J . Phys. Chem. 1990, 94, 1925-1932
1925
Ellipsometric Study of the Physical States of Phosphatidylchoilnes at the Air-Water Interface Daniel Ducharme? Jean-Joseph Max, Christian Salesse,t and Roger M. Leblanc* Centre de recherche en photobiophysique, Uniuersitt du Qutbec 6 Trois- Rivieres, 3351 boul. des Forges, C.P. 500, Trois-RiuiPres, Qutbec, Canada G9A 5H7 (Received: January 23, 1989; In Final Form: September 6, 1989)
The ellipsometric isotherms of a homologous series of phosphatidylcholines at the air-water interface are reported for the first time. The ellipsometric angle, 8A, has proven to be very sensitive to the nature of the physical state of the film as well as to the physical-state changes that occur during film compression. At 17 f 1 OC, two compounds of the series, Le. L-a-dimyristoylphosphatidylcholine(DMPC) (C-14) and L-a-dipalmitoylphosphatidylcholine(DPPC) (C- 16) exhibit a phase transition while L-a-distearoylphosphatidylcholine(DSPC) (C- 18) and L-a-diarachidoylphosphatidylcholine(DAPC) (C-20) show only a solid-state behavior upon compression. This physical state is also observed for DPPC at 10 OC. A rodlike model was used to depict the different molecular behaviors exhibited by the film-forming molecules of this series. Equations relating variations of refractive indices, film thickness, and molecular area to film organization were obtained so that the evolution of the molecular properties of the film can be followed during monolayer compression. Calculated refractive indices and film thicknesses were obtained from experimental 8A. Results indicate that refractive indices are higher for a solid state than for a liquid expanded state. Moreover, in the same physical state, refractive indices slightly increase with chain length. It is observed that positive anisotropy is shown for the films in the solid state whereas negative anisotropy is exhibited by those in the liquid expanded state. These results strongly suggest that phase transition is made up of a mixture of bent conformers, which, by reduction of molecular area, favors all-trans conformation.
of these parameters must be established. This has been achieved I. Introduction by means of an improved rodlike modelI8 of a lipid molecule based Phospholipids have received a great deal of attention because on CPK atomic models. The model allows one to mimic the they form the major structural component of cellular membranes. variation of the ellipsometric angle as a function of the molecular Monolayers of these compounds at the air-water interface conarea during film compression. The calculated refractive indices stitute an important class of model compounds for studying inobtained from the model agree within 2% with theoretically teractions between biological membrane components. Furthercalculated values of a lipid bilayer of similar interchain distance more, some lipid monolayers exhibit interesting physicochemical and orientatione20 Results show that for a solid-state film, both properties such as a phase transition. refractive indices increase with decreasing interchain distance and In order to demonstrate the sensitivity of ellipsometry for increasing chain length. These results are in agreement with ref characterizing the physical states of a film, a particular homol20 and 21. Moreover, a positive anisotropy (n,,> n,; Le., the ogous series of phosphatidylcholine that concurrently allows one refractive index of the film parallel to the optic axis is larger than to study the chain length effect upon the ellipsometric angle has that which is perpendicular to the same axis) is shown for combeen selected. Thus, the gaseous (G), liquid expanded (LE), liquid pounds that exhibit a solid behavior, while the reverse is observed condensed (LC), and solid (S) states and the gaseous/liquid for those in the liquid expanded region which precedes the LE/LC expanded (G/LE) as well as the liquid expanded/liquid condensed transition. Furthermore, for L-a-dipalmitoylphosphatidylcholine (LE/LC) phase change are investigated. (DPPC), a significant increase in both refractive indices and optical For DPPC, the last phenomenon (LE/LC) has been the most birefringence characterize the passage from the liquid expanded controversia1.l" The present consensus is that in the main to the solid state. The latter observation strongly suggests that transition, the monolayer is nonhomogeneous and bipha~ic.'-~ A the phase transition must go through chain conformation isomsignificant contribution to the understanding of monolayers at the air-water interface has been made by the use of techniques such ( I ) Gaines, G. L., Jr. J . Chem. Phys. 1978, 69, 924. as fluorescence microscopy,l*I3 synchrotron X-ray refle~tivity,l~*~~ (2) Hard, S.; Neuman, R. D. J. Colloid Interface Sci. 1981, 83, 315. surface quasi-elastic light scattering,I6 and surface viscosity.17 (3) Middleton, S. R.; Pethica, B. A. Faraday Symp. 1981, 16, 109. (4) Albrecht, 0.; Gruler, H.; Sackmann, E. J. Phys. (Paris) 1978,39,301. However, not much work has been devoted to examining the ( 5 ) Cadenhead, D. A. Structure of Biological Membranes; In Abraevolution of film thickness and refractive indices during film hamsson. S..Pascher. I.. Eds.: Plenum Press: New York. 1977: DD 63-83. compression.'* (6) Baret, J. F.; Bois,'A. G;. Dupin, J. J.; Firpo, J. L. J.'Colloidinterface In that regard, ellipsometry due to its high sensitivity in the Sci. 1982, 86, 370. (7) Neuman, R. D.; Fereshtehkhou, S.; Ovalle, R. J. Colloid Interface Sci. submonolayer region can be employed. Ellipsometry is an optical 1984, 101, 309. nondestructive method based on the fact that the optical properties (8) Losche, M.; Mohwald, H. Colloids Surf. 1984, 10, 217. of the sample surface cause the polarization form of the reflected (9) Pallas, N. R.: Pethica. B. A. Lanamuir 1985, 1 , 509. beam to differ from that of the incident beam.Ig From ellipso(IO) Losche, M.; Mohwald, H. Rev. h i . Instrum. 1984, 55, 1963. (11) Losche, M.; Mohwald, H. Eur. Biophys. J . 1984, 11, 35. metric measurements, information about film thickness and re(12) Weis, R. M.; McConnell, H. M. Nature (London) 1984, 310, 47. fractive indices can be obtained. For thin nonabsorbing films, (13) McConnell, H. M.; Tamm, L. K.; Weis, R. M. Proc. Natl. Acad. Sci. only one of the ellipsometric angles, 6A, varies during film comU.S.A. 1988, 80, 5795. pression, while 6$ remains null. Thus, all the information (film (14) Kjaer, K.; Als-Nielsen, J.; Helm, C. A,; Laxhuber, L. A,; Mohwald, H. Phys. Rev. Lett. 1987, 58, 2:!24. thickness and refractive indices) is included in that angle. ( 1 5 ) Helm, C. A.; Mohwald, H.; Kiaer, K.; Als-Nielsen, J. Europhys. Lett. In order to explain the ellipsometric isotherms in terms of the 1987, 4, 697. optical parameters of the film, the contribution and weight effects (16) Sauer. B. B.: Chen. Y. L.: Zoarafi. G.; Yu. H. Lnnamuir 1988.4, 11 I . Author to whom correspondence should be addressed. ' Departement de chimie, CCgep de Shawinigan, 2263 boul. du CollPge, Shawinigan, PQ, Canada G9N 6V8.
*
lnstitut fur Organische Chemie, Johannes Gutenberg Universitat, Mainz 6500, FRG.
0022-3654/90/2094-1925$02.50/0
(17) Abraham, B. M.; Ketterson,J. B. Langmuir 1985, I , 708. (18) Den Engelsen, D.; De Koning, B. J. Chem. SOC.,Faraday Trans. 1 1974, 70, 1603. (19) Azzam, R. M. A,; Bashara, N. M. Ellipsometry and Polarized Light, 1st ed.; North-Holland: New York, 1977. (20) Huang, W. T.; Levitt, D. G. Biophys. J. 1977, 17, 11 1. (21) Den Engelsen, D. Surface Sci. 1976, 56, 272.
0 1990 American Chemical Society
1926 The Journal of Physical Chemistry, Vol. 94, No. 5, 1990
erism. The ellipsometric isotherms of L-a-dimyristoylphosphatidylcholine (DMPC) and DPPC at 17 OC are characterized by a long plateau that precedes the onset of surface pressure, while no such plateau is shown by those exhibiting a sole solid behavior. Besides indicating evidence of long-range molecular orientational order, these results also suggest that patches that form in the G/LE region are precursors of the forthcoming physical state of the film, as detected at the onset of the surface pressure. Ellipsometry thus provides a nonperturbative characterization of the G/LE region. The observation of the coexistence of the G and LE states for DMPC and DPPC is supported by the work reported in ref 1 1 and 16. Moreover, in the solid state, for an identical surface pressure, the experimental ellipsometric angle when plotted against the number of carbon atoms of each aliphatic chain shows a linear relationship.
Ducharme et al. TABLE I: DSPC Simulation with a CPK case d. 8, n, a 4.5 1.860 5.0 1.850 b 8.5 1.660 10.0 1.650 C 21.5 1.510 22.5 1.509 1.508 23.5 24.5 1.507
Model n. 1.850 1.860 1.650 1.660 1.544 1.546 1.547 1.548
-6P,den 1.73 1.85 I .73 1.85 1.71 1.76 1.81 1.85
t'
11. Theory For a thin uniaxial nonabsorbing film with the optic axis perpendicular to the surface, the following e x p r e s s i ~ nfor~ ~6A~ ~ ~ is valid up to first-order terms in d / X . 6A=A-
sin 4o tan @ono I
-
( n o / n 2 ) 2tan2 +o
).
where A and are, respectively, the filmed and bare substrate phase changes monitored by reflection, no and n2 are the refractive indices of air and water, respectively, d is the film thickness, n L is the refractive index perpendicular to the optic axis (parallel to the surface), and nlI is the refractive index parallel to the optic axis (perpendicular to the interface). As seen in eq I , at a given angle of incidence, 40,and a wavelength, A, the ellipsometric angle, 6A, is a function of three film parameters: d, n,, and n,. The number of unknowns is thus more than can be determined from the sole measurement of 6A, and for such a case, it has been s h o ~ that n ~the~ variation ~ ~ ~ of the angle of incidence fails to characterize the anisotropic properties of the film. Under these conditions, in order to obtain the pertinent information about the evolution of these parameters during film compression, an improved rodlike modelI8 was set up, which allowed these optical constants to be calculated as a function of the orientation of the molecules. Thus, the experimental ellipsometric isotherm is compared to the one that is calculated, and a least-squares fit allows the film parameters to be deduced. 111. Models CPK Model. As seen in Figures 2-4, as the molecular area IS reduced, 6A becomes more and more negative. Equation 1 shows that this behavior may be explained by (i) an increase of film thickness; (ii) an increase of both refractive indices; (iii) a combination of a change in anisotropy and increasing film thickness. By means of CPK atomic models, an estimate of film thickness and refractive indices that best fit the experimental 6A has been preliminarily calculated. For each film thickness ranging from 4 to 40 A by steps of 0.5 A, the refractive index of n , has been varied from I .34 to 1.80 by steps of 0.01. For each n, value, birefringence ( n l - n L ) has been increased from 0.001 to 0.1 by steps of 0.005. 6A has been calculated for each of the values d. n,, and nil. Then, from assumed film thickness, refractive indices that best fit the experimental 6A have been selected and are shown in Table I for DSPC. Polar head groups have been assumed not to be detected by ellipsometry. The values in case a have been obtained by assuming that (i) at the ellipsometric jump, molecules lie flat on the water surface and are vertically oriented at the end of compression: and (ii) for such a case n, is greater than n,, for (22) Ayoub, G. T.; Bashara, N. M. J . Opt. Sot. Am. 1978, 68, 978. (23) Dignam, M. J.; Moskovitts, M.; Stobie, R. W. Trans. Faraday SOC. 1971. 67, 3306. (24) Antippa, A. F.: Leblanc, R . M.; Ducharme, D. J . Opr. SOC.A m . A 1986, 3, 1974.
Figure 1, Rodlike model of a phospholipid molecule: Do,diameter of one chain; D,,total length of the molecule; D,,length of the polar head group; X Y Z . coordinate axis: 8, angle with respect to the vertical Z axis.
the flatly oriented molecule whereas refractive indices are permuted for the vertically oriented molecules. For the flatly oriented molecules, a film thickness of 4.5 A (measured from CPK model) requires very high refractive indices to fit the experimental 6A at the jump. As the refractive indices are permuted (at the end of compression), a film thickness of 5 A, which is certainly not consistent with the CPK model for a vertically oriented molecule, satisfies the experimental ellipsometric angle. It must be concluded that although these values fit the experimental 6A, they are not physically acceptable. A similar situation is obtained in case b, where the film thickness of flatly oriented molecules has been increased; however, the conclusion regarding film thickness at the end of compression for a vertically oriented molecule is identical with that of case a. Since neither case a nor case b is a physically acceptable length value, it has been assumed that the molecules are already oriented when the ellipsometric jump takes place (case c). As the molecules become more and more vertically oriented, i.e. as d increases, n,, increases at the expense of n,. Theoretical refractive indices of a lipidic bilayerz0 have been used to evaluate a realistic film thickness consistent with the experimental 6A. Although the above calculations with CPK models set the range of physically acceptable optical parameters, variation of refractive indices and film thickness with the orientation of film-forming molecules is of the utmost importance. A geometric model presented in the next section, which gives the relation between the ellipsometric angle and molecular area (two experimental values), allows this information to be obtained. Geometric Model. In the model, as shown in Figure 1, the aliphatic chains and the polar head group are included in a rectangular parallelepiped whose width and length are 2 0 , and D,, respectively. Do may vary between the diameter of one chain and the center to center interchain distance. The polar head group, with length D2, is assumed to be fully extended in the subphase and to have the same cross section perpendicular to the long axis
The Journal of Physical Chemistry, Vol. 94, No. 5, 1990 1927
Ellipsometric Study of Phosphatidylcholines
of the molecule as do the two aliphatic chains together (Do X 2Do). Furthermore, due to its high degree of hydration, this hydrophilic moiety has a refractive index that is assumed to be equal to that of the subphase and is thus not detected by ellipsometry. From a synchrotron X-ray reflectivity study at the air-water interface, Helm et alls also advocate this nondistinguishable character, which is, however, confined only to the choline group. As indicated in the same figure, the corners of the polyhedron have been rounded up to take into account the geometry of the CPK atomic models and to avoid sharp angles. To derive film thickness and molecular area, molecules are assumed to be uniformly arranged with parallel hydrocarbon chains. Experimentally, the film has shown to be isotropic in the XYplane; Le. the t ~ o - z o n polarizer e~~ settings at null do not substantially deviate from 90' (less than 0.15' as on the pure substrate), which is not to be expected for a uniformly oriented film. One can assume, then, that the film is composed of domains of parallel-oriented molecules whose orientation, inside domains, is randomly distributed at the same angle to the normal. The statistical average of these domains thus leads to isotropy in the surface plane; i.e. the uniaxial symmetry of the film is preserved around the vertical axis. To calculate film thickness, it was assumed that molecules, instead of rotating with respect to an axis of rotation perpendicular to the long axis of the molecule, roll on a spherical surface centered at D0/2 above the polar head group. For geometric reasons, orientation may only take place from a given angle 8. However we only consider angle variations from 70 to 0". The limiting case for which 8 = 90" is not considered since the stable photometric signal is preceded by a fluctuating one; i.e. the ellipsometric jump angle (the onset of a stable detector signal which allows measurement to be taken) suggests that the orientation sets in at angles smaller than 90'. From that angle onward, as the molecules become more and more vertically oriented, the variation of the molecular area as well as the refractive indices of the film with respect to 0 may be obtained. In the model, the structure of the molecules is considered to be independent of orientation whatever its physical state. So, the geometric parameters Do,D,, and D2 as well as the molecular refractive indices n, and n, (along the long axis of the molecule for 8 = 0) are constants. Thus, the only variables are those that vary with respect to the reference axis: molecular area, A , film thickness, d, refractive indices of the film, nIland n,, as well as 8. The molecular area, A , may be obtained by projecting the cross section of the parallelepiped onto a plane parallel to the surface. One thus obtains A = 2D02/(cos 8)
and as deduced from Figure I , the variation of film thickness, d, as a function of the orientation is
d = (Dl - D2 - Do)COS 8 + Do
(3)
The variation of the refractive indices of the film, nl,and nL, with respect to orientation is obtained from molecular refractive indices through the following equationsI8
+ cos2 8)t, + t, sin2 8 nil =
(E, sin2 8 + t, cos 8 ) l I 2
(4)
where t, and t, are the dielectric constants (e = n2) of the film obtained for 8 = 0. The latter values are represented respectively by n, and n, in Tables IV-VI. The substitution of eq 2, 3, and 4 in 1 allows 611 to be calculated as a function of the molecular area A, where A is the variable, while Do, D,,D2,n,, and n, are the parameters. A program was developed to allow the parameters to be determined according to the least-squares method. A computer analysis has shown that if the geometric parameters are fixed, both refractive indices, n, and n,, can be accurately determined simultaneously (unpublished results). The procedure (25) De Smet, D. J. J . Opt. SOC.Am. A 1988, 5, 171
TABLE 11: Features of the Least-Squares Fits'
molecule
PhYS state
DMPC DPPC (17 "C)
LE LE S
DPPC (10 "C) DSPC DAPC
S S
S
n, f 0.001 n, i 0.002 1.442 1.470 1.491 1.496 1.499 1.502
1.429 1.420 1.509 1.522 1.523 1.529
mean error, dee.
max error,
0.001 0.006 0.002 0.004 0.008 0.009
0.017 0.012 0.006 0.008 0.013 0.017
dee.
OThe reader may refer to Tables IV-VI for further details.
then allows d, n,, and nIlto be calculated as a function of 8 for given Do, D,,and D 2values. For a given molecule in the solid state, i.e. DPPC, DSPC, and DAPC, the least-squares calculations are started with any two initial values randomly assigned to n, or n,. From previous CPK model calculations, the range of refractive indices was confined between 1.35 and 1.70. Successive iterations keep running until the calculated 6A value fits the experimental data at the molecular area at which 8 = 0 (2DO2). From these obtained values, d, n,, and nIl are calculated as a function of 8. The molecules that exhibit a liquid expanded state, i.e. DMPC and DPPC, are treated like those that show a solid-state behavior. The simulation considers that the molecule remains in this physical state (LE) throughout compression; i.e. the n, and n, values shown in Table IV are the extrapolated refractive indices should the film-forming molecules remain in the liquid expanded state. Since this state is followed by a phase transition, the best fit applies from the onset of the molecular area that characterizes this physical state to the beginning of phase transition. The phase transition and plateau region have not been fitted. In both cases (S and LE states), the maximum deviation between the experimental and calculated 6A-A is less than 0.02'. It is worth mentioning that the best fit is independent of the initial values assigned to either n, or n,. For a given molecule, these values may be changed at will and the final results will remain the same. Furthermore, since least-squares calculations of the 6A-A isotherms are independently carried out for the solid and the liquid expanded states, changes in film density that may occur as the film goes from the liquid expanded into the solid state are automatically taken into account through the variation of molecular area. Table I1 shows the features of the best fits where n, and n, are the refractive indices obtained for 8 = 0. The mean and maximum error apply to the fitted portion of the isotherm.
IV. Experimental Section The purity of DMPC, DPPC, L-a-distearoylphosphatidylcholine (DSPC), and L-a-diarachidoylphosphatidylcholine (DAPC) (Sigma Chemical Co., St. Louis, MO) was analyzed by thin-layer chromatography (TLC) and gas chromatography (GC).26 TLC showed only one spot, and G C analysis indicated 99.5 mol % for DMPC, DPPC, and DSPC and 99.0 mol % for DAPC. The molar weights used for DMPC, DPPC, DSPC, and DAPC are 677.6, 734.0, 801.1, and 846.3 g/mol, respectively. Reagent grade solvents were distilled before use. The lipids used were dissolved in a n-hexane-ethanol (9:1, (v/v)) mixture. The Langmuir trough has been previously de~cribed.~'The solutions were spread drop by drop with a 100-pL syringe (Precision Sampling Corp., Baton Rouge, LA) over the aqueous surface, stopping at approximately 10 cm from the moving barrier and 5 cm from the float. Prior to compression, the monolayer was allowed to stand 15 min to let the solvent evaporate. All isotherms were obtained with a spreading molecular area of over 300 A2/molecule. The compression speed was set at 5 A2/molecule-min. Deionized and prepurified water (Nanopure filter system, Barnstead, Boston, MA) was twice distilled in a quartz still (Model ( 2 6 ) Salesse, C.; Boucher, F.; Leblanc, R. M. Reo. Can.Biol. Exprl. 1988, 42, 123. (27) Ducharme, D.; Tessier, A,; Leblanc, R. M. Reo. Sci. Insrrum. 1987, 58, 571.
1928 The Journal of Physical Chemistry, Vol, 94, No. 5, 1990
> E
I
I
Ducharme et al.
-> E >
I
a
600
9 I-
5
k-
x
400
yj 200
?ia
3
-z
E
\
xE
I
I
I
50i t
I
1l
-
e
1.30; a
--
v)
-
I
I
I
0
E
a m
\
z E
E
It-- -
50
w
- 1.40 dz
W
a
2
U
30
=,>0.0033 molecule/A2) is used, the ellipsometric angle is observed slightly before the onset of surface pressure. However, the whole length of the plateau is not detected. If the geometric dimensions of a phospholipid molecule are considered, the molecular area for a flat water-lying molecule is approximately 300 A2/molecule. Thus, due to a lack of space, molecules are constrained to orient without the whole length of the plateau being detected. On the other hand, at low surface coverage, due to the provided free space, orientation may proceed gradually, which allows complete plateau detection. Due to the position of the probes (light beam and electrode), which are not too far away from the float ( 2 and 12 cm, respectively), more investigations are necessary to assess whether the plateaus are linked to float proximity detection. As previously mentioned, the film is assumed to be isotropic in the XY plane; i.e. the molecules must align and orient with parallel hydrocarbon chains in different directions with the same angle to the normal, in order to give rise to a film structure formation that can be detected by ellipsometry. The values of the measured ellipsometric angles in the plateau region clearly indicate the formation of an oriented stable film structure (film-forming molecules are oriented). The observation of the coexistence of the G/LE region is supported by the work of Losche
Ducharme et ai. et aL8 For DPPC dye-labeled monolayers between 200 and 100 A2/molecule, they noted the appearance of holes. These holes are surrounded by molecules. At large molecular area, their instability, i.e. variation in size due to a low molecular density environment, could explain the observed fluctuations in the photometric signal. In fact, in this region (>300 A2/molecule) 6A is either zero or unmeasurable. The fluctuations could come from the movement of holes and molecules under the light beam. However, as molecular density is increased, the holes should become smaller and less mobile. The molecular area at which the holes are constrained but yet not completely filled could correspond to the onset of the ellipsometric plateau onward. It is likely that during this process no changes in molecular orientation and refractive indices take place. The onset of the surface pressure would then correspond to the molecular area at which the filling of the gaps is completed. As shown in Figures 3 and 4,contrarily to DMPC and DPPC in the liquid expanded state, the ellipsometric isotherms of DPPC at 10 OC, as well as of DSPC and DAPC, do not exhibit a plateau before the onset of surface pressure. Since these films are in the solid state, we speculate that this behavior could be due to the formation of small domains of a few molecular diameters, which would be free either to move on the surface or to undergo fast breakup and reformation until the molecular area is sufficiently reduced to constrain the domains to condense into larger molecularly oriented structures. Ellipsometry thus provides a very sensitive nonperturbative characterization of the G / L E region.
VIII. Conclusions Several conclusions regarding the effect of the physical state of monolayers upon the ellipsometric angle may be drawn from this study. (1) For identical chemically structured compounds, the ellipsometric angle is very sensitive to the physical state of the film. ( 2 ) In the solid state, presumably due to similar refractive indices and optical birefringence, the ellipsometric angle decreases linearly with chain length. (3) Ellipsometry, through the two-zone measurements, provides valuable information about the overall orientation of the film. No substantial deviation from the two-zone indicates an isotropic structure of the film-forming molecules in the plane of the surface. (4)The fact that a long ellipsometric plateau is observed for a liquid expanded film, whereas no such plateau is shown for a solid film, indicates that ellipsometry can probe either the size or the kinetics of the disappearance of the holes. ( 5 ) The simple geometric model presented in this paper satisfactorily explains the ellipsometric behavior in the solid as well as in the liquid expanded state. The model shows that in the solid state higher refractive indices and positive anisotropy prevail whereas negative anisotropy and lower refractive indices are exhibited in the liquid expanded state. This strongly suggests that phase transition must go through acyl chain isomerism. Acknowledgment. This work was supported by the Natural Sciences and Engineering Research Council (NSERC) of Canada and Fonds pour la Formation de Chercheurs et I’Aide 2 la Recherche (FCAR). One author (D.D.) gratefully acknowledges le Cigep de Shawinigan and le Fonds FCAR for their support. Another author (C.S.) was supported by postgraduate scholarships from the NSERC, the Fonds FCAR, and the Fondation de I’UniversitC du Quebec. We also thank Mr. Gaetan Munger for his involvement in this work. Registry No. DMPC, 18194-24-6: DPPC, 63-89-8: DSPC, 816-94-4: DAPC, 61596-53-0; HZO, 7732-18-5.
