The Structure of Zwitterionic Phosphocholine Surfactant Monolayers

Q. S. Mu , X. B. Zhao and J. R. Lu , S. P. Armes , A. L. Lewis , R. K. Thomas ... Elias Pambou , Zongyi Li , Mario Campana , Arwel Hughes , Luke Clift...
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Langmuir 2006, 22, 5825-5832

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The Structure of Zwitterionic Phosphocholine Surfactant Monolayers M. Yaseen and J. R. Lu* Biological Physics Group, School of Physics and Astronomy, the UniVersity of Manchester, SackVille Street Building, SackVille Street, Manchester M60 1QD, UK

J. R. P. Webster and J. Penfold ISIS Facility, CCLRC Rutherford Appleton Laboratory, Chilton, Didcot, OX11 0QX, UK ReceiVed December 7, 2005. In Final Form: April 22, 2006 The structure of a zwitterionic phosphocholine (PC) surfactant monolayer adsorbed on the surface of water has been determined using neutron reflectivity in combination with H/D isotopic substitution. The most significant results of this study are the level of hydration of the PC headgroup and the lack of dehydration with increasing temperature and salt addition. The fraction of the alkyl chain (fc) immersed in water for all three chain isomers studied was found to be around 0.15, suggesting that the PC headgroup geometries influenced not only the headgroup hydration but also the degree of immersion of the alkyl chain in water. At the critical micelle concentration (CMC), the number of water molecules associated with the PC headgroup in CmPC (m ) 12, 14, 16) was on order of 15. This value was significantly greater than that obtained for nonionic and ionic surfactants with similar limiting area per molecule at the CMC (Acmc). However, the fraction of the chain immersed in water for the ionic and nonionic surfactants was much greater. This suggests that the unique surface biocompatibility of PC surfactants arises from their strong affinity for water, and the relatively low fraction of mixing with the alkyl chain arises from the higher structural order within the PC monolayer. As surface coverage decreased, the number of water molecules associated with each PC headgroup increased, but fc remained constant for all the surfactants. This observation was consistent with the small variation in the thickness of the headgroup region, and the entire layer changed little with surfactant concentration. This is attributed to the role of PC headgroup geometries to maintain the conformational order within the layer as packing density varies. Further structural analysis based on a kinematic approach showed that, as the chain length was increased from C12 to C14 to C16 at the CMC, the angle of tilt for the alkyl chain increased from 40° to 48° to 53°, respectively, whereas the thickness of the whole layer and that of the PC head region was largely constant. The almost vertical projection of the PC headgroup from these single alkyl chain surfactants is in sharp contrast to its strongly tilted conformation, as reported for dichain phospholipids such as dipalmitoyl glycerol phosphocholine (DPPC).

1. Introduction Information about the structural characteristics of the surfactant monolayer adsorbed at the air/water interface can be obtained from specular neutron reflectivity in combination with contrast manipulation using H/D isotopic substitution.1 Neutron reflectivity provides structural details of the surfactant layer at a level that is inaccessible to most other techniques. It has already been demonstrated in our previous work that, using chain deuterated surfactants2,3 in null reflecting water (NRW), neutron reflectivity can be used to determine the surface coverage over a wide range of surfactant concentrations. Further information about the structural conformation within the layer can be obtained by a combined use of varying the solvent contrast and selective deuterium labeling of the surfactant. For example, information about the distributions of the alkyl chain and headgroup regions and their positions relative to the solvent distribution at the interface can be obtained using appropriate deuterium labeling to the relevant segments inside the surfactant molecule. These advantages of neutron reflectivity in the study of soluble surfactant monolayers have been well exemplified in our previous work on a range of ionic and nonionic surfactants.4 * To whom all correspondence should be addressed. Tel: 44-1612003926, e-mail: [email protected]. (1) Thomas, R. K. Annu. ReV. Phys. Chem. 2004, 55, 391. (2) Yaseen, M.; Wang, Y.; Su, T. J.; Lu, J. R. J. Colloid Interface Sci. 2005, 288, 361. (3) Yaseen, M.; Lu, J. R.; Webster, J. R. P.; Penfold, J. Biophys. Chem. 2005, 117, 161.

Natural phospholipids play many important roles in biological systems.5 Lipids bearing phosphocholine (PC) mediate cell surface biocompatibility,6-8 among many other effects. To improve our understanding on the unique surface and interfacial properties of zwitterionic lipids, we present here a systematic study of the monolayer structure of single-chain zwitterionic PC surfactants adsorbed at the air/water interface. All the single-chain PC surfactants used in this work were entirely water soluble under the conditions studied. These studies will allow a direct comparison to be made with our previous work on nonionic9-11 and ionic12-14 surfactants. The most significant results from this study are the high level of hydration of the PC headgroups and the lack of dehydration with increasing temperature and salt addition. A further intriguing (4) Lu, J. R.; Thomas, R. K.; Penfold, J. AdV. Colloid Interface Sci. 2000, 84, 43. (5) Menger, F. M.; Chlebowski, M. E.; Galloway, A. L.; Lu, H.; Seredyuk, V. A.; Sorrells, J. L.; Zhang, H. Langmuir 2005, 21, 10336. (6) Chapman, D. Langmuir 1993, 9, 39. (7) Hayward, J. A.; Chapman, D. Biomaterials 1984, 5, 135. (8) Ishihara, K.; Nomura, H.; Mihara, T.; Kurita, K.; Iwasaki, Y.; Nakabayashi, N. J. Biomed. Mater. Res. 1998, 39, 323. (9) Lu, J. R.; Su, T. J.; Li, Z. X.; Thomas, R. K.; Staples, E. J.; Tucker, I.; Penfold, J. J. Phys. Chem. B 1997, 101, 10332. (10) Lu, J. R.; Thomas, R. K.; Lee, E. M.; Penfold, J.; Flitsch, S. L. Langmuir 1993, 9, 1352. (11) Lu, J. R.; Hromadova, M.; Thomas, R. K. Langmuir 1993, 9, 2417. (12) Lu, J. R.; Marrocco, A.; Su, T. J.; Thomas, R. K.; Penfold, J. J. Colloid Interface Sci. 1993, 158, 303. (13) Lyttle, D. J.; Lu, J. R.; Su, T. J.; Thomas, R. K.; Penfold, J. Langmuir 1995, 11, 1001. (14) Lu, J. R.; Purcell, I. P.; Lee, E. M.; Simister, E. A.; Thomas, R. K.; Rennie, A. R.; Penfold, J. J Colloid Interface Sci. 1995, 174, 441.

10.1021/la053316z CCC: $33.50 © 2006 American Chemical Society Published on Web 05/26/2006

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observation is the low fraction of the alkyl chain in water for all the PC surfactants studied, suggesting that the PC headgroup geometry influences not only the degree of hydration but also the fraction of the alkyl chain immersed in the solvent. These structural characteristics together with their invariance to temperature, salt addition, and pH are distinctly different from ionic and nonionic surfactants, and may be associated with their unique surface biocompatibility. Further detailed structural analysis has shown that the thickness of the PC headgroup regions and that of the entire PC surfactant layer vary little with surface coverage and change in the alkyl chain length. Although these observations imply that the alkyl chains would tilt in a manner similar to dichain phospholipids, such as dipalmitoyl glycerol phosphocholine (DPPC),15-17 the hydrophilic PC headgroups were found to orient vertically over the entire concentration range studied. This is in contrast to the varying PC headgroup conformations in DPPC layers, where the transition from parallel to vertical orientations was associated with the phase change from the liquid-expanded to the liquid-condensed (LC) state. 2. Experimental Section The neutron reflectivity measurements were made on the CRISP and SURF reflectometers18,19 at the Rutherford Appleton Laboratory near Oxford, UK, using the whole time-of-flight (TOF) method. The neutron wavelength range used was between 0.5 and 6.5 Å. The beam was inclined at 1.5° to the horizontal, specifically for liquid surfaces. The instrumental setup and parameters provide a wave vector (κ) range between 0.05 and 0.5 Å-1. In situations where the reflectivity is low, a further glancing angle of the incidence angle of 0.8° was used to extend the range of κ down to 0.02 Å-1. Prior to the reflectivity measurements, the solutions were poured into Teflon troughs, and a positive liquid meniscus was established in each trough. The samples were individually aligned using a laser beam that shared the same beam passage as the neutron beam. The instrument was calibrated with respect to the reflectivity measured at the air/D2O interface. A flat background, estimated by averaging the measured reflectivity over the κ range between 0.3 and 0.5 Å-1, was subtracted from each reflectivity profile. The background value was found to be around 2 × 10-6 in D2O and 4 × 10-6 in NRW. NRW is water containing 8.1% D2O and was also referred to as contrast-matched-to-air water. The neutron reflectivity profiles were analyzed using two different approaches: the optical matrix method20 and the kinematic approach.21 In the former, reflectivity was calculated from an optical matrix formula based on a structural model and was then compared with the measured one. This process was iterated until a good fit with meaningful physical significance was obtained. In the latter, structural analysis was carried out via a more direct partial structure factor approach,21 providing a more direct evaluation of structural parameters. Physical constants such as scattering lengths (Σbi),22 the full lengths (le), and volumes of surfactant chains and heads (V) used in the calculations23 are listed in Table 1, and from these parameters the scattering length densities (F) were calculated. (15) Brumm, T.; Naumann, C.; Sackmann, E.; Rennie, A. R.; Thomas, R. K.; Kanellas, D.; Penfold, J.; Bayerl, T. M. Eur. Biophys. J. 1994, 23, 289. (16) Naumann, C.; Dietrich, C.; Lu, J. R.; Thomas, R. K.; Rennie, A. R.; Penfold, J.; Bayerl, T. M. Langmuir 1994, 10, 1919. (17) Charitat, T.; Bellet-Amalric, E.; Fragneto, G.; Graner, F. Eur. Phys. J. B 1999, 8, 583. (18) (a) Penfold, J. Neutron, X-ray and Light Scattering; Lindner P., Zemb, Th., Eds.; Elsevier: New York, 1991; p 223. (b) Penfold, J. Rutherford Appleton Laboratory Internal Report, RAL-88-088; Rutherford Appleton Laboratory: Chilton, England, 1988. (19) Penfold J. Rep. Prog. Phys. 2001, 64, 777. (20) Born, M.; Wolf, E. Principles of Optics; University Press: Cambridge, UK, 1997. (21) (a) Crowley, T. L. Physica A 1993, 195, 354. (b) Crowley, T. L.; Lee, E. M.; Simister, E. A.; Thomas, R. K. Physica B 1991, 173, 143. (22) Sears, V. F. Neutron News 1992, 3, 26. (23) Tanford, C. J. Phys. Chem. 1972, 76, 3020.

Yaseen et al. Table 1. Physical Constants for CmPC Surfactants Used in the Data Analysis segments

Σbi × 10-5 (Å)

le (Å)

V (Å3)

F × 10-6 (Å-2)

C12H25- (hC12) C14H29- (hC14) C16H33- (hC16) -PO4-C2H4N+(CH3)3 (hPC) C12D25- (dC12) (98%D) C14D29- (dC14) (98%D) C16D33- (dC16) (98%D) -PO4-C2D4N+(CD3)3 (dPC) H2O D2O

-13.7 -15.4 -17.0 22.3 241.4 280.5 319.6 156.3 -1.7 19.1

16.7 19.3 21.8 11 16.7 19.3 21.8 11

350 400 450 297 350 400 450 297 30 30

-0.40 -0.39 -0.38 0.75 6.90 7.00 7.10 5.26 -0.56 6.35

The routes for the synthesis and purification of PC surfactants with different alkyl chain lengths (dCnhPC and hCnhPC) have been described in our previous work.2 The synthesis involved a two-step procedure. First, hydrogenated or deuterated n-alcohol was reacted with 2-chloro-2-oxo-1,3,2-dioxaphospholane (20% molar excess), to afford the product 2-n-alkyl-2-oxo-1,3,2-dioxaphospholane. This intermediate was then filtered to remove the amine salt and then purified through a silica flash column (40-60 mesh from Fluka) with petroleum ether and ethyl acetate in a 1:1 mixture. Subsequent removal of the solvent led to an intermediate product as a pale yellow oil. The intermediate product was then freeze-dried and reacted in a sealed thick-walled pressure tube with trimethylamine (3 times molar excess) at 75 °C for 24 h to afford the n-alkyl phosphocholine product. The final product was purified by flash silica column chromatography initially using chloroform then distilled methanol as the mobile phase solvent. The product was a white solid powder, with a typical yield of 75%. The routes for the synthesis and purification of head deuterated and fully deuterated (hCndPC and dCndPC) PC surfactants have also been described previously.3 In this case, n-alcohol was reacted with phosphoryl trichloride (POCL3; 20% molar excess) and stirred for 2-3 h. To this mixture, 2-bromo or 2-chloro ethanol (50% molar excess), the spacer, was added to form the phosphonate ester. To the purified and dried intermediate, trimethylamine (3 times molar excess) was then added in a sealed tube and left to react for 24 h at 75 °C to form the product. The final products were purified similarly using a flash silica column with appropriate solvent combinations and characterized using 1H and 31P NMR. The purity of the products and their stability against time have also been characterized previously using surface tension measurements.2,3 Surfactant solutions were prepared in D2O and NRW. The isotopes for C12PC used were alkyl chain deuterated dC12hPC, fully hydrogenated hC12hPC, head deuterated hC12dPC, and fully deuterated dC12dPC. For the longer chain PC surfactants, the chain deuterated dCnhPC and fully hydrogenated hCnhPC were used. The temperature was controlled to the required values of 25 °C, 35 °C, and 40 °C. These solutions were made at two specific concentrations, around the critical micelle concentration (CMC) and at a low concentration well below the CMC, to give an area per molecule (A) of 75-80 Å2. A similar set of solutions for the surfactant under 0.1 M NaCl was also prepared for structural determination.

3. Results and Discussion The CMC in pure water at 25 °C has been found to be 0.91 mM for C12PC, 0.14 mM for C14PC, and 1.2 × 10-2 mM for C16PC from surface tension.2,3 From surface tension, the area per molecule at the CMC (Acmc) is between 50 and 55 Å2. To compare the conformational order within the surfactant monolayer at different surface coverages, neutron reflectivity measurements were carried out at their CMCs and at lower concentrations to give an area per molecule of around 75 Å2. The structural analysis for C12PC used six isotopic contrast combinations involving dC12hPC/NRW, hC12dPC/NRW, dC12dPC/NRW, dC12hPC/D2O, hC12hPC/D2O, and hC12dPC/D2O. For the two longer chain

Structure of Zwitterionic PC Surfactant Monolayers

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homologues, only three contrasts of dCnhPC/NRW, dCnhPC/ D2O, and hCnhPC/D2O were used. 3.1. Structural Analysis of C12PC. A. Layer Structure Analysis Based on the Optical Matrix Approach. The use of the optical matrix24 method as a basis of the data analysis procedures for PC surfactant monolayers2,3 has been described in detail elsewhere. The structural parameters used in the fitting are the number of layers, the thickness (τ), and the scattering length density (F) for each layer. The interface can be divided into as many uniform sublayers as necessary to describe the scattering length density profile normal to the surface. In the case of surfactant adsorption on the surface of water, the resolution of the experiment usually limits the number of sublayers to three. In this work, both one- and two-layer models were used. From the single one-layer model for the reflectivity profiles measured in NRW, we can obtain the surface excess (Γ) or the limiting area per molecule (A) and the thickness. From reflectivity profiles measured on D2O, the number of water molecules (nw) associated with each surfactant head can also be estimated. The two-layer model provides more structural detail, and can provide an estimate about the fraction of the chain (fc) immersed in water. The relationship between A and τ in the case of applying a uniform layer model to a surfactant monolayer adsorbed from NRW is given as follows:

A)

∑bi Fτ

Figure 1. Simultaneous uniform layer model fits (shown as solid lines) to a set of reflectivity profiles for C12PC at 1.46 mM (just above the CMC) measured from dC12hPC (O), hC12dPC (4), and dC12dPC (b) in NRW. The corresponding two-layer model fits are also shown as dotted curves. For clarity, error bars are shown for dC12dPC only, but the error levels for the other two reflectivity profiles are similar.

(1)

where Σbi is the scattering length of the surfactant; τ and F were obtained from the best uniform layer fit to the measured reflectivity profile. The surface excess can then be calculated from the following equation:

Γ)

1 NaA

(2)

where Na is Avogadro’s number. The application of a two-layer model has previously been demonstrated for different surfactant systems,1,4,10 with the top sublayer consisting of alkyl chain only and the bottom sublayer, the remainder of the chain fraction, and all of the headgroup with any additional space being filled with water molecules. The scattering length density of the top chain sublayer is then given by

Fc )

(1 - fc)bc Aτc

(3)

head, and water. The volume restriction as defined by eq 5 implies the following relationships:

and the bottom sublayer in water is given by

Fh )

fcbc + bh + nbw Aτh

(4)

where the subscripts c, h, and w refer to the chain and head regions of the layer and water, respectively, τc and τh signify the top and bottom sublayer thickness, respectively, and fc is the fraction of the alkyl chain immersed in the bottom sublayer. It is usually further assumed that the headgroup region is spacefilling, and this leads to

Aτh ) fVc + Vh + nwVw

Figure 2. Simultaneous uniform layer model fits (shown as solid lines) to a set of reflectivity profiles for C12PC at 1.46 mM (above the CMC) measured from dC12hPC (O), hC12dPC (b), and hC12hPC (2) in D2O. The corresponding two-layer model fits are shown as dotted curves. For clarity, error bars are shown for hC12dPC only, but the error levels for the other two reflectivity profiles are similar.

(5)

where Vi is the appropriate molecular volume for the alkyl chain, (24) Heavens, O. S. Optical Properties of Thin Films; Dover: New York, 1955.

Aτh ) VTh

(6)

fVc + Vh + nVw ) Vcalc

(7)

Thus, if Vcalc is the calculated volume from theoretical values, and VTh is the total volume measured, then, within an error of (3 water molecules, Vcalc ) VTh. Figure 1 shows the set of reflectivity profiles measured in NRW for dC12hPC, hC12dPC, and dC12dPC, with all surfactant concentrations fixed at the CMC. Figure 2 shows the corresponding measurements in D2O. At constant concentration and constant area per molecule, the contrast was varied in a way to obtain more detailed information about the structure of the surfactant. Uniform one-layer fits are shown as continuous lines for the surfactant for the three different contrasts. From the one-

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layer model fits to the reflectivity profiles in NRW, Acmc for C12PC was found to be 50 ( 3 Å2 and is close to the values for nonionic C12E5 9 and ionic ammonium perfluorooctanoate.25 The total thickness of the layer was 20 ( 2 Å, and the thickness for the headgroup region was 15 ( 2 Å. The thickness for the sublayer immersed in the water was obtained from hC12hPC in D2O, and it was 14 ( 2 Å2, giving the number of water molecules associated with each PC head (nw) as approximately 15. This value of nw is higher than that for C12E5 at the CMC, where nw ) 10, but it is closer to that for ammonium perfluorooctanoate, where nw ) 12-13. These differences indicate a stronger affinity of PC headgroups for water. Given that the headgroup is immersed in water, the two-layer model as described above is more appropriate for describing the overall structure of the PC monolayers. The two-layer model, described by eqs 3-5, was subsequently used to extract information about the fraction of the alkyl chain associated with the headgroup region (fc). Acmc was again found to be 50 ( 3 Å2, and nw was 15 ( 3, consistent with the values obtained from the uniform layer model. The thickness was found to be 10 ( 2 Å for the alkyl chain layer in air and 15 ( 2 Å for the headgroup layer. The headgroup layer also contains part of the alkyl chain in water. Although the overall layer thickness is a little higher than that obtained from the single layer fit, the difference is within the error. The alkyl chain fraction immersed in water, fc, from the two-layer fit was found to be about 0.15 at the CMC. This amount of the alkyl chain in the water is unusually low compared to that observed for other surfactants such as nonionic C12E5,9 ionic SDS,14 and ammonium perfluorooctanoate,25 for which fc is about 0.2-0.4. This may be because the phosphorus atom is surrounded by four oxygen atoms that not only carry a negative charge but also contain lone pairs of electrons for hydrogen bonding to water in a three-dimensional framework, which would inhibit alkyl chain mixing. Thus, the PO4- group together with the oppositely charged choline group would provide the buoyant stability at the surface and maintain the constant thickness for the headgroup region. It should be noted that the two-layer model fit assumed a dry upper layer and a complete immersion of the bottom layer. Although the assumption simplified the data analysis, it could be regarded as unrealistic. However, given the same assumption was applied to the models used for other surfactant systems, the comparison of the lower fraction of immersion of the alkyl chain into water for the PC surfactants is valid. Tieleman et al.26 recently performed molecular dynamics (MD) simulations on C12PC micelles in water. They used 54 C12PC surfactant molecules, at 0.12 M and 0.46 M, both of which were well above the CMC. They showed that an ensemble consisting of 54 molecules and 1200 water molecules was a close representation of entropically driven micellization in the solution. Although micellization is dynamic and its size varies, the number of water molecules associated with each surfactant molecule was found to be around 22, and this is broadly consistent with the experimental data for C12PC micelles obtained from quasielastic light scattering and analytical ultracentrifugation.27 This value of nw is close to the value of 25 obtained from our model fit at 0.073 mM, but it is higher than the value of 15 obtained at the CMC. The differences must arise from the different packing and geometrical shapes between the planar and curved interfaces, and this is not unexpected. A value much closer to that of C12PC (25) Lu, J. R.; Ottewill, R. H.; Rennie, A. R. Colloids Surf., A 2001, 15, 183. (26) Tieleman, D. P.; van der Spoel, D.; Berendsen, H. J. C. J. Phys. Chem. B. 2000, 104, 6380. (27) Lauterwein, J.; Bo¨sch, C.; Brown, L. R.; Wu¨thrich, K. Biochim. Biophys. Acta 1979, 556, 244.

Yaseen et al.

for nw was obtained for DPPC at the air/liquid interface by Brum and co-workers.15 Neutron reflectivity measurements at the LC phase for this lipid indicate 14 water molecules at an area per molecule of 52 Å2. However, more interesting is the fact that they suggest a head thickness of 11.5 Å and conclude that the P-N dipole is close to the surface normal, an observation that is consistent with our results for C12PC at the CMC. The structure of the C12PC surfactant layers at a lower concentration of 0.073 mM (well below the CMC) was also determined under the same isotopic combinations. A was found to increase to 76 ( 3 Å2. The total thickness was 18 ( 2 Å and was slightly lower than the value of 20 ( 2 Å obtained at the CMC. From the two-layer model, the alkyl chain region was 10 ( 2 Å, and the headgroup region immersed in water was again 15 ( 2 Å. The number of water molecules was 25 ( 3 for the one-layer model and 27 ( 3 in the two-layer model. The fraction of the alkyl chain immersed in the water was again found to be 0.12-0.15. Comparison of the structure of the C12PC monolayers at the two concentrations suggests that A increases with a consequential increase in the number of water molecules, but the thickness of the layer under water and that of the entire layer show little change, which would indicate that the surfactant conformation is invariant in going from a close-packed environment at the CMC to a relatively more spacious environment. For each surfactant, the alkyl chain does not become more disordered with dilution, suggesting that the PC head is a stable anchor for the alkyl tail, which maintains its structural organization or conformation at both concentrations. This is implied from the constant thickness of the headgroup region and of the entire surfactant layer from this simple analysis. The application of the kinematic approach will provide further and more direct structural details of the PC surfactant layers and reinforce this deduction. B. Kinematic Approach. In this analysis, neutron reflectivity is separated into partial structure factors, each of which contains information about either the distribution of a given component across the interface or its relative location to another. Neutron reflectivity, R(κ), is related to the interfacial composition through the scattering length density perpendicular to the interfacial plane,1,14 F(z), by

16π2 |Fˆ (κ)|2 κ2

(8)

∫∞-∞ exp(-iκz)F(z)dz

(9)

R(κ) ) Fˆ (κ) )

where the scattering length density depends on the chemical composition, as shown in the following equation:

F)

∑mibi

(10)

where mi is the number density of the element, i, and bi is its scattering length. The analytical expression that relates reflectivity to the partial structure factors is shown as follows:

R(κ) )

16π2 2 [bc hcc + bh2hhh + bw2hww + 2bcbhhch + κ2 2bcbwhcw + 2bhbwhhw] (11)

where the subscripts c, h, and w denote the chain, head, and water, respectively. The self-term partial structure factors hcc, hhh, and hww contain information about the structural distributions for the alkyl chain, the head, and the water, respectively, while

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Table 2. (a) Structural Parameters Obtained from Uniform Layer Model Fits to C12PC under Different Contrasts. (b) Structural Parameters Obtained from Two-Layer Model Fits to C12PC under Different Contrasts (a) contrast

C (mM)

F× (Å-2)

τ(2 (Å)

A(3 (Å2)

(Γ ( 0.2) × 10-6 (mol m-2)

dC12hPC/NRW hC12hPC/D2O dC12hPC/D2O hC12dPC/NRW dC12dPC/NRW hC12dPC/D2O dC12hPC/NRW hC12hPC/D2O dC12hPC/D2O hC12dPC/NRW dC12dPC/NRW hC12dPC/D2O

1.46 1.46 1.46 1.46 1.46 1.46 0.073 0.073 0.073 0.073 0.073 0.073

2.71 4.41 4.58 2.01 3.77 5.88 1.95 4.75 4.75 1.35 2.71 5.75

20 14 22.5 15 21 14.6 18 14 20.2 14 20 14

49 50 52 50 50 50 75 73 75 75 73 75

3.41 3.32 3.21 3.30 3.32 3.32 2.21 2.27 2.20 2.22 2.27 2.22

106

nw ( 3 15 14 15 25 24 24

(b) contrast

C (mM)

F1 × 106 (Å-2)

τ1 ( 2 (Å)

F2 × 106 (Å-2)

τ2 ( 1 (Å)

A(3 (Å2)

(Γ ( 0.2) × 10-6 (mol m-2)

dh/NRW hh/D2O dh/D2O hd/NRW dd/NRW hd/D2O dh/NRW hh/D2O dh/D2O hd/NRW dd/NRW hd/D2O

1.46 1.46 1.46 1.46 1.46 1.46 0.073 0.073 0.073 0.073 0.073 0.073

4.31 -0.24 4.15 -0.22 4.88 -0.34 2.83 -0.15 2.75 -0.15 3.00 -0.35

10 10 10 10 9 10 10 10 10 10 10 10

0.75 4.35 4.88 2.01 2.71 4.75 0.48 4.82 5.05 1.36 2.05 4.88

15 15 15 15 15 15 15 15 15 15 13 14

49 49 49 52 48 49 75 75 75 76 71 77

3.42 3.42 3.42 3.19 3.50 3.42 2.21 2.21 2.21 2.19 2.35 2.16

the cross-term partial structure factors hch, hcw, and hhw contain information about their relative positions. Resolving the six partial structure factors requires six or more measurements of the reflectivity under different isotopic contrasts. In principle, any combination of six isotopic contrasts can lead to the resolution of the six partial structure factors, but some isotopic substitutions have a stronger influence on certain partial structure factors. For example, hcc is more reliably determined from dC12hPC/NRW, as is hhh from hC12dPC/NRW. The selective use of isotopic substitution to the surfactant and the solvent makes it possible to determine each of the partial structure factors unambiguously. The isotopic combinations selected for kinematic analysis at each concentration can be seen in Table 2. As described in our previous work,4 a Gaussian model is the most appropriate for representing the distributions of the alkyl chain:

n ) nmi exp

( )

-4z2 for all z σi2

(12)

nw ( 3 15 15 14 27 27 27

fc ( 0.05 0.14 0.17 0.17 0.16 0.15 0.15 0.13 0.16 0.15 0.16 0.12 0.13

and that of the head (δch) and can be related to the self partial structure factors through the following equation:

hch(κ) ) ([hcchhh]1/2 cos(iκδch)

(14)

For the distribution of water across the interface, a tanh distribution4 has been extensively used:

[21 + 21 tanh(z/ζ)]

n ) ns

(15)

From the fit of the water partial structure factor (hww) using eq 15, the width of the water distribution at the interface (ζ) can be obtained.11 The information about the relative position (δij) between surfactant chain or head distributions (hcc, hhh) to the center of water distribution (hww) can be obtained from the following equation:1,4

hij(κ) ) ([hiihjj]1/2 sin(iκδij)

(16)

where nmi is the maximum number density at the peak of the distribution, σi is the full width at 1/e of nmi, and z is the distance in the direction normal to the surface. For a Gaussian distribution, the partial structure factors for the chain and head can be written as

The cross distances between the centers of distributions, as determined from the partial structure factors, are related through the following equation:1,4

hii ) Γ2 exp(-κ2σi2/8)

The uncertainty in the signs in eqs 14 and 16 results from the phase problem, but, as shown, the constraint as given in eq 17 and the physical information such as the chain projection in the air help to provide a unique description for the interfacial structural distributions and their relative positions. The reflectivity profiles for the combinations of the six isotopic contrasts were analyzed together for C12PC. At a concentration around the CMC (1.46 mM), the use of eq 11 leads to the

(13)

It should be noted that σi, the full width of the Gaussian model, will be smaller than the thickness obtained from the corresponding uniform layer model (σI ) x3/2τ), but the area per molecule will be the same. The cross partial structure factor hch contains information about the relative distance between the center of the chain distribution

δcw ) δch + δhw

(17)

5830 Langmuir, Vol. 22, No. 13, 2006

Yaseen et al. Table 3. Structural Parameters Obtained from the Partial Structure Factor Analysis for Pure C12PC, C14PC, and C16PCa C × 10-3 A ( 3 (M) (Å2)

σh ( 2 (Å)

σc ( 2 ζww ( 1 δch ( 1 δcw ( 0.5 δhw ( 1 (Å) (Å) (Å) (Å) (Å)

1.46 0.073

49 75

14 12.5

0.135 0.005

51 77

(14 ( 3) 15.5 (12.5 ( 3) 14.5

0.0084 0.002

54 (14 ( 3) 15.5 78.5 (12.5 ( 3) 14.5

C12PC 7.5 7.5

7.5 6

8.5 6.5

1.5 1.5

C14PC 8 7

(7 ( 1.5) (7 ( 2)

8 6.5

(1 ( 1.5) (1 ( 1.5)

C16PC 7 6.5

(7 ( 1.5) (7 ( 2)

8.5 7

(1 ( 1.5) (1 ( 1.5)

15.5 14

a The width of the headgroup distribution in parentheses was obtained from a combined kinematic analysis to a set of three reflectivity profiles, where hCmdPC/NRW was not measured. The errors shown in parentheses indicate the range of variation beyond which the calculated partial structure factors showed obvious deviations from the measured ones.

Figure 3. Two self partial structure factors, hhh (a) and hcc (b), for C12PC. The solid lines were calculated using eq 14. The structural parameters obtained are given in Table 3.

again from the direct partial structure analysis, showing good agreement between all the parameters obtained. The surfactant at the surface of water does not reside on completely flat water. Instead, the thickness of the surfactant layer is roughened by capillary waves. This often results in layer broadening and makes it thicker than the actual fragment thickness. The broadening effect from capillary waves can be taken into account from the following equation:

σi2 ) li2 + ω2

Figure 4. Two cross partial structure factors, hch (a) and hcw (b), for C12PC. The continuous lines were calculated using eqs 14 and 16.

determination of the six partial structure factors: hhh, hcc, hww, hch, hcw, and hhw. Their subsequent analyses using eqs 13, 14, and 16 give the parameters σh, σc, ζs, δch, δcw, and δhw. The resultant self partial structure factors for the alkyl chain and the head (hcc and hhh) are shown in Figure 3, and the cross partial structure factors (hcw and hch) are shown in Figure 4. The structural parameters obtained are given in Table 3. The reliability of the structural analysis from the kinematic approach can be checked by comparing the cross distances following the relationship, as shown in eq 17. At 1.46 mM, |δch| + |δhw| ) 9 ( 1 Å, and this is compared with |δcw| ) 8.5 ( 1 Å from the direct partial structure analysis. At 0.073 mM, |δch| + |δhw| ) 7.5 Å, and this is compared with |δcw| ) 6.5 ( 1 Å,

(18)

where li is the intrinsic width of the distribution of the surfactant fragment projected normal to the surface, and ω is the roughness taking the form of the Gaussian distribution. If the PC head is considered to be fully extended along the normal to surface around the CMC, it is ∼11 Å. As it can be seen from Table 3, σh ) 14 Å; thus, a value of 8.7 Å is then obtained for ω from eq 18. The value of ω is more often estimated from surface tension variation. For a pure liquid surface, ω is inversely proportional to the square root of surface tension.28 If it is assumed that the same effect is followed at the surfactant adsorbed surface, it is straightforward to calculate ω for the PC surfactant surfaces. In the Gaussian model, the roughness of a pure water surface is about 6.5 Å for a surface tension of 72 mN/m. The surface tension drops to 40 mN/m at the CMC for C12PC, giving a ω value close to 8.5 Å. From eq 18, lh is again calculated to be 11 Å, consistent with the vertical projection of the PC headgroup. The value of ω for C12PC at the CMC is lower than the value of 11 Å found for the cationic surfactant, dodecyl trimethylammonium bromide (C12TAB).13 However, it is close to that for the C12 betaine surfactant, n-dodecyl-N,N-dimethylamino acetate,29 consistent with similar structural characteristics for the zwitterionic surfactant layers. As the roughness should contribute equally to the head (σh) and chain (σc), the corrected value of chain width (lc) was calculated to be 13 ( 2 Å. The angle of tilt, θ, of the carbon chain away from the surface normal can be obtained using

cos θ ) lc/le

(19)

where lc is the full width of the chain distribution at the half(28) Braslau, A.; Deutsch, M.; Pershan, P. S.; Weiss, A. H.; Als-Nielsen, J.; Bohr, J. Phys. ReV. Lett. 1985, 54, 114. (29) Hines, J. D.; Garrett, P. R.; Rennie, G. K.; Thomas, R. K.; Penfold, J. J. Phys. Chem. B 1997, 101, 7121.

Structure of Zwitterionic PC Surfactant Monolayers

Langmuir, Vol. 22, No. 13, 2006 5831

Table 4. Further Structural Parameters Obtained from the Removal of Roughness Arising from Capillary Wavesa C (mM)

γ σ c ( 2 lc ( 2 (mN/m) (Å) (Å)

σh ( 2 (Å)

1.46 0.073

40.5 58.0

15.5 14.0

C12PC 12.9 14 11 12.5

0.135 0.005

38.5 62.4

15.5 14.5

0.0084 0.002

41.5 51.8

15.5 14.5

lh ( 2 (Å) 11 10.3

ω(2 θ (Å) (°) 8.6 7.2

40 48

C14PC 12.8 (14 ( 3) (10.9 ( 3) 12.7 (12.5 ( 3) (10.4 ( 3)

8.8 7.0

48 48

C16PC 13 (14 ( 3) (11.1 ( 3) 12.3 (12.5 ( 3) (10 ( 3)

8.5 7.6

53 56

a The values in parentheses were obtained from combined fitting to a set of three reflectivity profiles where hCmdPC/NRW was not measured. The width of the headgroup distribution in parentheses was obtained from a combined kinematic analysis to a set of three reflectivity profiles where hCmdPC/NRW was not measured. The errors shown in parentheses indicate the range of variation beyond which the calculated partial structure factors showed obvious deviations from the measured ones.

height of the Gaussian distribution, and le is the extended full length of the alkyl chain. At the CMC, cos θ was found to be about 0.77, and θ ) 40° for the dodecyl chain. This is smaller than that for C12TAB13 (Acmc ) 48 Å2) with θ ) 54° but much smaller than that for the zwitterionic C12 betaine (A ) 48.5 Å2) with θ ) 60°. The angle of tilt for the dichain DPPC bilayer was reported to be about 40° at 30 mN/m and is, within the error, the same as the single-chain PC analogue.17 This is interesting considering that the dichain DPPC molecule has a longer chain and denser packing and stronger chain-chain interaction.30,31 At the low concentration of 0.073 mM, the headgroup region becomes slightly thinner, and this implies an increase in the tilt angle to some 50ο. The other parameters obtained using kinematic analysis are shown in Table 4. The analyses described above show that, in addition to the broadening effect from capillary waves and from structural disorder, the precise thicknesses for the alkyl chain, the PC head and the entire surfactant layer are dependent upon the exact description used. It is useful to compare the neutron reflection data from the planar surface with the NMR measurement from C12PC micelles. The radius of the C12PC micelles from NMR investigation32 was 22-23 Å, in good agreement with the planar layer thickness of some 20 Å determined from our neutron reflection work. It is also useful to compare the thickness of PC headgroups in the soluble surface monolayer with that determined from the dichain phospholipids such as DPPC. Researchers15,31,33-35 have reported the thickness of the PC headgroup in spread monolayers and bilayer stacks to be about 8 ( 2 Å. This is suggesting that the PC headgroup is rather parallel to the surface, although, in the LC phase, the thickness increases to 11.5 Å, as indicated previously.15 In addition to the different packing and hydrophobicity, the different conformation of the PC headgroups may arise from the different anchoring points. The attachment of PC onto the side hydroxyl group of the glycerol backbone in DPPC may structurally favor its tilting. 3.2. Structure of C14PC and C16PC Monolayers. To examine the effect of alkyl chain length on the monolayer structure, we (30) Johnson, S. J.; Bayerl, T. M.; Wo, W. H.; Noack, H.; Penfold, J.; Thomas, R. K.; Kanellas, D.; Rennie, A. R.; Sackmann, E. Biophys. J. 1991, 60, 1017. (31) Vacklin, H. P.; Tiberg, F.; Fragneto, G.; Thomas, R. K. Langmuir 2005, 21, 2827. (32) (a) Kallick, D. A.; Tessmer, M. R.; Watts, C. R. Magn. Reson. B 1995, 109, 60. (b) Tessmer, M. R.; Kallick, D. A. Biochemistry 1997, 36, 1971. (33) Fragneto, G. J. Phys.: Condens. Matter 2001, 13, 4973 (34) Tamm, L. K.; McConnell, H. M. Biophys. J. 1985, 47, 105.

have extended the neutron reflectivity measurements to C14PC and C16PC, using both alkyl chain deuterated and fully hydrogenated surfactants. The interest here was to examine whether an increase in the alkyl chain length affected the distribution of the PC headgroups. Although the contrast of hCmdPC/NRW (m ) 14 and 16) gives the best determination of headgroup thickness, in the absence of the head deuterated surfactant, the thickness for the headgroup region was estimated from the depth of immersion of the monolayer using hCmhPC/ D2O. The analysis gave a thickness of 14 ( 2 Å2 for C14PC and 13.5 ( 2 Å2 for C16PC. These values are within the error, the same as that for C12PC at the CMC using the same model treatment, indicating that an increase in alkyl chain length has little effect on the distribution of the PC headgroups. From the volume constraints calculated for the parts of the molecules submerged in water, nw for all three surfactants at their CMCs was found to be 15 ( 3. The uniform layer analysis further showed that the thickness of the alkyl chain region did not show any clear change with increase in the alkyl chain length. This together with the constant area per molecule at the CMC suggests an increasing alkyl chain tilting and packing density. The application of the two-layer model gave a sublayer of 10 ( 2 Å for the alkyl chain and 15 ( 2 Å for the head sublayer for C14PC and C16PC, similar to the division found for C12PC. The chain fraction immersed in water was also found to be 0.12-0.15 and showed no clear response to increasing alkyl chain length, suggesting that the hydrophilic PC headgroups control the packing and structural disorder within the surfactant monolayers. To examine the layer structure at a lower surface coverage, reflectivity measurements were made at 5 × 10-6 M for C14PC and at 2 × 10-6 M for C16PC. At these concentrations, A was found to be 78 ( 3 Å2 for both surfactants. As already observed, this range of surface coverage was achieved for C12PC at 0.073 mM. The results obtained from the one- and two-layer model fits for C14PC and C16PC were found to be remarkably similar to those shown for C12PC in Table 2a,b, with the total thickness from all three surfactants being around 20 ( 2 Å. From the two-layer model, the alkyl chain region was found to be 10 ( 2 Å, and the headgroup region in water was 14 ( 2 Å. These structural parameters are consistent with those obtained at the CMC, showing that, within the experimental error, the sublayer thicknesses and the fraction of alkyl immersion vary little with concentration or alkyl chain length. Alternative structural analysis based on the kinematic approach was also carried out for C14PC and C16PC. Although the reflectivity profiles were not measured for enough isotopic combinations at a given concentration to provide complete separation of the different partial structure factors, it was still feasible to obtain meaningful parameters through a simultaneous least-squares fitting to eq 11 using eqs 13, 14, and 16. The structural parameters obtained are also shown in Table 3 together with those for C12PC for direct comparison. Although the simultaneous fitting provided an estimate of σh, δch, and δhw, these parameters tended to have larger uncertainties. It was found that the structural parameters for C12PC fit the measured sets of reflectivity profiles from C14PC and C16PC reasonably well. The errors shown in Table 3 with these parameters reflected the variation within which the fits were still acceptable. The results thus show that, although not enough reflectivity profiles were measured for these longer chain surfactants, the simultaneous analysis still offered some insight into the surface structure. (35) Schalke, M.; Lo¨sche, M. AdV. Colloid Interface Sci. 2000, 88, 243.

5832 Langmuir, Vol. 22, No. 13, 2006

Yaseen et al.

4. Conclusions

Figure 5. A schematic representation of the main structural features for C12PC, C14PC, and C16PC at their CMCs from neutron reflection.

Comparison of the structural parameters shown in Table 3 clearly shows that, within a good approximation, all three surfactant monolayers at a given surface coverage can be fitted using a similar set of structural parameters. The width of the alkyl chain distributions for the Gaussian model is about 15.5 Å for C14PC and C16PC. After taking into account the roughness arising from capillary waves, the corrected thickness is about 13 Å and is the same as that for C12PC at the CMC, as already discussed. This, together with the constant fraction of immersion of the alkyl chain in water, means that the angle of tilt of the chain from the normal to the surface should also increase. It can be seen from Table 4 that the angle of tilt increases with alkyl chain length from around 40° for C12PC and 48° for C14PC to 53° for C16PC at the CMC. At low concentrations, where A ) 78 ( 3 Å2, the alkyl chain distributions become slightly more narrow, and the angle of tilt increases slightly. However, such increase is significantly lower than the values observed for ionic surfactants such as C12TAB.13 Although the width of the headgroup distribution was only indirectly determined, the values were sufficiently reliable to deduce the almost vertical structural conformations for the PC heads, as was observed for C12PC. The distances between the centers of distribution also show a good consistency and follow the relation as defined by eq 17 well. Most importantly, they are identical to those obtained from C12PC within an error of (2 Å. 3.3. Effects of Salt and Temperature. In the course of determining the conformational structure for PC surfactants, we also examined the effects of salt and temperature. For C12PC, each set of reflectivity measurements was made using the previously described six isotopic combinations at 25 and 40 °C, with and without 0.1 M NaCl and 0.1 M CaCl2. For C14PC and C16PC, each set of the reflectivity profiles was made using the standard three isotopic contrasts at 25 and 40 °C, with and without 0.1 M NaCl only. The same procedures as those already described were used to analyze the reflectivity profiles. Salt addition was found to lower the CMC to some extent,2 but salt and temperature were found to have little effect on layer structure, including the distributions for the head and alkyl chain and their relative locations. No measurable difference was found in the extent of alkyl chain immersion in water and the degree of PC hydration.

The main findings from this work are outlined schematically in Figure 5. Neutron reflectivity provides a reliable experimental approach to determine the detailed surface structure. The maintenance of the almost constant alkyl chain thickness is achieved by an increase in the angle of tilting of the alkyl chain length. The PC headgroup aligns almost vertically, and the area per molecule at the CMC is between 50 and 54 Å2 for the chain isomers studied. The whole surfactant layer is about 20 Å thick, and the extent of intermixing between the alkyl chain and water remains low at about 0.15. The PC headgroup remains strongly hydrated, and the level of hydration does not vary with increase in chain length, salt addition, or temperature increase. Comparison with the analogous dichain zwitterionic DPPC in its LC phase shows a similar thickness, hydration, and headgroup configuration. However, there is much more structural variation in the hydrophilic part upon change in concentration in the dichain lipid than in the single-chain PC. The zwitterionic CmPC surfactants (m ) 12, 14, 16) have a number of structural features similar to cationic CmTABs. In both cases, the alkyl chain thickness remains constant with increasing chain length. The almost constant layer thickness is maintained as a result of increased alkyl chain tilting. For both types of surfactants, the area per molecule does not vary much with alkyl chain length, showing the dominant effect of the headgroups in determining the surface packing. The headgroup layer thicknesses after removal of the contribution of surface roughness are around 10 Å for both types of headgroups, with the PC headgroup region being the thicker of the two. This shows that the headgroups are projected normal to the surface when adsorbed on the surface of water. However, the roughness revealed from CmTAB monolayers is greater and shows a steady increase with alkyl chain length. There is also a large extent of alkyl chain mixing with the headgroup and into the aqueous phase for CmTAB monolayers. In contrast, the roughness from CmPC monolayers remains almost constant, and the extent of alkyl chain mixing into water is very low and shows little variation with chain length. It is interesting to note that the nonionic headgroup of poly(ethylene glycol) binds to water through hydrogen bonding, but, in the zwitterionic PC head, hydration is mainly a consequence of a network of electrostatic interactions and hydrogen bonding, which provides a much higher biocompatibility. Zwitterionic surfactants also show similar features to nonionic surfactants, including little variation in surface tension and structural order with salt addition. Unlike nonionic surfactants, zwitterionic PC surfactants are invariant to temperature. The most striking characteristics of PC surfactants is their strong hydration and their ability to retain a high level of hydration against change in alkyl chain length or environmental conditions such as salt addition and temperature, and this ability is unique to zwitterionic PC surfactants. Acknowledgment. We thank EPSRC for support, and CCLRC for the provision of neutron beam time. Thanks to Haji M. Sharif and Javid Ali for their helpful discussion. Thanks also to Dr. T. J. Su for her helpful discussions. LA053316Z