Langmuir 1995,11,568-573
568
Effect of Subphase Composition on the Molecular Organization in Complex Monolayers of Pyrene-3-sulfonate and Dioctadecyldimethylammonium Bromide at the AirNater Interface Zlatica Kozarac,? Ramesh C. Ahuja," and Dietmar Mobius Max-Planck-Institut fur Biophysikalische Chemie, Postfach 2841,0-37018Gdttingen, Germany, and Rudjer BoBkovi6 Institute, Center for Marine Research, P.O. Box 1016, 41001 Zagreb, Croatia Received April 25, 1994. In Final Form: November 4, 1994@ Organization of complex monolayers of pyrene-3-sulfonate(PyS-)and dioctadecyldimethylammonium bromide (DOMA) at the airlwater interface has been studied by surface pressure and surface potential measurements, by fluorescence spectroscopy, and by reflection spectroscopy under normal and oblique incidence of light. Complex monolayers have been prepared using the cospreading technique, Le., by spreading mixed solutions containing the anchor lipid DOMA and PyS- in different molar ratios. It is deduced from various techniques that in the densely packed PyS-/DOMA complex monolayers, a densely packed submonolayer of b S - is formed under the head groups of the anchor lipid monolayer. Reflection spectroscopicmeasurements provide additionalinformation on the molecular arrangements at the interface and show that the pyrene chromophores do not lie parallel to the interface but are inclined. The influence of subphase composition on the monolayer organization and processes at the interface has also been studied. Excimer emission is observed in the mixed monolayers spread on the water subphase. Introduction of negatively charged counterions in the subphase leads to the dominance of monomer emission. The influence of the polysaccharide xanthan was investigated in more detail. The results are discussed in terms of a phenomenological model of molecular organization.
1. Introduction Pyrene and other polycyclic aromatic hydrocarbons (PAH-s)belong to the most ubiquitous and highly dangerous organic pollutants entering natural aquatic systems due to the anthropogenic influence and are listed as priority pollutants by the Environmental Protection Agency.l Recently, the interaction of pyrene and PAH-s with marine interstitial water organic colloids, as well as with several types of natural dissolved organic matter, has been examined by using fluorescence-quenching s p e c t r o s c ~ p yIt . ~was ~ ~ found that the binding of nonpolar organic pollutants to colloids as well as the partition coefficients to unfractionated and fractionated water soluble organic carbon, humic acid, and fulvicacid depends on the nature of organic matter. Pyrene has a significant affinity toward colloids having high lipid content.2 In an effort to get a better understanding of the complex phenomena of adsorption of pyrene and other PAH-s at phase boundaries, we have started a systematic physicochemical investigation of these compounds at the monolayerlsubphaseinterface. Pure pyrene when spread at the airlwater interface does not form a well-defined compact monolayer but instead forms crystallites which are pushed together in the compression process. Therefore, mixtures of pyrene with different lipids, which form stable monolayers, are more appropriate for the understanding of pyrene enrichment at the monolayerlwater interface. The behavior of pyrene a t different phase boundaries such as airlwater and lipid m~nolayerlwater,~ t Rudjer BoSkoviC Institute.
Abstract published in Advance ACS Abstracts, December 15, 1994. (1)Keith, H. L.;Telliard, W. A. Enuiron. Sci. Technol. 1979,13,416. (2)Chin, Yu.-P.; Gschwend, P. M. Enuiron. Sci. Technol. 1992,26, 1621. (3)Herbert, B. E.;Bertsch, P. M.; Novak, J. M. Enuiron. Sci. Technol. 1993,27,398. (4)Wistus, E.; Mukhtar, E.;Almgren, M.; Lindquist, S. E.Langmuir 1992,8,1366. @
at solid surfaces like y-alumina and ~ i l i c aclays,6 ,~ and zeolites,' and partitioning of pyrene and pyrenyl compounds at the mnicellelbulkinterface*andlor in vesicle^^-^^ has recently been studied by fluorescence spectroscopy. Fluorescence spectroscopy is a highly sensitive technique for the investigation and characterization of pyrene and its interactions with lipid monolayers a t the airlwater interface and in Langmuir-Blodgett films. Pyrene has a relatively long excited state lifetime, high quantum yield of fluorescence,and a pronounced tendency to form excimers. Furthermore, the structured monomer emission spectrum and the quantum yield of fluorescence are very sensitive tothe environment polarity and pyrenepyrene interactions. The proximity of pyrene molecules is easily detected by the presence of excimer emission. Excimers are molecular complexes existing only in the excited state. These excited state dimers are formed by the interaction of an unexcited pyrene with an excited pyrene during the excited state lifetime.13J4The spectral manifestation of this pyrene-pyrene interaction is that the monomer emission in the 360-430 nm range is replaced by a red-shifted broad structureless emission band with a maximum at ca. 480 nm. The location of pyrene molecules at the lipid monolayerlsubphase interface and the intermolecular association processes are expected to be influenced by the nature (dipole moment, (5)Mao, Y.; Thomas, J. K. Langmuir 1992,8,2501. (6)DellaGuardia, R.A,; Thomas, J. K. J . Phys. Chem. 1983,87,3550. (7)Suib, S.L.;Kostapapas, A. J . Am. Chem. Soc. 1984,106, 7705. (8) Tamori, K.; Watanable, Y.; Esumi, K. Langmuir 1992,8, 2344. (9)Almgren, M.; Grieser, F.; Thomas, J. K. J . Am. Chem. Soc. 1979, 101, 279. (10)Daems, D.; Van den Zegel, M.; Boens, N.; De Schryver, F. C. Eur. Biophys. J . 1986,12,97. (11)Blackwell. M.F.:Gounaris. K.; Barber, J. Biochim. Biophys. . _ Acta 1986,858,221. (12)Heureux, G. P.L.; Fragata,M. J.Photochem.Photobiol.B:Biology 1989,3,53. (13)Stevens, B.; Hutton, E. Nature 1960,186,1045. (14)Forster, Th. Angew. Chem. 1969,81, 364.
0743-746319512411-0568$09.00/00 1995 American Chemical Society
Effect of Subphase Composition on Molecular Organization charge) of lipid head groups and by the subphase composition. In this work we report on the interactions of pyrene and sodium 3-pyrenesulfonate (PyS-) with monolayers of dioctadecyldimethylammonium bromide (DOMA) at the aidwater interface. The characterization and investigation techniques are surface pressure-area and surface potential-area isotherms along with fluorescence and reflection spectroscopy. The influence of different electrolyes, particularly of polysaccharide xanthan on the molecular organization in complex monolayers, has also been studied. Xanthan was used as a model for carbohydrates which are known to be significant constituents of the natural surface microlayer (50-100 pm) at the natural aidwater interface.15J6
2. Experimental Section Dioctadecyldimethylammoniumbromide (DOMA)and dimyristoylphosphatidic acid (DMPA) were purchased from Sigma Chemical Co. and used as received. Sodium pyrene-3-sulfonate (PyS-1was obtained from Molecular Probes. The inorganic salts were of AR grade. Polyelectrolytes were obtained from Sigma Chemical Co. Chloroform (HPLC) was used as the spreading solvent and was obtained from Baker Chemicals. Deionized water from a Milli-Q system (Millipore Corp.) was used for preparing the subphase. Surface pressure-area (n-A) and surface potential-area (AV-A) isotherms have been measured on a rectangular thermostated Teflon trough enclosed in a plastic cabinet. Complex monolayers were prepared by spreading a chloroform solution of the two components (DOMA and PyS-) at the aidwater interface. It should be mentioned that PyS- itself is not surface active and goes into the subphase when spread alone at the air/ water interface. The monolayers were compressed by a movable Teflon barrier with compression velocity between 0.08 and 0.12 nm2/min/molecule. A Wilhelmybalance (20mm wide filter paper) was used to measure the surface pressure, and the surface potential was measured using a vibrating plate condenser. Fluorescence measurements have been performed by using a Perkin-Elmer luminescence spectrometer LS-5 modified for in situ measurements at the aidwater interface in combinationwith a rectangular Teflon trough.17 The monolayer was excited at 350 nm, and the emission spectrum was measured in the 375575 nm range. Fluorescence spectra have been normalized for a constant lipid density. Reflection spectroscopic measurements were performed under normal incidence oflight,ls and a modified instrument was used for the measurement of reflection spectra under oblique incidence using linearly polarized light.lgJO The reflection values are expressed as the difference AR in reflectivity from the surface covered with a monolayer and from the clean water surface.
3. Results and Discussion
3.1. PyS-/DOMA Cospread Monolayers on the Water Subphase. 3.1.I. Surface Pressure and Surface Potential-Area Measurements. The surface pressurearea (n-A) isotherms of DOMA monolayer (curve 1)and PyS-/DOMA monolayer, molar ratio 1:1, on pure water (pH = 5.6)are shown in Figure 1. When DOMAis spread at the aidwater interface, the original Br- counterions are dissolved in the subphase. The main counterion is (15)Hunter, K.A,; Liss, P. S. Organic sea surface films. In Marine Organic Chemistry; Duursma, E. K., Dawson, R., Eds.; Elsevier Sci. Publ. Co.: Amsterdam, 1981;p 259. (16)CosoviC, B.; VojvodiC, V. Mar. Chem. 1989,28,183. (17)Budach, W.Elektrostatische, dynamische und optische Eigenschaften von organisierten Monofilmen hinsichtlich des Aufiaus von Sensor-Schichtsystemen. PhD thesis, Giittingen, 1991. (18)Griiniger, H.; Mobius, D.; Meyer, H. J . Chem. Phys. 1983,79, 3701. (19)Mobius, D.;Orrit, M.; Griiniger, H.; Meyer, H. Thin Solid Films 1986,132,41. (20)Orrit, M.; Mobius, D.; Lehmann, U.; Meyer, H. J . Chem. Phys. 1986,85,4966.
Langmuir, Vol. 11, No. 2, 1995 569 "" 1
0.4
0.6 0.8 1 .o 1.2 Area / DOMA Molecule [nm' ]
4
Figure 1. Surface pressure-area (n-A) isotherms of the DOMA monolayer (curve 1) and the cospread PyS-/DOMA monolayer, molar ratio 1:l(curve 2), at the aidwater interface.
the strongly hydrated OH- ion, which does not bind strongly to the DOMA head group. Therefore, there is a strong repulsion between the positively charged quarternary ammonium head groups of DOMA resultingin an expanded isotherm. As the aredmolecule is reduced, the molecules are pushed together resulting in a transition from liquid expanded to liquid condensed phase. Morphological observations of the monolayer using a Brewster angle microscope do not show any change in the morphology during the whole compression process. The film remains homogeneous at the microscopic level with domain size less than 10 pm.21 The isotherm of the cospread PyS-/DOMA monolayer shows contraction with respect to DOMA until A = 0.85 nm2(15 rnNlm). Further compression until 0.75 nm21eads to a sharp increase in surface pressure. The area/DOMA molecule for the complex monolayer in the tightly packed state is higher than that for the pure DOMA monolayer. These results show that PyS- is tightly bound to DOMA as counterion in spite of lower concentration (nM) compared to that of OH- ion @MI. At large surface area values, the bound PyS- leads to a reduction in the repulsion between DOMA head groups resulting in condensation of the monolayer. As the area of PyS- is larger than that of DOMA, further compression behavior of the monolayer is dictated by the packing requirements of PyS-. The area per DOMA molecule in the condensed state in the complex monolayer at 40 mN/m is 0.75 nm2 compared to the expected value of 0.4nm2 for neutral double chained lipids. The expansion ofn-A isotherms as a result of lipid/ adsorbate interaction has usually been interpreted in terms of the penetration or incorporation of adsorbate molecules in the complex monolayers. However, this interpretation of the expansion is based only on the n-A measurements. It has been shown by us22,23that an isotherm expansion may be accounted for even if electrostatically adsorbed molecules are located underneath the lipid monolayer. The measured area value of 0.75 nm2 is less than the value of ca. 1 nm2 required if the pyrene molecules lie flat at the aidwater interface. Analysis of the n-A isotherms in terms of different pyrene conformations show that PyS- molecules cannot be (21)Ahuja, R.C.; Caruso, P. L.; Mobius, D. Thin Solid Films 1994, 242,195. (22)Mobius, D., Griiniger, H. Bioelectrochem. Bioenerg. 1984,12, 375. (23)Ahuja, R. C.;Caruso, P. L.; Mobius, D.; Wildburg, G.; Ringsdorf, H.; Philp, D.; Preece, J. A.; Stoddart, J. F. Langmuir 1993,9, 1534.
Kozarac et al.
570 Langmuir, Vol. 11, No. 2, 1995 Table 1. Values of Surface Potentials AV at 40 mN/m for DOMA and Cospread PyS-/DOMA Monolayers Spread on Water monolayer
surface potential (mV)
DOMA PyS-IDOMA, 1:5 PyS-IDOMA, 1:2 PyS-DOMA, 1 : l
995 870 870 810
incorporated in the hydrophobic part of the monolayer but that they are located underneath the densely packed head groups of the DOMA monolayer. Further evidence regarding this conclusion is obtained through the reflection spectroscopic data and will be discussed later in this paper. The ionic interaction between the cationic DOMA and anionic PyS- should also manifest itself in the surface potential data. Therefore, we measured the surface potential isotherms of DOMA and PyS-DOMA monolayers at the airlwater interface. The surface potential values for the densely packed DOMA and the cospread PyS-/DOMA monolayers are given in Table 1. It is seen that the surface potential of the PyS-/DOMA monolayers is considerably lower than that of the pure DOMA monolayer. The surface potential of a monolayer may be expressed through the following relation
L
S
I
0 0
Y
'"
400
450
500
550
Wavelength [nm] Figure 2. Fluorescence spectra of the cospread PyS-/DOMA monolayers at the aidwaterinterface. PyS-/DOMA molar ratios are indicated on the corresponding curves; subphase, water; surface pressure n = 30 mN/m. 14
AV = AVp + W ,
12
where AVp represents the contribution of the dipole moment of the lipid and qois potential due to charged head groups of the lipid and depends on the degree of dissociation and the density of the head groups and the nature and concentration of counterions in the subphase. The value of qomay be estimated from the Gouy Chapman theory.24 Upon specific adsorption of any counterion (in our case PyS-)at the monolayer/subphase interface, the value of qois expected to change. The values of AVgiven in Table 1are in qualitative agreement with this expectation. 3.1.2. Fluorescence Spectroscopy. Recently, mixed monolayers of pyrene with various lipids such as eicosanoic acid, dioleylphosphatidylcholine,dipalmitoylphosphatidylcholine,dihexadecyldimethylammonium bromide have been studied by fluorescence spectroscopy and by surface pressure-area mea~urements.~ These results show only monomer emission of pyrene regardless of the nature of the lipid, lipidpyrene ratio, and surface pressure. It was concluded4 that the pyrene molecules are located at the monolayerlsubphase interface and somehow do not show any interaction with each other. However, we were unable to reproduce the results reported above. We have found that mixed monolayers of DOWpyrene, molar ratio 1:1,show excimer emission although the signal is very weak (data not shown here). A considerably stronger excimer emission is obtained if pyrene is replaced by the negatively charged pyrene sulfonate (PyS-).This result is expected in view of the enhanced electrostatic interaction between the positively charged head group of DOMA and the negatively charged PyS- as discussed above. Fluorescence spectra of the PyS-/DOMA cospread monolayers with different molar compositions at the air1 water interface are shown in Figure 2. These spectra show both monomer emission with maxima at 380 and 398 nm and a broad intense excimer band in the region between 440 and 575 nm with the maximum at 480 nm. It is seen that the excimer emission depends strongly on the PyS-DOMA ratio (Figure 2) and on the PyS-DOMA surface density (Figure 3). The ratio of excimerlmonomer
10
(24) Davies, J. T.; Rideal, E. K. Interfacial Phenomena; Academic Press: New York, 1963.
I
8 6
4 2
. . . . . . . . . . . . . . . . . . . . . . 400
450
500
550
Wavelength [nm] Figure 3. Fluorescence spectra of the cospread PyS-/DOMA monolayers,molar ratio 1:1,at different stages ofcompression. Curves for surfacepressures in 20-40 mN/m range are identical;
subphase, water. emission increases by increasing the molar ratio of PySin the spreading solution as well as by increasing the surface density. It is noteworthy that excimer emission is observed even for monolayers with a rather low molar ratio of PyS- such as 1:5 (Figure 2). If the PyS- were bound statistically to the DOMA head groups, we would not expect any excimer emission as the average distance between two pyrene molecules is too large. The fact that excimer emission is nevertheless observed shows that bound PyS- is mobile and hops from a bound site to an empty site until it encounters another PyS-, leading t o the excimer emission. The excimer formation probability decreases with increasing distance or lower amount of PyS- per DOMA. If the average distance between the PyS- is so large that during the excited state lifetime of pyrene no encounter between two pyrene molecules is possible,only monomer emission is expected. Accordingly, the introduction of a spacer molecule with negatively charged or neutral head groups in the DOMA monolayer should lead to the suppression of PyS- mobility resulting in the reduction of excimer emission. We have therefore tested this hypothesis using a mixed monolayer of PyS-I DOMA with DMPA in a molar ratio of 1:l. Here DMPA is dimyristoylphosphatidicacid with a negatively charged
Effect of Subphase Composition on Molecular Organization
Langmuir, Vol. 11, No. 2, 1995 571
PyS'I DOMA ( 1:l )
400
450
500
550
400
Wavelength [nm] Figure 4. Fluorescence spectra of the cospread monolayers: (1) F'vS-IDOMA 1:l and (2) (F'vS-IDOMA 1:l)IDMPA. 1:l; subphase, water; surface pressuie n = 30 mN/m.
0.4
0.6 0.8 1.o 1.2 Area / DOMA Molecule [nm2 ]
1.4
Figure 5. Surface pressure-molecular area (n-A) isotherms of the cospread PyS-IDOMA monolayers, molar ratio 1:1, on water (curve 1)and 100 mg/L of xanthan (curve 2). phosphate head group. The results are shown in Figure 4. It is seen that PyS- is still bound to the monolayer showing, however, only monomer emission. A drastic reduction in the excimer emission confirms the validity of the molecular organization model presented above. 3.2. pYS-/DOMA Cospread Monolayers on Subphases Containing Different Electrolytes. 3.2.1. Surface Pressure-Area Isotherms. The surface pressure molecular area (rt-A) isotherms of cospread PyS-/DOMA monolayers on water and xanthan subphase, respectively, are shown in Figure 5. Xanthan is a water soluble polysaccharide biopolymer of microbial origin having polyelectrolyte properties. The repeat unit consists of five sugars along with two carboxyl groups on a cellulose backbone.25 DOMAmonolayers on the xanthan subphase exhibit the characteristics of a liquid expanded monolayer without a phase transition (data not shown). Such a phenomenon (monolayer expansion and disappearance of phase transition) indicates an interaction of xanthan with the monolayer and may be interpreted in this case as a partial incorporation of polyelectrolyte molecule into the DOMA monolayer. Cospread monolayers of PyS-/DOMA on both water and xanthan subphases show no phase transition. A n expan(25) Rinaudo, M.;Milas, M.Carbohydr. Polym. 1982,2,264.
450
500
550
Wavelength [nm] Figure 6. Fluorescence spectra of the cospread monolayers of PyS-/DOMA, molar ratio 1:1,spread on different polyelectrolyte solutions: (1)water, (2)80 mg/L ofpolyacrylamide, (3) 70 mgL of polystyrene sulfonate, and (4) 100 mg/L of xanthan; surface pressure n = 30 mN/m. sion of the isotherm on xanthan in comparison to water is observed up to 18 mNlm. In the densely packed region, a small contraction (ca. 0.07 nm2) is observed. Such behavior indicates that xanthan molecules penetrate partially into the mixed monolayer at lower surface densities and are squeezed out of the densely packed monolayer. 3.2.2. Fluorescence Spectroscopy. The presence of different polyelectrolytes in the subphase also influences the formation of pyrene excimers. Three different polyelectrolytes have been tested. Polyacrylamide which is a neutral molecule does not affect excimer formation in the film (Figure 6, curve 2). In the presence of negatively charged polystyrene sulfonate and polysaccharide xanthan, excimer formation is strongly reduced while the monomer emission is enhanced (Figure 6 , curves 3 and 4) in comparison with PyS-/DOMA monolayer spread on pure water. Here we discuss the influence of xanthan on complex PyS-/DOMA monolayers. Depending on the PyS-/DOMA ratio and the packing of the monolayer, excimers are formed since pyrene molecules are close enough to interact during the excited state lifetime of pyrene. In the presence of a negatively charged polyelectrolyte in the subphase, both PyS- and the polyelectrolyte interact with DOMA resulting either in a competitive desorption of PyS- in the subphase or in trapping of PySin the adsorbed polyelectrolyte sublayer. In the PyS-/ DOMA monolayer on water, the formation of excimers is dependent on the concentration and mobility of PyS(Figure 2). In the case of the PyS-/DOMA monolayer on xanthan solution, where predominantly monomer emission is observed, the fluorescence intensity depends on the PyS-/DOMA ratio and on the lipid packing (Figure 7). To gain a better understanding of the electrostatic interactions between the positively charged DOMA head group and the negatively charged adsorbate, we have investigated the competitive influence of simple electrolytes in the subphase on the PyS-IDOMA interactions. The electrolytes that were investigated were NaC1, NaF, NaZC03, and NaC104. We purposely did not choose NaBr and NaI as both Br- and I- are known to quench the pyrene fluorescence. The concentration of the electrolytes in each case was 1 mM. Thus, a mixed solution of PyS-/DOMA was spread a t the subphase solution, and fluorescence spectra were recorded. The results, presented in Figure
572 Langmuir, Vol. 11, No. 2, 1995
Kozaracet al.
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