pubs.acs.org/Langmuir © 2010 American Chemical Society
Sum-Frequency Spectroscopic Study of Langmuir Monolayers of Lipids Having Oppositely Charged Headgroups Woongmo Sung,† Sangjun Seok,† Doseok Kim,*,† C. S. Tian,‡ and Y. R. Shen‡ †
Department of Physics, Sogang University, Seoul 121-742, Korea, and ‡Department of Physics, University of California at Berkeley, Berkeley, California 94720, United States Received August 6, 2010. Revised Manuscript Received October 8, 2010
Sum-frequency vibrational spectroscopy, with the help of surface pressure-area (π-A) isotherm, was used to study lipid Langmuir monolayers composed of molecules with positively and negatively charged headgroups as well as a 1:1 neutral mixture of the two. The spectral profiles of the CHx stretch vibrations are similar for all monolayers in the liquidcondensed (LC) phase. They suggest a monolayer structure of closely packed alkyl chains that are nearly all-trans and well oriented along the surface normal. In the liquid-expanded (LE) phase, the spectra of all monolayers appear characteristic of loosely packed chains with significant gauche defects. The OH stretch spectra of interfacial water for both positively and negatively charged monolayers are significantly enhanced in comparison with a neutral water interface, but the phase measurement of SFVS indicates that OH in the two cases points toward the bulk and the interface, respectively. The enhancement results mainly from surface-field-induced polar ordering of interfacial water molecules. For a charge-neutral monolayer composed of an equal number of positively and negatively charged lipid molecules, no such enhancement is observed. This mixed monolayer exhibits a wide range of LC/LE coexistence region extended to very low surface pressure and its CHx spectral profile in the coexistence region resembles that of the LC phase. This result suggests that in the LC/LE coexistence region, the mixed monolayer consists of coexisting LC and LE patches in which oppositely charged lipid molecules are homogeneously mixed and dispersed.
1. Introduction In biological applications such as nonviral transfection and manufacturing of the bio membrane which reacts with specific kinds of proteins or molecules, detailed knowledge of the electrostatic condition at the interface is essential.1-3 From a theoretical perspective, the Gouy-Chapman theory has been used to describe electrostatics at the interfaces of electrolyte solutions. Application of the theory, however, is limited as the Poisson-Boltzmann (PB) equation, the mathematical representation of the GouyChapman theory, can only be solved analytically for limited cases.4 As model systems for experimental studies of electrostatic interfacial problems near a membrane, Langmuir monolayers have been commonly used with surface charges controlled by the charged headgroups on the amphiphilic molecules.5 Many experimental techniques such as X-ray, neutron scattering, capacitance measurement of diffusion double layer have been employed to investigate such interfaces.6,7 More recently, counterion adsorption to the headgroups of phospholipids in Langmuir monolayer and charge inversion phenomenon were investigated by X-ray fluorescence and reflectivity experiments.8-11 Interfacial
surface charge density profile for different counterion species in aqueous solution was observed by AFM.12 Due to their large permanent dipole moment, water molecules can be rather easily reoriented by a local field and, therefore, can act as a sensitive probe of the local electric field. It is, however, challenging to probe interfacial water molecules in the presence of bulk water because the depth over which the electric field is appreciable is often less than several nanometers for a few-mM salt concentration.13 As a second-order nonlinear optical process, sum-frequency generation (SFG) from an isotropic bulk is forbidden but is allowed at surfaces or interfaces where inversion symmetry is broken.14,15 Thus sum-frequency spectroscopy has been developed to study electronic and vibrational spectra of various surfaces and interfaces, in particular, vibrational spectra of liquid interfaces.16-19 For example, Richmond and co-workers used sum-frequency vibrational spectroscopy (SFVS) to probe orientation of water molecules at the CCl4/water interface.20 Interaction of water molecules with self-assembled monolayers17 and adsorbed proteins21 at air/water interfaces has been studied. The specific ion effect on water structure near a monolayer of polymer adsorbed at an air/water interface has also been investigated.22
*Corresponding author. E-mail:
[email protected]. (1) Ben-Tal, N.; Honig, B.; Miller, C.; McLaughlin, S. Biophys. J. 1997, 73, 1717. (2) Murray, D.; Honig, B. Mol. Cell 2002, 9, 145. (3) Wurpel, G. W. H.; Sovago, M.; Bonn, M. J. Am. Chem. Soc. 2007, 129, 8420. (4) Butt, H. J.; Graf, K.; Kappl, M. Physics and Chemistry of Interfaces, 2nd ed.; Wiley-VCH: New York, 2006. (5) Roberts, G. Langmuir-Blodgett Films; Plenum Press, New York and London, 1990. (6) Bard, A. J.; Faulkner, L. R. Electrochemical methods - Fundamentals and Applications; Wiley & Sons: New York, 2001. (7) Schalke, M.; Losche, M. Adv. Colloid Interface Sci. 2000, 88, 243. (8) Bu, W.; Vaknin, D. J. Appl. Phys. 2009, 105, 084911. (9) Pittler, J.; Bu, W.; Vaknin, D.; Travesset, A.; McGillivray, D. J.; Losche, M. Phys. Rev. Lett. 2006, 97, 046102. (10) Vaknin, D.; Bu, W. J. Phys. Chem. Lett. 2010, 1, 1936. (11) Kewalramani, S.; Hlaing, H.; Ocko, B. M.; Kuzmenko, I.; Fukuto, M. J. Phys. Chem. Lett. 2010, 1, 489.
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(12) Besteman, K.; Zevenbergen, M. A. G.; Heering, H. A.; Lemay, S. G. Phys. Rev. Lett. 2004, 93, 170802. (13) Bu, W.; Vaknin, D.; Travesset, A. Langmuir. 2006, 22, 5673. (14) Boyd, R. W. Nonlinear Optics, 2nd ed.; Academic Press: New York, 2003. (15) Shen, Y. R. The Principles of Nonlinear Optics; Wiley & Sons: New York, 2003. (16) Guyot-Sionnest, P.; Hunt, J. H.; Shen, Y. R. Phys. Rev. Lett. 1987, 59, 1597. (17) Ye, S.; Nihonyanagi, S.; Uosaki, K. Phys. Chem. Chem. Phys. 2001, 3, 3463. (18) Gan, W.; Wu, D.; Zhang, Z.; Feng, R. R.; Wang, H. F. J. Chem. Phys. 2006, 124, 114705. (19) Miranda, P. B.; Shen, Y. R. J. Phys. Chem. B 1999, 103, 3292. (20) Brown, M. G.; Raymond, E. A.; Allen, H. C.; Scatena, L. F.; Richmond, G. L. J. Phys. Chem. A 2004, 104, 10220. (21) Wang, J.; Buck, S. M.; Chen, Z. J. Phys. Chem. B 2002, 106, 11666. (22) Chen, X.; Yang, T.; Kataoka, S.; Cremer, P. S. J. Am. Chem. Soc. 2007, 129, 12272.
Published on Web 10/26/2010
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Figure 1. Molecular structures and π-A isotherms of (a) DPTAP, (b) DMPG, and (c)1:1 DPTAP/DMPG, mixture.
In deducing polar orientation of interfacial water molecules, conventional SFVS relies on fitting of the observed SF intensity spectra. It was recently shown that this could lead to erroneous results. Recently, direct measurement of polar molecular orientations has become possible with the introduction of phase-sensitive sum-frequency spectroscopic techniques.23-27 In this report, we present a SFVS study of lipid monolayers adsorbed at air/water interfaces. Positively charged 1, 2-dipalmitoyl-3-trimethylammonium-propane (DPTAP) and negatively charged 1,2 dimyristoyl-sn-glycero-3-phospho-(10 -racglycerol) (DMPG) Langmuir monolayers as well as a 1:1 DPTAP/DMPG mixed monolayer were prepared. The structures of these monolayers with different areas per molecule were probed by the CHx stretch modes in the SF vibrational spectra, and the effects of their surface charges on interfacial water structure were monitored by the OH stretch bands of water. It was found that all monolayers exhibited in the CHx stretch region a spectral profile characteristic of layers of well-ordered alkyl chains in the liquidcondensed (LC) phase and more disordered alkyl chains in the liquid-expanded (LE) phase. In the case of charged monolayers, the surface electric field created by the surface charges significantly enhanced the OH spectrum through field-induced polar ordering of the interfacial water molecules. Positive and negative surface charges tended to reorient water molecules with their oxygen and hydrogen terminals, respectively, facing the interface. For the 1:1 DPTAP/DMPG mixed monolayer, the absence of net surface charges was reflected in the much weaker OH spectrum. Its CHx spectral profile in the LC/LE coexistence region resembled that of a monolayer in the pure LC phase, although the absolute intensity is weaker. This together with the very low surface pressure suggested that the monolayer was composed of coexisting LC and LE patches with positively and negatively charged lipid molecules homogeneously mixed and dispersed in the patches.
2. Experimental Section The lipid molecules were purchased from Avanti Polar Lipids. Their chemical structures are shown in the inset of Figure 1. DPTAP has a trimethylammonium headgroup (with pKa ∼9.5) and DMPG a phosphate headgroup (with pKa ∼2.2). Adsorbed (23) Shen, Y. R.; Ostroverkhov, V. Chem. Rev. 2006, 106, 1140. (24) Ostroverkhov, V.; Waychunas, G. A.; Shen, Y. R. Phys. Rev. Lett. 2005, 94, 046102. (25) Ji, N.; Ostroverkhov, N.; Tian, C. S.; Shen, Y. R. Phys. Rev. Lett. 2008, 100, 096102. (26) Tian, C. S.; Ji, N.; Waychunas, G. A.; Shen, Y. R. J. Am. Chem. Soc. 2008, 130, 13033. (27) Nihonyanagi, S.; Yamaguchi, S.; Tahara, T. J. Chem. Phys. 2009, 130, 204704.
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on pure water (pH ∼5.7 for being exposed to air), the headgroups of DPTAP and DMPG are nearly all protonated and deptronated, respectively, forming positively and negatively charged monolayers.28,29 For a fully compressed Langmuir monolayer, the surface charge density is expected to be about e/40 A˚2. The lipid molecules were dissolved in pure chloroform (HLPC grade, Aldrich) with a concentration of ∼1.2 mM, and then spread onto ultrapure water (18.2 MΩ-cm) in a Langmuir trough. In the case of 1-hexadecanol, a small flake of 1-hexadecanol solid was deposited on water, and the molecules spread spontaneously to form a full monolayer. The π-A isotherms for the lipid monolayers in Figure 1 were obtained using a barrier speed of ∼0.58 A˚2/ min per molecule. The barrier in the Langmuir trough was controlled to maintain a constant surface pressure during the sum-frequency measurement. The trough was kept at room temperature (21 C) during measurements. The SFVS experiment was conducted using a picosecond Nd: YAG laser (Continuum PY61-10, 40 ps pulse width, 10-Hz repetition rate) together with a LiNbO3-based optical parametric generator/amplifier (OPG/OPA) system pumped by the laser.30,31 The OPG/OPA system provided the tunable IR input from 2.5 to 4 μm, and the second harmonic of the Nd:YAG laser the visible input (532 nm). Typical input energies were 1 mJ/pulse for visible and ∼200 μJ/pulse for tunable IR input, and incident angles were βvis = 45 and βIR = 60, respectively. The two beams overlapped within a beam diameter of ∼200 μm on the sample surface The SF output in the reflection direction was spatially and spectrally filtered and detected by a photomultiplier tube. Typically, the spectra were taken at 5 cm-1 interval, and each data point was the average of signals accumulated over at least 200 laser shots. The spectra were normalized against the sum-frequency spectrum from a z-cut quartz sample. Input/output polarization combination of SSP (denoting S-, S-, and P-polarized SF output, visible input, and IR input, respectively) was employed in all measurements except for the case of 1:1 mixture of DPTAP/DMPG where PPP polarization combination was also used.
3. Result and Discussion The surface pressure-area (π-A) isotherms of DPTAP, DMPG, and 1:1 DPTAP/DMPG Langmuir monolayers are displayed in Figure 1. Those for the first two are similar to the ones reported in the literature.32-34 The rapid rise of the surface pressure as A decreases below ∼50 and ∼60 A˚2/molecule in the cases of DMPG and 1:1 DMPG/DPTAP mixture, respectively, is a signature that the monolayers have entered the liquidcondensed (LC) phase. Collapse of the two monolayers occurs at ∼47 and ∼43 A˚2/molecule, respectively. The nearly constant pressure portion of each curve describes the liquid-condensed (LC)/liquid-expanded (LE) coexistence region, which connects to the LE phase as A further increases. For the DPTAP monolayer, the LC/LE coexistence region is not clearly defined, but the system is expected to be in pure LC and LE phases for A e 50 and g80 A˚2/molecule, respectively. At a given area per molecule, the surface pressure for charged DPTAP and DMPG monolayers are significantly higher than the neutral 1:1 DMPG/DPTAP mixture. Clearly, Coulomb repulsion between charged headgroups (28) Lide, D. R. CRC Handbook of Chemistry and Physics; CRC Press: Boca Raton, FL, 2000-2001. (29) The pKa values for DMPG and DPTAP headgroups indicate that both headgroups should be nearly completely ionize in water at pH ∼5.6. This was confirmed by SFVS measurements with pH titration showing that the OH SFG is maximum at pH 5.6 for both samples. (30) Sung, J.; Kim, D. J. Phys. Chem. C 2007, 111, 1783. (31) Jeon, Y.; Sung, J.; Bu, W.; Vaknin, D.; Ouchi, Y.; kim, D. J. Phys. Chem. C 2008, 112, 19649. (32) Lee, K. Y. C. Annu. Rev. Phys. Chem. 2008, 59, 771. (33) Gzyl-Malcher, B.; Filek, M.; Brezesinski, G. Langmuir. 2009, 25, 13071. (34) Garidel, P.; Blume, A. Chem. Phys. Lipids. 2005, 138, 50.
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Figure 2. SSP Sum-frequency spectra of (a) DPTAP/water, (b) DMPG/water, (c) 1:1 DPTAP/DMPG, on water, and (d) 1-hexadecanol/ water with the SSP polarization combinations. Solid lines are fits using eq 1. Red and blue lines describe Im χ(2) components of CHx (rþ, rFRþ, r-), and OH stretch resonances, respectively.
in the cases of DPTAP and DMPG is the reason behind the higher surface pressure. Figure 2 shows the SFVS spectra in the CH- and OH- stretch region for DPTAP, DMPG, and 1:1 DMPG/DPTAP Langmuir monolayers (all with a surface area per molecule of ∼50 A˚2) in comparison with that of 1-hexadecanol monolayer on water. With such a surface density, all three monolayers are presumably in the LC phase, and the alkyl chains of the closely packed molecules are well aligned along the surface normal with little gauche defects.35 Indeed, all SSP spectra in Figure 2 exhibit two prominent peaks at 2875 and 2934 cm-1 that can be identified as CH3 symmetric stretch (rþ) and Fermi resonance (rFRþ) between CH3 stretch and its bending overtone; they are characteristic of alkyl chains with nearly all-trans conformation.36-39 We note that in the case of DPTAP, we would expect the trimethyl group associated with the headgroup to also contribute to the CH3 stretch spectrum. However, we found the CHx spectra for DPTAP and DMPG monolayers are essentially the same in the LC phase (after eliminating interference from the OH spectrum as we shall discuss later), suggesting that the trimethyl contribution is negligible. Similar result was reported in a previous study on CTAB (centro-methyl ammonium bromine that also has a trimethyl group in the headgroup) and SDS (sodium dodecyl sulfate) monolayers on water.27 We believe that the trimethyl group is oriented in such a way that their symmetric axis is close to the surface plane in order to minimize its interaction with water and maximize the interaction of Nþ with water. As a result, its contribution to the SSP SF signal becomes small. In addition,
because the group is buried at the lipid/water contact region, the local field factor at the trimethyl group appears to be ∼0.7 times smaller than those at the methyl terminal of the alkyl chain, making the contribution of the trimethyl contribution to the SF signal relatively even weaker.37 A more careful analysis of the spectra in this region, to be discussed later, yields three other CHx stretch modes: CH3 antisymmetric stretch (r-: 2958 cm-1), CH2 symmetric stretch (dþ: 2855 cm-1) and CH2 antisymmetric stretch (d-: 2920 cm-1). Contribution to the spectra from interfacial water comes in the range between 3000 and 3750 cm-1. As often seen in SF vibrational spectra of water interfaces, two broad bands peaked at ∼3200 and ∼3420 cm-1 appear resulting from the inhomogeneously broadened OH stretch modes of hydrogenbonded interfacial water molecules.40,41 The strengths of the OH spectra for charged DPTAP and DMPG monolayers are much stronger than those of the neutral 1:1 DMPG/DPTAP and 1-hexadecanol monolayers, indicating that the charged lipid headgroups can effectively enhance polar ordering of the interfacial water molecules in the former cases.42 The much weaker OH spectrum of the 1:1 DMPG/DPTAP charge-neutral monolayer is typical of that of a neutral water interface. This seems different from that of the DPPC(dipalmitoylphosphatidylcholine)/water interface reported recently. The zwitterionic headgroup of DPPC is supposed to be neutral.43 but the DPPC/water spectrum suggested that the interfacial water molecules were appreciably polar-ordered. It was proposed that water molecules between cationic and anionic parts of the headgroup could have polar oriented to give rise to the strong OH spectrum.43 However,
(35) Kaganer, V. M.; Mohwald, H.; Dutta, P. Rev. Mod. Phys. 1999, 79, 779. (36) Zhuang, X.; Miranda, P. B.; Kim, D.; Shen, Y. R. Phys. Rev. B 1999, 59, 12632. (37) Sefler, G. A.; Du, Q.; Miranda, P. B.; Shen, Y. R. Chem. Phys. Lett. 1995, 235, 347. (38) Lu, R.; Gan, W.; Wu, B. H.; Chen, H.; Wang, H. F. J. Phys. Chem. B 2004, 108, 7297. (39) MacPhail, R. A.; Straws, H. L.; Snyder, R. G. J. Phys. Chem. 1984, 88, 334.
(40) Wei, X.; Miranda, P. B.; Zhang, C.; Shen, Y. R. Phys. Rev. B 2002, 66, 085402. (41) Du, Q.; Superfine, R.; Freysz, E.; Shen, Y. R. Phys. Rev. Lett. 1993, 70, 2313. (42) Watry, M. R.; Tarbuck, T. L.; Richmond, G. L. J. Phys. Chem. B 2003, 107, 512. (43) Chen, X.; Hua, W.; Huang, Z.; Allen, H. C. J. Am. Chem. Soc. 2010, 132, 11336.
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Table 1. Fitting Parameters for Sum-Frequency Spectra of DPTAP/Water, DMPG/Water, C16OH/Water, and (DPTAPþDMPG)/Water Aq (arb. units) -1
mode (wavenumber)
Γq (cm )
DPTAP
DMPG
C16OH
DPTAPþDMPG
dþ, 2855 cm-1 rþ, 2875 cm-1 d-, 2920 cm-1 rFRþ, 2934 cm-1 r-, 2958 cm-1 OHicelike, 3200 cm-1 OHliquidlike, 3420 cm-1
9.4 6.8 10 7.8 9.0 130 130
0.9 ( 0.4 4.5 ( 0.2 -0.8 ( 0.8 4.6 ( 0.4 -2.9 ( 1.2 80 ( 2 44 ( 2.6
0.9 ( 0.7 5.0 ( 0.3 -0.8 ( 0.8 4.9 ( 0.6 -3.0 ( 0.3 -57 ( 5 -38 ( 3.2
2.1 ( 0.5 6.6 ( 0.4 -0.1 ( 1.1 6.5 ( 1.0 -3.3 ( 1.3 3.5 ( 9.1 1.4 ( 12.
1.9 ( 0.3 6.2 ( 0.4 0.5 ( 1.0 5.4 ( 0.6 -3.1 ( 0.7 23 ( 15 12 ( 10
it could also be that cationic and anionic groups have different degrees of ionization. In our case, at pH ∼ 5.7, the headgroups of both DMPG and DPTAP are nearly all-ionized29 and the two molecules must be homogeneously dispersed, yielding a monolayer that is truly charge-neutral. Between the SF intensity spectra of DPTAP and DMPG in Figure 2a,b, there are also some clear differences, namely, a dip at 2950 cm-1 and a much stronger background in the former in comparison with the latter. They must result from opposite polar orientations of the interfacial water molecules in the two cases. In the spectral region where CH and OH modes overlap, interference of the two contributions modifies the intensity spectrum, and is expected to be different when the OH contribution switches sign because of change of the polar orientation. For quantitative analysis, we assume each spectrum is composed of discrete resonances and use the following expression to fit the SF spectra.23 2 Aq ð2Þ X Iðω ¼ ωIR þ ωVIS Þ χNR þ ωIR - ωq þ iΓq q ð2Þ
ð2Þ
¼ jχNR þ χR j2
ð1Þ
where, χ(2) NR denotes the nonresonant contribution and ωq, Aq, and Γq are the frequency, the amplitude, and the damping constant of the qth resonance, respectively. The amplitude Aq is related to the resonant amplitude aq of the nonlinear hyperpolarizability R(2) of the molecules in the same way as is χ(2) related to R(2) by ð2Þ
χi, j, k Ns
X ξ, η, ζ
ð2Þ Æði^3 ξÞðj^3 ηÞðk^ 3 ζÞæRξ, η, ζ
ð2Þ
where (i,j,k) and (ξ,η,ζ) refer to the lab and molecular coordinates, respectively, and Ns is the surface molecular density. We note that in the OH stretch region, the assumption of discrete resonances is only an approximation as the inhomogeneously broadened OH resonances actually form a continuum. Results of our fitting are shown in Figure 2, together with the deduced Im χ(2) spectrum that directly characterizes the resonances in each case. Two OH, three CH3 (rþ, r-, and rFRþ), and two CH2 (dþ and d-) modes were used in the fitting. The fitting parameters are listed in Table 1. It is seen that Aqs of the OH bands for the cases of DPTAP and DMPG are much larger than those for the two other cases because of surface-field-induced polar ordering of interfacial water molecules. However, they must be of opposite signs for the fitting to be satisfactory. As seen in eq 2, the opposite signs correspond to the opposite polar orientations of OH (water dipole pointing in opposite directions). This is expected because DPTAP and DMPG have oppositely charged headgroups that create oppositely directed surface fields. Physically, the positive surface charges of the DPTAP monolayer should tend to reorient OH of interfacial water molecules toward the water bulk, opposite in Langmuir 2010, 26(23), 18266–18272
direction to the orientation of the alkyl chain or CH3. However, we notice that Aqs of CH3 (rþ) and the OH bands appear to have the same sign. This is because the signs of aqs of the CH3(rþ) and OH modes are opposite, as has been proven in our phase-sensitive SFVS measurement [See also ref 27]. In this respect, the negatively charged DMPG monolayer on water should induce OH of interfacial water molecules toward the interface, and Aqs of CH3 (rþ) and the OH bands should have opposite signs, as was indeed observed. The dip at 2950 cm-1 and the appreciable background around it in the DPTAP/water spectrum of Figure 3a results mainly from interference of the positive CH3(r-) peak with the negative CH3(rþ) peak and the negative shoulder of the OH band around 2950 cm-1. Such an interference effect in SFVS spectra was observed before, and used to deduce the relative orientation of the molecules.17,21,27 To further confirm the results of spectral analysis, we measured directly the phase of SF output by recording its interference with a SF reference beam.26 The latter was generated from a y-cut quartz plate in the path of the IR- and visible input beams that generated SF signal from the sample. The collinearly propagating signal and reference beams should interfere. By inserting a variable retardation plate in the reference beam path to vary the phase of the reference beam relative to the signal, we obtained an interference fringe pattern that allowed us to determine unambiguously the phase of the SF signal. We did the phase measurement at four IR frequencies in the spectra; 2940 cm-1 (near the CH3 Fermi resonance), 3050 cm-1 (left shoulder of the lower-frequency OH band), 3200 cm-1 (near the peak of the lower-frequency OH band), 3500 cm-1 (near the peak of the higher-frequency OH band). Figure 3 shows the fringe patterns obtained from (a) DPTAP/water, (b) DMPG/water, and (c) from a z-cut quartz plate (served as another reference) in the sample position. Phase reversal between (a) and (b) is clearly seen from the fringe patterns at all frequencies except 2940 cm-1 (black circles) where the SF signal is dominated by the mode of CH3(rþFR) in both cases. Together with the measured SF intensity in Figure 3c, the phase information allows determination of the real and imaginary parts of the sum-frequency signal at these frequencies. The results agree with those shown in Figure 2 obtained from fitting. This phase reversal of OH modes provides direct evidence that the net polar orientation of water molecules at the DPTAP/water interface is opposite to that of the DMPG/water interface. Inversion of polar orientation of water molecules at charged surfactant/water interfaces was also observed recently by Tahara’s and Allen’s groups using phase-sensitive SFG measurement.43,44 We now focus on what we can learn about the structure of the lipid Langmuir monolayers from SF vibrational spectra with the help of the π-A isotherm in Figure 1. Presented in Figure 4 are the SSP spectra of DPTAP and DMPG Langmuir monolayers on water for four different surface areas per molecule prepared by (44) Nihonyanagi, S.; Yamaguchi, S.; Tahara, T. J. Am. Chem. Soc. 2010, 132, 6867.
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Figure 3. Measured interference fringes of the sum-frequency output from (a) DPTAP/water, (b) DMPG/water, and (c) z-cut quartz.
Figure 4. SSP Sum-frequency spectra from (a) DMPG/water and (b) DPTAP/water with different surface occupation areas per molecule.
compression of the monolayers. In the DMPG case, the π-A isotherm indicates that the monolayer is in the LC phase for A < 60 A˚2/molecule, in the LC-LE coexistence region between roughly 60 and 80 A˚2/molecule (characterized by the plateau on the π-A curve), and in the LE phase for A > 80 A˚2/molecule. The SF spectra in Figure 4a exhibit corresponding characteristic features in different regions. At 50 A˚2/molecule, it appears to be dominated by the CH3 stretch modes, indicating that the alkyl chains are closely packed and nearly all-trans without much gauche defect, a clear signature of a monolayer in the LC phase.16 At 60 A˚2/molecule, the spectral profile remains unchanged, indicating that the monolayer is still in the LC phase, although the spectral intensity is reduced because of the reduced surface density. At 80 and 120 A˚2/molecule, the monolayer is in the LE phase. The CH3 modes in the spectra decrease appreciably while the CH2 (dþ) mode at 2855 cm-1 becomes clearly visible, denoting the presence of significant gauche defects in the alkyl chains. The larger surface area per molecule allows the chains to have room for defects to form. Such behavior is typical of Langmuir monolayers with long alkyl chains in the LE phase, and is reflected in the SF spectrum. (See ref 16). Note that each lipid molecule has two alkyl chains, and therefore the surface area per chain is half the value of the surface area per molecule.) In the DPTAP case, the π-A curve 18270 DOI: 10.1021/la103129z
does not have a plateau to clearly mark the LC-LE coexistence region, and the SF spectrum (Figure 4b) can be used to help. At 50 A˚2/molecule, the spectrum exhibits prominent CH3 modes, indicating that the monolayer is mainly in the LC phase. At 60 A˚2/ molecule, the intensity of the CH3 modes drops appreciably and the CH2(dþ) mode grows to a comparable level, which is an indication that the monolayer is well into the LC-LE coexistence region with a significant fraction of the DPTAP molecules in the LE phase. At 80 and 120 A˚2/molecule, the spectra appearing similar to those of DMPG show that the monolayer must be in the LE phase. The strong background under the CHx peaks in Figure 4b comes from the shoulder of the neighboring strong OH band. As depicted in Figure 5a, the SF SSP spectra of the 1:1 DPTAP/ DMPG Langmuir monolayer appear very different from those of DPTAP and DMPG monolayers at the same surface areas per molecule. At 60 A˚2/molecule, the monolayer is clearly in the LC phase since the state is above the plateau in its π-A curve in Figure 1, and its SSP SF spectrum does resemble that of a DPTAP Langmuir monolayer (with small amount of defects) in the LC phase. At 80 and 100 A˚2/molecule, the monolayer is in the LC/LE coexistence region characterized by the plateau of the π-A curve in Figure 1. In the coexistence region, the monolayer is composed of coexisting LC and LE patches. The spectra at 80 and 100 A˚2/ Langmuir 2010, 26(23), 18266–18272
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Figure 5. Sum-frequency spectra from 1:1 DPTAP/DMPG, on water with different surface areas pre molecule. (a) SSP polarization combination and (b) PPP polarization combination. Table 2. Fitting Parameters for SSP Sum-Frequency Spectra of 1:1 DPTAP/DMPG Mixture on Water with Varying Surface Area Aq (arb. units) -1
mode (wavenumber)
Γq (cm )
120 A˚
100 A˚
80 A˚2
60 A˚2
dþ, 2855 cm-1 rþ, 2875 cm-1 d-, 2920 cm-1 rFRþ, 2934 cm-1 r-, 2958 cm-1
9.4 6.8 10 7.8 9.0
0.8 ( 0.1 1.5 ( 0.1 1.2 ( 0.2 1.7 ( 0.2 -1.1 ( 0.2
0.6 ( 0.1 2.3 ( 0.1 1.0 ( 0.1 2.1 ( 0.1 -1.1 ( 0.1
0.3 ( 0.2 2.6 ( 0.1 0.7 ( 1.2 2.4 ( 0.1 -2.0 ( 0.2
-0.2 ( 0.2 3.5 ( 0.2 1.1 ( 0.3 3.0 ( 0.2 -2.6 ( 0.2
2
2
Table 3. Fitting Parameters for PPP Sum-Frequency Spectra of 1:1 DPTAP/DMPG Mixture on Water with Varying Surface Area Aq (arb. Unit) mode (wavenumber)
Γq (cm-1)
120 A˚2
100 A˚2
80 A˚2
60 A˚2
dþ, 2855 cm-1 rþ, 2875 cm-1 d-, 2920 cm-1 rFRþ, 2934 cm-1 r-, 2958 cm-1
9.4 6.8 10 7.8 9.0
-0.7 ( 0.1 -0.4 ( 0.1 0.9 ( 0.2 -1.0 ( 0.1 2.0 ( 0.1
-0.3 ( 0.2 -1.1 ( 0.1 0.6 ( 0.2 -1.4 ( 0.2 2.2 ( 0.1
-0.4 ( 0.3 -1.4 ( 0.1 0.9 ( 0.4 -1.8 ( 0.3 3.5 ( 0.1
-0.4 ( 0.4 -1.5 ( 0.1 1.1 ( 0.3 -2.3 ( 0.2 4.5 ( 0.1
molecule, however, still resemble that of a monolayer in the pure LC phase, though with reduced intensities. This can be understood if the LE phase has a much lower surface density than the LC phase so that the observed SF spectrum is dominated by the LC patches. At 120 A˚2/molecule, the spectrum appears more like that of a DMPG monolayer in the LE phase.45 It suggests that the monolayer must be either in the LE-dominated LC/LE coexistence region or in the pure LE phase. To be sure that the SF spectra for 1:1 DPTAP/DMPG Langmuir monolayers of
