Vesicles from Salt-Free Cationic and Anionic Surfactant Solutions

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Langmuir 2003, 19, 10635-10640

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Articles Vesicles from Salt-Free Cationic and Anionic Surfactant Solutions Jingcheng Hao,*,†,‡ Weimin Liu,‡ Guiying Xu,† and Liqiang Zheng† Key Laboratory of Colloid and Interface Chemistry (Shandong University), Ministry of Education, Jinan 250100, People’s Republic of China, and State Key Laboratory of Solid Lubrication, Lanzhou Institute of Chemical Physics, Chinese Academy of Sciences, Lanzhou 730000, People’s Republic of China Received February 20, 2003. In Final Form: September 22, 2003

Vesicles from salt-free cationic and anionic (catanionic) surfactant aqueous solutions were prepared. The phase behavior and the phase transition in aqueous solutions of 100 mM tetradecyltrimethylammonium hydroxide (TTAOH) with fatty acids (decanoic acid (DA), lauric acid (LA), myristic acid (MA), and palmitic acid (PA)) were investigated. In this case, the solutions do not contain excess salts because the counterions H+ and OH- can form water. For the four systems of TTAOH/DA/H2O, TTAOH/LA/H2O, TTAOH/MA/H2O, and TTAOH/PA/H2O, one finds with increasing concentration of fatty acid a low viscous L1 phase, a viscous L1 phase, a L1/LR phase in which the birefringent LR phase is on the top of the viscous L1 phase, and finally a more or less transparent viscoelastic LR phase with the typical feature of unilamellar and multilamellar vesicles. The microstructures and the rheological properties of the unilamellar and multilamellar vesicles were determined by using freeze-fracture transmission electron microscopy and rheological measurements. Both unilamellar and multilamellar vesicles coexist in the birefringent LR phase. The unilamellar vesicles have diameters ranging from about 30 nm to more than 200 nm, and the multilamellar vesicles have diameters about 250 nm but are relatively rare. The complex viscosity (|η*|) with 100 mM TTACn (n ) 10, 12, 14, and 16) at a frequency (ν ) 0.01 Hz) was found to be increasingly linear with the carbon number of the fatty acids.

Introduction al.1

reported the spontaneous vesicle Since Kaler et formation in aqueous mixtures of single-tailed cationic and anionic surfactants, a lot of work about the vesicle phases that occur in cationic-anionic surfactant mixtures has been done.2-7 When a cationic surfactant solution and anionic surfactant solution are simply mixed, the strong reduction in area per headgroup resulting from ion paring induces formation of molecular bilayers at low concentrations. At the right mixing ratios, vesicles may be established spontaneously and are thermodynamically stable species.1,6,8 In the absence of electrostatic repulsion stabilizing cationic-anionic surfactant systems, in other words, one of the surfactants is present in a small excess, the cationic-anionic surfactant bilayers usually sponta* To whom corresponding should be addressed. Tel.: +86-5318366074(o). Fax: +86-531-8564750(o). E-mail: [email protected]. † Shandong University. ‡ Lanzhou Institute. (1) Kaler, E. W.; Murthy, A. K.; Rodriguez, B. E.; Zasadzinski, J. A. N. Science 1989, 245, 1371-1374. (2) Tomasic, V.; Popovic, S.; Vincekovic, N. F. J. Colloid Interface Sci. 1999, 215, 280-289. (3) Mao, M.; Huang, J.; Zhu, B.; Ye, J. J. Phys. Chem. B 2002, 106, 219-225. (4) Villeneuve, M.; Kaneshina, S.; Imae, T.; Aratono, M. Langmuir 1999, 15, 2029-2036. (5) Shioi, A.; Hatton, T. A. Langmuir 2002, 18, 7341-7348. (6) Marques, E.; Khan, A.; Miguel, M.da G.; Lindman, B. J. Phys. Chem. 1993, 97, 4729-4736. (7) Salkar, R. A.; Mukesh, D.; Samant, S. D.; Manohar, C. Langmuir 1998, 14, 3778-3782. (8) Bergstrom, M.; Pedersen, J. S.; Schurtenberger, P.; Egelhaaf, S. U. J. Phys. Chem. B 1999, 103, 9888-9897.

neously form closed vesicles.5-9 The cationic-anionic surfactant systems can produce a precipitate when the stoichiometry between the cationic and anionic surfactants is exactly 1.10 When the vesicles are prepared from the aqueous solutions of cationic-anionic surfactants, their surface ionic charge from the excess surfactant is shielded by the excess salt that is produced from the counterions of the two surfactants. This is very important for the understanding of the macroscopic properties of the vesicle phases. Charged vesicle phases without being shielded can be prepared by mixing small amounts of ionic surfactants to the L3 phase formed from a zwitterionic surfactant and cosurfactant.11 It is theoretically argued that the vesicles are formed in these systems by the influence of the charge density on the Gaussian bending constant.12 It was shown recently, however, that shearing forces that result from the mixing cationic-anionic aqueous solutions might also be responsible for the formation of the vesicles.10,11 Vesicles are not formed when the L3 phase (the L3 or sponge phase is a thermodynamically stable isotropic liquid consisting of a surfactant membrane in aqueous solution) was ionic charged without shear forces; instead a well-defined LR phase (the LR phase is a vesicle phase or a stacked bilayer phase) is produced.11 Very recently, flat nanodisks of finite (9) Bergstro¨m, M.; Pederson, J. S. Langmuir 1998, 14, 3754-3761. (10) Horbaschek, K.; Hoffmann, H.; Hao, J. J. Phys. Chem. B 2000, 104, 2781-2784. (11) Bergmeier, M.; Hoffmann, H.; Thunig, C. J. Phys. Chem. B 1997, 101, 5767-5771. (12) Jung, H. T.; Coldren, B.; Zasadzinski, J. A.; Iampietro, D.; Kaler, E. W. Proc. Natl. Acad. Sci. U.S.A. 2001, 98, 1353-1357.

10.1021/la030065q CCC: $25.00 © 2003 American Chemical Society Published on Web 11/22/2003

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size13 and regular hollow icosahedra14 in salt-free catanionic surfactant solutions were observed by using freezefracture transmission electron microscopy (FF-TEM). However, the unilamellar and multilamellar vesicles which coexist in salt-free cationic-anionic single-tailed surfactant aqueous mixtures of fatty acids and alkyltrimethylammonium hydroxide have not been completely prepared.15,16 Vesicle phases that are ionic charged and not shielded by the absence of the excess salt have interesting rheological properties. These vesicle phases are highly viscoelastic solutions and have a yield stress value. In the present work, we report the formation of vesicles from salt-free cationic-anionic surfactant concentrated solutions (g100 mM). To prepare the salt-free cationicanionic surfactant vesicles, we mixed fatty acids (decanoic acid, lauric acid, myristic acid, and palmitic acid) and the hydroxide-exchanged form (TTAOH, C14H29N+ (CH3)3OH-) of tetradecyltrimethylammonium bromide (TTABr). In this case, the solutions do not contain excess salt because of only the OH- and H+ counterions. In comparison to the situation with the excess salt NaBr where we had mixed TTABr and sodium laurate (SL),10 the phase sequence is much simpler and there is no precipitation at the equimolar concentrations for cationic and anionic surfactants. Experimental Section Chemicals. TTABr and SL were purchased from Fluka and recrystallized three times from the mixed solvent of ether/ethanol. TTAOH stock solution were prepared from TTABr solution (120 mM) by strong base anion exchanger (Ion exchanger III, Merck) at 40 °C, bromide ions cannot be detected by AgNO3 in the TTAOH stock solution (Ag+ + Br- f AgBrV), so the ion exchange with hydroxide is >99%. The critical micelle concentration, cmc, of TTAOH was determined by using surface tension measurements, and the cmc is 0.0018 mol‚L-1. Decanoic acid (DA), lauric acid (LA), myristic acid (MA), and palmitic acid (PA) were also from Fluka. The ionic impurities are sodium (0.1% molar fraction) and calcium (0.05% molar fraction). The four fatty acids are quasiinsoluble in water, however, they can be mixed with TTAOH solution into clear solutions during heating to 70 °C. Methods. The phase diagram of 100 mM TTAOH with the variable fatty acid concentrations (in the following text, we only show the phase diagram of TTAOH/lauric acid/H2O system) was established by observing the solutions in the test tubes at 25 °C. The samples were homogenized by mixing and heating to about 70 °C for a few minutes, and then the hot solutions were cooled to room temperature with permanent shaking during cooling. The solutions were allowed to equilibrate for at least 4 weeks at 25 °C. The solutions do not contain any trace of insoluble materials. The conductivity measurements were performed on a microprocessor conductivity meter, LF 2500 CON, from WTW (Germany). The rheological measurements were carried out with a Bohlin CS 10 stress-controlled rheometer using a cone plate measuring system and a double gap system. The lowest possible stress value amounts to 3 mPa. The viscoelastic properties of LR phase samples with 100 mM TTACn (n ) 10, 12, 14, and 16) were determined by the oscillatory measurements from 0.01 to 10 Hz, whereby alternatively the strain amplitude or the stress amplitude can be kept constant. The microstructure of the samples with the birefringence between the polarizers was examined by FF-TEM. A small (13) Zemb, Th.; Dubois, M.; Deme, B.; Gulik-Krzywick, Th. Science 1999, 283, 815-819. (14) Dubois, M.; Deme, Gulik-Krzywick, B.; Dedieu, J. C.; Vautrin, C.; Desert, S.; Perez, E.; Zemb, Th. Nature 2001, 411, 672-675. (15) Hao, J.; Hoffmann, H.; Horbaschek, K.J. Phys. Chem. B 2000, 104, 10144-10153. (16) Horbaschek, K.; Hoffmann, H.; Thunig, C. J. Colloid Interface Sci. 1998, 206, 439-456.

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Figure 1. Phase diagram of the mixtures of 100 mM TTAOH with 0.5 mM KCl and the variable lauric acid. The conductivity data (9) and the phase volumes (O) of the two phases with increasing amounts of LA at 25 °C are also included in the phase diagram. amount of sample (∼4 µL) was placed on a 0.1 mm thick copper disk covered with a second copper disk. The copper sandwich with the sample was frozen by plunging this sandwich into liquid propane which had been cooled by liquid nitrogen. For fracturing and replication, a freeze-fracture apparatus (Balzer BAF 400, Germany) was used at a temperature of -140 °C. Pt/C was deposited at an angle of 45°. The replicas were examined with a Zeiss CEM 902 transmission electron microscope operated at 80 kV. All the experiments were described in the following were done at 25 °C unless specified otherwise.

Results and Discussion Phase Behavior of the TTAOH/Lauric Acid/Water System. The sequence of phases of 100 mM TTAOH with 0.5 mM KCl with increasing amounts of LA at 25 °C was shown in Figure 1. The conductivities and the phase volumes of the two phases were also included in Figure 1. A 100 mM TTAOH micellar solution (L1 phase, consisting of small spherical and rodlike micelles) that had been prepared via an ion exchange procedure from TTABr is mixed with lauric acid up to the point of neutrality. Because of the low solubility of lauric acid in water at room temperature, the solutions mixed with lauric acid were heated to 70 °C for a few minutes and cooled to room temperature with permanent shaking during cooling. Then the samples were allowed to equilibrate for at least 4 weeks at 25 °C. In comparison to the phase diagram which we had obtained for mixed TTABr and SL,10 the phase diagram of the TTAOH/lauric acid/water system is much simpler. With the increase of lauric acid, one can observe a L1 phase, a viscous L1 phase, a two-phase L1/LR region in which the birefringent LR phase is on the top of a highly viscous L1 phase, and finally a birefringent and transparent viscoelastic LR phase with the typical feature of unilamellar and multilamellar vesicles which were determined by the following FF-TEM micrographs and the rheological measurements. The two-phase L1/LR region is between 0.89 and 0.94 of the molar ratios of TTAOH to LA (rTTAOH/LA). No precipitates were found at the equimolar concentrations of TTAOH and LA. From Figure 1, one should also note that the existence range of the vesicular phase only starts very close to the equimolar ratio of TTAOH to lauric acid with rTTAOH/LA g 0.94. When rTTAOH/LA g 0.105, the excess lauric acid cannot be solubilized by the TTAL solution, and a two-phase region of vesicles and insoluble

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Figure 2. Freeze-fracture TEM micrographs of two samples of 100 mM TTAL (a, the bar represents 0.14 µm) and 100 mM TTAP (b, the bar represents 0.35 µm). The vesicle solutions could be kept for more than 1 year without any change at 25 °C, showing that once formed, these vesicle phases are stable. The sample is in aqueous solution containing 20 wt % glycerol to avoid crystallization during the freeze process.

Figure 3. Phase behavior and phase transition of 50 mM TTAL LR phase with unilamellar and multilamellar vesicles in addition of TTABr or SL. The conductivity data (9) are also included.

lauric acid exists. Thus, we add the highest excess concentration of lauric acid up to 5 mM in 100 mM TTAOH solution. The conductivity data included in Figure 1 show that the conductivity of 100 mM TTAOH with 0.5 mM KCl solutions at an excess of TTAOH decreases with increasing LA concentrations, which indicates that the addition of LA to TTAOH solutions leads to the neutralization of OHby the proton of LA and thus to the exchange of OH- for L- as counterions of TTA+. The conductivity of 100 mM TTAL vesicular solution with 0.5 mM KCl amounts to about 18.6 µS‚cm-1, which is a very small value compared with the conductivity value of 100 mM TTAOH with 0.5 mM KCl (κ ) ∼7.5 mS‚cm-1). With 100 mM TTAL and excess lauric acid (cLA e 105 mM) solutions, conductivities with the order of 10 µS‚cm-1 are much lower than that of 0.5 mM KCl (κ0.5 mM KCl ) ∼0.1 mS‚cm-1). The conductivity data provide clear evidence for the formation of vesicles within the birefringent LR phase of TTAOH/lauric acid solutions. The unilamellar and multilamellar vesicles demonstrated by the following FF-TEM measurements

enclose a part of the solvent and the salt (here is K+ and Cl- ) and consequently present the enclosed charge carriers from contributing to the conductivity of the solutions. As a consequence the conductivity of the vesicular LR phase solutions may become lower than that of 0.5 mM KCl solution. The pH values vary from 12-13 at excess cationic TTAOH solutions (L1-phase and viscous L1-phase regions) to 7 at 100 mM TTAL vesicular solution, which is near equimolarity of cationic and anionic surfactants. When we change the chain of the fatty acid, i.e., C9H19COOH, C13H27COOH, and C15H31COOH, the phase diagrams have not been changed. However, the existence ranges of the vesicular phases of TTAOH/MA/H2O and TTAOH/PA/H2O systems start closer to the equimolar ratio of TTAOH to MA or TTAOH to PA than that of TTAOH/LA/H2O system. The vesicular solution of 100 mM TTAD is perfectly clear except in the LR phase. Microstructures of the Lr Phase. The birefringent and transparent viscoelastic LR phase solutions at the equimolar ratio of TTAOH to fatty acid (C9H19COOH, C11H23COOH, C13H27COOH, and C15H31COOH) contain

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Figure 4. Viscosity variation (b) for 100 mM TTAOH with increasing amounts of CH3(CH2)10COOH at 25 °C. The phase volumes (O) of the two phases are included in the phase diagram. There are significant changes in the viscosity at the phase transitions such as from globular to rodlike micelles and from rodlike micelles to vesicles.

unilamellar and multilamellar vesicles, which is demonstrated by FF-TEM micrographs. The typical FF-TEM micrographs of two LR phase samples with 100 mM TTAL and TTAP are shown in Figure 2. From the FF-TEM micrographs of the two samples, one can observe the features of the vesicles: both unilamellar and multilamellar vesicles coexist, primarily unilamellar vesicles form in the vesicular solutions, and these unilamellar vesicles have the rather polydisperse distribution. These unilamellar vesicles have diameters ranging from about 30 nm to more than 200 nm. The multilamellar

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vesicles are about 250 nm in size but are relatively rare. In comparison, the viscoelastic LR phase in the system of 100 mM cetyltrimethylammonium hydroxide (CTAOH) with 3-hydro-2-naphthoic acid (HNC) consists of the densely packed multilamellar vesicles.16 As demonstrated in the present experiments, we produced the viscoelastic unilamelaar and multilamellar vesicles from the concentrated salt-free cationic-anionic surfactant aqueous solutions (g100 mM). In the process of continuous work, we studied the extremely dilute saltfree cationic-anionic surfactant aqueous solutions by using FF-TEM micrographs. Equilibrium among unilamellar vesicles, multilamellar vesicles, and disks was observed. The disks have nearly monodisperse radii. Effect of TTAB or SL on TTAL Vesicle Phase. Figure 3 shows the phase behavior and the phase transition of the unilamellar and multilamellar vesicles with 50 mM TTAL in addition of TTAB and SL. The conductivities with the addition of TTAB and SL, respectively, are also included in Figure 3. When TTAB or SL was added to 50 mM TTAL vescoelastic vesicle solution, the unilamellar and multilamellar vesicles could be transferred into a two-phase L1/LR region, a viscous L1 phase, and then the L1 phase. The conductivities increase with the addition of TTAB or SL on both sides. Viscosity of the TTAOH/Lauric Acid/H2O System. The viscosity variation of the TTAOH/lauric acid/water system was illustrated in Figure 4 using the complex viscosity (|η*|) at a frequency ν ) 0.01 Hz. In Figure 4, the sequence of phases and the phase volumes of the two phases are also indicated. One can find that the solutions of 100 mM TTAOH mixed with LA have low viscosity values when LA concentrations are up to 70 mM. At an LA concentration higher than 75 mM, the viscosity

Figure 5. Rheograms for four vesicular LR phase solutions of 100 mM TTAD (a), 100 mM TTAL (b), 100 mM TTAM (c), and 100 mM TTAP (d). Plots of storage modulus G′, loss modulus G′′, and complex vesicosity |η*| as a function of the angular frequency are shown.

Vesicles from Salt-Free Surfactant Solutions

Figure 6. Complex viscosities (|η*| at ν ) 0.01 Hz) increase linearly as a function of carbon number in the fatty acids.

increase is particularly pronounced upon approaching the boundary of the L1 phase/viscous L1 phase. When 100 mM TTAOH was mixed with lauric acid at low concentrations, the low viscous CH3(CH2)13N+(CH3)3--OOC(CH2)10CH3 spherical micelles form. With increase of lauric acid concentrations, spherical micelles grow to viscous rodlike micelles and the viscosity obviously increases. The viscosity value reaches a maximum of 39 Pa‚s in the viscous L1 phase region. After LR phase is entered, the viscosity strongly increases because of the formation of unilamellar and multilamellar vesicles. The viscosity is much higher than that in the viscous L1 phase and has more or less a constant viscosity value, |η*| ) 536 Pa‚s or so, at ν ) 0.01 Hz. Rheological Properties of 100 mM TTACn (n ) 10, 12, 14, 16) Vesicular Phases. The results of the oscillatory measurements of 100 mM TTACn (n ) 10, 12, 14, 16) vesicular phases are shown in Figure 5. All rheograms of 100 mM TTACn (n ) 10, 12, 14, 16) show the characteristic features of viscoelastic vesicle solutions similar to other systems.15,16 The 100 mM TTAD and 100 mM TTAL are very clear solutions, however, 100 mM TTAM and 100 mM TTAP are slightly turbid solutions. All rheograms in Figure 5 show the characteristic features of unilamellar vesicles and multilamellar vesicles, which are demonstrated by FF-TEM micrographs in Figure 2. The complex viscosity, |η*|, decreases over the whole frequency range from 0.01 to 10 Hz with a slope of -1. The storage modulus (G′) and loss modulus (G′′) are almost frequency independent of frequency. The storage modulus

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(G′) has a value of 10 Pa for 100 mM TTAD, 30 Pa for 100 mM TTAL, 80 Pa for 100 mM TTAM, and 100 Pa for 100mM TTAP. These values of the storage modulus (G′) are almost about a decade higher than those of the loss modulus (G′′), which are similar to the other vesicle systems.15,16 The viscosity change of 100 mM TTACn (n ) 10, 12, 14, 16) solutions with different chain fatty acid is illustrated in Figure 6 using the complex viscosity at a frequency ν ) 0.01 Hz. From Figure 6, it is found that the complex viscosities, |η*|, increase linearly as a function of carbon number in the fatty acids. The yield stress values of the viscoelastic LR phase samples of 100 mM TTACn (n ) 10, 12, 14, 16) are large enough to suspend small air bubble in the solutions, which can easily be recognized by the fact that much small air bubbles that are dispersed in the four samples do not rise. Thus, these fluids with large yield stress values behave like the Bingham fluids. Ideally these viscoelastic vesicle solutions can be considered as Bingham bodies. In the case of a Bingham body, the shear stress has a critical value, P21,critical, which is the lowest shear stress necessary to produce viscous flow:

P21 ) P21,critical + ηBγ21 For P21 < P21,critical, the system behaves according to Hook’s law, that is to say, elastically. If P21 > P21, critical, plastic deformations occur and the system begins to flow. To determine the yield stress value, P21,critical, the deformation of the vesicular solutions was measured in dependence of the applied stress for two different intervals of time. For example, the yield stress values of 100 mM TTAD and 100 mM TTAM at T ) 25 °C were measured and are shown in Figure 7. The yield stress values were determined to be 0.6 Pa for 100 mM TTAD and 0.8 Pa for 100 mM TTAM, respectively. The yield stress values show the deformation of the viscoelastic LR phase solutions, and so it is already quite high for these viscoelastic LR phase solutions formed by TTAOH and fatty acids. Comparison between TTAOH/Lauric Acid/Water System and TTABr/SL/Water System. The LR phase of the TTABr/SL/water system is a very low viscous somewhat turbid solution, and the LR phase contains vesicles that are demonstrated in ref 10. However, the LR phase of the TTAOH/lauric acid/water system is a very viscous solution and the phase contains unilamellar and multilamellar vesicles.

Figure 7. Plot of the yield stress against the shear rate to demonstrate the yield value with 100 mM TTAD (a) and 100 mM TTAM (b) at 25 °C.

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The rheological properties of the birefringent LR phase of the TTABr/SL/water system are quite different from those of the vesicular phase in the TTAOH/lauric acid/ water system. In the TTABr/SL/water system, the complex viscosities, |η*|, decrease over the whole frequency range, however, the complex viscosity is about 10 decades lower than that in the case of the 100 mM TTAOH/lauric acid/ water system. The reason is that in the case of TTABr/ SL/water (55 mM/45 mM), the vesicles are strongly charged by the excess of 10 mM TTABr, but the charge density of the vesicles is shielded by the excess salt NaBr. Consequently storage modulus (G′) and complex viscosity (|η*|) are low. In the case of the TTAOH/lauric acid/water system, however, the vesicles are charged to some extent by the dissociation of C11H23COO-, but there is no excess salt NaBr to shield this charge density. Therefore storage modulus (G′) and complex viscosity (|η*|) are increased by 10 decades in comparison with the samples of TTABr/ SL/water. Rheological properties of 100 mM TTACn (n ) 10, 12, 14, 16) vesicular phases behave in the same way as 100 mM CTAHNC, which consists of the densely packed multilamellar vesicles.16 These differences are the result of the different ionic strength in the TTABr/SL/water system and TTAOH/ lauric acid/water system. In the TTABr/SL system, the excess salt NaBr shields the charges of the mixed surfactants, which makes the system more lipophilic. The repulsive interaction disappears and the bilayer collapses to a dense precipitate because the charges are shielded in the presence of the salt NaBr. However, in the salt-free system, there are some surface ionic charges remaining

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on the layer of the vesicles that is due to dissociation of the TTAL molecules and the charges are not shielded. To compare the viscoelastic unilamellar and multilamellar vesicles from the concentrated salt-free cationic/ anionic surfactant aqueous solutions (g100 mM), in process of the continuous work we studied the extremely dilute salt-free cationic-anionic surfactant aqueous solutions by using FF-TEM micrographs. We observed the equilibrium among unilamellar vesicles, multilamellar vesicles, and disks from the dilute salt-free cationic/anionic surfactant aqueous solutions. Such equilibrium with the nearly monodisperse radii of disks is a very rare system, which is only possible for the system with a spontaneous curvature and the positive Gaussian bending constants.13,17 The interesting experimental results could be reported shortly. Acknowledgment. J. Hao gratefully acknowledges support of this work by the NFSC (20243007), by the Program of Hundreds of Talents of the Chinese Academy of Sciences, by the Alexander von Humboldt-Foundation because Dr. Hao was an AvH Fellow guided by Professor Dr. Heinz Hoffmann at Bayreuth University (Germany), and by Shandong University. W. Liu acknowledges support by the NSFC (50275142) and by MST (2202AA302609). LA030065Q (17) Jung, H. T.; Lee, S. Y.; Kaler, E. W.; Coldrem, B.; Zasadzinski, J. A. Proc. Natl. Acad. Sci. U.S.A. 2002, 99, 15318-15322.