Langmuir 1992,8, 397-402
397
Effects of Micellization and Surface Adsorption on 2HNMR 2'1 Relaxation Times of n-Alkylphosphocholine Surfactants? Sigrid C. Kuebler and Peter M. MacdonaW Department of Chemistry and Erindale College, University of Toronto, Toronto, Ontario, Canada M5S 1A2 Received June 26,1991. I n Final Form: October 25, 1991 The motions of the choline group of a series of n-alkylphosphocholine surfactants have been studied in the monomer, micellar, and surface-bound monolayer states by means of deuterium nuclear magnetic resonance (*H NMR) spectroscopy. For this purpose, the dodecyl-, tetradecyl-, and hexadecylphosphocholine (DDPC, TDPC, and HDPC) homologues were deuterated in the methyl groups of the choline moiety and their 2H NMR TI relaxation times were measured. The choline methyl group is only slightly hindered in its movement at surfactant concentrations below the critical micelle concentration (cmc),but becomes increasingly more restricted as the supramolecular organization advances from the monomer to the micellar and further to the surface-bound monolayer state. The cmc of DDPC was determined by exploiting the difference in the TI relaxation times of the monomer versus the micellar state. The motional correlation times and the associated activation energies of HDPC bound as a monolayer to the surface of polystyrene particles indicate that the choline methyls of HDPC experience a motional environment akin to that of a bilayer of phosphatidylcholine in the thermotropic gel state.
Introduction Surfactant adsorption at a solid/liquid interface is an important means of modifying surface properties such as charge, wettability, and biocompatibility. Amphoteric surfactants have been found to be especially useful in applications requiring biological contact. They contain both positive and negative charges in their hydrophilic polar head group regions, and in their general properties they behave like nonionic surfactants.' The amphoteric polar groups found in natural biological membranes are of particular interest because of their biomimetic propertiesa2 Recently, we synthesized and characterized the properties of a homologous series of amphoteric surfactants called n-alkylphosphocholines.3The polar head region of these surfactants consists of the phosphocholine group, and the hydrophobic region consists of an n-alkyl chain. Phosphocholine is present in biological membranes as the polar group of the commonly occurring membrane lipid phosphatidylcholine. The n-alkylphosphocholines undergo micellization at concentrations similar to those of other amphoteric surfactants of identical alkyl chain lengths. The C16-homologue hexadecylphosphocholine (HDPC) binds to the surface of polystyrene particles with high affinity, and a t saturation coverage appears to form a monolayer of approximately erect surfactant molecule^.^ Most recently, we have demonstrated that the deuterium nuclear magnetic resonance (2HNMR) spectrum of HDPC, deuterated in the methyls of the choline quaternary nitrogen (HDPC-y-dd and bound as a monolayer at the surface of polystyrene particles, can be employed to monitor particle surface charge.5 However, many of the important details of this response, including the dynamics ~ _ _ _ _
* To whom correspondence should be addressed. + Supported by grants from the National Science and Engineering Research Council (NSERC)of Canada and the Ontario Centre for Materials Research (OCMR). (1)Attwood, D.;Florence, A. T. Surfactant Systems; Chapman and Hall: London, 1983. (2)Hayward, J. A.; Chapman, D. Biomaterials 1984,5, 135. (3)Macdonald, P.M.;Rydall, J. R.; Kuebler, S. C.; Winnik, F. M. Langmuir 1991, 7 , 2602. (4) Macdonald, P. M.; Yue, Y.; Rydall, J. R. Langmuir, in press. ( 5 ) Yue, Y.; Rydall, J. R.; Macdonald, P. M. Langmuir, in press.
of the surface-bound phosphocholine surfactant, need to be determined. Nuclear magnetic resonance provides a useful probe of molecular dynamics through measurement of the spinlattice (2'1) and spin-spin (2'2) relaxation times. Deuterium NMR is particularly useful in this regard because relaxation is dominated by a single mechanism (quadrupolar relaxation) which allows a straightforward quantitative interpretation of the data in terms of an effective correlation time for molecular motion. We report here deuterium NMR spin-lattice (TI) relaxation time measurements of several n-alkylphosphocholines-y-ds in various of the physical states assumed by surfactants, including monomers free in aqueous solution, micellar aggregates in aqueous suspension, and monolayers adsorbed at the surface of polystyrene particles in isotropic aqueous solution. We demonstrate that the mobility of the phosphocholine head group of the surfactant becomes increasingly restricted as the supramolecular organization progresses from monomer to micelle to monolayer. Furthermore, we show that the 2H NMR TI relaxation time may be used to determine the critical micelle concentration (cmc), and that the motional correlation times obtained for phosphocholine surfactant adsorbed to polystyrene particles are consistent with a monolayer arrangement of the surfactant molecules.
Materials and Methods The syntheses of dodecylphosphocholine (DDPC), tetradecylphosphocholine (TDPC), and hexadecylphosphocholine (HDPC)have been described previ~usly.~ Deuteron labels were introduced into the methyl groups of the choline quaternary nitrogen by replacing methyl iodide with methyl-& iodide (Aldrich Chemicals, Milwaukee, WI) in the final reaction step, thereby yielding DDPC-r-de, TDPC-?-de, and HDPC-?-de. Homodisperse,emulsifier-free polystyrene latex was made according to the method of Kotera et al.6in a single stage process, using KzSzOeas the initiator. The styrenemonomer was vacuum distilledprior to use. After reaction the formed latexwas cleaned using 10cycles of centrifugationand redispersion (including sonication). The number-averageparticlediameter was determined to be 520 & 10 nm from scanning electron micrographs. The ~
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( 6 ) Kotera, A.; Furusawa, K.; Takeda, Y., Kolloid Z. Z. Polym. 1970, 239, 617.
0743-7463/92/2408-0397$03.00/00 1992 American Chemical Society
Kuebler and Macdonald
398 Langmuir, Vol. 8, No. 2, 1992
particle surface charge density was determined to be -2.2 0.1 pC/cm2 from conductometric titrations. Surfactant binding to the polystyrene particles was performed as follows. Aliquota of aqueoussolutionsof surfactant wereadded to a 16 w t % polystyrene latex at 23 "C(totalweight of latex was typically 0.7 g) such that the final surfactant concentration equalled 8.0 mM. During the binding period the surfactant/ polymer mixtures were periodically gently vortexed to maintain homogeneity. After equilibration for 4 days, the polystyrene particles were separated from the aqueous supernatant by centrifugation (7OOOg,10 min), the supernatant was removed, and the condensed latex was transferred to an NMR tube for measurement as described below. The concentration of polystyrene particles in the pellet fractions was equal to 42 w t %. 2H NMR spectra were obtained at 45.98 MHz on a Chemagnetics CMX300 NMR spectrometer. The quadrupole echo technique' was employedfor the polystyrene pellet using quadrature detection and complete cycling of the pulse pairs.* Particulars regarding the 90° pulse length (2.0 ps), the interpulse delay (40 ps), the recycle delay (100 ms), the spectral width (10 kHz), the data size (2k), and the number of acquisitions (16 OOO) are those noted in parentheses. For the surfactant monomer and micelle solutions, a single pulse excitation was used with parameters as given above except the recycle delay (5 s) and the number of acquisitions (400). For measurements of the TI relaxation time of surfactant monomersor micelles,the standard inversion recovery sequence shown below was employed. (180,-7-90,-acquire), In the case of surfactant adsorbed to polystyrene particles, an inversion pulse followed by the quadrupole echo sequence as shown below was employed. (180,-r90,-r1-90,-~,-acquire),
Results Figure 1compares the 2H NMR spectra of HDPC-r-d6 in different physical states. The uppermost spectrum (A) was obtained with HDpc-7-d~in aqueous solution at a concentration above ita cmc. The spectrum consists of two narrow resonance lines. The resonance at 0 Hz is attributed to the natural abundance of deuterium in water (HDO) since the spectrometer frequency was referenced to D2O. The resonance at -79 Hz is assigned to HDPCy d 6 . The narrow line width of these resonances, relative to the spectra of HDPC-?-de in other physical situations, is a consequence of the rapid isotropic tumbling motions of the molecules in aqueous solution. Despite the geometric size differences between monomers and micelles of HDPC, both are sufficiently small that their rates of rotational tumbling are fast enough to average to zero the quadrupolar interactions which would broaden the 2H NMR resonance lines in more motionally restricted environments. Consequently, there are no discernable differences between the 2H NMR spectra of HDPC-y-d, in the monomeric versus the micellar state. However, as will be shown below, the 2H NMR T I relaxation time is capable of differentiating between the monomericand the micellar states. The lower spectrum (B) in Figure 1 was obtained with H D p c - 7 - d ~adsorbed to the surface of polystyrene particles in aqueous dispersions. The spectrum consists of a superposition of two narrow resonances upon a broad, axially symmetric Pake pattern. At the HDPC concentration used here the surface of the polystyrene particles is saturated with bound surfactant, leaving a small excess in s ~ l u t i o n . This ~ excess "free" HDPC-7-d~remains trapped in the interstices between polymer particles and, (7) Davis, J.; Jeffrey, K. R.; Bloom, M.; Valic, M. I.; Higgs, T. P. Chem. Phys. Lett. 1976, 42, 390. (8) Griffin, R. G. Methods Enzymol. 1981, 72, 108.
KHz
I I I I ~ l l ' l ~ l l l ' ~ l l l l ~ l l l l ~ l l ' l ~ ' l l l ~ l , l l ~ l l l l ~ l l l l
2.5 2
1.5
1
0.5
0
-0.5
-1
-1.5
-2-2.5
Figure 1. 2HNMR spectra of HDPC-r-ds (A) in aqueoussolution at a concentration of 1 x 10-2 M, Le., above the cmc, and (B) adsorbed as a monolayer on the surface of polystyrene particles
in aqueous dispersion.
along with the natural abundance deuterium in water, gives rise to the two narrow resonances. The broad Pake pattern arises from "bound" H D p c - r - d ~ .This spectral line shape is characteristic of deuterons in a motionally restricted environment such as that of the closely packed HDPC surface monolayer. The quadrupole splitting in such a spectrum corresponds to the separation (Hz) between the two maxima in the Pake pattern, and in this instance equals 575 Hz. Since the size of the quadrupole splitting measured here is only a fraction of the static splitting expected for these types of deuterons (125 kHz), the HDpc-7-d~deuterons must be experiencing rapid but anisotropic motional averaging. It is likely that this is brought about by a combination of overall long-axis rotations of the entire surfactant molecule and individual bond rotations internal to the choline group. Figure 2A shows typical resulta obtained for DDPCY'd6 in solution using the inversion recovery sequence. Incrementing the delay r in the pulse sequencemodulates the intensity of the signal in a fashion which can be related to the TIof the particular resonance using eq 1,where I, In 11, - I,] = In 2
+ In [I,] - ?/TI
(1)
is the intensity at infinite delay r and I, is the intensity at any particular value of r. A plot of In [(IT- Im)/-2.1J versus 7 should be linear for a single effective relaxation time while the slope will equal -UTI. It is evident from Figure 2A, for example, that the resonance line from DDpc-7-d~returns to its equilibrium intensity with a faster rate constant than does the resonance line from HDO. Figure 2B shows typical results obtained using the inversion pulse-quadrupole echo combination to measure the T I relaxation time of HDPC-r-d, bound as a mono-
Effects of Micellization and Surface Adsorption
A
Langmuir, Vol. 8, No. 2,1992 399 10.
-4
I
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, I ,
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I
,
I
1
I
/ I , , , , I
I
0.3
0.2
0.1
0
0.5
0.4
TAU (sec)
Figure 3. Modulation of the spectral intensity with the value of T in the inversion recovery method. The intensity at a particular value of T , I,, is plotted according to a normalized version of eq 2 as a function of the value of T for the cases of DDPC-?-deat 2.76 X lW M in aqueous solution (squares),DDPC?-de at 8.72 X M in aqueous solution (triangles),and HDPCy-ds adsorbed as a monolayer on the surface of polystyrene particles in aqueous solution (circles). All measurements were at 25 O C . The solid lines were obtained by regression analysis, and the slope of any one line is proportional to -UTI.
B
KHz
Figure 2. Stack plots of the 2HNMR spectra of (A) DDPC-r-ds in aqueous solution at a concentration of 2.76 X M and (B) HDPC-?-deadsorbed as a monolayer on the surfaceof polystyrene particles in aqueous dispersion. The plots show the modulation of the spectral intensity as a function of the value of the delay T in the inversion recovery sequence used to measure the TI relaxation time. layer at the surface of polystyrene particles. I t is obvious that the bound HDPC-y-d6 has a very different rate of longitudinal relaxation than either the free HDPC-yd6 or the HDO. It is important to note that there appears to be no anistropy in the value of TIacross the broad Pake pattern of the bound HDpc-7-d~;Le., the Pake pattern is modulated in intensity but undergoes no distortion in line shape for different values of T. Plots of In [(IT- 14/-21m1versus T are shown in Figure 3 for various physical states assumed by the n-alkylphosphocholines. In each instance there was a linear relationship between the logarithm of the intensity and the length of the delay. Hence, in all cases the relaxation behavior of the deuterons is determined by a single effective relaxation time. The data plotted in the figure are for DDPC-yd6 at a concentration below its cmc, DDPC-yd6 a t a concentration above its cmc, and HDPCy-ds bound as a monolayer at the surface of polystyrene particles. A comparison of the slopes of these lines shows that a decrease in the value of the T1 relaxation time accompanies the progress of the supramolecular organization of the surfactants from monomer to micelle to monolayer. The difference in the 2H NMR TIrelaxation time for DDPC-y-d, monomers versus micelles may be exploited to determine the surfactant cmc. Figure 4 shows the dependence of the TIrelaxation time on the concentration of DDPC-y-ds for a range of concentrations spanning the cmc. Below the cmc the TIrelaxation time equalled
c
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Figure 4. Determination of the cmc of DDPC from 2HNMR TIrelaxation time measurements. The TIrelaxation time of DDPC-y-ds in aqueous solution is plotted as a function of the log of the DDPC concentration. The solid line indicates the change in the mole fraction of surfactant present in micellar form as a function of DDPC concentration,and was determined as described in the text. The cmc corresponds to the concentration at which 50% of the surfactant exists in micellar form. approximately 200 ms, while above the cmc the TI relaxation time equalled approximately 100 ms. In the region of the cmc the TIrelaxation time changed smoothly between the two extreme values as a function of concentration. From the midpoint of the inflection we estimate that the cmc of D D p c - 7 - d ~in water at 25 "C equals 1.4 X 10-3 M, a value which is in excellent agreement with that determined previously for this surfactant using a fluorescent probe t e ~ h n i q u e . ~ The homologous n-alkylphosphocholines have cmc values which decrease by a factor of 10 for each additional pair of methylene segments added to the alkyl chain.3 Because of the need for extensive signal averaging a t low concentrations, 2HNMR TIrelaxation time measurements therefore become impractical as a means of determining the cmc of such species as TDPC and HDPC. However, we did compare the 21' relaxation time for allthree available homologous n-alkylphosphocholines at a concentration above the cmc of all three homologues (1X M). These were found to equal 94 ms (DDPC), 80 ms (TDPC), and 68 ms (HDPC).
Kuebler and Macdonald
400 Langmuir, Vol. 8, No. 2, 1992
.\ ,
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Figure 5. Arrhenius plot of the dependenceof the TIrelaxation time of “surface-bound”HDPC-yds on temperature (squares). Shown as well is the dependence of the *HNMR quadrupole splitting on temperature (circles).
the correlation time in eq 2 represents an integral over a correlation function, rather than a characteristic time scale for any one particular motion. Thus, different types of motion will influence the observed spin-lattice relaxation rate in proportion to their contribution to a spectral density function evaluated at the Larmor frequency and at twice the Larmor frequency. In the following, we will attempt to rationalize our deuterium spin-lattice relaxation data by considering the different types of motion experienced by the phosphocholine surfactants, and by evaluating their individual correlation times in the different physical environments to which the surfactants have been subjected. Below the cmc surfactant monomers undergo rapid isotropic tumbling of the entire molecule, in addition to rapid internal bond rotations and conformational flexing. The latter motions are expected to occur with correlation times in the picosecond range. A correlation time for molecular tumbling may be estimated using the Debye relation (3) where R is the radius of the particle under consideration, q is the solution viscosity, and kT is the Boltzmann temperature. For a surfactant monomer in solution an effective hydrodynamic radius may be estimated using the Stokes-Einstein relation T~
In contrast to the rapid isotropic motional averaging experienced by surfactant monomers and micelles in solution, HDPC bound to polystyrene particles experiences predominantly anisotropic motional averaging. Hence, one obtains the axially symmetric Pake pattern shown in Figure 1. The quadrupole splitting corresponds to the separation (Hz) between the two maxima in the Pake pattern and equalled 575 Hz at 25 “C. Between 40 and 15 “C the quadrupole splitting increases from 530 to 777 Hz, indicating increasing motional hindrance with decreasing temperature as shown in Figure 5. The T1 relaxation time of surface-bound H D p c - 7 - d ~deuterons was 40 ms at room temperature, which is significantly shorter than that of n-alkylphosphocholine monomers or micelles. The temperature dependence of the TI relaxation time of surface-bound H D p c - 7 - d ~is shown in Figure 5 in the form of an Arrhenius plot. Between the temperatures of 15 and 40 “C the T1 relaxation time increased from 31 to 59 ms. The fact that for the surface-bound H D p c - 7 - d ~ the TI relaxation time increased with increasing temperature indicates that the motions contributing to the relaxation fall in the so-called fast limit regime. From the slope of the Arrhenius plot, one estimates an activation energy for these motions of approximately 20 kJ/mol.
Discussion NMR relaxation time measurements provide a nonperturbing means of probing molecular dynamics. 2H NMR is particularly advantageous because the signals arise from a specified location in the molecule, and because the relaxation behavior is dominated by a single mechanism, the quadrupolar interaction. Moreover, 2H NMR is sensitive to a wide range of time scales of motion. For small molecules in nonviscous solution the motional correlation times usually fall into the fast correlation time limit (w,,T,
