Langmuir 1993, 9, 1534-1544
1534
Molecular Organization via Ionic Interactions at Interfaces. 1. Monolayers and LB Films of Cyclic Bisbipyridinium Tetracations and Dimyristoylphosphatidic Acid Ramesh C. Ahuja,' Pier-Lorenzo Caruso, and Dietmar Mobius Max-Planck-Institut fiir biophysikalische Chemie, Am Fassberg, Postfach 2841,D-3400 Gottingen, FRG
Gerald Wildburg and Helmut Ringsdorf Institut fur Organische Chemie, Universitat Mainz, J.-J.Becher- Weg 18-20, 0-6500 Mainz, FRG
Douglas Philp, Jon A. Preece, and J. Fraser Stoddart School of Chemistry, The University of Birmingham, Edgbaston, Birmingham B15 2TT,U.K. Received December 30, 1992. In Final Form: March 10, 1993 Organization of nonamphiphilic tetracation cyclobis(paraquat-p-phenylene) ,BBP4+,at the lipid/water interface using cospreading and adsorption techniques has been investigated. Dimyristoylphosphatidic acid (DMPA) was used as the anchor lipid. The cospreading technique involves the spreading of a mixed solution of the lipid and BBP4+ while the adsorption technique utilizes diffusion-controlled adsorption of BBP4+at the lipid/water interface. Surfacepressure (A)-, surface potential (AV-,and surface reflection (AR)-area isotherms along with UV spectroscopy techniques were used for characterization of the organization parameters. The r - A isotherms of the cospread DMPA/BBPQ+monolayers show expansion at low A and converge toward the DMPA isotherms at high A. The cospread monolayer isotherms show maximum expansion at a 1:l DMPABBP4+molar ratio. This type of behavior has usually been interpreted in terms of penetration (at low A ) into and squeezingout (at high A ) of the adsorbate from the hydrophobic part of the lipid monolayer. On the basis of conclusive evidence from AV and AR measurements, we propose a model which explainsthe expansion and convergence of isotherms without invoking penetration of adsorbate into the lipid monolayer. This model also anticipates the 1ipid:adsorbate ratio at which maximum expansion of isotherms and the formation of a compact Langmuir monolayer of adsorbate is expected and observed. The cospread monolayers were found to be stable (no loss of BBPQ+)with time and could be transferred to quartz substrates using the vertical dipping method. The relative merits of the cospreading and adsorption techniques are discussed. Adsorption kinetics of BBP4+at the DMPA/ water interface are rationalized in terms of the Nernst diffusion layer model. The diffusion coefficient for BBP4+was calculated to be 3.75 X 10-6 cm2/s,giving a Nernst layer thickness of 0.033 cm. 1. Introduction
Molecular recognition processes are central to both the formation and the functioning of supramolecular assemblies.'" The spatial organization and intermolecular forces between the components of the assembly determine the selectivity of the self-assembly process and the stability of the resulting structure. Whereas molecular recognition in synthetic systems610 occurs almost exclusively in a homogenous phase, it is basically an interfacial phenomenon in biological systems.11 Hence, synthetic supramolecular systems capable of forming highly organized twodimensional arrays at interfaces are of interest not only for applications in molecular devices but also for investigations of physicochemical processes occurring a t the monolayer/subphase interface as models for the understanding and elucidation of biomembrane systems. The properties and functions of these ordered supramolecular (1) Whitesides, G. M. Angew. Chem., Znt. Ed. Engl. 1990,29, 1209. ( 2 ) Lehn, J. M. Angew. Chem., Znt. Ed. Engl. 1990,29, 1304. (3) Seebach, D. Angew. Chem., Znt. Ed. Engl. 1990,29, 1320. (4) Philp, D.; Stoddart, J. F. Synlett 1991, 445. (5) Lehn, J. M. Angew. Chem., Znt. Ed. Engl. 1988,27,89. (6) Cram, D. J. Angew. Chem., Znt. Ed. Engl. 1988,27, 1009. (7) Stoddart, J. F. Annu. Rep. h o g . Chem. 1988,80, 353. (8) Diederich, F. Angew. Chem., Znt. Ed. Engl. 1988,27,362. (9) Schneider, H. J. Angew. Chem. 1991,30, 1417. (IO) Cram, D. J. Nature 1992,356, 29. (11) Lindsey, J. S. New J. Chem. 1991, 15, 153.
assemblies are governed not only by the chemical nature of their components but also by the organizational parameters such as surface density, relative orientation of various components, and aggregation at the molecular and supramolecular levels. In order to organize nonamphiphilic functional ligands (adsorbate) a t the aidwater interface, electrostatic (dipole/ dipole, ion/ion, ion/dipole) ,multiple hydrogen bonding,'2 or charge-transfer (CT) interactions could be used. The interplay and combination of several of these forces offer the possibility to utilize even very weak attractive forces (e.g., CT interactions) for achieving specific binding without sacrificingthe overall stability of the self-assembly. Thus, the use of a combination of available forces within a system allows the system to adapt in such a way that, in the first step, a relatively nonspecific, namely, ionic, interaction is used for the enrichment and defined organization of a ligand (ad+Jrbate) at the interface. In the second step, a weaker, more specific interaction, which does not disrupt significantly the well-defined organizational setup by the first strong nonspecific interaction, contributes significantly to the final recognition process. In the present paper, we report, as the first step of such a process, the results of our investigations on the organization of a tetracationic cyclophane, namely, cyclo(12) Etter, M. C. Acc. Chem. Res. 1990,23, 120.
0743-746319312409-1534$04.00/0 0 1993 American Chemical Society
Ionic Interactions at Interfaces
Langmuir, Vol. 9, No. 6, 1993 1535
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phiphilic anchor and nonamphiphilic ligand is spread at the air/water interface and the organizational parameters are controlled through appropriate choices of surface pressure, temperature, and nature and composition of the subphase. No additional adsorbate molecules are thus present in the subphase. The techniques used for the characterization and investigation of the organized monolayers of DMPA and BBP4+are surface pressure- and surface potential-area isotherms along with UV reflection spectroscopy at the aidwater interface. 2. Experimental Section
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Figure 1. Chemical structures of amphiphile dimyristoylphosphatidic acid (DMPA) and tetracationic cyclobis(paraquat-pphenylene) (BBP4+)along with the CPK model of BBP4+and the corresponding molecular areas.
bis(paraquat-p-phenylene),lS15 BBP4+,at the negatively charged amphiphilic anchor lipid (dimyristoylphosphatidic acid, DMPA) monolayers. We have chosen this cyclophane because it has a cavity (ca. 0.3 nm2) in the center which has been shown to be a versatile host for a-electron-rich aromatics exhibiting charge-transfer (CT) interactions, with the guest molecules fitting into the tight space of the cavity.13J6 The structures of the two components to be discussed, namely, the BBP4+and the DMPA, are given in Figure 1. Both the cospreading and the adsorption techniques have been applied to prepare the complex monolayers at the air/water interface. The use of electrostatic interactions for organizing water-soluble adsorbates to an amphiphilic anchor monolayer has already been demonstrated in the case of charged cyanine dyes,17 porphyrins,la19 phthalocyanines, and tetracyanoquinodimethane.20 The disadvantages of the adsorption technique are that the nonamphiphilic ligand (adsorbate)has to be water soluble, fairly high concentrations of adsorbates in the subphase are required, and the adsorption time is quite long. In addition, adsorbate multilayer formation as a result of adsorbate/adsorbate interactions is possible. Therefore, the transferred monolayers have uncontrolled composition. These problems may be overcome by employing the socalled cospreading technique.21p22 In this technique, a mixed solution with a well-defined molar ratio of am(13)Odell, B.; Reddington, M. V.; Slawin,A. M. Z.; Spencer, N.; Stoddart, J. F.; Williams, D. J. Angew. Chem., Int. Ed. Engl. 1988,27, 1547. (14)Ashton, P. R.; Odell, B.; Reddington, M. V.; Slawin, A. M. Z.; Stoddart, J. F.; Williams, D. J. Angew. Chem., Int. Ed. Engl. 1988,27, 1550. (15)Reddington, M. V.; Slawin, A. M. Z.; Spencer,N.; Stoddart, J. F.; Vicent, C.; Williams, D. J. J. Chem. SOC.1991,630. (16)Ashton, P.R.; Goodnow, T.; Kaifer, A. E.; Reddington, M. V.; Slawin, A. M. Z.; Spencer, N.; Stoddart, J. F.; Vicent, C.; Williams, D. J. Angew. Chem., Int. Ed. Engl. 1989,28,1396. (17)Kirstein, S.;Miihwald,H.; Shimomura,M. Chem.Phys.Lett. 1989, 164,303. (18)Griiniger, H.; Miibius, D.; Meyer, H. J. Chem. Phys. 1983, 79, 3701. (19)M6bius, D.;Griiniger, H. R. Bioelectrochem. Bioenerg. 1984,12, 375-392. (20)Barraud, A.; Lesieur, P.; Richard, J.; Ruaudel-Teiaier, A.; Vandevyver, M. Thin Solid Film 1985,133,125. (21)Yonezawa,Y.;Miibius, D.;Kuhn, H. Ber. Bunsen-Ges.Phys. Chem. 1986,90,1183. (22)Cordroch, W.; Miibius, D. Thin Solid F i l m 1992,210/211,135137.
The chemical structure and Corey-Pauling-Koltun (CPK) model of cyclobis(paraquat-p-phenylene), a cyclic bisbipyridinium tetracation with (PF6-)4 as the counterion,hereafter referred to as BBP4+, are shown in Figure 1. The synthesis of the macrocycle has been described by the Stoddart group.l3 The anchor phospholipids dipalmitoylphosphatidicacid (DPPA)and dimyristoylphosphatidic acid (DMPA) were used as received from Sigma. Chloroform (HPLC),acetonitrile, methanol, and mixtures thereof were used as spreading solventsand were obtained from Baker Chemicals. The water used for the subphase was obtained from a Milli-Q filtration unit of Millipore Corp. Surface pressure (T)- and surface potential (Am-area isotherms were measured on a Fromherz-type round trough enclosed in an insulating cabinet which was flushed with N2 during the measurements. The temperature of the subphase was kept constant at 21 "C. A Wilhelmy balance (15-mm-widefilter paper) was used to measure the surface pressure, and the surface potential was measured using the vibrating condenser method as described in ref 23. The isotherms were measured in discontinuous mode; i.e., the barrier was moved in either 2 cm2 or 1 mN/m steps, whichever occurred earlier. Then after a relaxation of 5 s, the ?r and AV values were recorded. UV-vis reflection (surface-enhanced) spectroscopy at the aidwater interface was done under normal incidence of light using the apparatus described in ref 18. The reflection values given here are the differences in the reflectivity of the monolayer-covered water surface and of the bare water surface.
3. Results and Discussion 3.1 Characterization of the Amphiphilic Anchor Monolayer. The chemical structure of the amphiphilic molecular anchor DMPA is shown in Figure 1. The phosphate head group of DMPA has two dissociable protons with pK values of 3 (singly ionized) and 8 (doubly ionized).% Thus, a monolayer of DMPA at an electrolytic subphase will be charged depending on the pH and the nature and concentration of ions in the subphase. The surface pressure (a)-and surface potential (Am-area (A) isotherms of DMPA at the air/water interface (pH 5.6) are shown in Figure 2. Additionally, the area-normalized surface potential (AVN= AV*A) as a function of the area per molecule is shown by the dashed curve. It is seen that AVN exhibits negative values till the molecular area reaches 0.82 nm2. A further decrease in the area per molecule leads to a sharp increase in AVN. The surface potential shows large fluctuations for A > 0.9 nm2. These fluctuations arise due to the coexistence of large domains of high and low lipid density in the liquid expanded (LE) phase. These erratic fluctuations disappear on increasing the pressure or with the establishment of the fluid phase. The negative values of AVNat larger molecular areas show the dominance of negative head group charge on the AVN values. The sharp increase in AVN at a values close to (23)Kuhn,H.; Miibius, D.; Biicher,H. Physical Methods of Chemistry; John Wiley & Sons: New York, 1972;Vol. 1, Part 3B,p 656. (24)Triiuble, H.;Teubner,M.; Wooley, P.; Eibl, H.-J. Biophys. Chem. 1976,4,319.
Ahuja et al.
1536 Langmuir, Vol. 9, No. 6, 1993 "I
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Figure 3. Surface potential contributions of different parts of the amphiphilicanchor DMPA as estimatedthrough the analysis of data in section 3.1.
zero and below 1mN/m has also been observed in many other lipid monolayer systems and is most probably due to the monolayer front, which is formed near the moving barrier, coming under the surface potential detector. However, in the LC phase or molecular area below A = 0.8 nm2 (1mN/m) and till A = 0.66 nm2 (3 mN/m), the surface pressure and AVNvalues increase linearly. For the values of A between 0.66 nm2 (3 mN/m) and 0.52 nm2 (5mN/m) in the fluid/solid coexistence region, the value of AVN remains constant. A further decrease of the area to 0.43 nm2 (15 mN/m) leads again to a linear increase in AVN. The final step of compression till A = 0.4 nm2 (45 mN/m) does not lead to any further change in AVN. The surface potential of an amphiphilic lipid monolayer at the air/water interface may be described2&%as a linear combination of three different terms: AV=AVo+AVp+\kO (1) The AVOterm is the measured value of the surface potential for the clean aidwater interface and is usually in the range of -300 to -500 mV. This term arises because of the asymmetry of the aidwater interface which leads to the orientation (with oxygen pointing toward air) of water molecules.29 The second term, AVp, also called the dipole term, is due to the permanent dipole moment of the lipid molecule. This may further be subdivided into the head (25) Demchak, R. J.; Fort,Jr., T. J. J. Colloid. Interface Sei. 1974,46, 191.
(26) Taylor, D. M.; Oliveira Jr., 0. N.; Morgan, H. Chem. Phys. Lett. 1989.161. 147. (27) Taylor, D. M.; Oliveira Jr., 0.N.; Morgan, H. J. ColloidInterface Sci. 1990, 139, 608. (28) Oliveira Jr., 0. N.; Taylor, D. M.; Morgan,H. Thin Solid F'ilms 1992,210/211, 76. (29) Wilson, M. A.; Pohorille, A,; Pratt, L. R. J. Chem. Phys. 1988,88, 3281. ----I
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group and the tailregion dipole momenta. The third term, 90,also called the double layer potential, is due to charges in the lipid. It should be mentioned here that the vibrating condenser technique employed in the present work measures only the vertical component (to the aidwater interface) of the corresponding dipole momenta. The values of AV reported here are always with respect to the clean subphase interface; Le., AV = AVm - AVO,where AVm is the measured value of the surface potential for the air/monolayer/water interface. Now we will discuss the other two terms for the specific case of DMPA. The dipolar term may be expressed2"%Pm as
where A is the mean area per lipid molecule, I.LHthe dipole moment of the polar head group region, pw the contribution of oriented water molecules which constitute the hydration shell of the lipid head groups at the air/water interface, and I.LTthe dipole moment of the hydrophobic part, and for aliphatic chains it is only the terminal -CH3 group which contributes to this term. CH,ET, and cware the local dielectric constants for the head group, tail, and interfacial water regions, respectively. The vertical component of a C--H+ dipole directed away from the water surface at half the tetrahedral angle is 330 mD, and the local dielectric constant may be taken as 2.8. The surface potential corresponding to the -CH3 group can be calculated from the relation AV = I . L T / A Cthe O ~ value , of A per methyl group (hydrocarbon chain) being equal to 0.2 nm2. Thus, a value of +222 mV is obtained. As DMPA has two aliphatic chains with terminal -CH3 groups, the contribution of the -CH3 groups alone to the surface potential is +444 mV. Now we turn our attention to the head group region. Comparing the AV data of various phospholipids, it is observed that a significant contribution to the surface potential comes from the ester groups. The dipole moment of an ester group is 1.8 D;31 thus, a permanent dipole moment of 1.8(cos 01 + cos 02)D per DMPA is expected, where 01 and 02 are the angles subtended by the two ester groups to the interface normal. The values of 01,02, and the local permittivity are however not known; thus, it is rather meaningless to calculate the dipole term without making speculative assumptions. However, a reasonable estimate of the contribution of the totalhead group region to the surface potential may be made from the following analysis of the experimental data. From the AV-A isotherm of DMPA at pH 0.5 (data not shown), it is seen that the AV value (at 0.4 nm2/molecule) is +530 mV compared to that of +230 mV (cf. Figure 2) at the pure water (pH 5.6) interface. It may be reasonably assumed that, at pH 0.5, the head group of DMPA is totally protonated. It is possible that at pH 0.5 the DMPA monolayer is hydrolyzed to some extent. Taylor et aL2' have reported a surface potential value of +440 mV for the DPPA monolayer at pH 2. These researchers have indicated that monolayers of DPPA at pH 2 may still be weakly ionized which is in agreement with our measurementa as the surface potential value at pH 0.5 is 90 mV higher than that reported by Taylor et al.27at pH 2. Thus, the value of +530 mV at pH 0.5 may be considered as the DMPA dipolar contribution (AVp) at 0.4 nm2 to the surface potential. Subtracting the contribution of two methyl groups (+444mV) from the experimentally measured +530 (30) Vogel, V.; Mbbius, D. J. Colloid Interface Sci. 1988,126,408-420. (31) Seelig, J.; McDonald, P. M.; Scherer,P. G.Biochemistry 1987,26, 7536.
Ionic Interactions at Interfaces
Langmuir, Vol. 9, No. 6,1993 1537
mV, a value of +86 mV for the head group region (including the two ester groups, the phosphate head group, and the hydration shell) is obtained. The tail region contribution to the surface potential is thus roughly 6 times that of the head group region. The surface potential contributions of different parts of DMPA are summarized in Figure 3. Assuming that the dipolar term does not change with pH, the value of 90at pH 5.6 comes out (by subtracting the dipolar term from the AVvalue) to be -300 mV. Taylor et al.27 have given an estimated value of -200 mV for DPPA monolayers. But this estimate depends on the surface potential measurements done at pH 2 where the monolayer may still be weakly ionized. In addition, surface potential measurements of the DMPA monolayer at the NaCl subphase (data not shown) show that AV a t 0.4 nm2 increases by ca. 50 mV, as the NaCl concentration is increased by one decade. The AVvalue for the 1M NaCl subphase has been found to be +535 mV, confirming the estimated value for 90to be ca. -300 mV. It is interesting to know if this estimated value of 90fits in the framework of the Gouy-Chapman theory32according to which (3) where k is Boltzmann's constant, T the absolute temperature, u the surface charge density equal to ea/A with e as the electronic charge, a the degree of dissociation of the head group, A the area per molecule, c the ionic concentration of the subphase (MI, and e the relative permittivity of the subphase. Assuming t = 80, and c = 3.16 pM (proton concentration at pH 5-61,a value of a = 0.1 is obtained for 90= -300 mV, implying that only 10% of the phosphate groups are negatively charged in the densely packed DMPA monolayer at the pH 5.6 subphase. This is in contrast to the value of a = 0.5 obtained by Miller et al. for the DMPA monolayer at the 5.5 subphase containing 1mM NaC1.33 Having analyzed the surface potential data in terms of electrostatic and dipolar components, it willbe interesting to investigate what component, namely, dipolar or ion/ ion, contributes to the binding of BBP4+at the monolayer/ water interface and to what extent it does so. 3.2. DMPA/BBP*+Cospread Monolayers. 3.2.1. Surface Pressure-Area Isotherms. The interaction of tetracationic cyclophane BBP4+ with DMPA was followed by investigating the changes in PA and AV-A isotherms. A mixed solution of DMPA (solvent, CHCld CHsOH (3:l)) and BBP4+(solvent, CH3CN) in the desired molar ratio of the two components was spread a t the air/ water interface, and the isotherms were measured as described in the Experimental Section. No additional BBP4+or any other ions were present in the subphase. The influence on P-A isotherms as a result of binding of BBP4+to DMPA, at various ratios of the two components, is shown in Figure 4. It is seen tht the T-A isotherms of BBP4+/DMPAmonolayers deviate quite strongly from the reference DMPA isotherm (cf. Figure 4, curve e). The isotherms show expansion (cf. Figure 4, curves a-c) as well as contraction (cf. Figure 4, curve d) behavior depending on the relative molar ratio of DMPA and BBP4+. The changes in the isotherms show that the tetracationic BBP4+ molecules are trapped a t the interface by interaction with the negatively charged head group of DMPA. In order to get a quantitative estimate of isotherm changes as a result of DMPA/BBP4+interactions, the dataof Figure 4 and other data not shown have been replotted in Figure ~~
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(32) McLaughlin, S. Curr. Top. Membr. Tramp. 1977,9,71. (33) Miller, A.; Helm, C. A.; M(lhwald, H. J. Phys. 1987,48,693-701.
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Figure 5. PA (area per molecule for a DMPA/BBP4+monolayer minus the area per molecule for DMPA; data taken from Figure 4 isotherms) as a function of the DMPABBP4+molar ratio at various surface pressures as indicated on the curves.
5. In this figure, we show the dependence of AA (obtained by subtracting the area per molecule of DMPA on pure water from the area per molecule in the corresponding DMPA/BBP4+ isotherm a t the same value of a) on the DMPABBP4+ molar ratio a t various values of P. The isotherms in Figure 4 and the data in Figure 5 reveal two major features. The first major feature of all the isotherms is that the area per molecule deviation (AA) from the reference isotherm is most pronounced a t low T. As the surface pressure is increased, the deviation from the reference isotherm decreases and finally the isotherms tend to converge toward the area per DMPA molecule (cf. Figure 5, T = 40 mN/m) without BBP4+. Thus, the presence of BBP4+ contributes only little to the area per molecule at 40 mN/m; the additional maximum area due to DMPA/
Ahuja et al.
1538 Langmuir, Vol. 9, No. 6,1993
BBP4+interaction is only 0.04 nm2per DMPA (cf. Figure 5,40 mN/m curve at molar ratio 1:l). This represents an expansion by ca. 10% at 40 mN/m compared to that by 60% at 3 mN/m (Figure 5, curve 3 mN/m, molar ratio 1:l) for the isotherm corresponding to the monolayer with a DMPA:BBP4+molar ratio of 1:l. This type of behavior, i.e., expansion at low a and convergence a t high a to the reference lipid area in isotherms, has been reported in many adsorption s t ~ d i e s ~and ~ l has ~ - ~usually ~ been interpreted in terms of penetration of adsorbate molecules into the lipid monolayer (leading to an expansion of the isotherm) at low a and squeezing out of the adsorbate moleculesfrom the lipid monolayer (leadingto convergence of the isotherms) a t high a. However, this explanation has usually been advanced purely on the basis of a-A isotherms, and no additional supporting evidence for the penetration and squeezing out of the adsorbate molecules from the lipid monolayer has been given. The second major feature of the isotherms is the dependence of the expansion of isotherms on the DMPA BBP4+molar ratio. It is clear (cf. Figures 4 and 5) that the maximum expansion, Le., the maximum value of AA, is shown by the monolayer with a DMPABBP4+ molar ratio of 1:1, This is true for all values of the surface pressure. As the DMPA:BBP4+molar ratio is increased, the value of AA and thereby the expansion decrease. For monolayers with molar ratios larger than 4:1, finally a contraction (negative values of AA; cf. Figure 5, curves for 1, 2, and 3 mN/m) of the monolayer with respect to the reference (without BBP4+)DMPA isotherm is observed. 3.2.2. LipidIAdsorbateInteraction Model. The two major features of the isotherms, namely, the expansion (at low a)and convergence (at high T )of isotherms together with both expansion and contraction of the isotherms depending on the DMPA:BBP4+molar ratio, cannot be accounted for in terms of the penetration and squeeze out model as discussed in the literature for other systems. Therefore, a qualitative model for lipid/adsorbate interaction (strongly supported by analysis of data obtained from other measurements such as AV-A and AR-A isotherms as discussed below) is proposed here. The CPK model of BBP4+(cf. Figure 1) and other data obtained by show that the molecular area of the Stoddart BBP4+should be ca. 1.7 nm2, The fact that the maximum expansion due to DMPA/BBP4+interaction (molar ratio 1:l; Figure 5, curve at 40 mN/m) a t 40 mN/m is ca. 10% leads to the conclusion that the BBP4+molecules could not be present in the lipid monolayer and rather should be located underneath the head groups of the densely packed DMPA monolayer a t high a . Such a structure with a densely packed DMPA monolayer supporting a densely packed BBP4+ monolayer underneath would require roughly 4.1 DMPA molecules (are per DMPA molecule at 40 mN/m, 0.4 nm2; cf. Figure 2) per BBP4+ molecule. Four molecules of DMPA contribute to the binding and retention of BBP4+with four positive charges on the tetracationic BBP4+and altogether occupy 1.6 nm2. The extra space of ca. 0.1 nm2 per BBP4+could be filled by a neutral molecule or DMPA. The four DMPA molecules through ion/ion interactions with BBP4+would also force the macrocycle to be parallel to the air/water interface. Molar ratios larger than 4:l (e.g., 8 1 ) thus denote (34)Peschke, J.; MBhwald, H. Colloids Surf. 1987,27, 305-323. (35) Ivanova, M. G.; Verger, R.; Bois, A. G.; Panaiotov, 1. Colloids Surf. 1991, 54, 279-296. (36) Anelli, P. L.;Ashton, P. R.; Ballardini, R.; Balzani, V.; Delgado, M.; Gandolfi, M. T.; Goodnow, T. T.; Kaifer, A. E.; Philp, D.; Pietraszkiewicz, M.; Prodi, L.; Reddington, M. V.; Slawin,A. M. Z.; Spencer, N.; Stoddart, J. F.; Vincent, C.; Williams, D. J. J. Am.Chem. Soc. 1992,114, 193-218.
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Figure 6. Schematic view of the DMPAIBBP'+ cospread monolayer at the &/water interface during the compression process. The DMPABBP4+molar ratios are (a) 1:l and (b)8:l (2' = 294 K).
an oversupply of DMPA (not all DMPA molecules in the monolayer get complexed with BBP4+)while molar ratios smaller than 4:l (e.g., 1:l) correspond to an undersupply of DMPA (less than four DMPA molecules available for complexation with one BBP4+). Our hypothesis is that the negatively charged phosphate head group of DMPA complexes with the positively charged pyridinium moiety of BBP4+ possibly already in the spreading solution or immediately after spreading at the air/water interface. Initially in the expanded state,the DMPA molecules attach themselves randomly to the BBP4+ molecules, with the minimum of one DMPA per BBP4+necessary to hold the BBP4+at the aidwater interface. This will thus lead to an expansion (each DMPA/BBP4+complex occupying at least 1.7 nm2 available area and further expansion due to the triply charged DMPA/BBP4+complex) of the DMPA isotherm at high molecular areas. Thus, the maximum expansion, according to this model, should occur for the DMPABBP4+ molar ratio 1:1, as this allows for the maximum number of DMPA/BBP4+ complexes. The isotherms for molar ratios larger than 4 1 (oversupply of DMPA) should show contraction behavior while the isotherms for DMPABBP4+molar ratios smaller than 4 1 (undersupply of DMPA) should show expansion behavior. The compression of the monolayer, as schematically shown in Figure 6 (for two typical DMPABBP4+molar ratios, 1:l and 8:1), could be envisaged as follows: As the monolayer is compressed to high surface pressures, DMPA molecules are forced together to fill the space between them to make a compact lipid monolayer, losing any extra BBP4+molecules (cf. Figure 6a; DMPABBP4+molar ratio 1:l) during the compression process. Thus, at a high surface pressure corresponding to the solid condensed phase of the lipid monolayer, only those BBP4+molecules are retained which are electrostatically bound to the negatively charged DMPA. It follows that the maximum loss of BBP4+during the compression process should occur
Ionic Interactions at Interfaces
"1
Langmuir, Vol. 9, No. 6, 1993 1539
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0.2
0.4
0.6
0.8
10
12
AredDMPA Molecule [ nm2 ] Figure7. Surfacepotential (normalizedto the amount of BBP4+ spread and at constant DMPA density)-area isotherms of the
m
86
4
3
2
1
Molecular Ratio DMPNBBP [-]
cospread DMPA/BBP'+ monolayers at the &/water interface. DMPA:BBP+ molar ratios are indicated on the corresponding curves.
Figure 8. Surface potential of the DMPA/BBP'+ cospread monolayers as a function of the DMPABBP4+molar ratio. The value at molar ratio corresponds to the pure DMPA monolayer (surface pressure 40 mN/m, T = 294 K).
for the molar ratio 1:l while no loss of BBP4+occurs for DMPABBP4+ molar ratios 141 (cf. Figure 6b). The predictions of this working model are amply confirmed by the isotherm data presented in Figures 4 and 5,where it is seen that the maximum isotherm expansion does occur at a molar ratio of 1:l and that isotherms corresponding to the other DMPABBP+ molar ratios show the predicted behavior. However, to substantiate the model, additional informationregarding the density, orientation, and nature of the interaction of BBP4+molecules at various stages of the DMPA/BBP4+isotherm is necessary. To obtain this information,surface potential-area and surface reflection (equivalent to optical absorption)-area isotherms were measured and are discussed below. 3.2.3. Surface Potential-Area Isotherms. As discussed above, the electrostatic contribution to the surface potential of DMPA at the aidwater interface is ca. -300 mV. It is thus expected that the adsorption of tetracationic BBP4+ to the negatively charged DMPA should be reflected in the AV-A isotherm. As a result of charge compensation,the surface potential AV should be shifted toward more positive values, the magnitude of the positive shift being dependent on the stoichiometryof the negative and positive charges. The AV-A isotherms corresponding to the FA isotherms (cf. Figure 4) are shown in Figure 7. It should be mentioned that the data presented in Figures 4 and 7 were obtained simultaneously. The AV values shown in Figure 7 have been normalized to the amount of BBP4+spread and are presented at constant density (AV*A)of DMPA. This was achieved by dividing the measured AV data by the amount of BBP4+spread and by multiplying with the molecular area. The normalization to BBP4+was done with the purpose of finding out the change in AVper BBP4+molecide. The following features of the AV-A isotherms are noteworthy: (a) AV values for the DMPA/BBP4+system are positive compared to the reference DMPA, (b) AV-A isotherm profiles show large but systematic changes as a function of the DMPA BBP4+molar ratio, (c) the negative dip at ca. 0.85 nm2 in the AV-A reference DMPA isotherm disappears as the molar fraction of BBP4+ is increased, (d) the absolute values of AV at A = 0.45 nm2 show saturation for the 4:l molar ratio, (e) at larger molar ratios (oversupply of DMPA),AVdecreases compared to the maximum reached for the 4:l molar ratio. These results are in conformity with the present model as discussed below. The compensation of negative charges of DMPA by positive charges of BBP4+leads to the positive shift of
AV. This is also the reason for the disappearance of the negative dip in AVin the reference DMPA isotherm. Now let us consider a typical isotherm separately. At a molar ratio of 4:l (cf. Figure 7),the normalized AVvalues show a small negative dip at 0.81nm2, further compression leads to a sharp jump in AV, and then the potential remains essentially unchanged till 0.6 nm2. For A < 0.6 nm2 and till 0.45nm2,the surface potential increases, and for A < 0.45 nm2, it slightly decreases. As there is no excess of BBP4+,no loss of BBP4+occurs during the compression process. This has the consequence that AV remains essentiallyconstant (withslight changes) with compression till a compact monolayer of DMPA is formed. For the DMPABBP4+molar ratio 1:l (cf. Figure 7), there is no negative dip in AV and the maximum of AV is at A = 0.81 nm2just before the u starts to increase. The negative dip in AV disappears as a result of overcompensation (one negative charge of DMPA per four positive charges of BBP4+)of the negative charge of DMPA by BBP4+. The maximum in AV at A = 0.81 nm2 is also because of this reason. Further compression below A = 0.81 nm2 leads only to a continuous slow decreaseof normalized AVvalues. That the BBP4+molecules are lost during the compression process is also clearlyevident from the systematicallylower values of AV for the molar ratios 3:1, 2:1, and 1:l. The maximum loss of BBP4+is expected to occur for the molar ratio 1:l (cf. Figure 6a) which is confirmed by the lowest values of AV as shown in Figure 7. The dependence of surface potential (at 40 mN/m) on the DMPABBP4+molar ratio is shown in Figure 8. The point in the left-hand corner at 230 mV corresponds to the pure DMPA and represents the reference value. It is seen that as the DMPABBP4+molar ratio is increased, the value of AV (ca. 530 mV) remains constant till the molar ratio 41 is reached. A further increase of DMPA leads to a linear decrease in AV. For DMPABBP4+molar ratios less than 41 (undersupply of DMPA) where there is an excess of BBP4+,loss of BBP4+occurs (cf. Figure 6a) during the compression process. The loss of BBP4+ however becomes less and less as the DMPABBP4+molar ratio is increased till 41 where no loss of BBP4+occurs. The final structure in the condensed state at 40 mN/m however is the same (for molar ratios 41,3:1,2:1,and l:l), leading to a constant value of the surface potential. For DMPABBP4+molar ratios larger than 4:l (undersupply of BBP4+or excess of DMPA), there is no loss of BBP4+ (all BBP4+molecules are complexed with DMPA) during the compression process (cf. Figure 6b) but some parts of
0)
(0))
Ahuja et
1540 Langmuir, Vol. 9, No. 6, 1993
E
t
50 7
40
,
I
500
-
30 -
s
v)
F
a
20 -
z
9)
8
r
-al
0.8:
E
E
Y
f
41.
'c
.-c0
10-
fJY
0.60.4-
0.2-
0
01 0.3
I
0.4
I
I
I
0.5
0.6
0.7
I
0.8
1-100
0.9
2
0.0-
al
I
AredDPPA Molecule [nm' ]
Figure 9. Surface pressure (T)- and surface potential (AWarea isotherms of DPPA (solid lines) and the cospread DPPA/ BBP'+ (molar ratio 1:l) (dashed lines) monolayers at the air/ water interface (2' = 294 K).
the DMPA monolayer are not complexed as a result of the undersupply of BBP4+, leading to a decrease in the AV with increasing amount of DMPA. The maximum value of the magnitude of positive shift in AV as a result of DMPA/BBP4+interaction is ca. 300 mV (maximum value in Figure 8 is 530 mV and the reference value is 230 mV) which is in good agreement with the anticipated value of 300 mV as discussed above in section 3.1. 3.2.4. DPPA/BBP4+Cospread Monolayers. Further strong evidence in support of the above discussed model is obtained through investigations of DPPA/BBP4+ cospread monolayers. DPPA is identical to DMPA except for the fact that the DPPA aliphatic chain is two CH2 units longer (14CH2 units) than that of DMPA (12 CH2 unite). This has the consequencethat the phase transition temperature of DPPA is shifted toward higher temperatures by ca. 20 OC. The *-A and AV-A isotherms of DPPA (solid lines) and DPPA/BBP4+(molar ratio 1:1, dashed lines) are respectively shown in Figure 9. It is seen that the DPPA isotherm at 21 OC does not show any fluid and pronounced fluid/solidcoexistenceregions during the compression process as compared to DMPA (cf. Figure 2)but goes directly into the solid phase. In addition, the surfacepotential-area isotherm profiles are rather erratic and not well behaved. Additional evidence such as Brewster angle microscopy of the spread monolayer of DPPA at the aidwater interface reveals that immediately after spreading large solid domains are formed. These domains come together during the compression process. The surface potential of DPPA at 40 mN/m is 230 mV which is equal to that of DMPA (cf. Figure 2). It means that the chain length has no influence on the surface potential which is in agreement with the other reports." Thus, the surface potential analysis of DMPA as summarized in Figure 3 may be taken to be valid also for DPPA. The T-A isotherm of the DPPA/BBP4+(1:l)cospread monolayer also shows expansion but not to the extent as shown by the DMPA/BBP4+system (cf. Figure 4,curve a). Thus at r = 5 mN/m, the DPPA/BBP4+monolayer shows expansion by 10% while the DMPA/BBP4+monolayer at the same surface pressure shows expansion by 60%. The expansion shown by the DPPA/BBP4+monolayer at 5 mN/M is similar to that shown by the DMPA/ BBP4+ monolayer at 40 mN/m. The reason for this different behavior lies in the fact that DPPA when spread shows the formation of solid domains (the state reached by DMPA at high T ) which adsorb the BBP4+but do not show any expansion as a result of enhanced interchain interactions. That the BBP4+ molecules adsorb to the
0.4
0.6
0.8
10
12
AredMolecule [nm2 ] Figure 10. Surface reflection (AR at 266 nm)-area isotherms of cospread DMPA/BBPC+ monolayers at the &/water interface. The values of AR have been normalized to the amount of spread BBP'+ and are presentad at constant density of DMPA. DMPA BBP*+molar ratios are indicated on the correspondingcurve8 (T = 294 K).
DPPA monolayer is shown clearly by the positive shift of AV by ca. 250 mV (cf. Figure 9, dashed curve), the disappearance of the negative dip in the AV-A profiie, and surface reflection at 265 nm (data not shown). These results show that the expansion of the isothermsas aresult of lipid/adsorbate interaction also depends on the lipid/ lipid interactions and the state of the lipid monolayer. This result is in contradiction with the penetration and squeeze out model according to which no significant difference between DMPA and DPPA is to be expected. 3.2.5. Surface Reflection-Area Isotherms. Additional and direct evidence for the DMPA/BBP4+ interactions may be obtained by measuring the UV reflection spectrum of BBP4+at the monolayer/water interface. This methodlo detects only those molecules which are at the interface and resonantly contribute to enhanced surface reflection. For example,the DMPA monolayer contributes to the AR only through scattering and a change in the refractive index of the aidwater interface while BBP+ resonantly reflects and absorbs the UV radiation. The W spectrum of BBP4+in acetonitrile shows a peak at 263 nm. The r - A and AR (at 265 nm)-A isotherms of DMPA/ BBP4+at different molar ratios were measured simultaneously. As a reference, the AR-A isotherm for DMPA was also measured. The value of AR for DMPA at 0.43 nm2 was found to be 0.0464%. This value was then subtracted from the AR values for different DMPABBP+ molar ratios to get the reflection value for BBP4+ alone. The results thus obtained are shown in Figures 10 and 11. The signal to noise ratio of our reflection apparatus in the 250-300-nm range is not as good as in the visible range because of the low UV output of the lamp used. The AR values presented in Figure 10have been normalized to the amount of BBP4+ spread and are at constant DMPA density. This was achieved by dividing the measured AR values by the amount of BBP4+spread and by multiplying with the area per molecule. The results show that AR in general shows a sharp increase (similarto A V-A isotherms) at A = 1 nm2 and then either remains constant or slightly decreases during further compression. The dependence of normalized (at constant surface density) Ai? on A is particularly informative. Thus, it is expected that, in the absence of any orientational change, aggregation, or loss of BBP4+, the normalized AR should remain constant during the whole compression process. The presence of aggregation may be detected through changes in reflection spectra at different surface densities. It was found that
Langmuir, Vol. 9, No.6,1993 1641
Ionic Interactiom at Interfaces
0.6 0.8 1 .o 1.2 AredDMPA Molecule [nm2] Figure 11. Surfacereflection (AR at 266nm)-area isothermsof mapread DMPA/BBP+ monolayers at the aidwater interface. The valuesof A23 are presented only at constant densityof DMF'A. DMPABBP+ molar ratios are indicated on the corresponding curves (T= 294 IO. 0.4
0.00 m
I
I l l 1
I
I
I
0
4 3 2 Molecular Ratio DMPNBBP I-]
I
6
I
Figure 12. Surface reflection (AR at 266 nm) of the DMF'A/ B B P cospread monolayers as a function of the DMF'ABBP4+ molar ratio. The value at molar ratio corresponds to the pure DMF'A monolayer (surface pressure 40 mN/m). the reflection spectrum of the DMPA/BBP4+system does not change with increasing surface pressure or surface density, ruling out aggregation effects. The fact that AR remains almost constant during the compression below A = 0.8 nm2 indicates that BBP4+ molecules do not change orientation. In addition, the relative values of AR at different DMPA:BBP4+ molar ratios show a systematic change in AR,again showing that maximum loss of BBP4+ occurs for the molar ratio 1:l (also cf. Figure 6a). Both these facts, namely, no change in orientation of BBP4+ and the correspondenceof AR and AVvalues to the DMPA BBP4+molar ratio, are strong evidence for the proposed model. The penetration model cannot account for these results as according to it the maximum value of AR should be observed for the 1:l molar ratio and the adsorbate molecules undergo orientational changes during the penetration and squeeze out phases. The AR-A isotherm profides for molar ratios higher than 4 1 (oversupply of DMPA) are shown in Figure 11. The A23 values have been normalized only for the constant density of DMPA; i.e., the measured AR values were multiplied by the area per molecule. It is seen that, as the relative amount of DMPA increases, the value of AR decreases proportionally. This is expected since more and more DMPA remains uncomplexed as a result of an undersupply of BBP4+ (cf. Figure 6b). The results of Figures 10 and 11 are summarized in
Figure 12 where AR values (at 40 mN/m) as a function of the DMPA/BBP4+molar ratio are shown. It is seen that AR values remain constant as the DMPABBP4+ molar ratio is changed from 1:l to 41,showing that the final structure and composition of the DMPA/BBP+ monolayer are the same at 40mN/M. A further increase of the DMPA BBP4+molar ratio (>4:1)leads to a linear decrease in AR, showing that the density of BBP4+molecules at the air/ water interface decreases. The dependence of AR (cf. Figure 12;sensitive to optical absorption properties of BBP4+) and AV (cf. Figure 8; sensitive to electrostatic interactions between the DMPA and BBP4+) on the DMPABBP4+ratio is the same, confirming the validity of the proposed model using different techniques. This meana that, for a compactDMPA/BBP4+monolayer,each BBP4+ molecule requires ca. 4 DMPA molecules which corresponds to a lipid area of ca. 1.7 nm2 in agreement with the area of BBP4+ based on model (cf. Figure 1) calculations. At DMPABBP4+ molar ratios below 41, BBP4+is in excess, leading to expansion of the isotherm at low surface pressures. The excess of BBP4+is, however, lost during the compression process, leading to a constant value of AR (at 40 mN/m) independent of the DMPA BBP4+molar ratio up to a 4 1 molar ratio. For DMPA BBP4+molar ratios >41,the lipid is in excess,thus leading to relative compression of the DMPA isotherms while no loss of BBP4+occurs during the compression process. In this case, parta of the DMPA monolayer are, however, without B B W , leading to a decrease of AR with increasing molar fraction of DMPA. These results, namely, the expansion and contraction of the isotherms as well as the existence of a particular value of the DMPABBP4+ molar ratio (4.1:l)showing maxima in AR and AV values, confirm the validity of the present model. The molar ratio at which the maxima in AV and AR are observed may be given by the following general relation: [molar ratio],
=
adsorbate molecular area --Am lipid molecular area A,
(4)
For the particular case of DMPA and BBP4+,A m = 1.7 nm2 and AL = 0.4 nm2, thus giving M& = 4.1:l. This relation has been found to be valid also for other systemss7 such as DMPA/porphyrin4+ where M& = 8 1 has recently been found to show maxima in AR and AV, with the porphyrin4+molecular area being ca. 3.2 nm2. 3.2.6. Stability of Cospread Monolayers. The stability (noloss of ligand moleculeswith time) of the complex monolayers is quite important for the transfer of these monolayers to substrates. In order to find out if the electrostatic interactions between DMPA and BBP4+are sufficient to hold the BBP4+molecules as the monolayer/ water interface, the stability of the DMPA/BBP4+(molar ratio 4:l) cospread monolayer was investigated. The cospread monolayer was compressed to 26 mN/m and the surface pressure and AR (at 265 nm) were monitored at constant surface area for a period of 5-6 h. The results of thisexperiment are shown in Figure 13. The subphase temperature was 29 OC. It is seen that though the surface pressure relaxes to some extent, the A23 values however remain stable over a long time period. As the AR signal is only due to BBP4+,it may be concluded that the surface density of BBP4+ molecules remains constant at the monolayer/water interface. The relaxation in surface pressure may possibly be attributed to slight changes in the water level (over 5-6 h) in the trough or reorganization (37) Ahuja, R.C.; Caruso,P.L.; Mbbiue, D. ManueCript in preparation.
1542 Langmuir, Vol. 9,No.6,1993
Ahuja et al.
-5 "L - j o'20
E e! 3
L 0.15 [> 9
I
p!
lol
n. 200 a,
fcn
0.10
i'
n
g
-s
50
100
150
200
250
300
Time [min] Figure 13. Stability of the DMPA/BBP'+, molar ratio 4 1 , cospread monolayer as a function of time. The monolayer was compressedto 25mN/m,andtherelaxationofthesurfacepressure (d and surface reflection (AR at 265 nm) were monitored at constant surface area (T= 302 K). 1.6
0.0 o 200
0.14
A
250
300 ,
350
400
2
1:
Wavelength [nm]
Figure 14. Spectra of DMPA/BBP'+, molar ratio 4 1 ,cospread monolayers: (a)opticalabsorptionof two monolayerstransferred to a quartz substrate, (b) optical reflection spectrum of a single monolayer at the air/water interface (surface pressure 25 mN/ m)
\
0.05
0.00
1.4
nM
Iu
0 0
0 cc -
B
30-
VI
/700nM
r
40-
.
of DMPA. From Figure 2, it is seen that the isotherm profile of DMPA is quite steep in the surface pressure region 10-40 mN/m, making the relaxation of surface pressure reasonable. 3.2.7. Transfer of Cospread Monolayers. An important aspect of the Langmuir-Blodgett technique is the preparation of a complex monolayer at the aidwater interface and its subsequent transfer to the substrate. Thus, we have attempted to transfer the DMPA/BBP4+ (molar ratio 4:l)cospread monolayer to quartz substrates. The optical reflection spectrum of the cospread monolayer (surface pressure 25 mN/m) at the air/water interface is shown in Figure 14 (curve b). The spectrum shows a maximum at 266 nm which is close to the spectrum of BBP4+ in water. A single monolayer on each side of the quartz plate was then transferred by using the vertical dipping method in the upstroke (dipping out of the substrate from subphase) mode. The optical absorption of the transferred monolayers was then measured and is shown in Figure 14 (curve a). The peak in the spectrum is now at 264 nm, showing a slight blue shift. This may be because of the change in medium. Using the vertical dipping procedure, we have been able to transfer 40 multilayers. The optical density a t the peak position was found to vary linearly (data not shown) with the number of monolayers, indicating good quality transfer of DMPA/ BBP4+cospread monolayers. The X-ray diffraction data (Bragg peaks) of the transferred multilayers (data not shown) shows a periodicity of 51 A, which corresponds to the bilayer thickness of the DMPA/BBP4+ system.
100 nM
" I 0
50
100
150
200
250
300
Time [min]
Figure 15. Adsorption kinetics (surface pressure versus time) of BBP+at the DMPA monolayer/subphaseinterfaceat different concentrationsof BBP4+in the subphase. BBP'+ concentrations in the subphase are indicated on the corresponding curves (T= 294 K).
3.3. DMPA/BBP4+Adsorbed Monolayers. BBP4+ is only slightly soluble (ca. 4 pM) in water. Thus, it is possible to adsorb and organize BBP4+ at the DMPA/ water interface through adsorption from the subphase. It is of interest then to compare the DMPA/BBP4+monolayers obtained using the techniques of cospreading and adsorption. 3.3.1. Kinetics of Adsorption of BBP4+ at the DMPA/Water Interface. The investigation of kinetics of adsorption a t the monolayer/subphase interface is ~ important to obtain a basic understanding of the role of various parameters such as the lipid surface density, lipid charge, lipid dipole moment, diffusion coefficient of the adsorbate, adsorbate charge, and nature and composition of the subphase in determining the properties of the adsorbed monolayer. We have employed surface reflection and surface potential techniques to study the adsorption processes. The surface reflection technique is ideally suited for this purpose aa the molecules in the subphase do not contribute to the signal. The kinetics of adsorption were measured in the following way: The trough was filled with the subphase containing a definite concentration of BBP4+. Then a solution of DMPA was spread on this subphase, and the monolayer was compressed to 42 mN/m quickly. The monolayer compression time was ca. 5 min. Subsequently the surface pressure A and surface reflection AR (at 265 nm) were monitored simultaneously at constant surface area for a period of 5 h. The results of the kinetics measurements are shown in Figures 15 and 16 for surface pressure and surface reflection, respectively. From Figure 15,it is seen that for the reference subphase (pure water), the surface pressure of DMPA relaxes quite strongly. The value of A decreased from the initial value of 42 mN/m to ca. 19 mN/m over a period of 5 h. The relaxation of the surface pressure of the pure DMPA monolayer is expected from the steep isotherm profile (cf. Figure 2)in the le40 mN/m range and the very short monolayer compression time (5 min). As the concentration of BBP4+ in the subphase is increased from 75 to 200 nM, the rate of relaxation of r decreases and the final pressure value increases. For BBP4+concentrations above 300 nM, the values of A first decrease and then tend to increase. The final surface pressure for the DMPA monolayer on a subphase containing 700 nM BBP4+was higher than the initial surface pressure. It is clear that the surface pressure kinetics is a result of two processes, namely, a decrease in
Ionic Interactions at Interfaces
Langmuir, Vol. 9, No. 6,1993 1543
0.20-
* a
o . o o l , , , , , l , , , ~1 0
50
100
, , , , , , , , , 1 , , , , 1 , , , , ~
150
200
250
300
Time [min]
Figure 16. Adsorption kinetics (surface reflection at 265 nm versus time) of BBP4+ at the DMPA monolayer/subphase interface at different concentrations of BBP4+in the subphase. BBP4+concentrations(nM) in the subphase are indicated on the corresponding curves (2' = 294 K). u of the reference DMPA and an increase in u as a result of BBP4+ adsorption. The increase in surface pressure subsequent to adsorption has usually been interpreted in terms of penetration of the adsorbate into the monolayer. However, model studies using the cospreading technique as discussed above in this paper rule out the interpretation of these results in terms of penetration. The surface reflection kinetics (cf. Figure 16) are much more informative from the adsorption point of view as the change in AR reflects only changes in BBP4+ surface density at the interface. The contribution of the DMPA monolayer to AR is seen in the curve obtained on pure water. The value of AR for the DMPA/water system remains constant over a time period of 5 h in contrast to surface pressure (cf. Figure 15). As mentioned above, ca. 5 min elapses between the spreading of the monolayer and the beginning of reflection measurements. Therefore, at time 0 as shown in Figure 16, there is already some reflection signal caused by the adsorbed BBP4+molecules. This initial signal is especially high for higher BBP4+ concentrations. Extrapolation of the AR signal to zero value does not lead to any fixed offset time since the AR signal shows nonlinear dependence on the density of DMPA. Fortunately, only the slope of the linear part of adsorption kinetics is required for the evaluation of relevant parameters. It is to be noted that, except for the case of 75 nM, the reflection AR reaches practically the saturation value (AR = 0.18%) in all cases. The dependence of slopes d(AR)/dtof the linear part of adsorption kinetics on the BBP4+concentration in the subphase is shown in Figure 17. It is clear that d(AR)/dt varieslinearly with BBP4+ concentration. The adsorption kinetics may be rationalized in terms of the Nernst diffusion layer model.% According to this model, the number of adsorbate molecules reaching the interface per unit time per unit area is given by the following relation: D = $0 - c )
0.0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
Concentration [pM] Figure 17. Dependence of a(AR)/at (slopes of the linear parta of the absorption kinetics curves shown in Figure 16) on BBP'+ concentration in the subphase, the linear relationship with the
slope being 99.13 L/(mol.min).
trostatic interaction energy >>kT),they are trapped there, and therefore c may be set equal to zero. The surface density t~ is related to AR through a constant fl which is a property of the adsorbate but also depends on the orientation of the adsorbate at the interface. Thus, eq 5 may be written as
It follows from the above that the slope d(AR)/dtshould be proportional to the bulk concentration of BBP4+which is in agreement with the results presented in Figure 16. Now we will try to get the value of /3 which relates the value of AR with the surface density of BBP4+ at the interface. From the molecular area of BBP4+ which is equal to 1.7 nm2,the surface density for a compact BBP4+ monolayer is given by u = 1/A. Thus, the value u = 9.77 X mol/m2 is obtained with the corresponding AR = 1.426 X 1o-Swhich in turn (0 = u/AR)gives fl = 6.85 X 1o-L m2/mol. The slope of the straight line in Figure 17 is 99.13 L/(mohmin). The value of d2a/(at dco) may be calculated to be 1.13 X 10-4 cm/s which is equal to D/6. In order to get the value of 6, we have to know the value of the diffusion coefficientD. An approximatevalue of D can be calculated from the Stokes-Einstein equation according to which
where u is the surface density of the adsorbate (BBP4+), co the bulk concentration of BBP4+in the subphase, c the concentration of BBP4+at the interface, 6 the thickness of the diffusion layer, and D the diffusion coefficient of BBP4+. Once the BBP4+ molecules reach the interface within binding distance of the lipid head groups (elec-
D = kT/6?rqa (7) where k is the Boltzmann constant, T the absolute temperature, q the viscosity of the subphase, and a the radius of the molecule. Strictly speaking this relation is valid only for diffusion of spherical molecules in spherically symmetric space. The present situation is however quite different. The BBP4+molecules are more like a slab with a cavity (ca. 0.3 nm2)rather than a sphere and close to the interface move under the influence of an electric field of the charged DMPA monolayer. But our purpose here is just to get an estimate of the diffusion coefficient. Thus, taking q = 1 cp for water at 298 K and a = 0.572 nm, the value of D = 3.75 X 10-6 cm2/s is obtained. Putting this value of D in the expression for D/6, a value of 0.033 cm is obtained for 6. For comparison values of 6 = 0.024 and 0.05 cm have been r e p ~ r t e d . ~ ~ $ ~ * ~ ~ 3.4. Adsorbed versus Cospread Monolayers. It is interesting to compare the merits of complex monolayers of DMPA/BBPQ+obtained through the techniques of
(38) Bockris, 3. 0.M.;Reddy, A. K.Modern Electrochemistry, 3rd ed.; Plenum/Rosetta New York, 1977; Vol. 2, p 1056.
(39)Physical Chemistry, an Aduanced Treatise, Eyring, H.,Ed.; Academic Press: New York, 1970; Vol. SA,p 247.
1644 Langmuir, Vol. 9, No. 6,1993
adsorption and cospreading. First of all it should be mentioned that the cospreadingtechnique does not require the adsorbate to be water soluble. Second, a minimal amount of adsorbate BBP4+is required; e.g., the amount of BBP4+ that was spread for the 4 1 molar ratio DMPA/ BBP4+cospread monolayer was equivalent to 7 nM in the subphase compared to that of 700 nM required for the adsorption technique. The adsorption time for obtaining a ligand monolayer varied between minutes and hours depending on the concentration of BBP4+in the subphase. No adsorption time is required in the cospreading technique. The surface density of the BBP4+monolayer is the same for both techniques as revealed by surface reflection (AR= 1.84 X 103)measurements. Another disadvantage of the adsorption technique is the uncertainty of monolayer composition such as aggregation of adsorbate molecules through adsorbate/adsorbate interactions at the interface. Finally, the composition of transferred monolayers obtained via adsorption is also not under experimental control. 4. Conclusions The present investigations have shown that, using the cospreading technique, it is possible to organize the nonamphiphilic cyclic bisbipyridinium tetracation (BBPI+) in the form of a compact and oriented monolayer at the aidwater interface. It has been shown that DMPA/BBP4+ interactions at the monolayer/water interface lead to both expansion and contraction of P A isotherms depending on the surface pressure and DMPABBP4+molar ratio. On the other hand, DPPA/BBP4+interactions do not lead to significant changes in the P A isothermsthough surface potential and reflection data show the adsorption of BBPI+. The surface potential results show a maximum for the DMPABBP4+ molar ratio 41. This corresponds to a structure with a densely packed monolayer of the cyclic tetracation below the densely packed anchor DMPA monolayer. Surface reflection data also lead to the same conclusion. In addition, these results show that BBP4+is
Ahuja et al.
forced by the four negatively charged DMPA anchor molecules to adopt an orientation so that the BBP4+plane is parallel to the aidwater interface. The molecular ratio at which the maximum values for the surface potential and UV reflection measurements are observed is determined by the molecular area ratio of the components (area of nonamphiphilic ligand per area of anchor lipid). It has been established that the expansion of isotherms as a result of lipid/ligand interaction does not mean penetration of cyclic ligands into the hydrophobic part of the lipid monolayer followed by squeezing out at high surface pressure as discussed in the literature for other systems. The adsorption of BBP4+from the aqueous subphase to the DMPA monolayer/subphase interface was also investigated, and it was found to be diffusion limited. The adsorption kinetics data have been analyzed in terms of the Nernst diffusion layer model, yielding a diffusion layer thickness of 0.033 cm assuming a diffusion coefficient for BBP4+to be 3.75 X 1o-B cm2/s. The merits of the cospreading technique, which allows for the organization of functionallyactive nonamphiphilic ligands at the air/water interface, are the following: a minimal amount of adsorbate is required, nonamphiphilic ligand does not have to be water soluble, long adsorption times are avoided, monolayers of defined composition are obtained, and the resulting monolayers are quite stable and can be transferred to substrates using the vertical dipping technique. The host/guest interactions of the oriented cyclic tetracation monolayers with correspondingr-electron-rich guest molecules will be reported in the near future.
Acknowledgment. We thank Dr. Jordan Petrov and Gernot Overbeck for a critical reading of the paper. Financial support of this work through the Bundesministerium fiir Forschung und Technologie via Grant 03M40080D is gratefully acknowledged. Partial funding of this work through the British Council is also gratefully acknowledged.
