Antievaporative Mechanism of Wax Esters: Implications for the

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Antievaporative Mechanism of Wax Esters: Implications for the Function of Tear Fluid Riku O. Paananen, Antti H. Rantamak̈ i, and Juha M. Holopainen* Helsinki Eye Lab, Department of Ophthalmology, University of Helsinki, Helsinki 00029, Finland S Supporting Information *

ABSTRACT: The tear film lipid layer (TFLL) is considered to act as an evaporation barrier and to maintain the tear film intact between blinks. In vitro methods have, however, failed to reproduce this evaporation-retarding effect. Wax esters (WEs) are a major component of the TFLL. Close to their bulk melting temperature, WEs have been found to retard the evaporation of water, but the nature of this mechanism has remained unclear. We studied the interfacial organization of WE films by measuring their isochors and isotherms and evaporation-retarding effect, and we imaged these films by Brewster angle microscopy (BAM). Behenyl palmitoleate (BP) was used as a representative WE because it resembles the WEs found in meibum. At low temperatures, BP forms solid monolayer crystals in which the molecules are organized in a bulk-like extended conformation. Within approximately 3 °C below the bulk melting temperature, these solid monolayer domains coexist with a fluid monolayer film. At temperatures above the bulk melting temperature, BP forms a completely fluid monolayer in which the molecules are in a hairpin conformation. A fluid hairpin monolayer of BP does not significantly retard evaporation, whereas a solid monolayer decreases evaporation by >50%. The results provide a molecular-level rationale for the evaporation-retarding properties of WEs close to their melting temperature.

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INTRODUCTION The traditional view of the tear film lipid layer (TFLL) consists of two layers.1 The polar lipids (phospholipids) reside at the water interface, and nonpolar lipids (wax esters, cholesteryl esters, and triglycerides) form a layer on top of the polar lipids.2,3 The polar lipids aid the nonpolar lipids in spreading rapidly over the aqueous surface of the tear film instead of forming aggregates or droplets. The lipid layer is thought to stabilize the tear film as well as to retard the evaporation of the tear fluid. In vivo studies in rabbits and humans have found that an intact TFLL causes a 4- to 20-fold reduction in evaporation rate,4−6 but in vitro studies have not been able to reproduce this effect.7−11 Condensed monolayers of saturated alcohols and fatty acids efficiently retard evaporation from the surface of water.12 The ability to retard evaporation is based on the van der Waals interactions between adjacent acyl chains and the low free volume inside the hydrocarbon portion of the monolayer, which prevents the diffusion of water molecules through the monolayer.13 Wax esters (WEs) have been found to retard evaporation close to their bulk melting temperature.14 Because WEs form a major part of meibum, it is likely that these lipids are at least partially responsible for the alleged evaporationretarding properties of the TFLL. The unsaturated structure of these WEs and the fact that they only retard evaporation close to their melting temperature suggest that the antievaporative structure formed by WEs differs from the one formed by saturated alcohols and fatty acids. We hypothesized that the evaporation-retarding ability of WEs can be explained by their surface behavior at different temperatures. In the present study, we investigated the © 2014 American Chemical Society

structures formed by behenyl palmitoleate (BP), a typical WE found in meibum, at the air−water interface by measuring isochors and isotherms coupled to Brewster angle microscopy (BAM) experiments. We also associated the structural changes to the evaporation-retarding properties of BP.

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EXPERIMENTAL SECTION

BP was obtained from Nu-Chek-Prep (Elysian, MN), and 1octadecanol, 1-docosanol, 1-hexacosanol, and 1-triacontanol were obtained from Sigma-Aldrich (St. Louis, MO). All lipids were stored at −20 °C until use. Lipids were dissolved in chloroform, and only fresh samples were used. PBS buffer was used as the subphase in all experiments. Isochor Measurements. BP was spread at the air−buffer interface of a KSV minitrough (Helsinki, Finland) in a 1 mM chloroform solution to a final mean molecular area of 30 Å2. The chloroform was allowed to evaporate for 10 min. Trough temperature was controlled using a Lauda ECO E4 thermostat (Lauda, Germany). Trough temperature was changed at a rate of 2 °C/min from 20 to 42 °C, and the change in surface pressure was recorded using a Wilhelmy plate. A slower temperature ramp rate (0.7 °C/min) had no effect on the results. BAM images were also captured at 1 °C intervals using a KSV NIMA microBAM (Helsinki, Finland). The BAM aperture was kept closed when images were not recorded in order to avoid heating the film with the laser beam. The surface tension of pure buffer varied linearly with temperature over the tested temperature range. A linear fit to the buffer surface tension was used to correct this effect from the measured isochor. Isotherm Measurements. WEs were spread at the air−buffer interface of a KSV minitrough (Helsinki, Finland) filled with PBS Received: March 10, 2014 Published: May 2, 2014 5897

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buffer in a 1 mM chloroform solution. The film was compressed at a rate of 10 (Å2/molecule)/min, and changes in surface pressure were recorded. BAM images were recorded during the compression isotherms. During compression, the subphase temperature was maintained within 36 ± 1 °C. Each isotherm was recorded three times to ensure the repeatability of the measurement. Thickness Measurements. Average image intensities were measured from five BAM images at different exposure times between 0.1−125 ms for each film using ImageJ software.15 A linear fit to the exposure time−image intensity data was used to obtain a value for average image intensity per unit time. Film reflectance was determined from the intensity per unit time data using a calibration procedure described in the Supporting Information (Figure S1). At Brewster angle, a first-order approximation for the reflectance of a thin film at the air−water surface can be written

R ∝ d2

Figure 1. Isochor of behenyl palmitoleate measured at a mean molecular area of 30 Å2 with BAM images showing the film appearance at different temperatures. Note that the exposure time is longer in images b and c than it is in images a, d, and e: (a) black, water surface; gray, solid monolayer; white, aggregates; (b, c) black, water surface; gray, fluid monolayer; white, solid monolayer; and (d, e) black, fluid monolayer; gray, solid monolayer; white, aggregates. The scale bar is 500 μm.

(1)

where d is the thickness of the film. This allows for the measurement of relative film thicknesses even when the optical properties of the film are unknown.16 Therefore, film thickness can be calculated by

d = A R − R bg

(2)

where R is the reflectance of the surface, Rbg is the background reflectance of the PBS subphase, and A is a constant. Solid monolayers of 1-octadecanol, 1-docosanol, 1-hexacosanol, and 1-triacontanol were used to determine values of 11.6 ± 0.4 × 103 Å (±SE) for A and 1.3 ± 0.2 × 10−6 for Rbg, as described in the Supporting Information (Figure S2). Evaporation Measurements. Evaporation measurements were conducted as described previously.17 During the experiments, the trough temperature was maintained within 37 ± 1 °C using a thermostat. Measurements were conducted 1 °C below the bulk melting temperature of the lipid in order to achieve efficient spreading of the lipids. Evaporation reduction by the lipid layer was calculated as

E=1−

JR e J = 1 − sat sat Jw Cs − C∞ HR

(3)

where J is evaporation rate from the surface containing a lipid film and Jw is evaporation rate from a pure water surface. Re is the environmental evaporation resistance for mass transfer by diffusion and convection, Csat s is the saturated water vapor concentration at the water surface, Csat ∞ is the saturated water vapor concentration far from the surface, and HR is the relative humidity far from the surface. Re, sat 3 Csat s , and C∞ were determined to be 1.9 ± 0.3 s/cm, 43 ± 6 g/m , and 19 ± 2 g/m3, respectively (see the Supporting Information).

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RESULTS Isochor. A representative isochor and corresponding BAM images are shown in Figure 1. At 24 °C, BP forms solid monolayer domains and three-dimensional aggregates, which do not spread on water surface, as seen with BAM (Figure 1a). As the temperature is increased to 34.5 °C, the surface pressure starts to increase, and a fluid monolayer is seen spreading around the solid domains by BAM (Figure 1b). As the temperature is increased further, the whole surface is covered by the fluid layer, and the surface pressure increases to a value of 2.3 mN/m (Figure 1c). Solid domains can be seen coexisting with the fluid monolayer (Figure 1d). Heating the film above 38 °C causes the solid domains to melt into bright, mobile droplets, and a plateau is observed in the surface pressure (Figure 1e). This corresponds to the bulk melting temperature of BP.14 Isotherm. Representative compression isotherm and compressibility data are shown in Figure 2 with the related BAM images along the compression isotherm. On the basis of the BAM images (Figure 2Ai), zero surface pressure, and zero

Figure 2. (A) Compression isotherm of behenyl palmitoleate with representative BAM images along the isotherm. Note that the exposure time is longer in images i and ii than it is in images iii−v: (i, ii) black, water surface; gray, fluid monolayer; (iii−v) black, fluid monolayer; gray, solid monolayer; white, aggregates. The scale bar is 500 μm. (B) Reciprocal compressibility of BP. The temperature during the measurements was 36 °C.

reciprocal compressibility (Figure 2B), a fluid monolayer coexists with a gas-phase monolayer at 65 Å2/molecule. As the film is compressed, an increasing proportion of the interface is covered by the fluid monolayer until it completely covers the surface at 50 Å2/molecule (Figure 2Aii). At this point, the surface pressure starts to increase, and reciprocal compressibility increases to 2.5 mN/m as the film undergoes a transition 5898

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reflecting the increased coverage of the solid phase. At a mean molecular area of 10 Å2, full coverage of the solid layer is exceeded, and no further reduction of evaporation occurs when the amount of lipid is increased.

to the solid phase (Figure 2Aiii). The solid domains grow in fractal shapes, but they show a homogeneous intensity, suggesting that the solid film is of uniform thickness. At a mean molecular area of 18 Å2, the solid domains fuse to form a homogeneous film, as observed with BAM (Figure 2Aiv). This transition is accompanied by an increase in surface pressure to 9 mN/m and a 30 mN/m peak in reciprocal compressibility (Figure 2B). At even lower mean molecular areas there is a plateau in surface pressure, and by BAM the film is observed to collapse (Figure 2Av). Interestingly, this collapse appears to be reversible, as virtually identical isotherms are obtained by subsequent compression−expansion cycles (not shown). Thickness Measurements. Average reflected intensities and calculated film thicknesses are given in Table 1. On the

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DISCUSSION According to the traditional view, one function of the TFLL is to reduce the evaporation rate from the ocular surface. However, its lipid composition does not match the current view of evaporation-retarding lipids.14 The aim of the current study was to further investigate the structures formed by WEs, a major component of meibum, at the air−water interface and to explain the evaporation-retarding properties of these compounds close to their melting temperature.14 BP was chosen because it structurally resembles the WEs found in meibum19 and has been found to retard evaporation at physiologically relevant temperatures.14 Understanding the behavior of individual components of the TFLL at a molecular level gives insight into the structure and function of this complex physiological system. As BP is spread from solvent to the air−water interface at 20 °C, it does not readily spread, but instead it forms solid monolayer crystals and three-dimensional aggregates (Figures 1a and 4a), similar to what has previously been observed for

Table 1. Reflectance and Thickness of Films (±SE) Determined from BAM Imagesa compound 1-octadecanol 1-docosanol 1-hexacosanol 1-triacontanol behenyl palmitoleate (solid) behenyl palmitoleate (fluid)

reflectance (×10−6) 4.4 6.6 9.9 11.5 14.3

± ± ± ± ±

1.1 1.6 2.4 2.7 3.4

2.0 ± 0.5

determined thickness (Å) 20 27 34 37 42

± ± ± ± ±

4 4 5 5 6

theoretical thickness (Å)18 21.6 26.7 31.8 36.8

± ± ± ±

0.2 0.2 0.3 0.3

9±4

All films, excluding the fluid BP film, were measured at a mean molecular area of 18 Å2. Theoretical thickness is calculated according to Tanford,18 assuming an all-trans conformation. a

basis of film reflectance, the thickness of a solid monolayer of BP was approximated to be 42 ± 6 Å (±SE), and the thickness of a fluid BP monolayer, 9 ± 4 Å. However, the value determined for the fluid monolayer thickness is likely an underestimate because the hydrocarbon packing density of the fluid monolayer is lower than that in the calibration lipids, which leads to lower reflectance. Evaporation Measurements. Evaporation reduction caused by films of BP with different mean molecular areas is represented in Figure 3. At high mean molecular area, the whole film exists as a fluid monolayer, and the evaporation rate through the film is similar to the evaporation rate from a pure air−water interface. This implies that the fluid monolayer does not significantly retard evaporation. When the mean molecular area is smaller than 50 Å2, the film starts to retard evaporation,

Figure 4. Schematic representation of the molecular orientation of BP over different temperature ranges. Ts, surface melting temperature; Tb, bulk melting temperature.

WEs below their melting temperature.14 This behavior is similar to long n-alkanes (>30 carbons), which form mono- and multilayers with perfect crystal symmetry when spread at the air−water interface because of the cohesive interaction between hydrocarbon chains.20 However, these monolayer crystals are not stable and tend to aggregate into three-dimensional aggregates, exposing the water surface and losing their evaporation-retarding properties. Heating the film to 35 °C, below the bulk melting temperature of BP (38 ± 1 °C), causes BP molecules to detach from the solid crystal and spread as a fluid monolayer on the water surface (Figures 1b and 4b). This phenomenon can be rationalized by simple thermodynamic arguments. The change in Gibbs energy at the melting point is zero; therefore, in bulk material

ΔS =

ΔH Tb

(4)

where ΔS is the entropy change, ΔH is the enthalpy of melting, and Tb is the bulk melting temperature. Upon melting at the air−water interface, molecules of the liquid phase occupy the air−water interface, and changes in Gibbs energy can be given as

Figure 3. Evaporation reduction capacity of BP (mean ± SD) at 37 °C as a function of mean molecular area. 5899

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Langmuir ΔG = ΔH − ΔSTs − πa

Article

⎡ a (a − a ) ⎛ R ⎤−1 Re ⎞ Re e 2 1 ⎢ ⎜ ⎟ − + 1⎥ E= ⎜ ⎟+ ⎢⎣ a(a 2 − a1) ⎝ R k,2 ⎥⎦ R k,1 ⎠ R k,1

(5)

where Ts is the surface melting temperature, π is the surface pressure, and a is the mean molecular area of the fluid monolayer spread at the air−water interface. Assuming that the molecules spreading in the fluid monolayer are very disordered, the entropy and enthalpy changes during melting at the air− water interface are approximately equal to those of bulk. Therefore, surface melting temperature is given by ⎛ πa ⎞⎟ Ts = Tb⎜1 − ⎝ ΔH ⎠

(7)

where ai is the mean molecular area and Rk,i is the evaporation resistance of the solid (2) or fluid (1) phase (derivation in Supporting Information). By using eq A8 in the Supporting Information, values of 2.7 ± 0.9 and 0.1 ± 0.1 s/cm are obtained for the evaporation resistances of the solid and fluid BP monolayer, respectively. According to the isotherm measurements, the mean molecular areas of the solid and fluid films are 18 and 50 Å2. Evaporation reduction predicted using these values is shown in Figure 3. The measured values are in agreement with the predicted behavior except for mean molecular areas close to 18 Å2. This can be explained by small imperfections in the film coverage, as a small decrease in coverage from 100 to 98% results in a large decrease in evaporation reduction from 59 to 41% according to eq 6. Evaporation reduction is not increased as the amount of WEs is increased beyond monolayer coverage, likely because BP does not seem to form multilayers in an ordered manner (Figure 2Av). According to a previous study on evaporation reduction by WEs,14 WEs only retard evaporation 1−8 °C below their bulk melting temperature. The inability of WEs to retard evaporation at low temperatures was explained by the incomplete coverage of the interface by the solid WE islands. At high temperatures, the fluidity of the films was considered to allow rapid permeation of water molecules. Both of these conclusions are supported by the results of the current study. The results presented here also offer an explanation for the evaporation-retarding effect several degrees below the bulk melting temperature. It appears that in this temperature range solid and fluid phases coexist and their relative proportions depend on the mean molecular area of the film. Because the WE film spreads efficiently only in the fluid phase, but retards evaporation only in the solid phase, the coexistence of these phases allows the film to spread and completely cover the water surface with a solid film, leading to the evaporation-retarding effect. Evaporation resistance of lipid films can be roughly divided into two types: interfacial resistance and bulk resistance.27 Interfacial resistance is caused by a thin, organized layer, like a monolayer at the air−water interface. Water molecules are thought to permeate such layers through holes formed by random fluctuations in the hydrocarbon density, domain boundaries, impurities, or larger cracks in the film.28 In contrast to interfacial resistance, which requires a specific molecular organization, bulk resistance can be achieved by practically any lipid films that remain stable at the air−water interface. It is caused by slow diffusion of water molecules through the lipid layer. However, in order for the bulk resistance to be comparable to the evaporation resistance of ordered monolayers, fluid lipid films need to be thicker than 100 nm.9 Current results suggest that WEs adopts an extended, bulk-like conformation in the solid phase at the air−water interface, which allows the molecules to pack tightly in an organized interfacial layer and to retard evaporation by a similar mechanism as saturated fatty alcohols or fatty acids. The melting temperature of healthy meibum has been determined to be 32.5 ± 1 °C with a broad melting range.29 The temperature of the inner eyelid is approximately 35 to 36 °C.30 Healthy meibum is liquid at this temperature and

(6)

For Jojoba-like WEs, enthalpy of melting is on the order of 100 × 10−21 J/molecule.21 From the isochor and isotherm measurements, the surface pressure of the fluid monolayer appears to be approximately 2.3 mN/m, and the mean molecular area, 50 Å2. Using these values, the surface melting temperature of BP is predicted to be 34 °C, well in agreement with the observed behavior. Melting proceeds until the water surface is completely covered by the fluid monolayer. Heating the film above the bulk melting temperature causes the remaining solid monolayer domains to melt into liquid droplets, which coexist with the fluid monolayer (Figure 1c and 4c), a state known as pseudopartial wetting.22 Similar surface melting behavior has been reported for long chain alkanes and alcohols at the air−SiO2 interface.23,24 Isotherm and compressibility data measured at 36 °C (Figure 2) reveal details about the organization of the film in the temperature range where solid and fluid monolayers coexist. At high mean molecular area, BP appears to form a fluid monolayer that coexists with a gas phase. The mean molecular area in this fluid monolayer is approximately 50 Å2, and a likely conformation of the molecules in this state is the previously suggested hairpin conformation, with ester groups anchored to the water and hydrocarbon chains pointing generally upward in a disordered manner.25 Upon compression, the surface pressure increases as the film undergoes a transition to the solid state. The mean molecular area of the solid film is approximately 18 Å2, close to the cross-sectional area of a single hydrocarbon chain, suggesting that the solid film consists of WE molecules in a fully extended conformation. According to the BAM measurements (Table 1), the thickness of the condensed BP film is 42 ± 6 Å. Thirty eight-carbons-long Jojoba-like WEs containing a double bond in both hydrocarbon chains would form crystals with a long crystal spacing values of 43 or 48 Å, depending on the crystal structure.21 A saturated WE with 38 carbons is predicted to have a long crystal spacing of 51 ± 3 Å in a rectangular cell.26 The thickness of the condensed layer is therefore on the same order as would be expected from a monolayer of fully extended BP molecules. A limitation of the used method is that the optical properties of the WE film are assumed to be equal to a solid fatty alcohol monolayer. This is not likely to be entirely accurate, but according to the isotherms, the hydrocarbon packing density is similar and therefore the optical properties are also likely to be similar. The evaporation measurements (Figure 3) show that a fluid monolayer of BP does not significantly retard evaporation, similar to fluid phospholipid or fluid fatty alcohol monolayers.12,17 A solid BP monolayer completely covering the water surface reduces evaporation by 59 ± 7% (mean ± SD). Evaporation reduction, E, as a function of mean molecular area, a, can be predicted by 5900

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Langmuir therefore it will flow with ease from the meibomian gland to the surface of the tear film. The average ocular surface temperature is lower (32−35 °C) and decreases constantly during the interblink period.31−34 Therefore, meibum cools, possibly several degrees, as it moves to the ocular surface. It has been suggested that solidification of TFLL on the ocular surface would explain its remarkable stability between blinks that is observed in vivo.35 The phase transition temperature of meibum has also been found to be significantly higher in patients with meibomian gland dysfunction than it is in healthy controls,36 thereby producing a less stable film at the ocular surface. The results presented here cannot be directly applied to the physiological TFLL, as it is a complex structure formed by multiple lipid types including WEs, cholesteryl esters, and polar lipids.37,38 However, because a major portion of meibum consists of WEs similar to BP,19 the current results offer a possible mechanism to connect meibum’s melting temperature to the stability and evaporation-retarding ability of the TFLL. To our knowledge, evaporation retardation by meibomian lipids has been studied in vitro either close to room temperature or at temperatures higher than 35 °C,8−11 which might be one reason why significant evaporation-retarding effects have not been found. It is possible that the evaporation-retarding effect of the TFLL is also achieved by a thin interfacial layer, which would explain why a thin TFLL (100 nm).39,40 Because of the presence of other lipid species than WEs, the molecular organization of the TFLL is not likely to be identical to the one presented here. However, cholesterol esters have also been found to organize in an extended bulk-like conformation and to form interdigitated bilayers at the air−water interface.41 Future studies are underway to determine the effect of adding other major TFLL components to evaporation-retarding WE films.

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REFERENCES

(1) McCulley, J. P.; Shine, W. A compositional based model for the tear film lipid layer. Trans. Am. Ophthalmol. Soc. 1997, 95, 79−88 discussion 88−93.. (2) Rantamäki, A. H.; Telenius, J.; Koivuniemi, A.; Vattulainen, I.; Holopainen, J. M. Lessons from the biophysics of interfaces: lung surfactant and tear fluid. Prog. Retinal Eye Res. 2011, 30, 204−215. (3) King-Smith, P. E.; Bailey, M. D.; Braun, R. J. Four characteristics and a model of an effective tear film lipid layer (TFLL). Ocul. Surf. 2013, 11, 236−245. (4) Mishima, S.; Maurice, D. M. The oily layer of the tear film and evaporation from the corneal surface. Exp. Eye Res. 1961, 1, 39−45. (5) Iwata, S.; Lemp, M. A.; Holly, F. J.; Dohlman, C. H. Evaporation rate of water from the precorneal tear film and cornea in the rabbit. Invest. Ophthalmol. 1969, 8, 613−619. (6) Craig, J. P.; Tomlinson, A. Importance of the lipid layer in human tear film stability and evaporation. Optom. Vis. Sci. 1997, 74, 8−13. (7) Borchman, D.; Foulks, G. N.; Yappert, M. C.; Mathews, J.; Leake, K.; Bell, J. Factors affecting evaporation rates of tear film components measured in vitro. Eye Contact Lens 2009, 35, 32−37. (8) Herok, G. H.; Mudgil, P.; Millar, T. J. The effect of Meibomian lipids and tear proteins on evaporation rate under controlled in vitro conditions. Curr. Eye Res. 2009, 34, 589−597. (9) Cerretani, C. F.; Ho, N. H.; Radke, C. J. Water-evaporation reduction by duplex films: application to the human tear film. Adv. Colloid Interface Sci. 2013, 197−198, 33−57. (10) Miano, F.; Calcara, M.; Giuliano, F.; Millar, T.; Enea, V. Effect of meibomian lipid layer on evaporation of tears. J. Phys.: Condens. Matter 2004, 16, S2461. (11) Brown, S. I.; Dervichian, D. G. The oils of the meibomian glands. Physical and surface characteristics. Arch. Ophthalmol. 1969, 82, 537−540. (12) Rosano, H. L.; La Mer, V. K. The rate of evaporation of water through monolayers of esters, acids and alcohols. J. Phys. Chem. 1956, 60, 348−353. (13) Henry, D. J.; Dewan, V. I.; Prime, E. L.; Qiao, G. G.; Solomon, D. H.; Yarovsky, I. Monolayer structure and evaporation resistance: a molecular dynamics study of octadecanol on water. J. Phys. Chem. B 2010, 114, 3869−3878. (14) Rantamäki, A. H.; Wiedmer, S. K.; Holopainen, J. M. Melting points−the key to the anti-evaporative effect of the tear film wax esters. Invest. Ophthalmol. Vis. Sci. 2013, 54, 5211−5217. (15) Schneider, C. A.; Rasband, W. S.; Eliceiri, K. W. NIH Image to ImageJ: 25 years of image analysis. Nat. Methods 2012, 9, 671−675. (16) de Mul, M. N. G.; Mann, J. A. Determination of the thickness and optical properties of a Langmuir film from the domain morphology by Brewster angle microscopy. Langmuir 1998, 14, 2455−2466. (17) Rantamäki, A. H.; Javanainen, M.; Vattulainen, I.; Holopainen, J. M. Do lipids retard the evaporation of the tear fluid? Invest. Ophthalmol. Visual Sci. 2012, 53, 6442−6447. (18) Tanford, C. The Hydrophobic Effect: Formation of Micelles and Biological Membranes, 2nd ed.; Wiley: New York, 1980. (19) Butovich, I. A.; Arciniega, J. C.; Lu, H.; Molai, M. Evaluation and quantitation of intact wax esters of human meibum by gas-liquid chromatography-ion trap mass spectrometry. Invest. Ophthalmol. Visual Sci. 2012, 53, 3766−3781. (20) Weinbach, S. P.; Weissbuch, I.; Lahav, M.; Leiserowitz, L.; Kjaer, K.; Bouwman, W. G.; Als Nielsen, J. Self-assembled crystalline monolayers and multilayers of n-alkanes on the water surface. Adv. Mater. 1995, 7, 857−862. (21) Bouzidi, L.; Li, S.; Di Biase, S.; Rizvi, S. Q.; Narine, S. S. Lubricating and waxy esters, I. Synthesis, crystallization, and melt behavior of linear monoesters. Chem. Phys. Lipids 2012, 165, 38−50.

CONCLUSIONS We have described the surface behavior of BP, a weakly surface active WE, at different temperatures. We discovered that BP undergoes surface melting at the air−water interface approximately 3 °C below its bulk melting temperature. This leads to monolayer spreading and formation of a uniform solid monolayer at the air−water interface that efficiently retards evaporation. These results provide a detailed explanation for the finding that this kind of lipid only prevents evaporation close to its melting temperature. The results also provide insight into the possible evaporation-retarding mechanism of the TFLL. ASSOCIATED CONTENT

S Supporting Information *

Description of the BAM calibration procedure and derivation of eqs 3 and 7. This material is available free of charge via the Internet at http://pubs.acs.org.

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ACKNOWLEDGMENTS

The Finnish Eye Foundation and Sigrid Juselius Foundation supported this study.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: juha.holopainen@hus.fi; Tel.: +358-9-471 77197; Fax +358-9-471 75100. Notes

The authors declare no competing financial interest. 5901

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dx.doi.org/10.1021/la501678t | Langmuir 2014, 30, 5897−5902