A full description of coexisting phases includes their respective compositions, which are provided by the thermodynamic tie-lines. Fluorescence microscopy enables visualization of coexisting lipid phases in giant unilamellar phases, but the composition information is missing. For cholesterol-containing lipid mixtures, knowledge of the compositions of the coexisting phases is important for understanding the nature of "membrane rafts". We propose and demonstrate a new method, based on ESR spectroscopy, for determining tie-lines in regions of two-phase coexistence in a ternary lipid mixture. Over 100 different lipid compositions containing the spin-labeled phospholipid 16-PC in or near the two-phase coexistence region of the liquid-disordered and the gel phases of dipalmitoyl-PC/dilauroyl-PC/cholesterol (DPPC/DLPC/Chol) were studied to determine five tie-lines, spread over virtually the full range of this coexistence region. The method is based on the facts that (1) along a tie-line the ESR spectrum must be a superposition of the two ESR spectra from the respective single phases at the phase boundaries (connected by the tie-line) in a ratio given by the lever rule; (2) along a tie-line the partition coefficient, K(p), for the spin-label, which is also determined in our method, must be constant. We do find that K(p) for 16-PC is close to unity, but its value depends on the particular tie-line. The coexisting phases in equilibrium are characterized by the K(p) of the spin-label and its respective dynamic parameters obtained from fitting the ESR spectra to dynamical models.
The hydrated ternary lamellar lipid mixture of dipalmitoyl-PC/dilauroyl-PC/cholesterol (DPPC/DLPC/Chol) has been studied by electron spin resonance (ESR) to reveal the dynamic structure on a molecular level of the different phases that exist and coexist over virtually the full range of composition. The spectra for more than 100 different compositions at room temperature were analyzed by nonlinear least-squares fitting to provide the rotational diffusion rates and order parameters of the end-chain labeled phospholipid 16-PC. The ESR spectra exhibit substantial variation as a function of composition, even though the respective phases generally differ rather modestly from each other. The Lalpha and Lbeta phases are clearly distinguished, with the former exhibiting substantially lower ordering and greater motional rates, whereas the well-defined Lo phase exhibits the greatest ordering and relatively fast motional rates. Typically, smaller variations occur within a given phase. The ESR spectral analysis also yields phase boundaries and coexistence regions which are found to be consistent with previous results from fluorescence methods, although new features are found. Phase coexistence regions were in some cases confirmed by observing the existence of isosbestic points in the absorption mode ESR spectra from the phases. The dynamic structural properties of the DPPC-rich Lbeta and DLPC-rich Lalpha phases, within their two-phase coexistence region do not change with composition along a tie-line, but the ratio of the two phases follows the lever rule in accordance with thermodynamic principles. The analysis shows that 16-PC spin-label partitions nearly equally between the Lalpha and Lbeta phases, making it a useful probe for studying such coexisting phases. Extensive study of two-phase coexistence regions requires the determination of tie-lines, which were approximated in this study. However, a method is suggested to accurately determine the tie-lines by ESR.
The growth process of self-assembled monolayers (SAMs) of thiophene on Au(111) surfaces was revealed by Fourier-transform infrared reflection absorption spectroscopy (FT-IR-RAS). Thiophene spontaneously adsorbs from an ethanol solution onto the gold surface, and forms well-ordered SAM. FT-IR-RAS measurements and Langmuir adsorption isotherms revealed that thiophene SAMs possess two phases with different molecular orientations during SAM growth. In the primary phase, thiophene orients parallel to the gold surface. In the final phase, the molecular orientation changes from parallel to upright configurations with respect to the surface. This suggests that even molecules without alkyl-chain moiety can reorient during SAM growth. A transition of the molecular orientation is caused by a balance between thiophene-thiophene and thiophene-gold interactions.
2D-ELDOR spectroscopy has been employed to study the dynamic structure of the liquid-ordered (Lo) phase versus that of the liquid-crystalline (Lc) phase in multibilayer phospholipid vesicles without (Lc) and with (Lo) cholesterol, using end-chain and headgroup labels and spin-labeled cholestane. The spectra are in most cases found to be dramatically different for these two phases. Thus, visual inspection of the 2D-ELDOR spectra provides a convenient way to distinguish the two phases in membranes. Detailed analysis shows these observations are due to increased ordering in the Lo phase and modified reorientation rates. In the Lo phase, acyl chains undergo a faster rotational diffusion and higher ordering than in the Lc phase, whereas spin-labeled cholestane exhibits slower rotational diffusion and higher ordering. On the other hand, the choline headgroup in the Lo phase exhibits faster motion and reduced but realigned ordering versus the Lc phase. The microscopic translational diffusion rates in the Lo phase are significantly reduced in the presence of cholesterol. These results are compared with previous studies, and a consistent model is provided for interpreting them in terms of the differences in the dynamic structure of the Lo and Lc phases.
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