. (7) to be evaluated to give: s(t)= se +
2RTG2 p Dat m
1/2
(10)
where se represents the equilibrium interfacial tension. Dynamic interfacial tensions s(t) of a 2 ×10 − 5 wt.% Triton X-100 adsorbed at the dodecane–water interface were fitted to the short-time equation, Eq. (9), and the apparent diffusion coefficient Da was found to be 9.36 ×10 − 6 cm2/s. In the long-time limit, the dynamic interfacial tension s(t) is plotted against t − 1/2 according to
O.S. Sudah et al. / Colloids and Surfaces B: Biointerfaces 13 (1999) 195–202
199
Eq. (10) as shown in Fig. 3. The fitting procedure gives Da = 8.23 × 10 − 6 cm2/s. The value of Da obtained here is in agreement with that reported by Liggieri et al. [5].
3.2. Lysozyme The equilibrium static interfacial tensions of lysozyme adsorbed at the dodecane – water interface at varying protein concentrations were also measured and are shown in Fig. 4. As expected the tension s is observed to decrease with increasing protein concentration; however, the tension becomes constant when the bulk lysozyme concentration reaches approximately 2×10 − 4 wt.%. This leveling off of the tension s was also observed for proteins adsorbed at air – water and oil–water interfaces [8 – 10]. The kinetics of protein adsorption is more complex than that of surfactants and has been shown to be affected by the following processes: (1) solute transport by convection or diffusion or both from the bulk aqueous solution to the subsurface just below the interface, (2) adsorption and penetration from the sub-surface onto the interface, and (3) molecular conformational changes that are accompanied by unfolding and stretching of adsorbed molecules. Dynamic interfacial tensions s(t) of lysozyme adsorbing onto the dodecane/water interface were
Fig. 3. Dynamic interfacial tension s(t) of Triton X-100 surfactant plotted as a function of t − 1/2 at 2 × 10-5 wt.% bulk concentration.
Fig. 4. Equilibrium interfacial tension s of lysozyme at the dodecane – water interface plotted as a function of bulk protein concentration.
measured at different bulk lysozyme concentrations. The s(t) data of adsorbed protein layers have been successfully modeled as first-order relaxation processes [8–10], i.e., by plotting the dynamic interfacial tension s(t) in the following form: ln
s(t)−se t =− . s0 − se t
(11)
Here, se refers to the equilibrium interfacial tension, s0 denotes the interfacial tension at t=0, and t represents a characteristic time for the relaxation process. Fig. 5 shows a plot of ln ([s(t)− se]/[s0 −se]) versus time t for a 2× 10 − 4 wt.% lysozyme solution. It is observed that the data fall into two linear regimes with distinct slopes with time constants t1 = 2.3 and t2 = 5.3 h indicating that the adsorption of lysozyme is characterized by two different relaxation processes. This behavior was also observed by Grahams and Philips [8–10] for the adsorption of proteins onto the air–water interface. Grahams and Philips measured both the dynamic interfacial pressure P(t) (defined as s(t)− s0, where s0 refers to the interfacial tension of the dodecane–water interface with no protein) and the interfacial protein concentration G(t). They found that both P(t) and G(t) increase with time in regime 1; however, only P(t) increases while the surface concentration G(t) remains con-
200
O.S. Sudah et al. / Colloids and Surfaces B: Biointerfaces 13 (1999) 195–202
teristic time for the first relaxation process is seen to decrease with increasing bulk lysozyme concentration. The time decreases from 4.4 to 2.3 h as the protein bulk concentration increases from 8× 10 − 5 to 2× 10 − 4 wt.%; however, it remains practically unchanged when the bulk concentration increases from 2×10 − 4 to 1.8× 10 − 3 wt.%. Similar behavior is observed for the time constant t2; it decreases from 15.9 to 5.3 h when the lysozyme bulk concentration increases from 8×10 − 5 to 2× 10 − 4 wt.% and remains fairly constant at the two higher concentrations.
Fig. 5. A plot of ln ([s(t)−se]/[s0 − se]) versus time t for a 21 ×0 − 4 wt.% lysozyme solution. The data are characterized by a series of two first-order processes with time constants t1 =2.3 and t2 = 5.3 h.
stant in regime 2. They suggest that the first relaxation process is related to the transport and adsorption/penetration of protein molecules into the interface, while the second is related to the rearrangement and unfolding of adsorbed proteins to its equilibrium state at the interface. For a 2×10 − 4 wt.% lysozyme solution, our results show that the second process (protein rearrangement/unfolding) is at least twice as slow as the first (adsorption/ penetration). The dynamic interfacial tension s(t) and the characteristic times t1 and t2 for lysozyme adsorbing onto the dodecane – water interface at three different bulk protein concentrations were obtained and are displayed in Table 1. An examination of the data shows that t2 is consistently larger than t1 indicating that the second relaxation process is slower than the first. The characTable 1 Relaxation times t1 and t2 for lysozyme at different bulk concentrations Lysozyme concentration (wt.%)
8×10−5 2×10−4 1.8×10−3
Relaxation times (h) t1
t2
4.4 2.3 1.9
15.9 5.3 5.9
3.3. Binary lysozyme–Triton X-100 In this section, we consider the effect of nonionic surfactant Triton X-100 on the adsorption kinetics of lysozyme at the dodecane–water interface. The dynamic interfacial tensions of a lysozyme–Triton X-100 mixture with compositions 2×10-4/2×10 − 5 wt.% were measured and plotted according to Eq. (11) to determine the effect of nonionic Triton surfactant on the adsorption of lysozyme. Fig. 6 shows ln ([s(t)−se]/ [s0 − se]) versus time t for the lysozyme–Triton X-100 mixture. Similar to the adsorption behavior of the one-component lysozyme system (shown in Fig. 5), two relaxation processes are observed. The corresponding characteristic times are t1 = 2.4 and t2 = 15.2 h compared with 2.3 and 5.3 h in the absence of surfactants. We see that t1 remains unchanged at approximately 2.4 h and is unaffected by the presence of nonionic Triton surfactants. This result suggests that, in this protein–surfactant mixture, the rate of the first relaxation process is dominated by the adsorption/penetration of lysozyme into the interface. The presence of Triton surfactants has little effect on t1 since the rate of adsorption of Triton is much faster than that of lysozyme. Fig. 6 further shows that, for 1.5 BtB 4 h, there exists a period during which the interfacial tension s(t) increases with time before the second relaxation process commences. This ‘transition’ may be attributed to competitive adsorption between the protein and surfactant molecules leading to molecular displacement at the interface.
O.S. Sudah et al. / Colloids and Surfaces B: Biointerfaces 13 (1999) 195–202
201
Fig. 6. Variation of interfacial tension s(t) with time for a lysozyme – Triton 2× 10 − 4/2 ×10 − 5 wt.% binary mixture. The quantity ln ([s(t) −se]/[s0 − se]) is plotted as a function of time. The time constants are found to be 2.4 and 15.2 h, respectively.
Fig. 7. Variation of interfacial tension s(t) with time for a lysozyme – Triton 1.8× 10 − 3/2 ×10 − 5 wt.% binary mixture. The quantity ln ([s(t) −se]/[s0 − se]) is plotted as a function of time. The time constants t1 and t2 are found to be 2.1 and 9.7 h, respectively.
In contrast, the second relaxation process, i.e., the unfolding/rearrangement of protein molecules at the interface, is significantly affected by the Triton surfactant as indicated by the 3-fold increase in the characteristic time t2 from 5.3 to 15.2 h. It implies that the unfolding/rearrangement process is an extremely slow one before the protein molecules reach an equilibrium state.
Fig. 7 shows the variation of ln ([s(t)−se]/ [s0 − se]) versus time t for at a higher protein concentration with lysozyme–Triton X-100 composition 1.8×10 − 3/21.8× 10 − 3/2×10 − 5 wt.%. We found that Triton surfactants have little influence on the time constant t1 but significantly affected t2. Values of these time constants are shown in Table 2.
O.S. Sudah et al. / Colloids and Surfaces B: Biointerfaces 13 (1999) 195–202
202
Table 2 Relaxation times t1 and t2 at different concentrationsa Lysozyme concentration (wt.%)
Lys Lys Lys Lys a
2×10−4 2×10−4–Triton 2×10−5 1.8×10−3 1.8×10−3–Triton 2×10−5
Relaxation times (h) t1
t2
2.3 2.4 1.9 2.1
5.3 15.2 5.9 9.7
Triton X-100 concentration is fixed at 2×10−5 wt.%.
4. Conclusion In this work we studied the kinetics of adsorption of nonionic surfactant Triton X-100 and a model protein lysozyme onto the dodecane–water interface. It was found that, consistent with earlier studies, the adsorption of Triton is diffusion-limited. In contrast, the adsorption of lysozyme is not diffusion-limited. The adsorption kinetics can be described by a series of two first-order relaxation processes consistent with the results of Graham and Philips [8 – 10]. The first relaxation may be attributed to the adsorption/penetration of protein molecules into the interface while the second relaxation is related to the relaxation or unfolding of adsorbed protein molecules to their equilibrium state. The second relaxation is slower than the first as indicated by the respective characteristic time constants. In binary Triton X-100– lysozyme mixtures, the presence of surfactants has little or no effect on the time constant of the first relaxation but has a significant influence in impeding the second relaxation process.
.
Acknowledgements This research was partially supported by the Center for Advanced Food Technology, Rutgers University.
References [1] G. Kretzschmar, R. Miller, Adv. Colloid Interface Sci. 37 (1991) 97. [2] P. Joos, J. P. Fang, G. Serrien, J. Colloid Interface Sci. 151 (1992) 144. [3] Y.C. Chiew, Q. Jiang, J.E. Valentini, J. Colloid Interface Sci. 155 (1993) 8. [4] Y.C. Chiew, Q. Jiang, Langmuir 9 (1993) 273. [5] L. Liggieri, F. Ravera, A. Passerone, J. Colloid Interface Sci. 169 (1995) 226. [6] L. Liggieri, F. Ravera, E. Ricci, A. Passerone, J. Colloid Interface Sci. 169 (1995) 228. [7] J. Van Hunsel, G. Bleys, P. Joss, J. Colloid Interface Sci. 114 (1986) 432. [8] D.E. Graham, M.C. Phillips, J. Colloid Interface Sci. 70 (1979) 403. [9] D.E. Graham, M.C. Phillips, J. Colloid Interface Sci. 70 (1979) 415. [10] D.E. Graham, M.C. Phillips, J. Colloid Interface Sci. 70 (1979) 427. [11] S. Damodaran, S. Xu, Langmuir 8 (1992) 2021. [12] S. Damodaran, S. Xu, Langmuir 10 (1994) 472. [13] F. Uraizee, G. Narasimhan, Biotechnol. Prog. 8 (1992) 187. [14] G. Narasimhan, F. Uraizee, J. Colloid Interface Sci. 146 (1991) 169. [15] J.D. Andrade, J.J. Magda, B.C. Tripp, J. Colloid Interface Sci. 173 (1995) 16. [16] A.L. De Roos, P. Walstra, Food Rev. Int. 9 (1993) 503. [17] Y.C. Chiew, Q. Jiang, Am. Chem. Soc. 14 (1995) 183. [18] C.J. Radke, C.A. MacLeod, J. Colloid Interface Sci. 160 (1993) 435. [19] M.F. Fowkes, J. Phys. Chem. 67 (1963) 2538. [20] H.K. Livingston, C.S. Swingley, M. Nakonecznyj, J.J. Jasper, J. Phys. Chem. 74 (1970) 1535. [21] A. Ward, L. Tordai, J. Chem. Phys. 14 (1946) 453.