- Email: [email protected]
Diffusion of water in multilamellar vesicles of dialkyl and dialkyl ester ammonium surfactants
Colloids and Surfaces A: Physicochem. Eng. Aspects 228 (2003) 64–73
Diffusion of water in multilamellar vesicles of dialkyl and dialkyl ester ammonium surfactants Cecilia Groth, Johanna Bender, Magnus Nydén∗ Materials and Surface Chemistry, Chalmers University of Technology, S-412 96 Gothenburg, Sweden
Abstract The NMR self-diffusion technique is used for measuring diffusion of water in highly concentrated multilamellar vesicle solutions. The signal intensity of water, i.e. the water echo-decay, is monitored down to very small intensities thus providing an accurate measure of how water is diffusing in the solution. It is noted that a large curvature is dominating the functional form of the echo-decay indicating the presence of a large number of multilamellar vesicles. It is also concluded that in order to measure accurately the volume fraction of water inside and outside vesicles the experimental time scale can be changed. From a multiexponential fit to the echo-decay the fraction fast and the sum of all slow components can be extracted. When the apparent app fraction “vesicle water”, Pvw , is plotted versus the experimental time scale the graph produced is a good representation of the difference in how fast water diffuses over the different vesicle membranes. From an extrapolation to “zero time” the true fraction of trapped water can be extracted, i.e. a quantitative measure of the volume fraction of vesicles at a certain concentration of surfactant. © 2003 Elsevier B.V. All rights reserved. Keywords: Multilamellar vesicles; NMR; Surfactants
1. Introduction Cationic surfactants are used in a wide range of applications as different kind of surface treaters as fabric softeners or personal care products or as cleaner for hard surfaces and paper processing. The largest use for cationic surfactants is in the range of fabric softeners and due to efforts to make the surfactants more biocompatible the active component in softeners have changed from ordinary di(hydrogenated tallow)alkyldimethylammonium (DHTMAC) quar∗ Corresponding author. Tel.: +46-31-772-2973; fax: +46-31-160-062. E-mail address: [email protected] (M. Nyd´en).
ternary (dialkyl quats) to ester quaternaries (dialkylester) that are more easily biodegradable [1]. Softeners are thought to act on the surface of the fabric by neutralizing the negatively charged surface. To maximize the adsorption of ester quats a double tailed surfactant has proven to be most efficient. This results in a very low solubility of monomeric surfactant in water and a subsequent formation of vesicles in concentrations ranging from the dilute region up to approximately 25 wt.% surfactant. It is believed that at low concentrations vesicles are mostly unilamellar but at higher concentrations more multilamellar vesicles are formed. The size and size distribution of vesicles depends on the nature of the surfactant and properties like salt concentrations, temperature etc. but it may
0927-7757/$ – see front matter © 2003 Elsevier B.V. All rights reserved. doi:10.1016/S0927-7757(03)00307-8
C. Groth et al. / Colloids and Surfaces A: Physicochem. Eng. Aspects 228 (2003) 64–73
also depend on how they are prepared. For example, if a simple stirring process is employed, the mean size depends strongly on the speed of stirring, resulting in smaller size as the stirring rate is increased. The membrane thickness is more or less constant around 4 nm but depending on the concentration and/or means of preparation each vesicle can be built up by one bilayer up by hundreds of bilayers. Vesicles can be divided into different groups depending on their size and structure; small uni-lamellar vesicles (SUV) are 10–50 nm, large uni-lamellar vesicles (LUV) between 50 and 500 nm, giant uni-lamellar vesicles larger than 0.5 m, or multi-lamellar large vesicles (MLV), 1–50 m. As mentioned above the size and size distribution can to a large extent be controlled. Furthermore, since the surfactants normally used for making vesicles often are double tailed surfactants with a very low solubility in water, making a vesicle a rather static entity (as compared with for example a micelle in which the exchange dynamics between surfactants in the micelle and monomeric surfactants most often is very fast). In addition, since the inner part of a vesicle is hydrophilic and the membrane is hydrophobic, they are suitable for example as carriers for small molecules or as model systems for cell membranes, as templates for reactors or as surfactant delivery systems (softeners) [2]. One very important property of a vesicle solution is the restriction in diffusion of hydrophilic substances due to the presence of the vesicle wall. For vesicles with more or less perfect membranes, i.e. without large holes or dislocations, the transport of a hydrophilic substance through the membrane is given by the difference in concentration between the water phase and the membrane. If, however, the membrane contains holes or dislocations, diffusion through these might be the rate determining step in the diffusion process that occurs through the membrane of a vesicle [3–7]. PGSE NMR is a powerful technique for physicochemical investigations in complex mixtures. The technique can be used in systems with restricted diffusion in for example cells [8] or in general to interpret restricted diffusion in various geometries [9]. Marques et al. studied water diffusion in unilamellar catanionic vesicles by PFG-SE NMR [10–13]. Water diffusion and exchange in normal and reversed unilamellar vesicles has also been investigated by Olsson et al. where it was noted that the lifetime of a solvent
65
molecule inside vesicles varied with the composition of molecules in the bilayer [14]. In this paper we are investigating the difference in membrane permeability between one dialkyl quat and one dialkylester quat by measuring the self-diffusion of water molecules by PGSE NMR. By comparing the decrease in fraction of “trapped” water obtained after different diffusion times, conclusions about the effective membrane permeability are drawn. By increasing the diffusion time more water molecules will have exchanged between the inside and outside of a vesicle. The transport process of a much larger hydrophilic molecule polyethylene glycol (PEG) is also measured with the aim to understand the mechanism of diffusion of water and other molecules in a concentrated solution of uni- and multi-lamellar vesicles formed by cationic vesicles.
2. Experimental 2.1. Materials Arquad HC and Armosoft DEQ were a gift from Akzo Nobel Surface Chemistry, USA and used without further purification. Arquad HC is a di(hydrogenated tallow)dimethylammonium chloride (DHTDMAC) with alkyl chain lengths between 16 and 18 having a maximum amount of 2-propanol (IPA) of 1% and an active matter of 96%. Armosoft DEQ is the corresponding dialkylester quat to Arquad HC with a maximum amount of ethanol of 10–20% and hence no further additions of alcohol were made. Ethanol (99.5%, spectrographically pure) was bought from Kemetyl, calcium chloride (technical, 40 mesh) from Aldrich and PEG (molecular weight 950–1050 g mol−1 ) from Merck. All chemicals were used without further purification. Milli-Q water was used for all sample preparations. 2.2. Sample preparation Samples were prepared by a standard method according to Bell [15], utilizing a glass beaker with a Teflon propeller stirrer with a diameter of 40 mm connected to a Heidolph RZR 2050 motor unit. Surfactant and alcohol are weighted in a screw capped tube and mixed in a heated water bath. Milli-Q
66
C. Groth et al. / Colloids and Surfaces A: Physicochem. Eng. Aspects 228 (2003) 64–73
water was added into a flask, (in the case of salt addition some water was used for dissolving the CaCl2 ) and heated to 64–66 ◦ C and equilibrated for 5 min at the lowest stirrer speed. The speed was increased to 1000 rpm before starting the addition of the melted surfactant–alcohol mixture. The mixture was stirred for 15 min before the cooling process started. After the temperature had reached 40 ◦ C the dissolved CaCl2 was added in small portions with 20 s between each portion. The solution was stirred for another 15 min after the addition. When preparing highly concentrated vesicle solutions and if the viscosity became too high during the addition of surfactant, 1/5 of the salt solution is added to the dispersion. After approximately 20 s more of the surfactant mixture or salt solution is added depending on the viscosity. This was repeated until all surfactant solution was added. If some salt solution was left this was added during the cooling process (see above). Samples were equilibrated on an automatic seesaw 2–3 days before they were studied with light microscopy to investigate size and structure of vesicles, centrifuged and transferred to 5 mm NMR-tubes for NMR-diffusion studies. 2.3. Methods 2.3.1. Light microscopy Samples were observed by optical light microscopy (Olympus BH-2) in normal (size and shape) and polarized light. For large vesicles the typical Malteser cross pattern shown in liquid crystalline vesicles were observed. 2.3.2. NMR experiments NMR pulsed gradient spin-echo (PGSE) selfdiffusion experiments where performed on 500 MHz Varian Unity Inova spectrometer equipped with a diffusion probe by Doty Sci., Inc. All diffusion measurements were performed at 20 ◦ C. Approximately ten logarithmically spaced diffusion times (∆) between 10 ms up to 10 s were used. The pulsed field gradient strength was varied between 0.003 to 3.52 T m−1 . The basic Hahn-echo sequence was used for between 10 to 160 ms and above that the stimulated echo-sequence was used due to reasons of echo instability at long diffusion times when using a Hahn echo.
2.3.3. NMR self-diffusion in vesicles With the NMR self-diffusion technique it is possible to measure component resolved diffusion. The experiment results in a so-called echo-decay in which the logarithm of the NMR signal of interest normally is plotted versus the gradient strength squared or against k, an experimental factor containing the relevant tunable parameters. For free diffusion and when represented in this manner the echo-decay results in a straight line with the slope given by the self-diffusion constant. In many cases the diffusion is not free, for example due to the presence of barriers, which is often seen as a curved echo-decay in which (somewhat simplified) a large slope represents molecules that during the diffusion time (typically between 10 ms and 10 s) have moved a large distance. Consequently, a small slope represents molecules diffusing only a short distance during the same diffusion time. For a two-compartment system with different diffusion constants in the two compartments and no chemical exchange, the echo-decay is simply given by the sum of the individual echo-decays. If, on the other, molecules are allowed to exchange, the results strongly depend on the rate of exchange compared with the experimentally determined diffusion time. If the lifetime is much smaller than the diffusion time the echo-decay is a straight line where the slope is given by the weighted sum of the two diffusion constants such that the observed diffusion constant Dobs = p1 D1 + p2 D2 . If the lifetime of a molecule in a particular site is close to the experimental diffusion time the echo-decay may be quite a complicated function and for multi-compartment systems it can be quite impossible to extract quantitative data about lifetimes and diffusion constants. In this paper we have chosen to fit the echo-decay (which indeed is very complicated for all experiments in this work) to a sum of four exponents according to Eq. (1). The rationale for doing so is that other methods of evaluation have failed in describing the full echo-decay for all diffusion times. This should be no surprise when considering the complexity of this particular system; multi-lamellar vesicles with a distribution both in size and in number of lamellas and, in addition, chemical exchange between the inside and outside occurring on a intermediate time-scale. Regardless of the complexity displayed by the system and the value of ∆ we have noted that the initial slope
C. Groth et al. / Colloids and Surfaces A: Physicochem. Eng. Aspects 228 (2003) 64–73
of the echo-decay represents the diffusion of water outside vesicles; i.e. it is possible to estimate the apparent fraction of water trapped inside vesicles after a certain diffusion time, ∆. From here on in this app work this water is termed Pvw , and is obtained from app Pvw = (1−pfree ) where pfree is the fraction belonging to the largest diffusion coefficient in Eq. (1), thus representing the fraction water outside the vesicles. There the diffusion path of water is slowed down due to collisions with vesicles but not restricted to diffuse a certain distance during the diffusion time as is the case for water “trapped” inside. I/I0 = p1 e−kD1 +p2 e−kD2 + p3 e−kD3 + p4 e−kD4 (1) In Eq. (1), I is the intensity of the water peak, I0 the intensity at zero gradient strength, Dn the diffusion coefficient and pn the volume fraction of n. The echo-decay for a polydisperse vesicle sample can roughly be divided into two parts, where the slope at low k-values gives the diffusion coefficient for “free” water. At higher k-values the echo-decay reflects the restriction in diffusion for water inside the vesicles due to the presence of the membrane. It is worth noting that a larger degree of polydispersity (both in size and in number of membranes making up the full vesicle) gives a larger curvature in the echo-decay.
3. Results and discussion The structure of the formulated vesicle solutions was investigated by light-microscopy. When using a
67
polarized light source, all solutions displayed the characteristic Maltese cross patterns due to their lamellar structure in the membranes. All solutions also showed a large degree of polydispersity. Vesicles around 5 m are most frequent but also a few large vesicles between 20 and 30 m where detected. In addition, many small vesicles could be indirectly detected as small and rapidly diffusing entities, the size was, however, difficult to estimate due to the limited resolution. It is important to emphasize that no visible differences in vesicle size between different types of surfactants or at different concentrations could be detected (Fig. 1). In Fig. 2, echo-decays obtained at four logarithmically spaced ∆ are shown for the dialkyl quat and the dialkylester quat, both at 10 wt.% surfactant. All echo-decays are well described by Eq. (1). The diffusion coefficient obtained from the initial slope is around 1–2 × 10−9 m2 s−1 . In order to describe the full echo-decay, particular for those obtained at very long ∆, apparent diffusion constants on the order of 10−14 m2 s−1 are obtained, indicating the presence of very restricted water molecules with a very short rms-displacement. As mentioned above the justification for using Eq. (1) is not based on physical relevance of the equation but should rather be seen as a robust fitting routine with regards to a perfect description of the rather complex echo-decay at all diffusion times with the aim to extract a correct value for the fraction trapped water as a function of diffusion time, ∆. If no or only little exchange is occurring during a diffusion time of 10 ms (the shortest diffusion time used) a water molecule outside a vesicle has diffused
Fig. 1. Light microscopy images of a 10 wt.% dialkyl quat solution showing large differences in vesicle sizes within the sample. The image to the left shows a vesicle with a diameter of 35 m and to the right smaller vesicles around 3–10 m in diameter is shown.
68
C. Groth et al. / Colloids and Surfaces A: Physicochem. Eng. Aspects 228 (2003) 64–73
Fig. 2. Water echo-decays in a 10 wt.% dialkyl quat (a) and in (b) a 10 wt.% dialkylester quat solutions at four different diffusion times. Experimental settings where ␦ = 4 ms, g was linearly increased from zero to 3.5 T m−1 .
on average 6 m but a trapped molecule can only move within the boundaries of the vesicle. Thus, even though it is a very crowded surrounding for that type of water it will still diffuse quite a large distance compared with water inside vesicles as the diffusion time is increased. Therefore, at longer diffusion times, by allowing a molecule to exchange between the outside and inside during ∆, the time spent outside will clearly have the largest influence with respect to the total diffused distance. By increasing the diffusion time the fraction of water that had the chance to diffuse over the membrane is increased and the obtained amount of apparently trapped water will decrease. This is also what is seen in Fig. 2 for both the dialkyl quat and the
dialkylester quat. However, the two surfactants show app markedly different results with regards to Pvw . For app dialkyl quat at ∆ = 0.32 s, Pvw is around 0.05 and for the dialkylester quat it is around 0.001. This indicates that water passes the dialkylester quat membrane faster than the dialkyl quat membrane. At this stage it should, however, be realized that when comparing app Pvw for different surfactant vesicles, this property is app strongly dependent on the vesicle radius. Since Pvw depends on the volume of water inside compared with outside a small difference in size will have a large app impact on Pvw . In Fig. 3(a) and (b) echo-decays for = 10 ms and 5 s are shown together with the fits of Eq. (1).
Fig. 3. Water echo-decays for a 10 wt.% dialkyl quat sample. In figure (a) ∆ = 10 ms and in (b) ∆ = 5 s. Shown also as full lines are the best fit of Eq. (1). The obtained diffusion coefficients from the initial slope (i.e. the fastest obtained diffusion constant from Eq. (1)) is D = 1.82 × 10−9 (±1.4 × 10−10 ) for ∆ = 10 ms and D = 5.86 × 10−10 (± 7.6 × 10−12 ) m2 s−1 for ∆ = 5 s. The fraction of free diffusing water is Pfw = 0.64 (± 0.04) for ∆ = 10 ms and Pfw = 0.95 (± 0.003) for ∆=5 s.
C. Groth et al. / Colloids and Surfaces A: Physicochem. Eng. Aspects 228 (2003) 64–73
69
app
Fig. 4. Pvw vs. ∆ at four different concentrations, 7, 10, 15 and 20% with dialkyl quat (a) and 10, 15, 20 and 25% surfactant by weight with dialkylester quat (b). The full lines are the best fits of a simple exponential function that is regarded to describe the behavior reasonably well both for the dialkyl and diester quat.
In addition, it can be seen that a very good fit is obtained and the residuals are small and random. It was, however, noted that for some ∆, in particular for long ∆, residuals are not random indicating that in order to properly describe the full echo-decay at least four exponents have to be used. In order to test the validity of using Eq. (1) when evaluating the fraction of water in different domains we compare the obtained echo-decays at different ∆ between dialkyl and dialkylester quat. It is noted that for the dialkylester quat the echo-decay drops of to much lower signal intensities, indicating that water is diffusing more rapidly through the membrane in that system. app In Fig. 4 the fitted Pvw is displayed versus ∆, for dialkyl (Fig. 4(a)) and dialkylester quats (Fig. 4(b)) at four different concentrations. Also from this representation it is suggested that the dialkylester quat allows for faster water diffusion over the membrane as can app be seen as a larger change in Pvw as a function of than for the dialkyl quat. In addition, when comparing the different concentrations of surfactants we note that a reasonably accurate “zero-time” value may be obtained. This is a way to obtain the true volume fraction of trapped water in the vesicle since when extrapapp olating Pvw to zero time, the fraction water obtained is not modulated by chemical exchange between the outer and inner part of the vesicle. The fact that the two surfactants display a large difference in water permeability can be an effect of a larger disorder in ester membranes because of the ester group and/or a higher degree of un-saturation for
the alkyl chains in the dialkylester quat than for the normal dialkyl quat. It can also be due to differences in membrane defects, or, as previously mentioned, differences in size. With regards to membrane transport, two different possible diffusion mechanisms are discussed in the literature—by diffusion through the intact membrane (solubility mechanism) or through pores/defects in the membrane (pore mechanism). For diffusion through an intact membrane the hydrophobic part of the bilayer is referred to as an oil layer in which water is sparingly soluble. Permeating molecules must dissolve in the hydrophobic region, diffuse across and re-dissolve into the water phase on the other side of the membrane [4,5,7,16]. The partition coefficient of water in long chain hydrocarbons as n-hexadecan is Kw = 0.42 × 10−8 g water dm−3 alkane [17]. The diffusion coefficient, D, can be expressed as a function of the flux, J, and a concentration gradient across the membrane ∂C/∂X by Fick’s first law: ∂C J = −D (2) ∂X By integrating and introducing the permeability coefficient P, the partition coefficient K, and δ as the thickness of the membrane Eq. (2) yields: KD J P = = (3) δ ∆C Dw · KW · VW P = (4) d · Vsurfactant
70
C. Groth et al. / Colloids and Surfaces A: Physicochem. Eng. Aspects 228 (2003) 64–73
with d, thickness of the bilayer; Dw , diffusion coefficient of H2 O in the bilayer; Kw , partition coefficient of H2 O in the bilayer; VW , molar volume of H2 O and Vsurfactant , molar volume of surfactant. For a given thickness of the membrane, P is directly proportional to Dw Kw and by further assuming that water has the same diffusion coefficient in all liquid−crystalline bilayers the permeability is only dependent on changes in partition coefficient Kw or the solubility of water in the hydrophobic part of the membrane. The second model is the pore-mechanism [3,5–7] where molecules passes the membrane through defects (holes) produced by fluctuations within the membrane. 3.1. Addition of PEG In an attempt to separate the two different mechanisms by which water can diffuse over the membrane a large, hydrophilic molecule, PEG, Mw = 1000 g mol−1 , was added to the water phase before preparing the vesicle solution. If PEG is not allowed to diffuse over the membrane the presence of a significant amount of membrane defects can be neglected. Regarding the choice of the molecular weight we note that the polymer should have a rather fast diffusion so that the frequency by which it collides with the membrane is large enough. Thus the choice of 1000 in Mw is a polymer that has diffusion fast enough so that the entire volume of the membrane will, during the time scale of the experiment, be experienced by the polymer. Also it is much larger than water. Similarly to the case of water diffusion the echo-decays obtained at different ∆ where compared. The fraction, as obtained from a fit of Eq. (1) to the echo-decay, was of particular interest. In Fig. 5 the echo-decays for PEG added to formulations of both dialkyl and dialkylester quat are shown and displays a decreasing signal intensity at large k-values as ∆ is increased, indicating that also PEG diffuses over the membrane. By comparing the apparent trapped fraction obtained for PEG with the value obtained for water a qualitative difference between the permeability for PEG and water can be app estimated. For dialkyl quats, Pv PEG is 0.51 at ∆ = 0.1 app and Pvw for water 0.35. The higher values for PEG than water shows that water is exchanged faster than PEG during ∆. This is also shown for systems containing dialkylester quats. At this stage it should be noted that we have not observed any significant membrane
Fig. 5. PEG echo-decays obtained at 1 wt.% PEG and 10 wt.% surfactant at ∆ = 0.1 and 0.32 s for both dialkyl and dialkylester quat.
defects from microscopy images, vesicles there (small or large) appear to be rather intact. Considering that the hydrodynamic radius of the PEG is only around 10 Å the size of the holes should be much larger in order to function as an effective diffusion mechanism, but could still be much smaller than the lower limit of detection for the light microscope. Since also PEG can diffuse over the membrane it is difficult to determine the diffusion mechanism. As discussed above the other mechanism besides diffusion through membrane defects is the solubility mechanism. 3.2. Addition of salt Salt is added to vesicle formulations as a viscosity reducer. It is added during the cooling process when vesicles are formed and acts to create a concentration gradient of CaCl2 between inside and outside of the vesicles. By the osmotic pressure created over the membrane, water from inside vesicles is transported to the bulk to reduce the concentration gradient and vesicles are thereby decreasing in size. This is showed in Fig. 6 when the fraction of trapped water decreases as the salt concentration is increased. Taken together with the results from the PEG measurements we believe that if the membranes were full of defects the addition of salt would not display the large effects as seen from the echo-decays in Fig. 6. Ions cannot diffuse through the membrane by the solubility mechanism since solubility of ions in an alkane is very poor. The
C. Groth et al. / Colloids and Surfaces A: Physicochem. Eng. Aspects 228 (2003) 64–73
Fig. 6. Water echo-decays for dialkyl quat at 10 wt.% with different concentrations of salt.
poor solubility gives the system enough time to shrink the vesicles due to the large difference in osmotic pressure created over the vesicle membrane. Hence, water will diffuse out creating vesicles with much smaller size. 3.3. Phase separated samples Even though vesicle solutions were remarkably stable it was noted that after approximately 1 year some of the samples had phase separated into one lower and one upper phase. This was noted for both dialkyl and dialkylester quats. To establish that the dialkylester quat is chemically intact the two phases were freeze dried and analyzed by 1 H-NMR and compared with the original surfactant. This showed that the ester is very stable and no chemical degradation could be detected. The phase separation process can be explained in terms of differences in density between multilamellar and unilamellar vesicles. The density decrease as the number of bilayers increase, therefore, multi-layered vesicles will be predominately found in the upper phase and less multi-layered and unilamellar vesicles in the lower phase. Both phases were studied by diffusion NMR at two different ∆, 0.1 and 1 s and the results are shown in Fig. 7. Showed as an insert in the upper right corner of Fig. 7 are the echo-decays for the two phases at ∆ = 0.1 s where the diffusion results from the upper phase displays a much higher apparent volume fraction of trapped vesicle water,
71
Fig. 7. Water echo-decay for a fresh 10 wt.% dialkylester quat sample (open squares) and the echo-decay obtained for the same sample 1 year later (open triangels), which then has phase separated. Both echo-decays where obtained with ∆=0.1 s. The echo-decay displayed for the phase-separated sample was the calculated result from the individual echo-decays obtained from the upper and lower phase after weighing the individual results by their respective volume fractions, 15 and 85%. The individual echo-decays at ∆ = 0.1 s for the upper and lower phases are shown as an insert. app
app
Pvw = 0.4, than the lower phase, Pvw = 0.05. It is app also interesting to note that Pvw for both the upper and lower phase is not changing when ∆ is increased. app For the lower phase Pvw = 0.05 at both ∆, indicating that most water has been exchanged during ∆. For unilamellar vesicles, i.e. in the lower phase sample, the exchange is fast and during 0.1 s almost all water molecules in the vesicles have time to diffuse over the membrane so that most of the water appears as if it is free. In order for chemical exchange between the inside and outside of vesicles water inside vesicles located in the upper phase needs to be transported through many bilayers and, therefore, the exchange between inside and outside is much slower. The results show that practically all water in the lower phase is exchanged already after 0.1 s while in the upper phase almost no water is exchanged during 1 s. By adding the weighted echo-decay for the two phases, i.e. 0.15 for upper phase and 0.85 for the lower phase (from the measured volume fraction of the two phases in the phase separated sample) and comparing this weighted echo-decay with the echo-decay for the same sample before storage it is noted that they are practically identical (Fig. 7). This indicates that the vesicles are
72
C. Groth et al. / Colloids and Surfaces A: Physicochem. Eng. Aspects 228 (2003) 64–73
stable for a long time and it is also an indication that the size is not changing over 1 year. Further it gives an explanation for the rather complex echo-decay obtained from the vesicle solutions in this work. Water trapped in multilamellar vesicles need much more time to escape the inside. Depending on the amount of multilamellar vesicles the echo-decay displays a varying amount of water trapped in vesicles, i.e. it is to a very large extent the fraction multilamellar vesicles that determine the curvature of the echo-decay. If multilamellar vesicles are dominating the total number of vesicles the echo-decay will result in a large curvature when the logarithm of the water signal is plotted versus k, as it is done in all echo-decay plots in this work.
4. Conclusions The NMR self-diffusion technique has been shown to be a powerful technique for characterizing the self-diffusion process of water in crowded multilamellar vesicle solutions. By changing the experimental diffusion time, ∆, water diffusion can be monitored over almost three orders of magnitude with respect to time. It was found that in order to measure that, actual amount of water inside vesicles the echo-decay is simply fitted to a multi exponential decay in which the fastest component is extracted. The value of this diffusion coefficient must be verified to represent approximately the diffusion constant that is to be expected from solvent diffusion in a crowed solution of spherical objects. The fraction belonging to this diffusion constant then represents the fraction of water outside the vesicles. In order to find the true value for this fraction it must first be realized that the value is strongly dependent on the experimental diffusion time. For a reliable value it is noted and highly recommended that the time dependence of this fraction should to be carefully measured. Even though the NMR self-diffusion method at present can measure at very short diffusion times (10 ms in this work) it is noted that in order to find the fraction not modulated by exchange, i.e. in order for the term “apparent fraction trapped water” to reduce to “trapped fraction water”, the results must to be extrapolated to ∆ = 0 since during the first few milliseconds a large fraction of the water inside has already exchanged with water outside.
When applied to the two different vesicle-forming surfactants in this work the NMR self-diffusion technique reveals differences in membrane permeability. It is noted that when the membrane contains a surfactant with an ester function in the dialkyl chains water diffuse much faster than if the ester function is removed. This is well represented in a figure displaying how the apparent fraction trapped water is represented versus the experimental diffusion time, ∆. Since this work has focused on the development of the NMR method as such for measuring diffusion of water in multilamellar vesicle solutions we note that there are a number of unanswered questions regarding the differences in water diffusion between the two surfactants used in this work. In work to come we will investigate the effect of saturation/unsaturations of the alkyl chain. We also aim to correlate results from DSC measurements of the transition temperature to NMR self-diffusion data.
Acknowledgements We would like to thank Akzo Nobel Surface Chemistry for financially support and the Swedish NMR Center for spectrometer time.
References [1] P.E. Hellberg, K. Bergström, K. Holmberg, J. Surf. Deterg. 3 (2000) 81. [2] B.H. Robinsson, M. Rogerson, in: K. Holmberg (Ed.), Handbook of Applied Surface and Colloid Chemistry, vol. 2, Wiley, Chichester, 2002. [3] A. Carruthers, D.L. Melchior, Biochemistry 22 (1983) 5797. [4] J.d. Gier, Chem. Phys. Lipids 64 (1993) 187. [5] M. Jansen, A. Blume, Biophys. J. 68 (1995) 997. [6] R. Lawaczeck, Berichte., Ber. Bunsenges. Phys. Chem. -Chem. Phys. 92 (1988) 961. [7] S. Paula, A.G. Volkov, A.N. VanHoek, T.H. Haines, D.W. Deamer, Biophys. J. 70 (1996) 339. [8] P.W. Küchel, A. Coy, P. Stilbs, Magn. Reson. Med. 36 (1997) 637. [9] W.S. Price, Concepts Magn. Reson. 9 (1997) 299. [10] E.F. Marques, O. Regev, A. Khan, M.D. Miguel, B. Lindman, J. Phys. Chem. B 102 (1998) 6746. [11] E.F. Marques, O. Regev, A. Khan, M.D. Miguel, B. Lindman, J. Phys. Chem. B 103 (1999) 8353. [12] E.F. Marques, Langmuir 16 (2000) 4798.
C. Groth et al. / Colloids and Surfaces A: Physicochem. Eng. Aspects 228 (2003) 64–73 [13] E.F. Marques, Association and Microstructurembling Catanionic Systems, Lund University, 2002. [14] U. Olsson, K. Nakamura, H. Kunieda, R. Strey, Langmuir 12 (1996) 3045–3054. [15] J. Bell, Preparation of Dispersions for Testing as Fabric
73
Softeners, Akzo Nobel Chemicals, Surfactant America (USA), 1999. [16] D.D. Lasic, Liposomes: from Physics to Applications, Elsevier, Amsterdam, 1993. [17] P. Schatzberg, J. Polym. Sci. 10 (1965) 87.
Diffusion of water in multilamellar vesicles of dialkyl and dialkyl ester ammonium surfactants Cecilia Groth, Johanna Bender, Magnus Nydén∗ Materials and Surface Chemistry, Chalmers University of Technology, S-412 96 Gothenburg, Sweden
Abstract The NMR self-diffusion technique is used for measuring diffusion of water in highly concentrated multilamellar vesicle solutions. The signal intensity of water, i.e. the water echo-decay, is monitored down to very small intensities thus providing an accurate measure of how water is diffusing in the solution. It is noted that a large curvature is dominating the functional form of the echo-decay indicating the presence of a large number of multilamellar vesicles. It is also concluded that in order to measure accurately the volume fraction of water inside and outside vesicles the experimental time scale can be changed. From a multiexponential fit to the echo-decay the fraction fast and the sum of all slow components can be extracted. When the apparent app fraction “vesicle water”, Pvw , is plotted versus the experimental time scale the graph produced is a good representation of the difference in how fast water diffuses over the different vesicle membranes. From an extrapolation to “zero time” the true fraction of trapped water can be extracted, i.e. a quantitative measure of the volume fraction of vesicles at a certain concentration of surfactant. © 2003 Elsevier B.V. All rights reserved. Keywords: Multilamellar vesicles; NMR; Surfactants
1. Introduction Cationic surfactants are used in a wide range of applications as different kind of surface treaters as fabric softeners or personal care products or as cleaner for hard surfaces and paper processing. The largest use for cationic surfactants is in the range of fabric softeners and due to efforts to make the surfactants more biocompatible the active component in softeners have changed from ordinary di(hydrogenated tallow)alkyldimethylammonium (DHTMAC) quar∗ Corresponding author. Tel.: +46-31-772-2973; fax: +46-31-160-062. E-mail address: [email protected] (M. Nyd´en).
ternary (dialkyl quats) to ester quaternaries (dialkylester) that are more easily biodegradable [1]. Softeners are thought to act on the surface of the fabric by neutralizing the negatively charged surface. To maximize the adsorption of ester quats a double tailed surfactant has proven to be most efficient. This results in a very low solubility of monomeric surfactant in water and a subsequent formation of vesicles in concentrations ranging from the dilute region up to approximately 25 wt.% surfactant. It is believed that at low concentrations vesicles are mostly unilamellar but at higher concentrations more multilamellar vesicles are formed. The size and size distribution of vesicles depends on the nature of the surfactant and properties like salt concentrations, temperature etc. but it may
0927-7757/$ – see front matter © 2003 Elsevier B.V. All rights reserved. doi:10.1016/S0927-7757(03)00307-8
C. Groth et al. / Colloids and Surfaces A: Physicochem. Eng. Aspects 228 (2003) 64–73
also depend on how they are prepared. For example, if a simple stirring process is employed, the mean size depends strongly on the speed of stirring, resulting in smaller size as the stirring rate is increased. The membrane thickness is more or less constant around 4 nm but depending on the concentration and/or means of preparation each vesicle can be built up by one bilayer up by hundreds of bilayers. Vesicles can be divided into different groups depending on their size and structure; small uni-lamellar vesicles (SUV) are 10–50 nm, large uni-lamellar vesicles (LUV) between 50 and 500 nm, giant uni-lamellar vesicles larger than 0.5 m, or multi-lamellar large vesicles (MLV), 1–50 m. As mentioned above the size and size distribution can to a large extent be controlled. Furthermore, since the surfactants normally used for making vesicles often are double tailed surfactants with a very low solubility in water, making a vesicle a rather static entity (as compared with for example a micelle in which the exchange dynamics between surfactants in the micelle and monomeric surfactants most often is very fast). In addition, since the inner part of a vesicle is hydrophilic and the membrane is hydrophobic, they are suitable for example as carriers for small molecules or as model systems for cell membranes, as templates for reactors or as surfactant delivery systems (softeners) [2]. One very important property of a vesicle solution is the restriction in diffusion of hydrophilic substances due to the presence of the vesicle wall. For vesicles with more or less perfect membranes, i.e. without large holes or dislocations, the transport of a hydrophilic substance through the membrane is given by the difference in concentration between the water phase and the membrane. If, however, the membrane contains holes or dislocations, diffusion through these might be the rate determining step in the diffusion process that occurs through the membrane of a vesicle [3–7]. PGSE NMR is a powerful technique for physicochemical investigations in complex mixtures. The technique can be used in systems with restricted diffusion in for example cells [8] or in general to interpret restricted diffusion in various geometries [9]. Marques et al. studied water diffusion in unilamellar catanionic vesicles by PFG-SE NMR [10–13]. Water diffusion and exchange in normal and reversed unilamellar vesicles has also been investigated by Olsson et al. where it was noted that the lifetime of a solvent
65
molecule inside vesicles varied with the composition of molecules in the bilayer [14]. In this paper we are investigating the difference in membrane permeability between one dialkyl quat and one dialkylester quat by measuring the self-diffusion of water molecules by PGSE NMR. By comparing the decrease in fraction of “trapped” water obtained after different diffusion times, conclusions about the effective membrane permeability are drawn. By increasing the diffusion time more water molecules will have exchanged between the inside and outside of a vesicle. The transport process of a much larger hydrophilic molecule polyethylene glycol (PEG) is also measured with the aim to understand the mechanism of diffusion of water and other molecules in a concentrated solution of uni- and multi-lamellar vesicles formed by cationic vesicles.
2. Experimental 2.1. Materials Arquad HC and Armosoft DEQ were a gift from Akzo Nobel Surface Chemistry, USA and used without further purification. Arquad HC is a di(hydrogenated tallow)dimethylammonium chloride (DHTDMAC) with alkyl chain lengths between 16 and 18 having a maximum amount of 2-propanol (IPA) of 1% and an active matter of 96%. Armosoft DEQ is the corresponding dialkylester quat to Arquad HC with a maximum amount of ethanol of 10–20% and hence no further additions of alcohol were made. Ethanol (99.5%, spectrographically pure) was bought from Kemetyl, calcium chloride (technical, 40 mesh) from Aldrich and PEG (molecular weight 950–1050 g mol−1 ) from Merck. All chemicals were used without further purification. Milli-Q water was used for all sample preparations. 2.2. Sample preparation Samples were prepared by a standard method according to Bell [15], utilizing a glass beaker with a Teflon propeller stirrer with a diameter of 40 mm connected to a Heidolph RZR 2050 motor unit. Surfactant and alcohol are weighted in a screw capped tube and mixed in a heated water bath. Milli-Q
66
C. Groth et al. / Colloids and Surfaces A: Physicochem. Eng. Aspects 228 (2003) 64–73
water was added into a flask, (in the case of salt addition some water was used for dissolving the CaCl2 ) and heated to 64–66 ◦ C and equilibrated for 5 min at the lowest stirrer speed. The speed was increased to 1000 rpm before starting the addition of the melted surfactant–alcohol mixture. The mixture was stirred for 15 min before the cooling process started. After the temperature had reached 40 ◦ C the dissolved CaCl2 was added in small portions with 20 s between each portion. The solution was stirred for another 15 min after the addition. When preparing highly concentrated vesicle solutions and if the viscosity became too high during the addition of surfactant, 1/5 of the salt solution is added to the dispersion. After approximately 20 s more of the surfactant mixture or salt solution is added depending on the viscosity. This was repeated until all surfactant solution was added. If some salt solution was left this was added during the cooling process (see above). Samples were equilibrated on an automatic seesaw 2–3 days before they were studied with light microscopy to investigate size and structure of vesicles, centrifuged and transferred to 5 mm NMR-tubes for NMR-diffusion studies. 2.3. Methods 2.3.1. Light microscopy Samples were observed by optical light microscopy (Olympus BH-2) in normal (size and shape) and polarized light. For large vesicles the typical Malteser cross pattern shown in liquid crystalline vesicles were observed. 2.3.2. NMR experiments NMR pulsed gradient spin-echo (PGSE) selfdiffusion experiments where performed on 500 MHz Varian Unity Inova spectrometer equipped with a diffusion probe by Doty Sci., Inc. All diffusion measurements were performed at 20 ◦ C. Approximately ten logarithmically spaced diffusion times (∆) between 10 ms up to 10 s were used. The pulsed field gradient strength was varied between 0.003 to 3.52 T m−1 . The basic Hahn-echo sequence was used for between 10 to 160 ms and above that the stimulated echo-sequence was used due to reasons of echo instability at long diffusion times when using a Hahn echo.
2.3.3. NMR self-diffusion in vesicles With the NMR self-diffusion technique it is possible to measure component resolved diffusion. The experiment results in a so-called echo-decay in which the logarithm of the NMR signal of interest normally is plotted versus the gradient strength squared or against k, an experimental factor containing the relevant tunable parameters. For free diffusion and when represented in this manner the echo-decay results in a straight line with the slope given by the self-diffusion constant. In many cases the diffusion is not free, for example due to the presence of barriers, which is often seen as a curved echo-decay in which (somewhat simplified) a large slope represents molecules that during the diffusion time (typically between 10 ms and 10 s) have moved a large distance. Consequently, a small slope represents molecules diffusing only a short distance during the same diffusion time. For a two-compartment system with different diffusion constants in the two compartments and no chemical exchange, the echo-decay is simply given by the sum of the individual echo-decays. If, on the other, molecules are allowed to exchange, the results strongly depend on the rate of exchange compared with the experimentally determined diffusion time. If the lifetime is much smaller than the diffusion time the echo-decay is a straight line where the slope is given by the weighted sum of the two diffusion constants such that the observed diffusion constant Dobs = p1 D1 + p2 D2 . If the lifetime of a molecule in a particular site is close to the experimental diffusion time the echo-decay may be quite a complicated function and for multi-compartment systems it can be quite impossible to extract quantitative data about lifetimes and diffusion constants. In this paper we have chosen to fit the echo-decay (which indeed is very complicated for all experiments in this work) to a sum of four exponents according to Eq. (1). The rationale for doing so is that other methods of evaluation have failed in describing the full echo-decay for all diffusion times. This should be no surprise when considering the complexity of this particular system; multi-lamellar vesicles with a distribution both in size and in number of lamellas and, in addition, chemical exchange between the inside and outside occurring on a intermediate time-scale. Regardless of the complexity displayed by the system and the value of ∆ we have noted that the initial slope
C. Groth et al. / Colloids and Surfaces A: Physicochem. Eng. Aspects 228 (2003) 64–73
of the echo-decay represents the diffusion of water outside vesicles; i.e. it is possible to estimate the apparent fraction of water trapped inside vesicles after a certain diffusion time, ∆. From here on in this app work this water is termed Pvw , and is obtained from app Pvw = (1−pfree ) where pfree is the fraction belonging to the largest diffusion coefficient in Eq. (1), thus representing the fraction water outside the vesicles. There the diffusion path of water is slowed down due to collisions with vesicles but not restricted to diffuse a certain distance during the diffusion time as is the case for water “trapped” inside. I/I0 = p1 e−kD1 +p2 e−kD2 + p3 e−kD3 + p4 e−kD4 (1) In Eq. (1), I is the intensity of the water peak, I0 the intensity at zero gradient strength, Dn the diffusion coefficient and pn the volume fraction of n. The echo-decay for a polydisperse vesicle sample can roughly be divided into two parts, where the slope at low k-values gives the diffusion coefficient for “free” water. At higher k-values the echo-decay reflects the restriction in diffusion for water inside the vesicles due to the presence of the membrane. It is worth noting that a larger degree of polydispersity (both in size and in number of membranes making up the full vesicle) gives a larger curvature in the echo-decay.
3. Results and discussion The structure of the formulated vesicle solutions was investigated by light-microscopy. When using a
67
polarized light source, all solutions displayed the characteristic Maltese cross patterns due to their lamellar structure in the membranes. All solutions also showed a large degree of polydispersity. Vesicles around 5 m are most frequent but also a few large vesicles between 20 and 30 m where detected. In addition, many small vesicles could be indirectly detected as small and rapidly diffusing entities, the size was, however, difficult to estimate due to the limited resolution. It is important to emphasize that no visible differences in vesicle size between different types of surfactants or at different concentrations could be detected (Fig. 1). In Fig. 2, echo-decays obtained at four logarithmically spaced ∆ are shown for the dialkyl quat and the dialkylester quat, both at 10 wt.% surfactant. All echo-decays are well described by Eq. (1). The diffusion coefficient obtained from the initial slope is around 1–2 × 10−9 m2 s−1 . In order to describe the full echo-decay, particular for those obtained at very long ∆, apparent diffusion constants on the order of 10−14 m2 s−1 are obtained, indicating the presence of very restricted water molecules with a very short rms-displacement. As mentioned above the justification for using Eq. (1) is not based on physical relevance of the equation but should rather be seen as a robust fitting routine with regards to a perfect description of the rather complex echo-decay at all diffusion times with the aim to extract a correct value for the fraction trapped water as a function of diffusion time, ∆. If no or only little exchange is occurring during a diffusion time of 10 ms (the shortest diffusion time used) a water molecule outside a vesicle has diffused
Fig. 1. Light microscopy images of a 10 wt.% dialkyl quat solution showing large differences in vesicle sizes within the sample. The image to the left shows a vesicle with a diameter of 35 m and to the right smaller vesicles around 3–10 m in diameter is shown.
68
C. Groth et al. / Colloids and Surfaces A: Physicochem. Eng. Aspects 228 (2003) 64–73
Fig. 2. Water echo-decays in a 10 wt.% dialkyl quat (a) and in (b) a 10 wt.% dialkylester quat solutions at four different diffusion times. Experimental settings where ␦ = 4 ms, g was linearly increased from zero to 3.5 T m−1 .
on average 6 m but a trapped molecule can only move within the boundaries of the vesicle. Thus, even though it is a very crowded surrounding for that type of water it will still diffuse quite a large distance compared with water inside vesicles as the diffusion time is increased. Therefore, at longer diffusion times, by allowing a molecule to exchange between the outside and inside during ∆, the time spent outside will clearly have the largest influence with respect to the total diffused distance. By increasing the diffusion time the fraction of water that had the chance to diffuse over the membrane is increased and the obtained amount of apparently trapped water will decrease. This is also what is seen in Fig. 2 for both the dialkyl quat and the
dialkylester quat. However, the two surfactants show app markedly different results with regards to Pvw . For app dialkyl quat at ∆ = 0.32 s, Pvw is around 0.05 and for the dialkylester quat it is around 0.001. This indicates that water passes the dialkylester quat membrane faster than the dialkyl quat membrane. At this stage it should, however, be realized that when comparing app Pvw for different surfactant vesicles, this property is app strongly dependent on the vesicle radius. Since Pvw depends on the volume of water inside compared with outside a small difference in size will have a large app impact on Pvw . In Fig. 3(a) and (b) echo-decays for = 10 ms and 5 s are shown together with the fits of Eq. (1).
Fig. 3. Water echo-decays for a 10 wt.% dialkyl quat sample. In figure (a) ∆ = 10 ms and in (b) ∆ = 5 s. Shown also as full lines are the best fit of Eq. (1). The obtained diffusion coefficients from the initial slope (i.e. the fastest obtained diffusion constant from Eq. (1)) is D = 1.82 × 10−9 (±1.4 × 10−10 ) for ∆ = 10 ms and D = 5.86 × 10−10 (± 7.6 × 10−12 ) m2 s−1 for ∆ = 5 s. The fraction of free diffusing water is Pfw = 0.64 (± 0.04) for ∆ = 10 ms and Pfw = 0.95 (± 0.003) for ∆=5 s.
C. Groth et al. / Colloids and Surfaces A: Physicochem. Eng. Aspects 228 (2003) 64–73
69
app
Fig. 4. Pvw vs. ∆ at four different concentrations, 7, 10, 15 and 20% with dialkyl quat (a) and 10, 15, 20 and 25% surfactant by weight with dialkylester quat (b). The full lines are the best fits of a simple exponential function that is regarded to describe the behavior reasonably well both for the dialkyl and diester quat.
In addition, it can be seen that a very good fit is obtained and the residuals are small and random. It was, however, noted that for some ∆, in particular for long ∆, residuals are not random indicating that in order to properly describe the full echo-decay at least four exponents have to be used. In order to test the validity of using Eq. (1) when evaluating the fraction of water in different domains we compare the obtained echo-decays at different ∆ between dialkyl and dialkylester quat. It is noted that for the dialkylester quat the echo-decay drops of to much lower signal intensities, indicating that water is diffusing more rapidly through the membrane in that system. app In Fig. 4 the fitted Pvw is displayed versus ∆, for dialkyl (Fig. 4(a)) and dialkylester quats (Fig. 4(b)) at four different concentrations. Also from this representation it is suggested that the dialkylester quat allows for faster water diffusion over the membrane as can app be seen as a larger change in Pvw as a function of than for the dialkyl quat. In addition, when comparing the different concentrations of surfactants we note that a reasonably accurate “zero-time” value may be obtained. This is a way to obtain the true volume fraction of trapped water in the vesicle since when extrapapp olating Pvw to zero time, the fraction water obtained is not modulated by chemical exchange between the outer and inner part of the vesicle. The fact that the two surfactants display a large difference in water permeability can be an effect of a larger disorder in ester membranes because of the ester group and/or a higher degree of un-saturation for
the alkyl chains in the dialkylester quat than for the normal dialkyl quat. It can also be due to differences in membrane defects, or, as previously mentioned, differences in size. With regards to membrane transport, two different possible diffusion mechanisms are discussed in the literature—by diffusion through the intact membrane (solubility mechanism) or through pores/defects in the membrane (pore mechanism). For diffusion through an intact membrane the hydrophobic part of the bilayer is referred to as an oil layer in which water is sparingly soluble. Permeating molecules must dissolve in the hydrophobic region, diffuse across and re-dissolve into the water phase on the other side of the membrane [4,5,7,16]. The partition coefficient of water in long chain hydrocarbons as n-hexadecan is Kw = 0.42 × 10−8 g water dm−3 alkane [17]. The diffusion coefficient, D, can be expressed as a function of the flux, J, and a concentration gradient across the membrane ∂C/∂X by Fick’s first law: ∂C J = −D (2) ∂X By integrating and introducing the permeability coefficient P, the partition coefficient K, and δ as the thickness of the membrane Eq. (2) yields: KD J P = = (3) δ ∆C Dw · KW · VW P = (4) d · Vsurfactant
70
C. Groth et al. / Colloids and Surfaces A: Physicochem. Eng. Aspects 228 (2003) 64–73
with d, thickness of the bilayer; Dw , diffusion coefficient of H2 O in the bilayer; Kw , partition coefficient of H2 O in the bilayer; VW , molar volume of H2 O and Vsurfactant , molar volume of surfactant. For a given thickness of the membrane, P is directly proportional to Dw Kw and by further assuming that water has the same diffusion coefficient in all liquid−crystalline bilayers the permeability is only dependent on changes in partition coefficient Kw or the solubility of water in the hydrophobic part of the membrane. The second model is the pore-mechanism [3,5–7] where molecules passes the membrane through defects (holes) produced by fluctuations within the membrane. 3.1. Addition of PEG In an attempt to separate the two different mechanisms by which water can diffuse over the membrane a large, hydrophilic molecule, PEG, Mw = 1000 g mol−1 , was added to the water phase before preparing the vesicle solution. If PEG is not allowed to diffuse over the membrane the presence of a significant amount of membrane defects can be neglected. Regarding the choice of the molecular weight we note that the polymer should have a rather fast diffusion so that the frequency by which it collides with the membrane is large enough. Thus the choice of 1000 in Mw is a polymer that has diffusion fast enough so that the entire volume of the membrane will, during the time scale of the experiment, be experienced by the polymer. Also it is much larger than water. Similarly to the case of water diffusion the echo-decays obtained at different ∆ where compared. The fraction, as obtained from a fit of Eq. (1) to the echo-decay, was of particular interest. In Fig. 5 the echo-decays for PEG added to formulations of both dialkyl and dialkylester quat are shown and displays a decreasing signal intensity at large k-values as ∆ is increased, indicating that also PEG diffuses over the membrane. By comparing the apparent trapped fraction obtained for PEG with the value obtained for water a qualitative difference between the permeability for PEG and water can be app estimated. For dialkyl quats, Pv PEG is 0.51 at ∆ = 0.1 app and Pvw for water 0.35. The higher values for PEG than water shows that water is exchanged faster than PEG during ∆. This is also shown for systems containing dialkylester quats. At this stage it should be noted that we have not observed any significant membrane
Fig. 5. PEG echo-decays obtained at 1 wt.% PEG and 10 wt.% surfactant at ∆ = 0.1 and 0.32 s for both dialkyl and dialkylester quat.
defects from microscopy images, vesicles there (small or large) appear to be rather intact. Considering that the hydrodynamic radius of the PEG is only around 10 Å the size of the holes should be much larger in order to function as an effective diffusion mechanism, but could still be much smaller than the lower limit of detection for the light microscope. Since also PEG can diffuse over the membrane it is difficult to determine the diffusion mechanism. As discussed above the other mechanism besides diffusion through membrane defects is the solubility mechanism. 3.2. Addition of salt Salt is added to vesicle formulations as a viscosity reducer. It is added during the cooling process when vesicles are formed and acts to create a concentration gradient of CaCl2 between inside and outside of the vesicles. By the osmotic pressure created over the membrane, water from inside vesicles is transported to the bulk to reduce the concentration gradient and vesicles are thereby decreasing in size. This is showed in Fig. 6 when the fraction of trapped water decreases as the salt concentration is increased. Taken together with the results from the PEG measurements we believe that if the membranes were full of defects the addition of salt would not display the large effects as seen from the echo-decays in Fig. 6. Ions cannot diffuse through the membrane by the solubility mechanism since solubility of ions in an alkane is very poor. The
C. Groth et al. / Colloids and Surfaces A: Physicochem. Eng. Aspects 228 (2003) 64–73
Fig. 6. Water echo-decays for dialkyl quat at 10 wt.% with different concentrations of salt.
poor solubility gives the system enough time to shrink the vesicles due to the large difference in osmotic pressure created over the vesicle membrane. Hence, water will diffuse out creating vesicles with much smaller size. 3.3. Phase separated samples Even though vesicle solutions were remarkably stable it was noted that after approximately 1 year some of the samples had phase separated into one lower and one upper phase. This was noted for both dialkyl and dialkylester quats. To establish that the dialkylester quat is chemically intact the two phases were freeze dried and analyzed by 1 H-NMR and compared with the original surfactant. This showed that the ester is very stable and no chemical degradation could be detected. The phase separation process can be explained in terms of differences in density between multilamellar and unilamellar vesicles. The density decrease as the number of bilayers increase, therefore, multi-layered vesicles will be predominately found in the upper phase and less multi-layered and unilamellar vesicles in the lower phase. Both phases were studied by diffusion NMR at two different ∆, 0.1 and 1 s and the results are shown in Fig. 7. Showed as an insert in the upper right corner of Fig. 7 are the echo-decays for the two phases at ∆ = 0.1 s where the diffusion results from the upper phase displays a much higher apparent volume fraction of trapped vesicle water,
71
Fig. 7. Water echo-decay for a fresh 10 wt.% dialkylester quat sample (open squares) and the echo-decay obtained for the same sample 1 year later (open triangels), which then has phase separated. Both echo-decays where obtained with ∆=0.1 s. The echo-decay displayed for the phase-separated sample was the calculated result from the individual echo-decays obtained from the upper and lower phase after weighing the individual results by their respective volume fractions, 15 and 85%. The individual echo-decays at ∆ = 0.1 s for the upper and lower phases are shown as an insert. app
app
Pvw = 0.4, than the lower phase, Pvw = 0.05. It is app also interesting to note that Pvw for both the upper and lower phase is not changing when ∆ is increased. app For the lower phase Pvw = 0.05 at both ∆, indicating that most water has been exchanged during ∆. For unilamellar vesicles, i.e. in the lower phase sample, the exchange is fast and during 0.1 s almost all water molecules in the vesicles have time to diffuse over the membrane so that most of the water appears as if it is free. In order for chemical exchange between the inside and outside of vesicles water inside vesicles located in the upper phase needs to be transported through many bilayers and, therefore, the exchange between inside and outside is much slower. The results show that practically all water in the lower phase is exchanged already after 0.1 s while in the upper phase almost no water is exchanged during 1 s. By adding the weighted echo-decay for the two phases, i.e. 0.15 for upper phase and 0.85 for the lower phase (from the measured volume fraction of the two phases in the phase separated sample) and comparing this weighted echo-decay with the echo-decay for the same sample before storage it is noted that they are practically identical (Fig. 7). This indicates that the vesicles are
72
C. Groth et al. / Colloids and Surfaces A: Physicochem. Eng. Aspects 228 (2003) 64–73
stable for a long time and it is also an indication that the size is not changing over 1 year. Further it gives an explanation for the rather complex echo-decay obtained from the vesicle solutions in this work. Water trapped in multilamellar vesicles need much more time to escape the inside. Depending on the amount of multilamellar vesicles the echo-decay displays a varying amount of water trapped in vesicles, i.e. it is to a very large extent the fraction multilamellar vesicles that determine the curvature of the echo-decay. If multilamellar vesicles are dominating the total number of vesicles the echo-decay will result in a large curvature when the logarithm of the water signal is plotted versus k, as it is done in all echo-decay plots in this work.
4. Conclusions The NMR self-diffusion technique has been shown to be a powerful technique for characterizing the self-diffusion process of water in crowded multilamellar vesicle solutions. By changing the experimental diffusion time, ∆, water diffusion can be monitored over almost three orders of magnitude with respect to time. It was found that in order to measure that, actual amount of water inside vesicles the echo-decay is simply fitted to a multi exponential decay in which the fastest component is extracted. The value of this diffusion coefficient must be verified to represent approximately the diffusion constant that is to be expected from solvent diffusion in a crowed solution of spherical objects. The fraction belonging to this diffusion constant then represents the fraction of water outside the vesicles. In order to find the true value for this fraction it must first be realized that the value is strongly dependent on the experimental diffusion time. For a reliable value it is noted and highly recommended that the time dependence of this fraction should to be carefully measured. Even though the NMR self-diffusion method at present can measure at very short diffusion times (10 ms in this work) it is noted that in order to find the fraction not modulated by exchange, i.e. in order for the term “apparent fraction trapped water” to reduce to “trapped fraction water”, the results must to be extrapolated to ∆ = 0 since during the first few milliseconds a large fraction of the water inside has already exchanged with water outside.
When applied to the two different vesicle-forming surfactants in this work the NMR self-diffusion technique reveals differences in membrane permeability. It is noted that when the membrane contains a surfactant with an ester function in the dialkyl chains water diffuse much faster than if the ester function is removed. This is well represented in a figure displaying how the apparent fraction trapped water is represented versus the experimental diffusion time, ∆. Since this work has focused on the development of the NMR method as such for measuring diffusion of water in multilamellar vesicle solutions we note that there are a number of unanswered questions regarding the differences in water diffusion between the two surfactants used in this work. In work to come we will investigate the effect of saturation/unsaturations of the alkyl chain. We also aim to correlate results from DSC measurements of the transition temperature to NMR self-diffusion data.
Acknowledgements We would like to thank Akzo Nobel Surface Chemistry for financially support and the Swedish NMR Center for spectrometer time.
References [1] P.E. Hellberg, K. Bergström, K. Holmberg, J. Surf. Deterg. 3 (2000) 81. [2] B.H. Robinsson, M. Rogerson, in: K. Holmberg (Ed.), Handbook of Applied Surface and Colloid Chemistry, vol. 2, Wiley, Chichester, 2002. [3] A. Carruthers, D.L. Melchior, Biochemistry 22 (1983) 5797. [4] J.d. Gier, Chem. Phys. Lipids 64 (1993) 187. [5] M. Jansen, A. Blume, Biophys. J. 68 (1995) 997. [6] R. Lawaczeck, Berichte., Ber. Bunsenges. Phys. Chem. -Chem. Phys. 92 (1988) 961. [7] S. Paula, A.G. Volkov, A.N. VanHoek, T.H. Haines, D.W. Deamer, Biophys. J. 70 (1996) 339. [8] P.W. Küchel, A. Coy, P. Stilbs, Magn. Reson. Med. 36 (1997) 637. [9] W.S. Price, Concepts Magn. Reson. 9 (1997) 299. [10] E.F. Marques, O. Regev, A. Khan, M.D. Miguel, B. Lindman, J. Phys. Chem. B 102 (1998) 6746. [11] E.F. Marques, O. Regev, A. Khan, M.D. Miguel, B. Lindman, J. Phys. Chem. B 103 (1999) 8353. [12] E.F. Marques, Langmuir 16 (2000) 4798.
C. Groth et al. / Colloids and Surfaces A: Physicochem. Eng. Aspects 228 (2003) 64–73 [13] E.F. Marques, Association and Microstructurembling Catanionic Systems, Lund University, 2002. [14] U. Olsson, K. Nakamura, H. Kunieda, R. Strey, Langmuir 12 (1996) 3045–3054. [15] J. Bell, Preparation of Dispersions for Testing as Fabric
73
Softeners, Akzo Nobel Chemicals, Surfactant America (USA), 1999. [16] D.D. Lasic, Liposomes: from Physics to Applications, Elsevier, Amsterdam, 1993. [17] P. Schatzberg, J. Polym. Sci. 10 (1965) 87.