Langmuir 1990,6, 1647-1655
1647
Surface Analysis of Lipid Layers at Air/Water Interfaces Judy B. Chung, Robert E. Hannemann, and Elias I. Franses* School of Chemical Engineering, Purdue University, West Lafayette, Indiana 47907 Received September 12, 1989.I n Final Form: April 17, 1990 In order for the surface layer composition of lipid mixtures to be probed, Langmuir-Blodgett (L-B) films were deposited from surface layers of spread films or of aqueous dispersions of lung surfactant components. The composition and density of the deposited L-B films were determined by thin-layerchromatography/opticaldensitometry (TLC/OD) or attenuated total internal reflection Fourier transform infrared spectroscopy (ATR-FTIR). In FTIR, L-a-dipalmitoylphosphatidylcholine(DPPC) with perdeuterated chains (d-DPPC) was also used for the hydrocarbon stretching bands in mixtures to be better separated. TLC/OD and IR results indicated that DPPC and PG (phosphatidylglycerol) were the main components of the sheep lung surfactant used. Results from monitoring changes in the surface layer composition during surface layer compression with the use of the above two analysis methods were consistent. L-B films from DPPC-PA (palmitic acid) and d-DPPC-PA showed no enrichment of DPPC in the surface layer following compression from a surface pressure of II = 10 to II = 50 dyn/cm. However, depositions from spread films of DPPC-PG and d-DPPC-PG on water at 25 “C showed a significant enrichment of DPPC, from initially 50 to over 80 mol 5% as the surface pressure increased from 20 to 60 dyn/cm. The difference in the behavior of DPPC-PA and DPPC-PG films may be due to the difference in the solution nonidealities of the two mixtures. This hypothesis is supported by the surface pressure-area (II-A) isotherms of the mixtures. The II-A isotherm of a sheep lung surfactant dispersion resembled that of the DPPC-PG mixture, in that two plateaus (at II = 50 and 70 dyn/cm) were observed. As suggested from the isotherm data, enrichment of DPPC in the surface layer of the lung surfactant dispersion was measured. L-B depositions from spontaneously adsorbed and subsequently compressed surface layers of sheep lung surfactant dispersions showed a significant enrichment of DPPC, from -55 to -80 wt ’%,as the layer was compressed from Jl = 40 (uncompressed surface layer) to 70 dyn/cm. The results indicate the central role of DPPC in controlling the extraordinarily low surface tensions of aqueous mixtures of lung surfactants and supports the previously proposed “squeeze-out”hypothesis for surface layers of mixed lipids.
Introduction Lack of an adequate supply of lung surfactant can lead to respiratory distress syndrome, which afflicts many prematurely born infants.’ The key components of lung surfactants, which are phospholipids such as phosphatidylcholines (PC), phosphatidylglycerols (PG), and phosphatidylethanolamines (PE),could potentially be used as an exogenous lung surfactant replacement for treating RDS. For this reason, study of their surface properties has received much attention.2-6 The key to finding a consistent and effective replacement is to understand the physicochemical mechanism of how the multicomponent lung surfactant uniquely decreases the surface tension at the alveolar air/fluid interface to less than 10 dyn/cm (“superlow”surface tension) upon surface compression. No soluble hydrocarbon surfactant is known to reduce the surface tension of water to less than 20 dyn/cm at 25 “C with or without surface compre~sion.~ Although DPPC (dipalmitoylphosphatidylcholine)can produce low surface tensions upon compression, it works well only when in mixtures with PG or other lipids and proteins.2* Thus, it is important to determine the changes in the surface composition and density of the various lung phospholipid surface layers which are responsible for the unique tension behavior. * Author to whom all correspondence should be addressed (317)494-4078. (1)Avery, M. E.; Mead, J. Am. J. Dis. Child. 1959,97,517. (2)Notter, R. H.; Morrow, P. E. Ann. Biomed. Eng. 1975,119. (3)Notter, R.; Shapiro, D. Pediatrics 1981,68,781. (4)Notter, R.; Finkelstein, J. J. Appl. Physiol. 1984,57,1613. ( 5 ) Avery, M. E.; Taeusch, H. W.; Floros, J. N. Engl. J . Med. 1986,315, 825. (6)Jobe, A.; Ikegami, M. Am. Rev. Respir. Dis. 1987,136,1256. (7)Shinoda, K.;Nakagawa, T.; Tamamushi, B.; Isemura, T. Colloidal Surfactants-Some Physicochemical Properties; Academic Press: New York, 1963,pp 106,181. 0743-7463/90/2406-1647$02.50/0
A widely accepted hypothesis regarding the surface composition is the “squeeze-out”mechanism in which nonDPPC components are selectively removed from the surface layer as the surface is compressed. Surface layers of PG alone are incapable of sustaining as large surface pressures as DPPC. After surface compression, the resulting DPPC-enriched surface layer shows tensions lower than 10 dyn/cm.8-12 This hypothesis has been based mostly on evidence from surface pressure ll-surface area A isotherms&12and has been proposed earlier for other mixed lipids.13-15 Such isotherms provide only indirect measures of the surface density and composition, because they can be affected by dissolution or collapse of the surface layer and by the presence of various surface-active components in the surface layer other than DPPC or PG.16J7 The ability to directly probe the surface for changes in molecular composition and surface density during compression should help elucidate the mechanism of lung surfactant function. Two methods which allow in situ measurement of the surface layer composition a t various surface pressures are the use of radioactive tracers18J9 and external reflection (8)Watkins, J. C. Biochim. Biophys. Acta 1968,152,293. (9)Trauble, H.; Eibl, H.; Sawada, H. Naturwissenschaften 1974,61, 344. (10) Hildebran, J. N.; Goerke, J.; Clementa,J. A. J.Appl. Physiol. 1979, 47,604. (11)DeFontanges, A.; Bonk, F.; Taupin, C.; Ober, R. Colloids Surf. 1985,14,309. (12)Boonman, A.; Machiels, F. H. J.; Snik, A. F. M.; Egberts, J. J. Colloid Interface Sci. 1987,120,456. (13)Schulman, J. H.; Hughes, A. H. Biochem.J. 1935,29,1243. (14)Schulman, J. H. Trans. Faraday SOC.1937,33,116. (15)Florence, R. T.; Harkins, W. D. J. Chem. Phys. 1938,6,856. (16)Horn, L.W.;Gershfeld, N. L. Biophys. J. 1977,18,301. (17)Notter, R. H. In Pulmonary Surfactant;Robertson,B., VanGolde, L. M. G., Batenburg, J. J., Eds.; Elsevier Science Publishers: Amsterdam, 1984;p 17. (18)Tajima, K.;Gershfeld, N. L. Biophys. J . 1985,47,203.
0 1990 American Chemical Society
1648 Langmuir, Vol. 6, No. 11, 1990 infrared (ER-IR)spectroscopy.20,21The main advantages of the radioactive tracer method are the following: (i) high sensitivity, (ii) atom specificity, and (iii) no disturbance to the surface. There are some difficulties, however, in using this method for dispersions due to the required calibration procedure. In order for the surface concentration to be determined, the contribution to the signal by the radioactive molecules which are present in the bulk phase close to the surface must be subtracted from the total radioactivity measured.Is This bulk-phase concentration of the dispersed insoluble material, such as lung surfactant and its components, is not reliably known. Another major hurdle in using the radioactive tracer method is the usual unavailability of radioactively labeled natural lung surfactants. Recently, a method t o record an ER-IR spectrum of phospholipids at the air/water interface was developed for qualitative structural studies of the surface monolayers.20j21This method could possibly be extended to quantitative analysis of surface layers. However, because the method is still in its initial stages of development, more studies are required before routine quantitative analysis becomes feasible. Methods for sampling the surface microlayer in lakes have been reviewed.22 Another viable method to probe the surface layers, and the focus of this paper, is to sample the surface layers a t various surface pressures by physically separating some representative samples of the surface layer and analyzing them for their density and molecular composition. The Langmuir-Blodgett (L-B) deposition technique is wellknown, involving transferring insoluble monolayers to solid s u b s t r a t e ~ . ~ 3T7h~e~surface layers of spread or spontaneously adsorbed layers can be directly sampled at various surface pressures by the L-B deposition technique. In this paper, we report results of two methods by which such deposited films are analyzed for their compositions and surface densities: (i) thin-layer chromatography (TLC)/optical densitometry (OD)25-27 and (ii) attenuated total internal reflection Fourier transform infrared (ATRFTIR) s p e c t r o s ~ o p y From . ~ ~ ~these ~ ~ measurements, substantial enrichment of the surface layer in DPPC for binary and multicomponent lung surfactant mixtures is directly shown for the first time, providing support for the occurrence of the "squeeze-out" mechanism in mixed lipid layers and helping clarify the roles of the various lipid components in the tension-lowering process. Experimental Section Materials and Sample Preparation. Synthetic L-adipalmitoylphosphatidylcholine (DPPC) (99+ % ) and ammonium salt L-a-phosphatidylglycerol (PG) from egg lecithin, with various chains (palmitic, C15H31, oleic, C15H29,and others), were obtained from Sigma Chemical Co. Palmitic acid was obtained from Fluka Chemical Co. L-CY-DPPC with perdeuterated hydrocarbon tail groups was obtained from Avanti Polar Lipids. These lipids were used without further purification. Sheep lung surfactant in saline (16 mg/mL) from lung lavage of (19) Muramatsu, M. In Surface and Colloid Science;Matijevic, E., Ed.; Wiley-Interscience: New York, 1973; Vol. 6, p 101. (20) Dluhy, R. A.; Cornell, D. G. J . Phys. Chem. 1985,89, 3195. (21) Mitchell, M. L.; Dluhy, R. A. J . Am. Chem. SOC.1988, 119, 712. (22) Guanski, H.; Goupil, D. W.; Baier, R. E. In Atmosphen'c Pollutants in Natural Waters; Eisenreich, S . J., Ed.;Ann Arbor Science Publishers: Ann Arbor, 1981; p 165. (23) Langmuir, I. Trans. Faraday SOC.1920, 15, 62. (24) Blodgett, K. B. J . Am. Chem. SOC.1935,57, 1007. (25) Tsai, M. Y.; Marshall, J. G. Clin. Chem. 1979, 25, 682. (26) Tsai,M. Y.; Cain, M.; Josephson, M. W. Clin. Chem. 1981,27,239. (27) Dugan, E. A. Liq. Chromat. 1985, 3. (28) Okamura, E.; Umemura, J.; Takenaka, T. Biochem. Eiophys. Acta 1985,812, 139. (29) Lotta, T. I.; Laakkonen, L. J.; Virtanen, J. A,; Kinnunen, P. K. J. Chem. Phys. Lipids 1988, 46, 1.
Chung et al. healthy adult ewes30was provided by Dr. A. Jobe (UCLA Medical Center). All other chemicals were of reagent grade. Distilled water was drawn through a Millipore Milli-Q fourcartridge system for further purification. Spreading solutions were prepared by dissolving the lipids in a spreading solvent of 9:l (v/v) hexane/ethanol. DPPC dispersions were prepared by magnetically stirring DPPC in water at temperatures above 45 "C for about 30 min to better homogenize the dispersion. The dispersions were then cooled to 25 O C . Samples were used within 48 h after preparation to avoid or minimize effects of lipid hydrolysis.31 Isotherms. A Joyce-Loebl computer-controlled Langmuir trough with a Wilhelmy balance was used to record II-A isotherms. The surface area could vary from 1000 to 200 cm2 with a constant-perimeter barrier made of PTFE (Teflon) tape. The spread monolayers were prepared by applying known volumes of the spreading solution on the water surface and letting the solvent evaporate. Lung surfactant surface layers were formed by layering 2.0 mL of the sheep lung surfactant dispersion in saline (16 mg/mL) on the surface of the water in the trough. Spontaneous adsorption of only a small fraction of the total surfactant was presumed to occur from the dispersion at the air/ liquid interface. Isotherms were recorded at 25 "C. Langmuir-Blodgett (L-B) Depositions. After the appropriate surface layer was formed, depositions were made at various controlled surface pressures by the L-B technique with the same Langmuir trough which was used to record the II-A isotherms. The ratio of surface area decrease (AA) at constant II t o the deposition area of the substrate (Asubt)is defined as the transfer ratio TR. The transfer ratio is unity if there is a perfect transfer of the monolayer to the substrate. Transfer ratios greater than 1 indicate collapse, dissolution, or desorption of portions of the monolayer into the subphase. Speeds of deposition could be controlled from 5 to 50 mm/min. The temperature was controlled to f3 "C or better. The instrument was housed in a clean room (class 100-1000) which was equipped with a laminar flow hood (class 100). For film analysis by the thin-layer-chromatography technique, depositionswere made on 3 X 1 in. precleaned plain glass microscope slides from Fisher Scientific. The slides were further cleaned under vacuum in a Harrick Scientific plasma cleaner. The substrate area of deposition was 25 cm2. Unless otherwise specified, all depositions on glass were made by first lowering and then raising the substrate once through the air/water interface. For analyzing the surface by infrared spectroscopy, depositions were made on silicon attenuated total internal reflection (ATR) plates from Harrick Scientific or Wilmad Glass Co. These plates were 50 X 10 x 2 mm, single-pass, and of trapezoidal shape with an angle of incidence of 45". The number of internal reflections was 25. The substrate area of deposition was 9.6 cm2. Depositions on the ATIR plates were made as the plate was raised through the interface once. All depositions were made at 25 "C. Film Dissolution and Thin-Layer Chromatography (TLC). The mixtures which were present in certain deposited L-B films were separated by TLC. The materials from the L-B depositions were removed from the substrate by multiple rinses with the spreading solvent. Maximum recovery of the deposited material from the substrate was achieved by rinsing the glass slides with 40 mL of the spreading solvent for 5 min in a 50-mL Teflon beaker (from American Scientific Products). When known amounts of DPPC, PG, and palmitic acid (PA) were directly applied to the slides and then washed with the above technique, 80-9096 of the surfactant applied was detected by TLC. For this reason, only results obtained with this technique are reported. When the glass slides were broken and immersed in the spreading solvent overnight, the percent surfactant recovery ranged from 40% to 70%, probably because of excessive adsorption on the glass fragments. Using the Teflon beaker was crucial for minimizing surfactant adsorption on the container, since washing the slides in glass beakers yielded only 15-25 % recovery. After sufficient evaporation of the solvent (to (30) Jobe, A.; Ikegami, M.; Glatz, T.; Yoshida, Y.; Diakomanolis, E.; Padbury, J. J . Clin. Invest. 1981, 67, 370. (31) Chung, J. B.; Shanks, P. C.; Hanneman, R. E.; Franses, E. I. Colloids Surf. 1590, 43, 223.
Langmuir, Vol. 6, No. 11, 1990 1649
Surface Analysis of Lipid Layers Table I. Calibration Data of the L-B/TLC/OD Method for One-Component Films on Glass Slides n, OWtP ameM,b no. of dyn/cm TR pg/cmz pg/cm2 measurements DPPC 30 50 60 70 70
25
1.0 1.0 3.0 3.0
0.21 f 0.02 0.22 f 0.02 0.23 f 0.02 0.24 f 0.02
C
0.17 f 0.03 0.15 i 0.03
0.05-0.18
4
4 2
0.1-0.3 0.44 f 0.05
3
30
0.21 f 0.02 0.22 f 0.02
0.12d f 0.1 0.14d f 0.1
2 2
20
1.1
0.18 f 0.02
0.12 f 0.01
3
PA 4 From isotherm. b OD/area of deposition. Deposition at constant area. Area of deposition corrected for AA-t behavior of PG at constant II.
about 0.1 mL), the rinsed material was loaded on a 10 X 20 cm TLC plate (silica gel H with 5% ammonium sulfate from Analtech). The Teflon beaker was rinsed profusely with the solvent, and the evaporation/applicationstep was repeated 3 times to ensure that all of the deposited material was transferred to the TLC plate. Results of control experiments are given in the Results section. Plates were developed in a chamber containing 506:6 ethanol/chloroform/ammonium hydroxide (v/v/v).27 The developed TLC plates were allowed to dry for -5 min, sprayed with 1:l (v/v) sulfuric acid/water, and charred at 300 "C on a hot plate (type 2200 Thermolyne from Curtis Matheson Scientific Inc).26 A clear separation between DPPC (Rf = 0.4) and PG (R, = 0.9) was observed. Rf is the ratio of the distance traveled by the sample spot divided by the distance traveled by the solvent. R, values of PA and PG were very similar, and the two spots overlapped on the TLC plates. Thus, compositional analyses of binary DPPC-PG or DPPC-PA mixtures are possible with this method, but analyses of ternary DPPC-PG-PA mixtures are not. Concentrations of the TLC spots were determined with optical densitometry (OD)2"27 at 525 nm with a Helena Laboratories transmission densitometer (Quick Scan Jr.), which also allowed for density integration. The deposited amounts were determined by comparison of the integrated intensities of the TLC spots of the deposited samples with those of standards. Solutions of standards were prepared by dissolution of known amounts of DPPC, PG, and PA in the same solvent, which was used for spreading. The optical density was linear with concentration up to about 20 pg for DPPC and up to 5 pg for PG and PA. Because the specific optical density per 1pg of sample varied from plate to plate by up to 50%, depending on the amount of acid sprayed and the length of time on the hot plate, it was necessary to calibrate each plate separately. Further details are given elsewhere.32 Infrared Spectroscopy. L-B films on solid substrates were also analyzed by ATR-FTIR. L-B films which had been deposited onto silicon ATR plates were directly examined in a Digilab FTS-40 Fourier transform infrared spectrometer equipped with a TGS detector. The Harrick horizontal sampling infrared reflection spectroscopy attachment was used. For each spectrum, 64 interferograms were collected; spectral resolution was 4 cm-'. The spectrum of each ATR plate was used as a background. The frequency determination was accurate to better than *0.5 cm-'. Absorbance bands were integrated with a Digilab program. Results and Discussion Results from L-B/TLC/OD. Table I compares the surface densities of one-component films as estimated from a II-A isotherm to those determined via the L-B/TLC/ OD method. We recovered 60-80% of the deposited L-B films at ll below the collapse pressure following the abovedescribed wash/transfer procedure for all three lipids. (32)Chung, J. B. Ph.D. Thesis, Purdue University, 1989.
40
I
'
0
I
I
I
1
I
20
40
60
80
II (dyn./cm) Figure 1. Composition of L-B films deposited from mixed spread
PG 1.8 2.0
100
films of DPPC-PG, initially 4951 mol/mol, at various surface pressures. Composition was determined from TLC/OD.
Recovery of films which were directly applied to the glass slides ranged from 80 % to 90 % , which shows that desorption and redissolution were nearly complete. The results indicate that the method samples the surface layer at II values below the collapse pressure reasonably well. At 11 1 6 0 dyn/cm, however, the D P P C monolayer collapsed, as evidenced by the monolayer instability and by the dramatic increase in the transfer ratio (TR) to nearly 3. After the collapse, since the surface layer was no longer uniform, the deposition on the L-B plate may be difficult to control. Hence, at the large J I values the determination of surface density becomes more uncertain with this sampling method, as indicated by the erratic percent recovery of DPPC of 22-12596 a t II = 60 and 70 dyn/ cm. Results from TLC/OD analysis of L-B films which were deposited from a mixed DPPC-PG (initially 51:49 mol/ mol) spread surface film are shown in Figure 1. Substantial enrichment of DPPC in the surface layer was observed with surface compression. At low surface pressures (II 5 30 dyn/cm), thecomposition was about the same as the one on the L-B layer. This and Table I rule out the possibility of selective desorption and suggest that the L-B/TLC method provides a representative sample of the mixed monolayer. The relative amount of DPPC in the surface layer increased from 50 96 to 80 96 as the surface pressure was increased from 20 to 60 dyn/cm. PG monolayer films collapse a t -40 dyn/cm." As the surface is compressed, PG, which is the more easily collapsible or less stable component of the film mixture, is selectively removed or "squeezed out" of the surface layer, leaving a layer which is richer in DPPC. A mass balance indicates that both DPPC and PG are partially expelled from the surface. However, the expelled material contains more PG than DPPC, resulting in a net DPPC enrichment of the surface layer after the indicated surface compression. A discwion of why the squeeze-out effect is more pronounced with lipids containing unsaturated chains can be found in ref 15. In a similar set of experiments with DPPC-PA spread films, initially 26:74 mol/mol (or 5050 wt/wt), there were no significant changes in the mole percent of DPPC (Figure 2). Even after the surface had been compressed to 50 dyn/ cm, which is above the collapse pressure of PA, the DPPC mole fraction was within experimental error of the initial value, with depositions normally done minutes after film formation. These results also suggest that the LB-TLC method obtains a representative sample of the mixed monolayer. When depositions were made 1 h after the surface had been compressed, however, a small DPPC enrichment from 26 to 33 5% was consistently measured. T h e difference in t h e behavior of DPPC-PA and DPPC-PG mixtures is probably due to the extent of miscibility between the components in the two mixed films. Due to their identical hydrocarbon tails, DPPC and PA should be more miscible than DPPC and PG, and the
Chung et al.
1650 Langmuir, Vol. 6 , No. 11,1990 40
I
0
20
40
60
80
(dyn/cm)
Figure 2. Composition of L-B films deposited from a DPPCPA, initially 2674 mol/mol, mixed spread film at various surface pressures. Composition was determined from TLC/OD. Open circles represent depositions made immediately (minutes) and the closed circles represent depositions made 1h after the surface pressure was changed. former mixture apparently behaves as a more ideal twodimensional mixture than the latter. If DPPC and PA are miscible, then their mixed surface film is homogeneous, and the composition of the film is expected to remain uniform at all surface densities, even after partial film collapse. The small increase in the DPPC content after a delayed deposition may be due to a preferential loss of PA from the surface layer during compression. This slight loss is probably due to dissolution of PA, which has a finite solubility in the subphase. Surface layers adsorbed from dispersions could also be analyzed after L-B deposition. In order to study a system a t conditions which were closer to those in vivo, L-B depositions were made from surface layers spontaneously adsorbed from an aqueous dispersion of sheep lung surfactant. Weight percent units were used because the molecular weights of the various components of the lung surfactant sample were not known. The main component of the lung surfactant sample was DPPC, as identified from TLC and as is usually observed in natural ~ u r f a c t a n t . ~ ~ Optical densitometry results indicated that the bulk lung surfactant contained 50-60 w t 5% DPPC. T h e rest consisted of PG and possibly other components which are eluted as PG.30 Therefore, for TLC purposes, binary mixtures of DPPC-PG are reasonable representations of this lung surfactant. The concentration of PG present in the deposited film after one dipping cycle was below the detection limits (-2 pg) of our optical densitometer. For this reason, multiple-layer depositions were made for DPPC-PG layers. After three cycles, which took -40 min, PG spots were detectable and could be quantified. Then, the relative amounts of DPPC to PG were determined. Spontaneous adsorption of lung surfactant molecules, with no surface layer compression, resulted in an initial surface pressure of 40 dyn/cm. The transfer ratio (as measured from the area change) for the deposition at 40 dyn/cm was zero. At 60 dyn/cm, the transfer ratio for the first cycle was nearly 2, with deposition occurring both on the way up and on the way down. The TR for the second and third cycle was 1,with deposition occurring only on the way up. The L-B depositions from the uncompressed surface layers contained 55 wt 7;DPPC, which compares well with the composition of the bulk sample. This value is consistent with a reported value of 53 % for an uncompressed sample by Hildebran e t a1.,'0 who made their estimate by comparing the E A isotherms of a dog lung surfactant with those of lipid mixtures containing known amounts of DPPC. Surface layers which were deposited after the surface had been compressed by 70 7; (- 2 min) to yield II = 60 dyn/cm contained nearly 90 w t 9; DPPC, consistent with a value of -90% estimated by Hildebran et d.l0with the above-mentioned method. When the transfer
3000
ZWO
2600
2400
no0
2000
1600
1600
Wavenumber (em-')
Figure 3. ATR-FTIR spectra of DPPC (A),PG (B),PA (C),and d-DPPC (D)samples applied to silicon ATR plates. The spectra have been vertically shifted for clarity. ratio was not 1, only the relative amounts of surfactant components could be reliably determined with this method. During depositions from a dispersion, the substrate may pick up particles from the dispersion in addition to the L-B layer itself. The observed zero transfer ratio from the deposition at II = 40 dyn/cm indicates that either (i) most if not all of the detected material from this deposition is due to the bulk dispersion particles or (ii) adsorption from the solution during deposition was fast enough to reestablish the surface layer after the substrate was removed. Nevertheless, if only the dispersed particles were detected in our method, the composition should remain constant at all II values. The increase in the relative amount of DPPC at higher surface pressures indicates that the surface layer was mainly probed and that significant enrichment of DPPC occurred in the surface layer upon compression. Evidence from TLC/OD indicates, therefore, that in mixed spread films which mimic lung surfactants and in surface layers which are adsorbed from a natural lung surfactant dispersion, non-DPPC components are preferentially expelled, or squeezed-out, with surface compression. The main sources of error in the L-B/TLC/OD method of analysis may arise from (i) the L-B sampling of the surface, (ii) effects of collapsed microstructures, (iii) incomplete or selective washing and transfer of the deposited material from the glass slides to the Teflon container, (iv) inaccuracies of loading of the fluid sample to the TLC plate, and (v) the calibration and integration procedures of the OD measurements. Our results indicate t h a t source (iii) is minor. T h e sources iii-v can be eliminated if the L-B film could be directly analyzed without having to first dissolve the film and then load the lipid to a TLC plate. R e s u l t s by ATR-FTIR Spectroscopy. A viable method to reduce the above errors iii-v is to examine the deposited films directly by depositing the surface film onto an ATR plate and analyzing for ita composition and surface density by FTIR spectroscopy. The ATR mode was used because it is much more sensitive than the transmission mode, which has poor signal-to-noise ratio for one monolayer. P u r e Components. Spectra of bulk DPPC, PG, PA, and d-DPPC (with perdeuterated hydrocarbon tails) were recorded to examine how well the individual components could be distinguished in mixtures (Figure 3). The band assignments and their frequencies are listed in Table 11. The largest and most important IR bands in the spectra of the undeuterated compounds are the carbonyl C=O stretching band, occurring at 1700-1740 cm-l, and the
Langmuir, Vol. 6, No. 11, 1990 1651
Surface Analysis of Lipid Layers Table 11. Observed Bands of ATIR-FTIR of P u r e Lipids wavenumber (hm-1) for vibration mode'
DPPC
PA
PG
d-DPPC
u(C=O) dCHd 4CHd u(methy1)
1736 2850 2917 2956
1703 2850 2917 2956
1739 2854 2923 2956
1735 2091 2194 2213
"x I
41
1.60
Y,
d
stretching; s, symmetric; as, antisymmetric. 0.60
d 4 a
a
d
I4
0.0 0.0
p
H
3.00
8.00
9.00
12.00
x 10. (CH,+CH./cms)
Figure 5. Calibration curves of integrated absorbance of the CH2
R
a W
+
E
I 01 0
/
J 1
I
I
J
3
4
6
e..
2
p x l O b o (mol/cm*)
Figure 4. Surface pressure-surface density isotherms of DPPC (A) and (I-DPPC (B)spread films. T h e compression rate was 10 cm2/s.
hydrocarbon stretching bands (antisymmetric and symmetric), occurring from 2800 to 3000 cm-1.33-35 A shift from 1736 to 1703 cm-l was observed in the PA carbonyl stretching band (acid) from that of DPPC (ester), as previously reported.33 The intense C=O band, which was seen in the bulk palmitic acid spectrum (Figure 3C), was not detected in the spectra of L-B-deposited palmitic acid monolayers. Instead, a weak band was found near 1700 cm-l. This phenomenon has been observed before with stearic acid monolayers and has been attributed to short-range image force fields a t semiconductor surfaces.% This effect is expected to occur only a t very short (- 1bond length) distances from the surface of the solid substrate. The peak of the CH2 antisymmetric stretching band of PG occurred a t a higher wavenumber (by 6 cm-l) than that of D P P C (Table 11). No shift was observed in the frequency of the methyl group. This shift may be due to the smaller number of trans hydrocarbon chain conformations in the PG hydrocarbon According to the manufacturer, the hydrocarbon chains of the PG sample which was used here contain one or more double bonds (oleyl, etc.), which apparently reduce the trans conformations of the hydrocarbon chains compared to those of DPPC chains.34~35~37 d-DPPC showed a slightly different surface behavior than DPPC (Figure 4). d-DPPC is a less efficient surfactant than DPPC, in that 28% more surface density of d-DPPC is required t o reach II = 25 dyn/cm. The isotherms of the two phospholipids are qualitatively similar, in that they both show two plateaus, the higher plateau occurring a t II values greater than 60 dyn/cm. The d-DPPC (perdeuterated chains) spectrum shows large shifts in the stretching bands of the hydrocarbon tail groups from that of DPPC (Table 11). These shifts are (33) Bellamy, L. J. Advances in Infrared Group Frequencies; Methuen & Co.: Great Britain, 1968. (34) Fringeli, U. P.; Gunthard, Hs.H. In Membrane Spectroscopy;Grell, E., Ed.; Springer-Verlag: Berlin, 1981; Vol. 31, p 273. (35) Amey, R.; Chapman, D. In Biomembrane Structure and Function; Chapman, D., Ed.; Verlag-Chemie: Weinheim, 1984; p 199. (36) Kimura, F.; Umemura, J.; Takenaka, T. Langmuir 1986, 2, 96. (37) Shah, D.; Schulman, J. H. J. Lipid Res. 1967,8, 227.
CH3 or CD2 + CD3 stretching regions vs the number density of those groups in the L-B films. Films were deposited from DPPC, PG, PA, and d-DPPC spread monolayer films at various surface pressures. Data at pressures at which films were deposited with TR = 1 were used. The densities of the CH and CD groups in the deposited film were determined from the surface density p and the molecular structures (see text). T h e surface density (p! was determined from the I I - p isotherms. Open circles, DPPC; triangles, PA; closed circles, d-DPPC.
due to the well-known isotope effect, in which, for diatomic molecular vibrations, the frequency is inversely proportional to the square root of the reduced mass p of the atom pair, C-H or C-D.3733 The reduced mass of a system of two bodies is defined as
where ml and m2 are the masses of the two bodies. The frequencies are related as follows:
where the subscript i denotes the isotope. The predicted frequency ratio is 0.73. For asymmetric stretching frequencies, this ratio is about 0.75.39 Both predictions compare well with the observed ratios 0.73-0.75. In addition to the frequency shift, the specific absorptivities of the CH2 and CD2 stretching bands are different.39140 The theory for polyatomic molecules or groups is complex.39 Based on simple theory for diatomic molecules, the integrated absorbance I is inversely proportional to the reduced mass39 (3) For CD2 vs CH2, the predicted ratio is 1/1.9. An intensity ratio of 1/2.3 was reported from data on ethane and hexade~terohexane.~~ The agreement is good, considering the simplifications of the theory. L-B films were deposited from spread monolayers of the pure components a t various II values in order to prepare calibration curves of integrated absorbance vs surface density. Only depositions with T R = 1, for which the deposition is most orderly, were used in preparing the calibration curve in Figure 5. Surface densities were (38) Colthup, N. B.; Daly, L. H.; Wiberly, S. E. Introduction to Infrared and Raman Spectroscopy; Academic Press: New York, 1975. (39) Pinchas, S.; Laulicht, I. Infrared Spectra of Labeled Compounds; Academic Press: London, 1971. (40) Sverdlov, L. M. Optics and Spectroscopy (Engl. Transl.) 1959, 7
I ,
1 ,
IL.
(41) Nyquist, I. M.; Mills, I. M.; Person, W. B.; Crawford, B. J.Chem. Phys. 1957,26, 552.
1652 Langmuir, Vol. 6, No. 11, 1990
sob0
nbo
erbo
rh
r;oo
Chung et al.
2doo
I&
I&
Wavenumber (cm-' )
Figure 6. ATR-FTIR spectra of L-B films deposited at different surface pressures n (dyn/cm): (A) DPPC spread film, n = 20; (B)DPPC-PA, initially 50:50 mol/mol mixed spread film, n = 20; (C) DPPC-PA mixed spread f i b , II = 50; (D) PA spread f i b ,
n = 20.
determined from the spread monolayer isotherms. The surface density unit which is used for DPPC is the number of CH2 CH3 groups in the molecule contributing to the hydrocarbon stretching region a t wavenumbers from 2800 to 3000 cm-l (34 for DPPC and PG and 15 for PA). dDPPC shows weak CHs-CH2 band intensities due to the four undeuterated hydrocarbon groups in its head group. For d-DPPC, the number (30) of CD2 + CD3 groups contributing to the stretching band from 2100 to 2300 cm-l was used. The C-D/C-H absorbance ratio was 1/(2.8 f 0.4), which is within experimental error of the ratio (1/ 2.5) determined from the spectra of equal amounts of dDPPC and PA applied to the ATIR plate after evaporation of a solvent. These values are not very different from the values of 111.9 and 112.3 quoted earlier. The wavenumber of the antisymmetric CH2 stretching band of DPPC shifted from 2921 f 0.5 for depositions made a t II = 10 dynlcm to 2919 cm-l for those made a t II = 15 dynlcm. This shift is consistent with previous measurements in the literature20128and corresponds to a change to more trans conformations of the DPPC hydrocarbon tails, which become more ordered. This shift also corresponds to the transition from the LE to the LC monolayer state,28observed by Mitchell and Dluhy with ER-IR by in situ measurements.20*21The observation of this shift in the L-B layer indicates that the L-B deposition process retains the monolayer structure which existed on the airlwater interface, and hence the method appears to provide good surface sampling. B i n a r y Spread F i l m Mixtures. Spectra of L-B depositions from an initially 5050 mollmol DPPC-PA spread film, Figure 6, indicate that the surface density increases with increasing surface pressure, as expected. The bands in the carbonyl region of the DPPC-PA spectra look like the combination (or sum) of the carbonyl bands from the spectra of the pure components (Figure 6, A and D), indicating qualitatively the presence of both DPPC and PA in the deposited mixed surface layer. No enrichment of DPPC between depositions at 20 and 50 dyn/ cm could be inferred from the spectra. It is difficult to obtain quantitative information on the relative amounts of DPPC and PA from these spectra, since the hydrocarbon stretching bands occur a t the same wavenumbers (Table 11)and the C=O region overlaps in part with a band from water, which may be present in the L-B film. Figure 7 shows the spectra of films deposited from an initially 50:50 (mol/mol) d-DPPC-PA surface film a t II = 10 and 40 dyn/cm. At n 1 45 dyn/cm, film collapse was observed visually. Although the total intensities of
Figure 7. ATR-FTIR spectra of L-B films deposited from dDPPC-PA (initially 4951 mol/mol) mixed spread films at surface pressures II (dyn/cm) of 10 (A) and 44 (B).
n
l
\
0
26
+
I
:.
I
I
60
76
100
126
Surface area (A'/molec)
Figure 8. E A isotherms of mixed spread films of d-DPPC-PA, initially 4951 mol/mol (A), and d-DPPC-PG, initially 51:49 mol/ mol (B). Surface layers were compressed at 10 cmz/s. the bands are slightly larger a t the higher pressure, calculations showed no significant changes in the relative amounts of d-DPPC and PA. Integration of the CH and CD stretching bands and use of the calibration curve of Figure 5 indicated 46 f 5 mol 96 d-DPPC a t the surface layer for both surface pressures. These numbers are equal (within experimental error) to the initial content of 50 mol % d-DPPC a t the surface and agree with the L-B/TLC/ OD results. Moreover, a slight DPPC enrichment (from 46 to 56 mol % a t II = 10 dynlcm and from 46 to 60 mol % a t II = 40 dyn/cm) was determined by IR when depositions were made 1 h after compression of the surface (cf. Figure 2). This loss of PA from the surface may be due to a slow dissolution rather than collapse of PA in the subphase, as PA has a higher solubility in water (-3 ppm a t 25 0C42) than DPPC. As mentioned earlier, DPPC and PA may form a uniform solution a t the surface layer. The 11-A isotherm of the d-DPPC-PA mixture (Figure 8, curve A) shows no second plateau a t the collapse pressure of PA (-45 dyn/cm), suggesting no selective collapse of PA. This is consistent with the hypothesis that a random (nonsegregated) lipid mixture is present a t the surface layer.43 As the surface layer is compressed beyond its collapse pressure, it may collapse as a mixture of uniform composition, with apparently no preferential removal of one component over the other. L-B films were also deposited from spread surface layers of DPPC-PG mixtures with a molar ratio of 49:51 a t (42) Tanford, C. The Hydrophobic Effect;Wiley and Sons: New York, 1980. (43) Gaines, G. L. Insoluble Monolayers at Liquid-Gas Interfaces; Interscience: New York, 1966.
Langmuir, Vol. 6, No. 11, 1990 1653
Surface Analysis of Lipid Layers I
0.02-
I
so00
I
I
I
ZSBO
29so
i.040
I
I
I
2WO
73M
I
?SO
I
I
2840
ZUZO
1
ZWO
Wavcnumkr (em-')
Figure 9. ATR-FTIR spectra for hydrocarbon stretching regions of L-B depositions from a DPPC-PG mixed spread f i (initially 4 9 5 1 mol/mol) at surface pressures rI (dyn/cm) of 20 (A), 25 (B), 40 (C),and 60 (D).
Figure 10. ATR-FTIR spectra of L-B films deposited from a d-DPPC-PG (initially 51:49 mol/mol) mixed spread film at surface pressures II (dyn/cm) of 20 (A), 40 (B), and 60 (C).
Table 111. Surface Layer Composition of DPPC-PG Spread Film. at Various II Values Determined by the Spectral Subtraction Method n, integrated absorbance, cm-1 mol % dyn/cm TR (2800-3000 cm-I) DPPC
Table IV. Surface Layer Composition of d-DPPC-PG Spread Film. at Various 11 Values Determined by Integration of CH2 and CD2 Stretching Bands integrated absorbance, cm-' d-DPPC PG
20 30 40 60 a
2.0 2.2 4.0 5.7
0.97 f 0.02 1.12 f 0.02 1.19 f 0.02 4.08 f 0.08
61 f 10 76 f 10 92 f 8 95f5
Initially 49 mol % DPPC.
surface pressures II ranging from 20 to 60 dyn/cm. The hydrocarbon stretching regions of the ATR-FTIR spectra of the deposited films are shown in Figure 9. The band intensity increased with increasing surface pressure, indicating that the surface density increases with surface compression. A shift in the antisymmetric stretching band from 2921 to 2919 cm-1 was observed from II = 20 to 30 dyn/cm. The wavenumber remained constant at 2919.3 cm-l for depositions a t n = 40 and 60 cyn/cm. This shift indicates the transition from a mixture of DPPC-PG to a predominantly DPPC surface layer with increasing n. The spectral subtraction method4 was used iteratively to calculate the amounts of DPPC and PG from the spectra of the pure components and the spectrum of the mixture. Table 111 shows the results. Because the hydrocarbon stretching bands of DPPC and PG overlap, the estimated uncertainty was high (estimated at ca. 10-2096). Even with this uncertainty, however, it is quite clear that the surface layer composition of DPPC increased significantly, from 49 to over 90 mol 5%. This result is consistent with the previous inferences from the frequency shift and the L-B/ TLC/OD results for similar layers, in which the DPPC content increased from 49 to 85 mol % as the surface pressure increased from 20 to 60 dyn/cm. The C = O bands were not used in the subtraction method, because they occurred at approximately the same frequencies for DPPC and PG (Table 11). It was observed that the higher the surface pressure the higher the mole percent of DPPC (Table III), because of the squeeze-out mechanism, and the faster the monolayer collapse. The latter leads to increased TR, which surely appears to correlate with percent DPPC, as pointed out by a reviewer. We have attributed no further importance to this correlation, because the importance of the transfer ratio is unclear for collapsing monolayers, whose behavior is time-dependent. (44) Cameron, D. G.; C a d , H. L.; Mantsch, H. H. J . Biochem. Biophys. Meth. 1979, 1 , 21.
n,
dyn/cm 10 20 30 40 60
(2100-2300
TR 0.5 1.0 3.0 3.0 5.0
cm-1) 0.14 f 0.03 0.34 f 0.01 0.48 f 0.01 0.61 f 0.01 1.83 f 0.02
(2800-3000 cm-1) 0.41 f 0.08 0.47 f 0.01 0.42 f 0.01 0.10 f 0.01 0.04 f 0.01
mol %
d-DPPC 51f5 69f3 77 f 4 95f5 97 f 3
Initially 51 mol % d-DPPC.
By the use of deuterated DPPC chains, the uncertainties arising from trying to separate overlapping bands by the spectral subtraction method can be eliminated. Figure 10 shows the spectra of L-B depositions from an initially 51: 49 (mol/mol) surface of a d-DPPC-PG spread film made a t n ranging from 20 to 60 dyn/cm. Increased intensities of the C=O stretching band indicate an increase in the total lipid surface density. The CH2 stretching bands a t 2800-3000 cm-' are mostly from PG and partly from the four CH2 groups of d-DPPC, which are not part of the hydrophobic tails. Both the absolute density of d DPPC and the density relative to that of PG increased with increasing n. The uncertainties in calculations from mixtures with d-DPPC, due to calibration and integration errors, were estimated to range from 5% to 10%. In Table IV, the compositions of the d-DPPC-PG surface layer a t increasing n are shown. As the surface was compressed from 10 to 60 dyn/cm, the DPPC content a t the surface increased from 51 to over 90 mol 7%. The results of Table IV agree well with those of Table 111. Hence, the approach of using the spectral subtraction method for separating overlapping bands, which differ by 6 cm-' (Table 11), appears to be reasonable and yields results consistent with those of the other methods. The E A isotherm of d-DPPC-PG spread film (Figure 8) shows evidence of immiscibility in the mixed film.Unlike the isotherm of the d-DPPC-PA mixture (Figure €9,this isotherm looks like a superposition of the d-DPPC and the PG isotherms. The monolayer transition of d-DPPC from liquid-expanded to liquid-condensed is observed a t 15 dyn/ cm (cf. Figure 4),as is the collapse of PG a t 40 dyn/cm. If surface layers are partially immiscible, then they may contain separate patches, some rich in DPPC and some rich in PG. Those patches which a r e rich in t h e components with the smaller collapse pressures are expected to collapse (or be "squeezed out") first.43
1654 Langmuir, Vol. 6, No. 11, 1990
Chung et al. A
0.064
i
0.05-
0.00
T2QO
(A)
,A r iaD0
3000
I
!
I
2600
2400
2200
I - - Y I 2000 la00
1600
3obo
& &
mi0
\V;iveiiiiiiilicr (ciiii' )
Table V. Results from IR Analysis of L-B Depositions from 500 ppm DPPC Dispersion in Water and from a Spread Film for Comparison integrated absorbance (2800-3000 cm-9, cm-l II,dyn/cm TR dispersion spread film 0 0.5 2.0 3.0
1.41 1.78 2.47 5.69
zpbo
2sbo
&o
=io
&o
zsbo
Wavenumber ( c d )
Figure 11. ATR-FTIR spectra of L-B films deposited from a 500 ppm DPPC dispersion in water at surface pressures II (dyn/ cm) of 0 (A), 10 (B),40 (C), and 60 (D).Depositions were made at 25 "C.
0 10 40 60
2oio
0 0.7 1.1 3.8
Dispersions. A surface layer which was produced by spontaneous adsorption from a 500 ppm DPPC dispersion was deposited a t various 11 values before and after compression. With no compression (II I 1dyn/cm), the measured transfer ratio was about zero, which would suggest no deposition of the surface layer. However, the ATIR-FTIR spectrum shows bands with wavenumbers and absorbances which are characteristic of DPPC with a surface density of p = 2.0 X 1010mol/cm2 (Figure 11, A). This surface density corresponds to a deposition from a DPPC spread monolayer a t II = 70 dyn/cm, if a transfer ratio of 1 were assumed. T h e deposited material is probably due in part to some entrainment of dispersed DPPC particles. As the surface pressure increased, the intensity of the bands increased, Figure 11 (B, C, and D), and the measured transfer ratios were nonzero (Table V), indicating deposition of the adsorbed surface layer. Possible entrainment of particles as well cannot be ruled out. Even though the low-11deposition may lead to quite variable densities depending on particle entrainment, the increase in p after surface compression to higher II was so much larger (4-fold) that it can be attributed to the surface layer (Table V). As with the L-B/TLC/OD analyses of adsorbed surface layers of the sheep lung surfactant dispersion, possible entrainment of particles is a minor effect, because the intensities correlate with the surface pressure. The spectra indicate the presence of a substantial surface density of DPPC molecules a t the surface layer at T < 42 "C. Even though the mechanism by which D P P C reaches t h e surface a n d adsorbs spontaneously is unclear, DPPC is responsible for the superlow tensions which were measured after compre~sion.~~ L-B depositions were also made from a sheep lung surfactant dispersion in water a t ll= 40 dyn/cm (surface was not compressed) and for surface compressions up to II = 70 dyn/cm. The IR spectra of the hydrocarbon regions for these depositions are shown in Figure 12. As suggested from the TLC/OD analyses, the IR bands indicate that DPPC and PG are the main components of these lung surfactants. As in the DPPC-PG mixed surface layer, a shift
Figure 12. ATR-FTIR spectra shown for the hydrocarbon stretching regions of L-B depositions from a sheep lung surfactant dispersion in water at surface pressures II (dyn/cm) of 40 (no compression) (A), 50 (B), 60 (C), and variable from 68 to 50 (minimum area) (D). Table VI. Surface Layer Composition of an Aqueous Sheep Lung Surfactant Dispersion at Various II Values Determined by the Spectral Subtraction Method integrated absorbance, cm-1
mol %
II,dyn/cm
TR
(2800-3000 cm-')
DPPC
40 (uncompressed) 50 60 70-50 (min. area)
0 2.0 2.0
1.28 f 0.03 1.90 f 0.04 1.86 0.04 1.74 f 0.03
59 f 10 69 f 10 69 f 10 76 f 10
a
a
*
Deposition at minimum area; see text.
from 2921 to 2919 cm-' in the antisymmetric stretching band was observed a t the higher surface pressures, indicating that mostly DPPC is present on the surface a t 70 dyn/cm. The deposition a t 70 dyn/cm was made by first compressing the surface layer of the lung surfactant dispersion to the minimum area of the trough (84% area reduction) and then raising the dipping substrate through the surface layer with no control of the surface pressure. II dropped from 70 to 50 dyn/cm during this deposition, evidently because some surfactant was removed and the area could not be further decreased. The surface layer composition was estimated by the spectral subtraction method, as with the DPPC-PG mixed spread films (Table VI). DPPC enrichment from 59 to 76 wt 5% was observed as the surface was compressed by 60 % ,showing the same trends as the L-B/TLC/OD results of lung surfactant depositions discussed earlier. The lung surfactant dispersion isotherm (Figure 13) showed a similar pattern as the d-DPPC-PG mixture, with the presence of a second plateau a t about II = 50 dyn/ cm during compression (and the usual hysteresis during expansion), indicating preferential removal of nonDPPC components at higher pressures. The combined evidence suggests that DPPC and PG mostly determine the key features of the II-A behavior of this natural lung surfactant and supports the use of such binary mixtures as models of the more complex lung surfactnt mixtures.45
Conclusions A method was developed to analyze the surface layer composition and density of natural lung surfactant systems and certain simple lipids a t various degrees of surface compression. This method involves transferring the surface layer to a solid substrate by Langmuir-Blodgett deposition (45) Morley, C. J. In Surfactant Replacement Therapy;Shapiro, D. L., Notter, R. H.,Eds.; Alan, R.Liss: New York, 1989;p 219.
Langmuir, Vol. 6, No. 11, 1990 1655
Surface Analysis of Lipid Layers 76
