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Structure of stratum corneum lipids characterized by FT-Raman spectroscopy and DSC. II. Mixtures of ceramides and saturated fatty acids
Chemistry and Physics of Lipids 89 (1997) 3 – 14
Structure of stratum corneum lipids characterized by FT-Raman spectroscopy and DSC. II. Mixtures of ceramides and saturated fatty acids Reinhard Neubert a,*, Willi Rettig a, Siegfried Wartewig b, Matthias Wegener a, Antje Wienhold a a
Department of Pharmacy, Martin-Luther-Uni6ersity Halle-Wittenberg, Wolfgang-Langenbeck-Str. 4, D-06120 Halle/Saale, Germany b Institute of Applied Dermatopharmacy, Wolfgang-Langenbeck-Str. 4, D-06120 Halle, Germany Received 3 February 1997; accepted 24 May 1997
Abstract Fourier transform (FT) Raman spectroscopy and differential scanning calorimetry (DSC) were used to study the thermotropic phase behaviour of mixtures of ceramides type IV (CER) and stearic acid (SA). For comparison the melting behaviour of SA was re-examined. The Raman spectra of all mixtures in the solid state show sharp bands associated with trans sequencies of the alkyl chain residues of both lipids. These features demonstrate that the hydrocarbon chains are highly ordered in the mixtures, too. The temperature dependence of the conformationally sensitive bands is used to estimate the degree of order in terms of the relative population of trans and gauche conformations. The DSC heating curves for the mixtures show two endothermic transitions which are typical for eutectic melting. The factor group splitting of the CH2 scissoring mode, arising from the orthorhombic subcell packing of SA, disappears in the course of the eutectic melting of samples with a SA content lower than 90 mol%. Both DSC and Raman spectroscopic studies reveal that CER and SA are immiscible in the solid state. The phase diagram of the system is a simple eutectic type one. The addition of SA to CER shifts the melting temperature of ceramides to lower values. However, though SA is a major component of stratum corneum (SC) it is not efficient enough to increase the fluidity of ceramides. © 1997 Elsevier Science Ireland Ltd. Keywords: Stratum corneum; Ceramides; Stearic acid; Phase behaviour; Raman spectroscopy; DSC
* Corresponding Author. Tel.: + 49 345 5525000; fax: + 49 345 5527021. 0009-3084/97/$17.00 © 1997 Elsevier Science Ireland Ltd. All rights reserved. PII S 0 0 0 9 - 3 0 8 4 ( 9 7 ) 0 0 0 4 9 - 2
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R. Neubert et al. / Chemistry and Physics of Lipids 89 (1997) 3–14
Fig. 1. Raman spectra of various mixtures of ceramides type IV and stearic acid at 40°C: (a) ceramides IV; (b) 48 mol% CER/52 mol% SA; (c) 19 mol% CER/81 mol% SA; (d) stearic acid in different spectral ranges: (i) low frequency range 100 – 250 cm − 1; (ii) 850–1550 cm − 1; and (iii) C–H stretching range.
1. Introduction For elucidating the very complex nature of stratum corneum (SC), the outermost layer of the skin, our leading concepts are: ‘Examining mixtures of well-defined SC lipids with the anticipation of characterizing their thermotropic phase behaviour as well as the molecular interactions between the components selected’ and ‘An ultimate goal to learn how these interactions can be influenced with respect to dermal and to transdermal drug delivery, respectively’. In earlier papers on ceramides type IV (CER) and CER/cholesterol mixtures (Wegener et al., 1996; Wartewig et al., 1997), we have demonstrated that the combina-
tion of differential scanning calorimetry (DSC) with Fourier transform (FT) Raman spectroscopic measurements offers the possibility to discuss the macroscopic behaviour of SC lipid mixtures, e.g. melting, in terms of molecular properties. This is based on the well known findings that Raman spectra of lipids are particularly sensitive to the conformational, packing, and dynamic changes involving hydrocarbon chains. Therefore, bilayer reorganizations within the SC may be monitored directly as a function of temperature by using this spectroscopic method (Bunow and Levin, 1980; Verma and Wallach, 1984; Levin and Lewis, 1990; Cao et al., 1995). In the literature, there are many reports on the func-
R. Neubert et al. / Chemistry and Physics of Lipids 89 (1997) 3–14
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Table 1 Relevant Raman bands at 40°C and assignments according to literature data (Dollish et al., 1973; Hendra et al., 1977; Kobayashi et al., 1986; Minoni and Zerbi, 1982; Brown et al., 1987; Kim et al., 1989; Lin-Vien et al., 1991) Assignments
Ceramides type IV (CER)
Stearic acid (SA)
56 mol%CER/44 mol% SA
LAM CH3 rocking, chain-end tt n(C–C) head nas(C –C) n(C–C), chain with g units ns(C–C) progression nS(C–C) CH2 twisting CH2 scissoring ns(CH2) nas(CH2)
168; 203 890 902 1063 1090 1110 1129 1296 1439; 1457 2848 2880
149; 162 894 910 1064 — 1105 1130 1298 1423;1439;1459 2847 2882
204 893 908 1065 1092 1111 1130 1297 1424; 1439; 1458 2849 2882
tions of ceramides (Elias, 1990; Holleran et al., 1991). However, only a few studies have been made on the role of saturated fatty acids in the bilayer of the SC and the interaction between ceramides and fatty acids. Therefore, in this paper we report on the thermotropic phase behaviour of CER/stearic acid (SA) mixtures studied by applying DSC and FT Raman spectroscopy. For the purpose of comparison we have also re-examined the melting behaviour of pure SA.
2. Materials and methods
2.1. Samples CER and SA (n-C17H35COOH) were purchased from Sigma (St. Louis. MO, USA) and used as received. The mixtures were casted from chloroform solution and dried under vacuum. Stable samples were prepared by annealing the lipids at 40°C for 24 h. The samples were kept in cylindrical glass tubes for Raman experiments and in aluminium pans for DSC measurements.
2.2. Differential scanning calorimetry The thermograms were recorded with a DSC-7 Perkin-Elmer differential scanning calorimeter, where the scan rate was usually 2 K min − 1. The transition temperature was determined as onset
temperature by extrapolation to the baseline of the most rapid rise in the excess heat capacity curve as a function of temperature. The enthalpy was determined from the area under the transition peak by comparison with that for a known standard (indium).
2.3. Fourier transform Raman spectroscopy The Raman spectra were acquired using a Bruker Fourier transform infrared spectrometer IFS 66 equipped with the Raman module FRA 106. A diode pumped Nd:YAG laser which emits at a wavelength of 1064 nm was used as the excitation source. The scattered radiation was collected at 180° to the source. Typical spectra were recorded at a laser power of 300 mW at sample location and a resolution of 4 cm − 1. In order to improve the signal to noise ratio, typically, 400 scans were co-added, corresponding to a measurement time of 10 min. The temperature dependence of the Raman spectra was studied in the range from 40 to 90°C (stability90.2°C). Temperature variations were performed by flowing air suitably heated onto the sample in a glass Dewar cell. After a temperature step, the sample was allowed to equilibrate for 15 min to stabilize the temperature before recording each spectrum. The manipulation and evaluation of the spectra, in particular, integration and curve fitting were carried out using the Bruker OPUS software. Generally, Raman intensities were determined as integrated band intensities.
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R. Neubert et al. / Chemistry and Physics of Lipids 89 (1997) 3–14
Fig. 2. Temperature dependence of the Raman spectra of stearic acid in the spectral ranges: (a) 850 – 1550 cm − 1; and (b) 2800 – 3000 cm − 1.
3. Results and discussion
3.1. Stearic acid The DSC thermogram for the SA sample exhibits an endothermic transition at (69.7 9 0.2) °C with an enthalpy of transition DH =(64.5 9 0.3) kJ mol − 1. The Raman spectrum of SA at 40°C, shown in Fig. 1, is in accordance with that of the most stable modification C reported in the literature (Kobayashi et al., 1986; Zerbi et al., 1987). The Raman bands relevant to our problem (Dollish et al., 1973; Kobayashi et al., 1986; Zerbi et al., 1987; Lin-Vien et al., 1991) are summarized in Table 1. Premelting phenomena in SA have already been reported by Zerbi et al. (1987). Our observations shown below conform to and supplement their results. (a) The doublet of the longitudinal acoustic mode (LAM 3) at 148 and 163 cm − 1 , characteristic of the double-layered orthorhombic subcell, shows unambiguously the planar all-trans conformation of alkyl chain residues (Minoni and Zerbi, 1982). The LAM 3 bands are observable up to 2° below the melting point and their wavenumbers are practically temperature independent, indicating the highly ordered structure
of hydrocarbon chains in the solid state. (b) In the 850–900 cm − 1 region, only the methyl rocking mode associated with the tt chain-end conformation appears as a sharp 894 cm − 1 band up to temperatures of 65°C. For temperatures above 65°C, the intensity of this band decreases and new weak bands rise at 875, 860, and 845 cm − 1, which belong to CH3 rocking modes of gt, gg and tg chain-end conformations, respectively (Brown et al., 1987; Kim et al., 1989). Finally, in the melt the methyl rocking modes associated with the various chain-end conformations produce a broad background. (c) No changes are observed for the asymmetric and symmetric C–C stretching bands in the temperature range between 40 and 65°C, indicating that the alkyl chain residue remains in trans conformations (3 or more trans bonds in sequence). Between 65°C and the melting point the intensity of these bands decreases drastically and the C–C stretching band owing to hydrocarbon chains with gauche units appears at 1083 cm − 1 (Fig. 2a). But, it should be noted however, that C–C stretching bands belonging to trans sequences are still detectable above 65°C. On melting the lamellae arrangement is lost and the resulting structure is a random mixture of gauche and trans bonds.
R. Neubert et al. / Chemistry and Physics of Lipids 89 (1997) 3–14
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Fig. 3. Intensity ratio I[nas(CH2)]/I[ns(CH2)] versus temperature for: (a) ceramides type IV; (b) 56 mol% CER/44 mol% SA; (c) 19 mol% CER/81 mol% SA; and (d) stearic acid.
(d) The factor group splitting of the CH2 scissoring vibration near 1430 cm − 1 (Hendra et al., 1977) disappears on melting near 69°C (Fig. 2a). The occurrence of this splitting proves, as known, that the orthorhombic subcell of SA comprises two
molecules. The splitting is useful for elucidating the mixtures, because CER do not show this effect. (e) The weak and rather broad Raman-active band of the symmetric CO stretching vibration of the dimer at 1638 cm − 1 remains unchanged up to
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R. Neubert et al. / Chemistry and Physics of Lipids 89 (1997) 3–14
Fig. 3 (continued)
65°C. In the course of melting this dimer band shifts to 1664 cm − 1 and remains clearly observable in the liquid state. This indicates that the hydrogen bonds between the head groups of the dimers become weaker during the melting, but, the SA dimer still exists in the melt.
(f) The distinct temperature-induced changes in the complex feature of C–H stretching vibrations are illustrated in Fig. 2b. In order to quantify this effect, the overlapping bands have been decomposed, as described in part I of this series (Wegener et al., 1996) and the temperature
R. Neubert et al. / Chemistry and Physics of Lipids 89 (1997) 3–14
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Fig. 4. Peak position of the symmetric stretching mode ns(CH2) versus temperature for: (a) ceramides type IV; (b) 56 mol% CER/44 mol% SA; (c) 19 mol% CER/81 mol% SA; and (d) stearic acid.
dependence of the integrated intensity of the symmetric and asymmetric CH2 stretching bands were determined accordingly. As shown in Fig. 3d, the normalized intensity ratio I(nas(CH2))/I(ns(CH2) starts decreasing substantially near 65°C with a collapse at melting. Further, it appears that the
band position of ns(CH2) is very sensitive for monitoring the trans/gauche transformation within the chain residue (see Fig. 4d). The peak position of the symmetric CH2 stretching mode shifts from 2847 cm − 1 to 2854 cm − 1 in the course of melting. The point of inflection of the relevant
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R. Neubert et al. / Chemistry and Physics of Lipids 89 (1997) 3–14
Fig. 4 (continued)
curve amounts to 67.5°C and coincides practically with the onset temperature of the DSC curve, when the different temperature regimes of the DSC and Raman scattering experiment are taken into consideration. In short, the spectroscopic data confirm that SA crystallizes into a orthorhombic subcell pack-
ing with two dimer molecules per unit cell. The highly ordered all-trans structure of the alkyl chains is present up to about 5°C below the melting point. The disorder connected with the premelting begins with the occurrence of gauche conformers near the CH3 end group of the alkyl chains. The hydrogen bonds between the head
R. Neubert et al. / Chemistry and Physics of Lipids 89 (1997) 3–14
groups are likewise involved in the premelting in the manner that they become weaker but still remain in the melt.
3.2. Mixtures of ceramides IV and stearic acid The DSC heating curves for various mixtures of CER and SA, presented in Fig. 5, show two endothermic transitions which are typical for eutectic melting. The first transition is associated with the melting of the eutectic mixture. The onset temperatures of these peaks vary between 62 and 66°C. The following peak arises from the
Fig. 5. DSC heating curves of various mixtures of ceramides type IV and stearic acid after storage at 40°C for 24 h: (a) ceramides IV; (b) 79 mol% CER/21 mol% SA; (c) 56 mol% CER/44 mol% SA; (d) 48 mol% CER/52 mol% SA; (e) 19 mol% CER/81 mol% SA; (f) 8 mol% CER/92 mol% SA; and (g) stearic acid.
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melting of the purely residual component. The end of this melting process is characterized by the offset temperature. Both onset and offset temperatures against the content of SA are depicted in Fig. 6. The full lines in the diagram represent schematically the eutectic type phase diagram indicating immiscibility of the two lipids in the solid state. The eutectic point is estimated to appear at a temperature of about 63.5°C and with a composition of 90 mol% SA and 10 mol% CER. Raman spectroscopic investigations support these observations. The dominant part of the Raman spectra of the mixtures consists of bands arising from alkyl chains of both components. The band assignments for a mixture selected is included in Table 1. Generally, it is not possible to distinguish between Raman bands belonging to alkyl chains of the two lipids. The exceptions are the longitudinal acoustic modes which appear at different wavenumbers for CER and SA. The major results of the Raman studies are the following. (a) Of particular interest is the fact that LAM bands of both lipid chains occur for each composition of the mixtures in the solid state (Fig. 1i). These bands are observable up to a few degree below the temperature of the first transition at 63.5°C. This demonstrates distinctly that the alkyl chains are also highly ordered in the mixtures. (b) The appearance of the sharp stretching bands nas(C–C), ns(C–C) and nas(CH2) in the case of all mixtures clearly verifies this behaviour (see Fig. 1b, c). Between the eutetic temperature and the temperature of the second transition bands belonging to trans sequences are still detectable. This observation suggests that above the eutetic temperature the alkyl chains of the residual component remain in the ordered state characterized by trans segments. (c) The CH3 rocking mode associated with the tt chain-end conformation occurs as a sharp band for all mixtures studied. In the course of both transitions the intensity of this band decreases and CH3 rocking bands belonging to gt, gg and tg chain-end conformations appear. Fi-
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R. Neubert et al. / Chemistry and Physics of Lipids 89 (1997) 3–14
Fig. 6. Phase diagram for mixtures of ceramides type IV and stearic acid.
nally, in the liquid state the various CH3 rocking modes form a broad band between 850 and 900 cm − 1. These features also indicate the ordered structure of the alkyl chains for temperature below both phase transition temperatures. (d) Using the evaluation procedures mentioned above the thermally-induced changes of the relative population of the trans and gauche conformers in terms of spectral data were determined. As examples, the temperature dependence of the normalized intensity ratio I[nas(CH2)]/I[ns(CH2)] and of the peak position of the symmetric CH2 stretching mode for two mixtures are displayed in Figs. 3 and 4, respectively. The plots for the 56 mol% CER/44 mol% SA sample nicely reveal two transitions in accordance with the DSC results. In the case of the 19 mol% CER/81 mol% SA mixture, we find only a broadening of the temperature profiles. However, the asymmetry in the shape of profiles hints two transitions separated by a few degrees. (e) An essential point is that the factor group splitting of the CH2 scissoring mode, arising from the orthorhombic subcell packing of SA, disappears in the course of the first transition at about 63.5°C for mixtures with a content of SA lower
than 90 mol% (Fig. 7). In the case of mixtures with a stearic acid content higher than 90 mol%, this splitting remains up to the complete melting of the system. This observation illustrates that the first transition reflects the eutetic melting of the mixture. In conclusion, DSC and Raman spectroscopic studies have shown that CER and SA are immiscible in the solid state. The hydrocarbon chains of both lipids exhibit a highly ordered structure in the solid state. The phase diagram of the system is simply a eutetic type one. The addition of SA to CER lowers the melting temperature of CER from 89°C to only 63.5°C, the eutectic temperature. However, a high amount of stearic acid is necessary to reach this lowest temperature. Based on these results it is assumed that though stearic acid is certainly a major component of the SC, it is not efficient enough to increase the fluidity of CER.
Acknowledgements This work was supported by the Deutsche Forschungsgemeinschaft (DFG), Sonderforschungsbereich 197, Project A8.
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Fig. 7. Temperature dependence of the factor group splitting of the CH2 scissoring mode for 19 mol% CER/81 mol% SA mixture: (a) Raman spectra; and (b) band positions.
References Brown, K.G., Bicknell-Brown, E., Ladjadj, M., 1987. Ramanactive bands sensitive to motion and conformation at the chain termini and backbones of alkanes and lipids. J. Phys. Chem. 91, 3436 – 3442. Bunow, M.R., Levin, I.W., 1980. Molecular conformations of cerebrosides in bilayers determined by Raman spectroscopy. Biophys. J. 32, 1007–1021. Cao, A., Liquier, J., Taillandier, E., 1995. Infrared and Raman spectroscopy of biomolecules. In: Schrader, B. (Ed.), IR and Raman Spectroscopy, Verlag Chemie, Weinheim, pp. 344 – 372.
Dollish, F.R., Fateley, W.G., Bentley, F.F., 1973. Characteristic Raman frequencies of organic compounds, Wiley, New York. Elias, P.M., 1990. The importance of epidermal lipids for the stratum corneum barrier. In: Osborne, D.W., Amann, A.H. (Eds.), Topical Drug Delivery Formulations, vol. 42. Marcel Dekker, New York, Basel, pp. 13 – 28. Hendra, H.P., Jobic, E.P., Marden, E.P., Bloor, D., 1977. The vibrational spectrum of polymethylene. III. Polarized Raman spectrum of single crystal texture polyethylene and a single crystal of C23H48. Spectrochim. Acta 33A, 445 – 452. Holleran, W.M., Mao-Qiang, M., Gao, W.M., Menon, G.K., Elias, P.M., Feingold, K.R., 1991. Sphingolipids are re-
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quired for mammalian epidermal barrier function. J. Clin. Invest. 88, 1338 – 1345. Kim, Y., Strauss, H.L., Snyder, R.G., 1989. Conformational disorder in the binary mixture n-C50 H102/n-C46H94: a vibrational spectroscopic study. J. Phys. Chem. 93, 485– 490. Kobayashi, M., Kobayashi, T., Cho, Y., Kaneko, F., 1986. State of molecular assembly and physical properties of crystalline long-chain compounds studied by vibrational spectroscopy. Makromol. Chem. Macromol. Symp. 5, 1– 20. Levin, I.W., Lewis, E.N., 1990. Fourier transform Raman spectroscopy of biological materials. Anal. Chem. 62, 1101A – 1111A. Lin-Vien, D., Colthup, N.B., Fateley, W.G., Grasselli, J.K., 1991. The handbook of infrared and Raman characteristic frequencies of organic molecules, Academic Press, Boston, MA.
.
Minoni, G., Zerbi, G., 1982. End effects on longitudinal accordion modes: fatty acid and layered systems. J. Phys. Chem. 86, 4791 – 4792. Verma, S.P., Wallach, D.F.H., 1984. Raman spectroscopy of lipids and biomembranes. In: Chapman, D. (Ed.), Biomembrane Structure and Function, Verlag Chemie, Weinheim, pp. 167 – 199. Wartewig, S., Neubert, R., Rettig, W., Wegener, M., 1997. Ceramides/cholesterol mixtures as characterized by FTRaman spectroscopy. Mikrochim. Acta [Suppl.] 14, 263 – 264. Wegener, M., Neubert, R., Rettig, W., Wartewig, S., 1996. Structure of stratum corneum lipids characterized by FTRaman spectroscopy and DSC. I. Ceramides. Int. J. Pharm. 128, 203 – 213. Zerbi, G., Conti, G., Minoni, G., Pison, St., Bigotto, A., 1987. Premelting phenomena in fatty acids: an infrared and Raman study. J. Phys. Chem. 91, 2386 – 2393.
Structure of stratum corneum lipids characterized by FT-Raman spectroscopy and DSC. II. Mixtures of ceramides and saturated fatty acids Reinhard Neubert a,*, Willi Rettig a, Siegfried Wartewig b, Matthias Wegener a, Antje Wienhold a a
Department of Pharmacy, Martin-Luther-Uni6ersity Halle-Wittenberg, Wolfgang-Langenbeck-Str. 4, D-06120 Halle/Saale, Germany b Institute of Applied Dermatopharmacy, Wolfgang-Langenbeck-Str. 4, D-06120 Halle, Germany Received 3 February 1997; accepted 24 May 1997
Abstract Fourier transform (FT) Raman spectroscopy and differential scanning calorimetry (DSC) were used to study the thermotropic phase behaviour of mixtures of ceramides type IV (CER) and stearic acid (SA). For comparison the melting behaviour of SA was re-examined. The Raman spectra of all mixtures in the solid state show sharp bands associated with trans sequencies of the alkyl chain residues of both lipids. These features demonstrate that the hydrocarbon chains are highly ordered in the mixtures, too. The temperature dependence of the conformationally sensitive bands is used to estimate the degree of order in terms of the relative population of trans and gauche conformations. The DSC heating curves for the mixtures show two endothermic transitions which are typical for eutectic melting. The factor group splitting of the CH2 scissoring mode, arising from the orthorhombic subcell packing of SA, disappears in the course of the eutectic melting of samples with a SA content lower than 90 mol%. Both DSC and Raman spectroscopic studies reveal that CER and SA are immiscible in the solid state. The phase diagram of the system is a simple eutectic type one. The addition of SA to CER shifts the melting temperature of ceramides to lower values. However, though SA is a major component of stratum corneum (SC) it is not efficient enough to increase the fluidity of ceramides. © 1997 Elsevier Science Ireland Ltd. Keywords: Stratum corneum; Ceramides; Stearic acid; Phase behaviour; Raman spectroscopy; DSC
* Corresponding Author. Tel.: + 49 345 5525000; fax: + 49 345 5527021. 0009-3084/97/$17.00 © 1997 Elsevier Science Ireland Ltd. All rights reserved. PII S 0 0 0 9 - 3 0 8 4 ( 9 7 ) 0 0 0 4 9 - 2
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R. Neubert et al. / Chemistry and Physics of Lipids 89 (1997) 3–14
Fig. 1. Raman spectra of various mixtures of ceramides type IV and stearic acid at 40°C: (a) ceramides IV; (b) 48 mol% CER/52 mol% SA; (c) 19 mol% CER/81 mol% SA; (d) stearic acid in different spectral ranges: (i) low frequency range 100 – 250 cm − 1; (ii) 850–1550 cm − 1; and (iii) C–H stretching range.
1. Introduction For elucidating the very complex nature of stratum corneum (SC), the outermost layer of the skin, our leading concepts are: ‘Examining mixtures of well-defined SC lipids with the anticipation of characterizing their thermotropic phase behaviour as well as the molecular interactions between the components selected’ and ‘An ultimate goal to learn how these interactions can be influenced with respect to dermal and to transdermal drug delivery, respectively’. In earlier papers on ceramides type IV (CER) and CER/cholesterol mixtures (Wegener et al., 1996; Wartewig et al., 1997), we have demonstrated that the combina-
tion of differential scanning calorimetry (DSC) with Fourier transform (FT) Raman spectroscopic measurements offers the possibility to discuss the macroscopic behaviour of SC lipid mixtures, e.g. melting, in terms of molecular properties. This is based on the well known findings that Raman spectra of lipids are particularly sensitive to the conformational, packing, and dynamic changes involving hydrocarbon chains. Therefore, bilayer reorganizations within the SC may be monitored directly as a function of temperature by using this spectroscopic method (Bunow and Levin, 1980; Verma and Wallach, 1984; Levin and Lewis, 1990; Cao et al., 1995). In the literature, there are many reports on the func-
R. Neubert et al. / Chemistry and Physics of Lipids 89 (1997) 3–14
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Table 1 Relevant Raman bands at 40°C and assignments according to literature data (Dollish et al., 1973; Hendra et al., 1977; Kobayashi et al., 1986; Minoni and Zerbi, 1982; Brown et al., 1987; Kim et al., 1989; Lin-Vien et al., 1991) Assignments
Ceramides type IV (CER)
Stearic acid (SA)
56 mol%CER/44 mol% SA
LAM CH3 rocking, chain-end tt n(C–C) head nas(C –C) n(C–C), chain with g units ns(C–C) progression nS(C–C) CH2 twisting CH2 scissoring ns(CH2) nas(CH2)
168; 203 890 902 1063 1090 1110 1129 1296 1439; 1457 2848 2880
149; 162 894 910 1064 — 1105 1130 1298 1423;1439;1459 2847 2882
204 893 908 1065 1092 1111 1130 1297 1424; 1439; 1458 2849 2882
tions of ceramides (Elias, 1990; Holleran et al., 1991). However, only a few studies have been made on the role of saturated fatty acids in the bilayer of the SC and the interaction between ceramides and fatty acids. Therefore, in this paper we report on the thermotropic phase behaviour of CER/stearic acid (SA) mixtures studied by applying DSC and FT Raman spectroscopy. For the purpose of comparison we have also re-examined the melting behaviour of pure SA.
2. Materials and methods
2.1. Samples CER and SA (n-C17H35COOH) were purchased from Sigma (St. Louis. MO, USA) and used as received. The mixtures were casted from chloroform solution and dried under vacuum. Stable samples were prepared by annealing the lipids at 40°C for 24 h. The samples were kept in cylindrical glass tubes for Raman experiments and in aluminium pans for DSC measurements.
2.2. Differential scanning calorimetry The thermograms were recorded with a DSC-7 Perkin-Elmer differential scanning calorimeter, where the scan rate was usually 2 K min − 1. The transition temperature was determined as onset
temperature by extrapolation to the baseline of the most rapid rise in the excess heat capacity curve as a function of temperature. The enthalpy was determined from the area under the transition peak by comparison with that for a known standard (indium).
2.3. Fourier transform Raman spectroscopy The Raman spectra were acquired using a Bruker Fourier transform infrared spectrometer IFS 66 equipped with the Raman module FRA 106. A diode pumped Nd:YAG laser which emits at a wavelength of 1064 nm was used as the excitation source. The scattered radiation was collected at 180° to the source. Typical spectra were recorded at a laser power of 300 mW at sample location and a resolution of 4 cm − 1. In order to improve the signal to noise ratio, typically, 400 scans were co-added, corresponding to a measurement time of 10 min. The temperature dependence of the Raman spectra was studied in the range from 40 to 90°C (stability90.2°C). Temperature variations were performed by flowing air suitably heated onto the sample in a glass Dewar cell. After a temperature step, the sample was allowed to equilibrate for 15 min to stabilize the temperature before recording each spectrum. The manipulation and evaluation of the spectra, in particular, integration and curve fitting were carried out using the Bruker OPUS software. Generally, Raman intensities were determined as integrated band intensities.
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R. Neubert et al. / Chemistry and Physics of Lipids 89 (1997) 3–14
Fig. 2. Temperature dependence of the Raman spectra of stearic acid in the spectral ranges: (a) 850 – 1550 cm − 1; and (b) 2800 – 3000 cm − 1.
3. Results and discussion
3.1. Stearic acid The DSC thermogram for the SA sample exhibits an endothermic transition at (69.7 9 0.2) °C with an enthalpy of transition DH =(64.5 9 0.3) kJ mol − 1. The Raman spectrum of SA at 40°C, shown in Fig. 1, is in accordance with that of the most stable modification C reported in the literature (Kobayashi et al., 1986; Zerbi et al., 1987). The Raman bands relevant to our problem (Dollish et al., 1973; Kobayashi et al., 1986; Zerbi et al., 1987; Lin-Vien et al., 1991) are summarized in Table 1. Premelting phenomena in SA have already been reported by Zerbi et al. (1987). Our observations shown below conform to and supplement their results. (a) The doublet of the longitudinal acoustic mode (LAM 3) at 148 and 163 cm − 1 , characteristic of the double-layered orthorhombic subcell, shows unambiguously the planar all-trans conformation of alkyl chain residues (Minoni and Zerbi, 1982). The LAM 3 bands are observable up to 2° below the melting point and their wavenumbers are practically temperature independent, indicating the highly ordered structure
of hydrocarbon chains in the solid state. (b) In the 850–900 cm − 1 region, only the methyl rocking mode associated with the tt chain-end conformation appears as a sharp 894 cm − 1 band up to temperatures of 65°C. For temperatures above 65°C, the intensity of this band decreases and new weak bands rise at 875, 860, and 845 cm − 1, which belong to CH3 rocking modes of gt, gg and tg chain-end conformations, respectively (Brown et al., 1987; Kim et al., 1989). Finally, in the melt the methyl rocking modes associated with the various chain-end conformations produce a broad background. (c) No changes are observed for the asymmetric and symmetric C–C stretching bands in the temperature range between 40 and 65°C, indicating that the alkyl chain residue remains in trans conformations (3 or more trans bonds in sequence). Between 65°C and the melting point the intensity of these bands decreases drastically and the C–C stretching band owing to hydrocarbon chains with gauche units appears at 1083 cm − 1 (Fig. 2a). But, it should be noted however, that C–C stretching bands belonging to trans sequences are still detectable above 65°C. On melting the lamellae arrangement is lost and the resulting structure is a random mixture of gauche and trans bonds.
R. Neubert et al. / Chemistry and Physics of Lipids 89 (1997) 3–14
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Fig. 3. Intensity ratio I[nas(CH2)]/I[ns(CH2)] versus temperature for: (a) ceramides type IV; (b) 56 mol% CER/44 mol% SA; (c) 19 mol% CER/81 mol% SA; and (d) stearic acid.
(d) The factor group splitting of the CH2 scissoring vibration near 1430 cm − 1 (Hendra et al., 1977) disappears on melting near 69°C (Fig. 2a). The occurrence of this splitting proves, as known, that the orthorhombic subcell of SA comprises two
molecules. The splitting is useful for elucidating the mixtures, because CER do not show this effect. (e) The weak and rather broad Raman-active band of the symmetric CO stretching vibration of the dimer at 1638 cm − 1 remains unchanged up to
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Fig. 3 (continued)
65°C. In the course of melting this dimer band shifts to 1664 cm − 1 and remains clearly observable in the liquid state. This indicates that the hydrogen bonds between the head groups of the dimers become weaker during the melting, but, the SA dimer still exists in the melt.
(f) The distinct temperature-induced changes in the complex feature of C–H stretching vibrations are illustrated in Fig. 2b. In order to quantify this effect, the overlapping bands have been decomposed, as described in part I of this series (Wegener et al., 1996) and the temperature
R. Neubert et al. / Chemistry and Physics of Lipids 89 (1997) 3–14
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Fig. 4. Peak position of the symmetric stretching mode ns(CH2) versus temperature for: (a) ceramides type IV; (b) 56 mol% CER/44 mol% SA; (c) 19 mol% CER/81 mol% SA; and (d) stearic acid.
dependence of the integrated intensity of the symmetric and asymmetric CH2 stretching bands were determined accordingly. As shown in Fig. 3d, the normalized intensity ratio I(nas(CH2))/I(ns(CH2) starts decreasing substantially near 65°C with a collapse at melting. Further, it appears that the
band position of ns(CH2) is very sensitive for monitoring the trans/gauche transformation within the chain residue (see Fig. 4d). The peak position of the symmetric CH2 stretching mode shifts from 2847 cm − 1 to 2854 cm − 1 in the course of melting. The point of inflection of the relevant
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Fig. 4 (continued)
curve amounts to 67.5°C and coincides practically with the onset temperature of the DSC curve, when the different temperature regimes of the DSC and Raman scattering experiment are taken into consideration. In short, the spectroscopic data confirm that SA crystallizes into a orthorhombic subcell pack-
ing with two dimer molecules per unit cell. The highly ordered all-trans structure of the alkyl chains is present up to about 5°C below the melting point. The disorder connected with the premelting begins with the occurrence of gauche conformers near the CH3 end group of the alkyl chains. The hydrogen bonds between the head
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groups are likewise involved in the premelting in the manner that they become weaker but still remain in the melt.
3.2. Mixtures of ceramides IV and stearic acid The DSC heating curves for various mixtures of CER and SA, presented in Fig. 5, show two endothermic transitions which are typical for eutectic melting. The first transition is associated with the melting of the eutectic mixture. The onset temperatures of these peaks vary between 62 and 66°C. The following peak arises from the
Fig. 5. DSC heating curves of various mixtures of ceramides type IV and stearic acid after storage at 40°C for 24 h: (a) ceramides IV; (b) 79 mol% CER/21 mol% SA; (c) 56 mol% CER/44 mol% SA; (d) 48 mol% CER/52 mol% SA; (e) 19 mol% CER/81 mol% SA; (f) 8 mol% CER/92 mol% SA; and (g) stearic acid.
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melting of the purely residual component. The end of this melting process is characterized by the offset temperature. Both onset and offset temperatures against the content of SA are depicted in Fig. 6. The full lines in the diagram represent schematically the eutectic type phase diagram indicating immiscibility of the two lipids in the solid state. The eutectic point is estimated to appear at a temperature of about 63.5°C and with a composition of 90 mol% SA and 10 mol% CER. Raman spectroscopic investigations support these observations. The dominant part of the Raman spectra of the mixtures consists of bands arising from alkyl chains of both components. The band assignments for a mixture selected is included in Table 1. Generally, it is not possible to distinguish between Raman bands belonging to alkyl chains of the two lipids. The exceptions are the longitudinal acoustic modes which appear at different wavenumbers for CER and SA. The major results of the Raman studies are the following. (a) Of particular interest is the fact that LAM bands of both lipid chains occur for each composition of the mixtures in the solid state (Fig. 1i). These bands are observable up to a few degree below the temperature of the first transition at 63.5°C. This demonstrates distinctly that the alkyl chains are also highly ordered in the mixtures. (b) The appearance of the sharp stretching bands nas(C–C), ns(C–C) and nas(CH2) in the case of all mixtures clearly verifies this behaviour (see Fig. 1b, c). Between the eutetic temperature and the temperature of the second transition bands belonging to trans sequences are still detectable. This observation suggests that above the eutetic temperature the alkyl chains of the residual component remain in the ordered state characterized by trans segments. (c) The CH3 rocking mode associated with the tt chain-end conformation occurs as a sharp band for all mixtures studied. In the course of both transitions the intensity of this band decreases and CH3 rocking bands belonging to gt, gg and tg chain-end conformations appear. Fi-
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Fig. 6. Phase diagram for mixtures of ceramides type IV and stearic acid.
nally, in the liquid state the various CH3 rocking modes form a broad band between 850 and 900 cm − 1. These features also indicate the ordered structure of the alkyl chains for temperature below both phase transition temperatures. (d) Using the evaluation procedures mentioned above the thermally-induced changes of the relative population of the trans and gauche conformers in terms of spectral data were determined. As examples, the temperature dependence of the normalized intensity ratio I[nas(CH2)]/I[ns(CH2)] and of the peak position of the symmetric CH2 stretching mode for two mixtures are displayed in Figs. 3 and 4, respectively. The plots for the 56 mol% CER/44 mol% SA sample nicely reveal two transitions in accordance with the DSC results. In the case of the 19 mol% CER/81 mol% SA mixture, we find only a broadening of the temperature profiles. However, the asymmetry in the shape of profiles hints two transitions separated by a few degrees. (e) An essential point is that the factor group splitting of the CH2 scissoring mode, arising from the orthorhombic subcell packing of SA, disappears in the course of the first transition at about 63.5°C for mixtures with a content of SA lower
than 90 mol% (Fig. 7). In the case of mixtures with a stearic acid content higher than 90 mol%, this splitting remains up to the complete melting of the system. This observation illustrates that the first transition reflects the eutetic melting of the mixture. In conclusion, DSC and Raman spectroscopic studies have shown that CER and SA are immiscible in the solid state. The hydrocarbon chains of both lipids exhibit a highly ordered structure in the solid state. The phase diagram of the system is simply a eutetic type one. The addition of SA to CER lowers the melting temperature of CER from 89°C to only 63.5°C, the eutectic temperature. However, a high amount of stearic acid is necessary to reach this lowest temperature. Based on these results it is assumed that though stearic acid is certainly a major component of the SC, it is not efficient enough to increase the fluidity of CER.
Acknowledgements This work was supported by the Deutsche Forschungsgemeinschaft (DFG), Sonderforschungsbereich 197, Project A8.
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Fig. 7. Temperature dependence of the factor group splitting of the CH2 scissoring mode for 19 mol% CER/81 mol% SA mixture: (a) Raman spectra; and (b) band positions.
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