© Copyright 1996 American Chemical Society
DECEMBER 11, 1996 VOLUME 12, NUMBER 25
Letters A Novel Langmuir Trough for Equilibrium and Dynamic Measurements on Air-Water and Oil-Water Monolayers Brent S. Murray* and Phillip V. Nelson Food Colloids Group, Procter Department of Food Science, University of Leeds, Leeds LS2 9JT, U.K. Received July 29, 1996. In Final Form: October 7, 1996X A new “Langmuir” trough is described which allows accurate, symmetric compression and expansion of a spread or adsorbed film of molecules. Compression/expansion of films is equally easy at an air-water (A-W) interface or oil-water (O-W) interface. Several different modes of operation of the trough are described which allow measurement of surface/interfacial pressure (π)-area per molecule (A) isotherms and measurement of dilatational rheology of interfacial films. Equlibrium and dilatational measurements are consistent with those obtained via other techniques. The π-A isotherms of spread films of the proteins, β-lactoglobulin (β-L) and bovine serum albumin (BSA) are considerably more expanded at the O-W interface compared to the A-W interface.
Introduction The response of a film of molecules at an interface to expansion and compression is a key factor determining the ease of formation and stability of a multitude of colloidal systems, such as emulsions and foams.1 For this reason many methods have been developed for monitoring the change in interfacial tension on expansion or contraction of an interface.2 In addition much effort has been expended in trying to interpret the resultant interfacial tension kinetics in terms of adsorption/desorption, aggregation/disaggregation, and intramolecular rearrangement of surfactant molecules.3 In the classic Langmuir trough, π-A isotherm experiments obtained for insoluble films provide information on the molecular interactions * Author for correspondence: tel, 44 (0)113 2332962; fax, 44 (0)113 2332982; e-mail,
[email protected]. X Abstract published in Advance ACS Abstracts, November 15, 1996. (1) Murray, B. S.; Dickinson, E. Food Sci. Technol. Int. 1996, in press. (2) Prins, A. In New Physico-Chemical Techniques for the Characterization of Complex Food Systems; Dickinson, E., Ed.; Chapman and Hall: Cambridge, 1995; p 214. Prins, A.; Berginke-Martens, D. J. M. In Food Colloids and Polymers: Stability and Mechanical Properties; Dickinson, E., Walstra, P., Eds.; Royal Society of Chemistry: Cambridge, 1993; p 291. (3) Miller, R.; Kretzschmar, G. Adv. Colloid Interface Sci. 1991, 37, 97.
S0743-7463(96)00748-2 CCC: $12.00
within such films under equilibrium conditions, which may be useful in interpreting the corresponding dynamic behavior of both soluble and insoluble surfactants. It is true to say, however, that up to now most experimental methods and data analysis have concentrated on the A-W interface, neglecting the O-W interface.1 One of the principal reasons for this seems to be the experimental difficulties associated with compressing and containing an O-W film using the conventional, rectangular-type trough arrangement.4 The new apparatus will first be described, then some π-A isotherms on proteins and some dynamic measurements on surfactants at an O-W and A-W interface will be presented. The results and technique will then be compared with previous work. The Novel Langmuir Trough: Description and Operation Figure 1 illustrates the essential features of the apparatus. A solid PTFE frame, or barrier, sits in a rectangular PTFE trough of dimensions 25 × 35 × 4 cm. The barrier has four, rigid solid sides 15 cm long and 4 cm high. The four sides are hinged at the corners so that (4) Brookes, J. H.; Pethica, B. A. Trans. Faraday Soc. 1964, 60, 208.
© 1996 American Chemical Society
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Figure 2. Relative area Ar versus drive movement, starting from maximum trough area.
of the oil layer is maintained constant by a siphon system. As a film is compressed, for example, the surface of the oil is continually sucked off via a peristaltic pump, to maintain a constant level. The film may be compressed at constant slow speed (for π-A isotherms), or be subjected to a sudden rapid expansion and the resultant π(t) behavior monitored. Also, the film may subjected to constant logarithmic expansion6 or sinusoidal expansion and contraction for measurement of dynamic dilatational moduli.7 Figure 1. Schematic illustration of the apparatus: view from side (a) and above (b). Key: a, trough walls; b, lower (aqueous) phase; c, barrier; d, upper (oil) phase; e, hydrophobic Wilhelmy plate; f, siphon; g, direction of stepper motor drive for compression.
they provide a continuous, leak-free enclosure. The walls have PTFE pegs underneath so that liquid (generally the aqueous phase) can flow under the walls to create a welldefined interfacial area inside the barrier. Two opposite corners of the barrier are connected to a geared down Powermax P22 stepper motor (Unimatic Engineers, London, U.K.). The stepper motor is driven via a PC interfaced to a Digiplan PDX13 stepper motor drive (Unimatic Engineers, London, U.K.). The drive movement is accurate to 0.01 mm. On driving the two corners together or apart the interfacial area inside the barrier is changed. The interfacial tension/pressure is measured via a Wilhelmy plate dipping into the interface, at the center of the film, suspended from a force transducer (Maywood Instruments, Basingstoke, U.K.). The maximum compression ratio is approximately 1:20, the limiting factor being avoidance of contact of the Wilhelmy plate with the barrier. The rhomboidal shape change on driving together the two opposite corners of the trough produces the change in area relative to the maximum area, Ar, illustrated in Figure 2. The experimental arrangement has recently been patented5 and further details are available from the author on request. For measurements at an O-W interface a 3-4 mm layer of oil is gently layered over the top of the aqueous phase. A hydrophobic plate, made from carbon black coated mica, was completely submerged beneath the surface of the oil layer and suspended at the O-W interface by two thin stainless steel wires. The plate made zero contact angle with the oil phase throughout the experiment. (Hydrophilic mica plates were used for A-W measurements.) On compression/expansion the oil and O-W interface are then completely contained within the barrier. The height (5) UK Patent Application 96 11936.7.
Materials and Methods Imidazole, potassium dihydrogen phosphate, disodium hydrogen phosphate, sodium chloride, and hydrochloric acid were all AnalR grade reagents from BDH Merck. Bovine serum albumin (prod. code A-7638, lot no. 14H9348), β-lactoglobulin (prod. code L-0131, lot no. 91H7005), n-heptane (99+%), and n-tetradecane (99%) were from Sigma Chemicals. Brij58 was from ICI Surfactants. All water used was from a Millipore alpha-Q purification system with a surface tension of 72.0 mN m-1 at 25 °C. Before protein was spread, the A-W interface was reduced rapidly to an Ar lower than that used in the subsequent π-A experiments. The interface was sucked clean with a vacuum line, the interface expanded, and the process repeated until π < 0.1 was obtained on compression. For the A-W interface spreading was then performed (see below). For the O-W interface the oil phase was added at this stage and the protein spread immediately afterward. The concentration of protein in spreading solutions (made up in water) was typically 0.2 mg mL-1. For spreading, a drop of spreading solution was slowly formed on the tip of the syringe and then the drop slowly lowered to touch the interface, the syringe tip raised, and the process repeated until all the solution had been spread. Spreading took 5-10 min and measurement of the π-A isotherm was begun 10 min after spreading. The protein was spread at high film area, generally at A > 25 000 Å2 per molecule. The relative molecular mass of BSA was taken as 66 500 Da8 and that of β-lactoglobulin (dimeric form) taken as 36 700 Da9 for calculation of the π-A isotherms. Films were compressed at constant speed in the linear region of Ar (i.e., starting at Ar ) 0.7, see Figure 2) so that dAr/dt ≈ 1.5 × 10-4 s-1. Below this compression rate no difference in (6) van Aken, G. A. In Food Macromolecules and Colloids; Dickinson, E., Lorient, D., Eds.; Royal Society of Chemistry: Cambridge, 1995; p 43. Bergink-Martens, D. J. M.; Bos, H. J.; Prins, A. and Schulte, B. C. J. Colloid Interface Sci. 1990, 138, 1. Voorst Vader, F.; van Erkens, Th.F.; van den Tempel, M. Trans. Faraday Soc. 1964, 60, 1170. (7) Lucassen, J.; van den Tempel, M. Chem. Eng. Sci. 1972, 27, 1283. Ting, L.; Wasan, D. T.; Miyano, K. J. Colloid Interface Sci. 1985, 107, 345. (8) van Holde, K. E. Physical Biochemistry; Prentice-Hall: Englewood Cliffs, NJ, 1971. (9) Fox, P. F., Ed. Developments in Dairy Chemistrys1 Proteins; Applied Science: London, 1982.
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Figure 3. Interfacial pressure (π) versus area per molecule (A) for BSA (circles) and β-L (triangles). Open symbols are for O-W films; filled symbols for A-W films. Dashed line is isotherm for A-W BSA film due MacRitchie.10 the π-A isotherms was observed; this was therefore considered to be slow enough that the π-A isotherm obtained represented the “true” equilibrium isotherm. Without further purification the n-tetradecane contained low levels of weakly surface active impurities, but these were completely dominated by spread protein and had negligible effect on the results obtained.
Results and Discussion Tests on spread films of long chain alcohols such as 1-octadecanol produced π-A isotherms in perfect agreement with the standard results for these films. These are not shown but it is apparent that the trough may be used as any other conventional Langmuir trough at the A-W interface. Results for Spread Protein Films. Figure 3 shows results for spread films of BSA and β-L at the A-W interface and the O-W interface. The oil phase was n-tetradecane. For BSA the aqueous phase was pH 7.4, 0.3 M NaCl, for comparison with the result of MacRitchie,10 obtained under the same conditions; for β-L the aqueous phase was 0.02 mol dm-3 imidazole buffer, adjusted to pH 7.0 with HCl. Measurements were made at 25 ( 0.3 °C. The results shown in Figure 3 are representative of those found by spreading 100-200 µL of protein solution; i.e., isotherms were independent of the amount of solution spread, indicative of reproducible spreading. The A-W isotherm for BSA is in good agreement with the result of MacRitchie,10 particularly at low π. At high π, the corresponding A is slightly lower. Isotherms for protein films are often displaced to higher A when they are compressed too quickly; in this work the films were compressed very slowly and the π-A curves were completely reproducible in that on re-expansion followed by immediate compression the same π-A curve was obtained. It was observed, however, that if a protein A-W film was left for 12 h or more and then re-compressed the π-A curve appeared to be slightly more expanded (e.g., A was increased by ca. 10% at π ) 15 mN m-1) on the first compression. But if this was immediately followed by a second and subsequent π-A experiments the original π-A curve (shown in Figure 3) was recovered. It may be that the protein film at the A-W interface is in a more aggregated state and continues to unfold very slowly after spreading. Depending on exactly how long the film is left before compression, a slightly more expanded π-A (10) MacRitchie, F. Adv. Colloid Interface Sci. 1986, 25, 341.
isotherm may be obtained, which may also explain the slight discrepancy with the result of MacRichie.10 The results in Figure 3 show that the π-A isotherms for both BSA and β-L are considerably more expanded at the O-W interface compared to the A-W interface, typically by a factor of about 1.7 in the low π (e.g., π < 2 mN m-1) region. For π > 7 mN m-1, however, β-L is apparently more compressible at the O-W interface, while for BSA the film is considerably more expanded up to the highest value of π measured. Interestingly, no aging effects were observed for the protein O-W isothermss leaving the films for 12 h or more did not result in an initially more expanded film on subsequent compression. The difference in behavior of the proteins at the two types of interface is significantsvery often similarity has been assumed due to lack of data11sbut the difference is perhaps not so unexpected. One can assume that the oil phase will be a better solvent than “air” for the more hydrophobic side chains of the amino acid residues in the protein polypeptide chain. Consequently, more unfolding of the polypeptide chain might be expected, resulting in a higher effective area per molecule. Other work is in preparation12 which also confirms this general picture for A-W and O-W protein films. Results for Adsorbed Surfactant Films. One of the design features of the new trough is the facility for rapid expansion/compression of adsorbed or spread films. To demonstrate the technique a low molecular weight, nonionic surfactant has been studied, well below the cmc: 10-8 mol cm-3 Brij 58. This is actually a mixture of surfactants but it has been studied many times before using a wide variety of techniques, most noticeably by Joos and co-workers.13,14 In particular, there are reliable data available at an O-W interface14swhich is not the case for most other surfactants. Figure 4 shows the behavior of 10-8 mol cm-3 Brij 58 solution at the A-W interface and at the n-heptanewater interface, temperature 21 ( 0.3 °C. After addition of the bulk phase(s) to the trough, π was monitored with time until a constant equilibrium value, πeq, was obtained. Then the interface was expanded to 1.1 times its initial area and the resulting change in π followed. In Figure 4 the kinetics of π are expressed in terms of the reduced variable, R, for comparison with the measurements of van Hunsel and Joos.14 Here
R ) (πeq - π)/(πeq - π0)
(1)
where π0 is the initial value of π after expansion of the interface. Α 10% change in area can be achieved in under 50 ms, but to aid comparison with other work the expansion speed was arranged so that the film was expanded by ∆A ) 0.1 in 1.00 s. R(t) may be analyzed in terms of a diffusional relaxation time, τD. For a diffusioncontrolled adsorption process the relaxation back toward equilibrium is given by14
R ) exp(t/τD) erfc(t/πD)1/2
(2)
so that the value of τD serves to characterize the kinetics of R(t) completely. The experimental results shown in Figure 4 have τD values of 21.8 and 10.0 s for the A-W (11) Graham, D. E.; Phillips, M. C. J. Colloid Interface Sci. 1980, 76, 227. Lucassen-Reynders, E. H. Food Struct. 1993, 12, 1. Clark, D. C.; Mackie, A. R.; Wilde, P. J.; Wilson, D. R. Faraday Discuss. 1994, 98, 253. (12) Murray, B. S.; Ventura, A.; Lallement. In preparation. (13) van Hunsel, J.; Joos, P. Colloid Polym. Sci. 1989, 267, 1026. Buqiang, L.; Joos, P.; Van Uffelen, M. J. Colloid Interface Sci. 1995, 171, 270. Horozov, T.; Joos, P. J. Colloid Interface Sci. 1995, 173, 334. (14) van Hunsel, J.; Joos, P. Colloids Surf. 1987, 25, 251.
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type Langmuir troughs and all drop/bubble methods. The effect is also expected to be greater for higher and more rapid changes in Ar. (Indeed, at the microscopic level, a combination of shear and dilatational stresses probably occurs for any step change in area, particularly in the vicinity of the boundaries containing the film.) Of the trough methods, only the elastic band barrier apparatus due to Benjamins et al.15 maintains constant shape on expansion/compression. The shearing imposed has the effect that after an imposed expansion, for example, relaxation of the film to the equilibrium surface pressure will include the shearing of film elements past one another. The behavior of π(t) may then also be influenced by the shear rheology of the film. With this particular method very small changes in Ar may be applied (providing the corresponding ∆π is large enough to measure), minimizing the effect. Several studies16 have illustrated the presence of gradients of π throughout polymeric films at high π, where the films often possess high film viscosity. This apparatus follows the convention of compressing the film symmetrically and measuring the π at the center of the film. This makes the measurements comparable with other measurements, and they are very consistent. Figure 4. Kinetics of reduced interfacial pressure change, R, for 10-8 mol cm-3 Brij 58 at (a) A-W interface and (b) n-heptane-water interface. Dashed lines are results due to van Hunsel and Joos.14
and O-W interfaces, respectively. These compare very favorably with the corresponding values of 22 and 7.5 s obtained by van Hunsel and Joos using different techniques.13,14 The above results suggest good agreement with other techniques, but certain specific features of the new method should be addressed. Because there is a change in shape on change in area, this means that films are also subjected to shear as well as dilatational stresses. The criticism may be leveled at all methods involving a shape change on change in area, including conventional rectangular-
Conclusion The new trough has been demonstrated as being capable of performing π-A measurements on spread films and dilatational measurements on adsorbed films, at both the A-W and O-W interface, which are in agreement with results from other techniques. For protein films, significant differences exist between films spread at the O-W and A-W interfaces, films being considerably more expanded at the O-W interface. LA960748O (15) Benjamins, J.; de Feijter, J. A.; Evans, M. T. A.; Graham, D. E.; Phillips, M. C. Faraday Discuss. 1976, 59, 218. (16) Malcolm, B. R. J. Colloid Interface Sci. 1985, 104, 520. Peng, J. B.; Barnes, G. T.; Abraham, B. M. Langmuir 1993, 9, 3574. Byattsmith, J. G.; Malcolm, B. R. J. Chem. Soc., Faraday Trans. 1994, 90, 493.
